Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
q STREPTOCOCCUS PNEUMONIAE CAPSULAR
POLYSAC~A~TnE GENES AND FLANKING REGIONS
BACKGROUND OF THE lNV~N-llON
The present application is a continuation-in-part of
co-pending U.S. Patent Application Serial No. 08/243,546,
filed May 16, 1994. The entire text and figures of which
disclosure is specifically incorporated herein by
reference without disclaimer. The government owns
certain rights in the present invention pursuant to grant
number AI28457 from the Public Health Service and
T32 AI07041-13 from the National Institutes of Health.
1. Field o~ the Invention
The present invention relates generally to the
~ields of bacterial capsule formation and the genes
responsible for polysaccharide synthesis. More
particularly, it concerns the genes and gene products
that direct the formation of the Streptococcus pneumoniae
serotype-specific polysaccharide capsule. The present
invention also includes the identification of non-type
specific gene sequences, flanking the capsule genes, and
their use for the directed expression of specific
serotypes of S. pneumoniae capsules.
2. De~cri~tion of the Related Art
Infections due to S. pneumoniae are among the top
ten causes of death in the United States. The normal
populations most affected are young children and the
elderly: pneumococcal pneumoniae, mainly affecting the
elderly, causes ~40,000 deaths per year among ~500,000
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cases and represents 60 to 80~ of all bacterial
pneumoniae; pneumococcal meningitis, with ~4000
cases/year, represents 11~ of the total meningitis cases
and has a fatality rate of ~30~ - greater than twice that
of the two other leading causes, N. m~n; ngi tidis and H.
influenzae; bacteremia, usually following pneumoniae or
meningitis, accounts for ~35,000 cases per year (~30%
fatal); and otitis media, the most frequent reason for
pediatric office visits after well-child care, is caused
by S. pneumoniae in ~50~ of cases (ACIP, 1981; ACIP,
1989; Austrian, 1984; Burke et al., 1971; Center for
Disease Control, 1978; Johnston and Sell, 1964; Koch and
Dennison, 1974).
Other populations have an even higher incidence of
pneumococcal infections: approximately 30~ of sickle cell
children will have severe pneumococcal infections in the
first three years of life and -35~ of those will die
(Overturf, et al., 1977; Powars, et al., 1981; Powars,
1975); in both adults and children with HIV infections,
5. pneumoniae is the major cause of invasive bacterial
respiratory disease (Janoff et al., 1992). Patients with
lymphomas, Hodgkins disease, multiple myeloma,
splenectomy, and other debilitating diseases or
immunologic deficiencies, are particularly susceptible to
serious pneumococcal disease, as are those with chronic
illnesses such as diabetes mellitus and heart disease.
Furthermore, strains of S. pneumoniae are emerging that
harbor resistances to multiple antibiotics, including
penicillin (Appelbaum, 1992; Jacobs et al., 1978;
Landesman et al., 1982).
The polysaccharide capsule of S. pneumoniae is the P
major virulence determinant of this organism. Despite
early studies of the genetics, pathogenesis, and
immunology of capsular polysaccharides, it r~m~; n.~
unclear why certain capsular types appear to have a
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greater capacity to cause disease. Of the more than 80
known capsular serotypes, 23 account for more than 90~ of
all pneumococcal infections.
In children, the most prevalent types are 3, 6, 14,
19, and 23, (Gray and Dillon, 1986), whereas in adults
typeæ 1, 3, 4, 6, 7, 8, 9, 12, 14, 18, 19, 23 prevail
(Finland and Barnes, 1977). In assays of opsono-
phagocytosis (Branconier and Odeberg, 1982; Giebink et
al., 1977; Knecht et al., 1970), complement activation
and deposition (Fine, 1975; Gordon et al., 1986;
Hostetter, 1986; Stephens et al., 1977; Winkelstein et
al., 1980; Winkelstein et al., 1976), and mouse virulence
(Briles e~ al., 1992; Briles et al., 1986; Knecht et al.,
1970; Mac~eod, 1965; Walter et al., 1941; Yother et al.,
1982), levels of virulence have frequently been found to
vary with the type of capsule expressed. For example,
isolates expressing type 3, 4, and 19 capsules are highly
resistant to phagocytosis, whereas those expressing types
6A, 14, 23 and 37 are significantly less resistant
(Branconier and Odeberg, 1982; Hostetter, 1986; Knecht et
al., 1970; Wood and Smith, 1949).
The importance of the capsule also results from the
fact that anti-capsular antibodies are highly protective
against infection. Nonetheless, the current
polysaccharide-based vaccine is not particularly useful
in some of the populations most affected by pneumococcal
disease, e.g., the very young and the elderly, because of
poor or absent immune response to polysaccharide
antigens.
The ability to produce improved vaccines and
therapies for pneumococcal infections will most likely be
the result of a better understanding of the basic
pathogenic mechanisms of the organism. This understanding
necessarily includes the genetic basis for the expression
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of serotype-specific polysaccharides and the role of
capsular type per se in pathogenesis.
Some 85 different serotypes of Streptococcus
pneumoniae, differing in the structure of the
polysaccharide produced, have been identified (van Dam et
al., 1990). The basis for the emergence of new capsule
types r~; n~ obscure. Whether influenced by mutation,
recombination, or immune selection, genetic exchange of
DNA is likely to have played a major role in the
evolution of capsule types. It is known that
pneumococcal capsule types can be changed through genetic
transformation in vitro (Dawson, 1930; Dawson and Sia,
1931; Langvad-Nielson, 1944; Avery et al., 1944).
Epidemiological studies suggest that a significant degree
of genetic exchange occurs in vivo (Crain et al., 1990;
Coffey et al., 1991; Versalovic et al., 1993). However,
the mechanism by which capsule types are exchanged is not
fuily understood.
Extensive study was made of the genetics of capsular
polysaccharide synthesis in S. pneumoniae using
spontaneous mutants with defects in biosynthetic
functions (Effrussi-Taylor, 1951; Ravin, 1960; Bernhei~er
and Wermundsen; 1972). The results of these studies
indicated that the genes for polysaccharide synthesis
were closely linked and could be transferred as a unit
during genetic transformation. A cassette-type model of
capsule type change based on this data has been proposed
(Taylor, 1949; Austrian et al., 1959; Bernheimer and
Wermundsen, 1972). According to the model, the
type-specific genes for each capsule type would be
present only in the genome of a strain of that capsule
type and would show little homology to the type-specific
genes of other capsule types. The type-specific genes
would be located in homologous sites in the different
chromosomes, clustered together between regions of highly
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homologous flanking DNA. During transformation,
recombination would occur in the flanking regions,
resulting in the replacement of the recipient's
type-specific region by that of the donor.
The clustering of capsule biosynthetic genes
proposed by the model is analogous to the organization
that has been observed in the gram negative bacteria
Escherichia coli (K antigens) (Roberts et al., 1988),
Neisseria meni~gitidis (Frosch et al., 1989), and
Haemophilus influenzae (Kroll et al., 1989). For each of
these organisms, the type-specific region encoding
biosynthetic functions (region 2) is flanked by highly
homologous regions necessary for polysaccharide
translocation (region 1) and modification (region 3).
Since H. influenzae, like S. pneumoniae, is naturally
transformable, it has been proposed that capsule type
change in this pathogen may occur by transformation with
the type-specific gene cluster from a different serotype
(Zwahlen et al., 1989).
The one exception to the cassette model of capsule
type change in S. pneumoniae is binary capsule formation.
When non-encapsulated mutants have been transformed with
chromosomal DNA from a strain of a different capsule
type, most of the encapsulated transformants express the
capsule type of the donor. However, at a frequency 10 to
100 times lower, encapsulated transformants are obtained
which express both capsules (Bernheimer and Wermundsen,
1972). In some of these transformants, the second set of
capsule genes is closely linked to the original set.
However, these strains are unstable, and, at high
frequency, lose the ability to produce the original
capsule type. In binary strains in which the acquired
capsule genes are unlinked to the original genes, binary
capsule production is stable (Bernheimer and Wermundsen,
1969). Elucidation of the mechanism of binary capsule
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type formation may be the key to understanding novel
capsule type creation in S. pneumoniae.
It is clear that a better understanding of the
genetics of capsular polysaccharide synthesis in
Streptococcus pneumoniae is needed. The identification
of type-specific capsular genes and the ability to
transfer them, singly or as a gene cassette, to desired
recipients, will elucidate the role of capsular types in
virulence and allow easy identification of S. pneumoniae
serotype. This ability will improve existing methods of
diagnosis, identifying not only the presence of S.
pneumoniae but also the capsular type of the invading
strain. Furthermore, it will allow construction of
strains producing elevated levels of capsular
polysaccharides for improved vaccines.
SUMMARY OF THE lNv~NllON
Cap~ular Poly~accharide Genes and Fl ~nk; n~ Region~
The present invention arises out of the discovery
and sequence characterization of a gene family that
confers on S. pneumoniae the ability to produce type-
specific capsules that define the serotype of theorganism. The inventors refer to this gene family as the
capsule synthesis or cps genes. These genes encode the
various enzymatic functions of capsule synthesis and
determine the particular structure of the capsule
polysaccharide that is produced, and thereby define
serotype. These genes, designated cpsB, cpsC, cpsE,
cpsD, cpsS, cpsU, cpsM, tnpA, and plpA, map to specific
DNA segments of sizes believed to range from about 0.5 kb
to greater than 10 kb that appear to be type-specific for
3 5 S. pneumoniae. Based upon the findings of the inventors,
many type-specific genes may be distinguished on the
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basis of restriction fragment length polymorphism (RFLP)
analysis.
The present invention also includes the discovery
and sequence characterization of non-type specific DNA
regions that flank both sides of the cps locus. These
flanking DNA segments can be used to identify the
location of cps flanking DNA from any strain of S.
pneumoniae. This invention thus provides the ability to
identify the cps locus within all strains and allows for
the subsequent isolation and characterization of all
genetic elements involved in determining S. pneumoniae
serotype.
The classification of S. pneumoniae strains is based
on serological analysis of cell surface structures. 85
distinct serotypes have been identified to date based on
the formation of surface molecules. The formation of the
cell surface of S. pneumoniae, and in particular its
polysaccharide capsule, has, until now, eluded
characterization at the molecular genetic level.
However, studies of the biosynthesis of the
polysaccharide capsule have revealed that at least some
of the genes are likely to include enzymes involved in
the preparation of the sugar backbones for incorporation
into the saccharide backbone, such as UDP-glucose
dehydrogenase.
As mentioned above, these polymorphic "type-
specific" sequence regions were found to be bounded or
flanked by "non-type specific" regions having sequence
elements that are apparently shared among the various
subtypes. These regions, referred to as the left and
right flanking regions, extend for, at least, 1 to 3 kb
on either side of the cps genes. Thus, in type 3, the
entire length of the capsule synthesis genes, including
the non-type specific flanking regions, and any DNA
=
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sequences in between, is greater than 9 kb. In other
capsule types the length is on the order of about 5 to 20
kb, with the maximum length being related to the
complexity of the polysaccharide encoded. Importantly,
it is these flanking regions that allow recombination and
integration o~ the type specific capsule genes to occur.
Thus, when a selected cps gene or genes is positioned
between the flanking regions, the resultant construct can
be stably integrated into a S. pneumoniae host.
The present discoveries concerning the cps gene
regions, the identification of conserved flanking
regions, and the construction of erythromycin resistant
insertions in adjacent, non-type specific DNA elements,
allows for the changing of capsular serotypes by
"cassetting-in" the biosynthetic genes for different
serotypes. This methodology may be employed for
generating high yield capsular polysaccharide producing
strains of different (heterologous) serotypes. ~or a
high yielding strain, the existing serotype biosynthetic
genes may be deleted and a different serotype's genes
inserted ("cassetted in"). These other serotypes may
come from strains where their natural genetic background
gives only poor or moderate levels of capsular
production.
As used herein, the term "gene cassette" or simply
"cassette", is intended to refer to any DNA segment
flanked by one, or both, or part of, the cps flanking
regions or a cps genetic element or DNA sequence which is
found between the flanking regions.
Prior to the present invention, the foregoing
underlying mechanism of genetic recombination of the
capsule synthesis genes was unknown, as were the specific
sequences'involved. A principal contribution of the
present inventors is the specific characterization o~ the
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individual genes and flanking regions, allowing for their
manipulation and individual transfer to hosts. Now,
discrete nucleic acid segments, or cassettes, containing
a cps flanking region, gene or genes, can be readily
prepared and easily used in transformation.
Hybridization Probes and Primer~
Accordingly, in certain embodiments the invention
concerns nucleic acid segments that hybridize with cps
genes and/or flanking regions. The nucleic acid segments
will generally be less than about 20 kb in length, and
preferably less than about 15 kb in length, or even 10
kb, and will comprise a non-type specific S. pneumoniae
15 cps gene flanking region, and/or a type-specific cps
gene, of sufficient length to allow hybridization with a
pneumococcal cps flanking region and/or gene. Nucleic
acid segments that are capable of hybridizing with the 5'
flanking region, the 3' flanking region, to both flanking
regions, to one or more of the genes designated cpsB,
cpsC, cpsE, cpsD, cpsS, cpsU, cpsM, tnpA and 'plpA, and
to one or more genes in combination with one or more
flanking regions, are encompassed by the invention.
Nucleic acid segments that include a first sequence
portion capable of hybridizing to the 5' cps gene
flanking region and a second sequence portion capable of
hybridizing with the 3' cps gene flanking region form one
aspect of the invention. Such nucleic acid segments may
be combined with one or more cps genes and may be
constructed to form a genetic unit in which the gene (or
genes) is located between the two flanking regions. The
gene(s) may be from a different cps serotype to the
flanking regions or they may be from the same cps
serotype to the flanking regions. Such genetic units may
be termed cassettes and may also encompasses the form of
a circular DNA segment, plasmid, cosmid, or phage. An
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-- 10
isolated fragment of DNA containing DNA such as might be
generated by restriction digestion, ligation, and/or PCR
methodologies would also be included. The nucleic acid
segment, or cassette, may also include other regions of
DNA, such as restriction or cloning sites, PCR primers,
promoters, antibiotic resistance genes, and the like, as
necessary or desired to make a functional genetic unit.
In order to have utility in connection with the
present invention, all that is required is that such a
nucleic acid segment or genetic unit include a region of
sufficient complementarity and size to allow selective
cross-hybridization with the target flanking region or
gene sequence.
In general, shorter and intermediate length nucleic
acid fragments will be useful as hybridization probes and
primers, and in particular, for use in PCR, where the
primers will allow generation of the entire intervening
cps sequence.
Thus flanking region and gene fragments on the order
of at least about 14-15, 20, 30, 40, 50, or 100 to 200,
contiguous complementary nucleotides are contemplated,
although sequences of 500, 1,000, or more, nucleotides in
length may also be used. The DNA segments may, of
course, be of any length within the stated ranges. This
is the me~n; ng of "about" in this context, with "about"
meaning a range longer or shorter than the stated length,
extending to the previously quoted longer and shorter
lengths (with about 14 or so still being the minimum
length). The ranges thus encompass 1 to 4, 1 to 9, 1 to
49, 1 to 99, and the like, nucleotides in length.
Longer nucleic acid segments and fragments having on
the order of up to 1,000, 2,000, 3,000, 5,000, 10,000,
15,000 or longer in length will also have particular
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utilltles in addition to their functlon in hybridization
embodiments. In particular, longer nucleic acid segments
and fragments including selected cps coding sequences may
be employed in the expression of recombinant proteins,
and nucleic acid segments that include one or more cps
gene sequences positioned between the flanking regions
(cassette constructs) may be used in gene transfer
embodiments as described above. The DNA segments may, of
course, be of any length within the ranges stated above,
so long as they function to achieve the desired ef~ect.
"About" in this context therefore indicates ranges of
from 1 to 999, or 1 to 4,999, and the like, nucleotides
longer or shorter than the stated length.
Nucleic Acid and Amino Acid Sequences
Exemplary flanking regions sequences, as disclosed
herein, are set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ ID
NO:3, SEQ ID NO:4 and SEQ ID l~0:6 (FIG. 7 and FIG. 8).
The 5' Cp5 gene flanking region is represented by SEQ ID
NO:l, SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO: 4. SEQ ID
NO:l, SEQ ID NO:2 and SEQ ID N0:3 corresponds to regions
of DNA sequenced in the upstream portion of the 5'
flanking region and SEQ ID NO:4 corresponds to the
downstream portion of the 5' flanking region and is
termed the "repeat" region. DNA between SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO:4 are also part of
the cps 5' flanking region. SEQ ID NO:1 is 300
nucleotides in length and corresponds to the last 180
nucleotides of cpsB and the 5' end of cpsC which begins
immediately (FIG. 6A and FIG. 7). SEQ ID NO:2 is 261
nucleotides in length and corresponds to a 3' end region
of cpsC (FIG. 6B and FIG. 7). SEQ ID NO:3 iS 262
nucleotides in length and corresponds to part of cpsE and
the 5' end of the repeat region (FIG. 6C and FIG. 7).
SEQ ID NO: 4 is 934 nucleotides in length, (nucleotide 1
through 934, FIG. 6Di, FIG. 6Dii and FIG. 8). The 3' cps
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gene flanking region is 1307 nucleotides in length, l
through 1307, SEQ ID NO:6 (nucleotide 5886 through 7192,
FIG. 6Ii, FIG. 6Iii, FIG. 6Iiii and FIG. 8).
The inventors have found that certain S. pneu~cniae
nucleotide sequences described in the scientific
literature correspond to stretches of sequences fror the
flanking regions of the present invention. For exam~le,
Guidolin et al. (1994) have sequenced 6, 322 base pairs of
the l9F S. pneumoniae serotype cps locus. Sequence
analysis indicated that this region contained six
complete open reading frames and one partial, which they
named cpsl9fA to cpsl9fG. Southern hybridization
revealed that cpsl9fA and cpsl9fB were conserved in 12
other S. pneumoniae serotypes tested, including serotype
3. cpsl9fC and cpsl9fD also hybridized to Type 3 S.
pneumoniae. The sequences for cpsB, cpsC and cpsE ~SEQ
ID NO:l, SEQ ID NO:2 and SEQ ID NO:3), as disclosed
herein, are about 99~ identical to cpsl9fB, cpsl9fC and
cpsl9fD respectively (Guidolin et al. 1994). However,
cpsE is truncated at the 3' end with respect to the l9F
gene (lacks ~280 nt). The site of the truncation is
adjacent to the region designated as the "repeat"
sequence ( SEQ ID NO: 4). Based on blotting data (see
Example 17) part of the repetitive element is in SEQ ID
NO:3. This sequence extends 132 nt upstream of the SacI
site at the start of SEQ ID NO: 4, as shown in FIG. 7.
Although Guidolin et al. (1994) have sequenced this area
in serotype l9f, they do not identify this sequence as
being a gene flanking region and do not suggest its use
as part of an S. pneumoniae capsular cassette.
Garcia et al. (1993) localized a 781 bp EcoRV
subfragment of a gene (cap3-1) that they proposed was
involved in the transformation to a capsulated phenotype.
The first 52 nucleotides of the 781 Garcia et al.
sequence correspond to nucleotides 883 to 934 of SEQ ID
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N0:4. The next 443 nucleotides of the Garcia et al.
sequence corresponds to nucleotides 1 to 443 o~ SEQ ID
N0:5. However, the r~ n'ng 286 nucleotides of the 78i
bp sequence do not show any homology with the instan~_
sequences. As wlth the above, this article does not
identify this sequence as being a gene flanking region
and do not suggest its use as part of an S. pneumoniae
capsular cassette. However, the same group (Arrecubleta
et al., 1994) using probes in Southern blotting,
identified the upstream region of their cap3A as a
sequence common to all pneumoniae strains tested.
The amino acid sequences described by Garcia et al.
(1993) include a putative protein of 138 amino acids
(CAP3-1), transcribed in the same direction as Cps~, and
another truncated open reading frame, transcribed ir the
opposite direction. The first 117 amino acid residues o~
CAP3-1 corresponds to the first 117 residues of CpsD (SEQ
ID NO:11) CAP3-1 is reportedly similar to the amino-
terminus o~ GDP-mannose dehydrogenase o~ Pseudomonas
aeruginosa. The r~m~; n; ng 21 residues of CAP3-1
described by Garcia et al. (1993) have no homology ~ith
the remaining 277 amino acid residues of CpsD, suggesting
that Garcia et al. had cloned a truncated gene in wnich
the 5' end of the cpsD gene was aberrantly fused to an
unidentified DNA sequence.
Pearce et al. (1994) described an S. pneumoniae
protein and corresponding nucleotide sequence, that they
proposed was a permease-like protein involved in peptide
transport. They termed this protein PlpA. Nucleotides
484 to 1307 of SEQ ID N0:6 correspond to nucleotides 843
to 1672 of the plpA described by Pearce et al. (199~),
and the amino acid SEQ ID N0:15 corresponds to resi~ues
282 to 557 of their PlpA. Previously, Pearce et al.
(1993) had described an S. pneumoniae protein they called
Expl. The amino acid sequence of Expl corresponds to
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-- 14
residues 49 through 209 of SEQ ID NO:15. The inventors
have found that the ~plpA located adjacent to the type 3-
specific genes lacks about one third of its 5' end when
compared to plpA genes located adjacent of the capsule
genes of other types such as that used by Pearce et al.
(1994). Neither Pearce et al. (1994) nor Pearce et al.
(1993) identify this sequence as being involved in
capsule synthesis in any way, nor do they suggest that it
forms part of a common DNA flanking region.
Although certain stretches of nucleotide sequences
may have been known in the art, their function,
relationship to capsule synthesis and, particularly,
their role as interchangeable flanking regions has rot
previously been described. An important feature of the
invention is that the functional characterization o_ the
flanking regions allows, for the first time, for the
exchange of S. pneumoniae type-specific capsule genes to
be manipulated and controlled. This is only possible in
light of the inventors discovery of the conserved cps
gene flanking regions. Nucleic acid segments, including
cassettes and plasmids, that include both 5' flanking
region sequences and 3' flanking region sequences are
thus one preferred embodiment of the invention. PCR
primers that have sequences corresponding to both
flanking regions form another preferred embodiment of the
invention.
Encoded within the upstream 5' flanking region (SEQ
ID NO:1, SEQ ID NO:2 and SEQ ID NO:3) are the partially
sequenced genes cpsB, cpsC and cpsE which encode for CpsB
(SEQ ID NO:7), CpsC (SEQ ID NO:8 and SEQ ID NO:9) and
CpsE (SEQ ID NO:10) (FIG. 6A, FIG. 6B, FIG. 6C and FIG.
7).
The invention also includes other cps gene
sequences, either alone or in combination with the
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flanking region sequences described above. The type-
specific portions of the polycistronic cps gene locus
operon, as disclosed herein, are encompassed within
nucleotides 1 through 4951, SEQ ID NO:5 (nucleotides 935
through 5885, FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii, FIG. 6Eiv,
FIG. 6Fi, FIG. 6Fii, FIG. 6Fiii, FIG. 6Fiv, FIG. 6Gi,
FIG. 6Gii, FIG. 6Giii, FIG. 6Hi, FIG. 6Hii, FIG. 6Hiii,
FIG. 7 and FIG. 8). seside the open reading frames ~or
the proteins, the sequences also contain putative
promoters that direct the transcription of the genes of
the cps locus. Other promoters, herein termed
~recombinant promoters~, may also be used to direct the
expression of the cps genes in accordance with the
invention.
The genes encoded within SEQ ID NO:5 and as
disclosed herein, include cpsD, cpsS, cpsU, cpsM and part
o ~ tnpA .
cpsD is 1277 nucleotides in length, 1 through '277
o:~ SEQ ID NO:5 (935 through 2211, FIG. 6Ei, FIG. 6Eii,
FIG. 6Eiii, FIG. 6Eiv and FIG. 8).
cpsS is 1267 nucleotides in length, 1277 through
2543 of SEQ ID NO:5 (2211 through 3477, FIG. 6Fi, FIG.
6Fii, FIG. 6Fiii, FIG. 6Fiv and FIG. 8).
cpsU is 1055 nucleotides in length, 2707 through
3761 of SEQ ID NO:5 (3641 through 4695, FIG. 6Gi, FIG.
6Gii, FIG. 6Giii and FIG. 8).
cpsM is 1194 nucleotides in length, 3758 through
4951 of SEQ ID NO:5 ~4692 through 5885, FIG. 6Hi, FIG.
6Hii, FIG. 6Hiii and FIG. 8).
- CpsS is just downstream of CpsD, only 15 nucleotides
separate a potential start codon for CpsS from the stop
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codon of CpsD. Other start codons for CpsS are at
nucleotides 1311 and 1355 (SEQ ID NO:5) . There is a
large non-coding reglon between cpsS and cpsU (nuc ~otide
2543 through 2707, SEQ ID NO:5) . Where as cpsD ar.~ cpsS
5 overlap by one nucleotide and cpsU and cpsM overla_ by 4
nucleotides. All four genes are transcribed in th_ same
direction and cpsD and cpsS are in the same readir.
frame.
Encoded within the 3' flanking region (SEQ ID `~0:6)
is the truncated sequence for plpA, designated 'plF',
which is 823 nucleotides in length, 484 through 13~- of
SEQ ID NO:6 (nucleotides 6370 through 7192, FIG. 6--,
FIG. 6Iii, FIG. 6Iiii and FIG. 8) . As mentioned a~ve
15 the 5 ' end is not present in the plpA gene of type ~ S.
pneumoniae. A partial transposase sequence, tnpA, _s
contained between cpsM and 'plpA. It is transcribe~ in
the opposite orientation to all other genes descri~ed
herein, and extends from nucleotide 480 through 1, SEQ ID
20 NO:6 to overlap with the cpsM gene nucleotide 4951
through 4902, SEQ ID NO: 5 a total of 531 nucleotides
(nucleotides 5836 through 6366, FIG. 6Ji, FIG. 6Ji and
FIG. 8) .
cpsD encodes a 394 amino acid sequence (SEQ I-
NO:11) which is homologous to that of the UDP-gluccse
dehydrogenase (HasB) from Streptococcus pyogenes. ~he
deduced amino acid sequence encoded by cpsS predic_s a
protein of 416 residues (SEQ ID NO: 12) which is
homologous to polysaccharide synthases. cpsU encodes a
306 amino acid sequence (SEQ ID NO: 13) which is
homologous to glucose-1-phosphate uridylyltransfer~ses
from several other bacterial species. cpsM encodes a 397
amino acid sequence (SEQ ID NO: 14) which has homolc~y
35 with phosphoglucomutases from several bacterial spe^ies.
However, it lacks approximately 25~ of the C-termi~al
present in other phosphomutases and may not encode ~
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functional protein. ~plpA encodes a 274 amino acid
sequence (SEQ ID NO:15) and tnpA encodes a 177 amino acid
sequence (SEQ ID NO:16).
Thus, in certain particular embodiments the present
invention concerns the individual cps genes, including
segments encoding sequences corresponding to one or more
of the cpsB, cpsC, cpsE, cpsD, cpsS, cpsU and cpsM genes.
tnpA and plpA gene constructs, when combined with other
cps genes and flanking regions, are also encompassed by
the invention. In further embodiments, the invention
concerns the respective proteins and polypeptides encoded
by the cpsB, cpsC, cpsE, cpsD, cpsS, cpsU, cpsM, tnpA and
plpA genes. The proteins, polypeptides and peptides of
the invention may be used in a variety of embodiments,
including, e.g., in immunological protocols to generate
antibodies that may, in turn, be used in diagnostic
embodiments to detect S. pneumoniae.
It should be noted that in the definition of the
genes and proteins, the term '~ cps" is not used to
indicate that the gene or protein concerned has a defined
role in capsule synthesis in all cases. Rather, it
indicates that the gene is located between the cps gene
flanking regions, i.e., within the cps operon, and in
close association with other cps genes. It should also
be noted that, the "S. pneumoniae gene region" refers to
all genetic elements associated with the cps genes,
including genes incorporated within the flanking regions.
A "genetic element" refers to any DNA that may encode for
a protein or polypeptide, regardless of functionality.
The utility of the cps genes rem~;n.~ that, e.g., they may
be used in the same diagnostic manner to identify S.
pneumoniae.
~ While the present disclosure is exemplified in part
through the cloning and sequencing of type 3 cps genes,
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the techniques are equally applicable to the cps genes of
other capsule serotypes, including any one of the 85
serotypes. For example, using the techniques developed
for the characterization of the type 3 cps gene region,
the inventors have proceeded to characterize the
restriction maps for the type 2 type 6B strains (Example
16, FIG. 11). As expected, the maps of the non-type
specific flanking regions were found to be identical from
serotype to serotype, whereas the maps for the cps gene
regions themselves were serotype specific.
Diagnostic Embodiments
The cps flanking region and gene sequences, and the
encoded proteins, may be employed in diagnostic
embodiments. For example, the amount of S. pneumoniae,
or S. pneumoniae serotype, present within a biological
sample, such as blood, serum or a swab from nose, ear or
throat, may be determined by means of a molecular
biological assay to determine the level of nucleic acid
complementary to the cps loci, or even by means of an
lmml~noassay to determine the level of one of the
polypeptides encoded by a gene from this locus. The cps
locus DNA segment used in molecular biological assays may
include the non-type specific segments such as the 5' and
3' flanking regions, SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:6 and any sequence in
between, or the region encoding various polypeptides,
such as those incorporated within SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, ID NO:5 and SEQ ID NO:6.
In a molecular biological method for detecting
S. pneumoniae, one would obtain nucleic acids from a
suitable sample and analyze the nucleic acids to identify
a specific nucleic acid segment complementary to the cps
loci (whether type- or non-type-specific). The nucleic
acid segment will generally be identified by sequence,
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-- 19
which method generally includes either; identifying a
transcript with a corresponding or complementary
sequence, e.g., by Northern or Southern blotting using an
appropriate probe or; identifying a transcript with .wo
or more shorter primers and amplifying with PCR
technology.
Blotting Technique~
To detect S. pneumoniae, as may be used to identify
a patient with otitis media, pneumococcal pneumonia or
pneumococcal meningitis, using a method based upon
hybridization and blotting techniques, one may use a
probe with a sequence as set forth in SEQ ID NO:l, SEQ I3
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID
NO:6, including any sequence in between, or an equivalent
thereof. This imparts an evident utility to the nucleic
acid segments of the present invention.
To conduct such a diagnostic method, one would
generally obtain sample nucleic acids ~rom the sample and
contact the sample nucleic acids with a nucleic acid
segment corresponding to the cps loci disclosed herein,
under conditions effective to allow hybridization of
substantially complementary nucleic acids, and then
detect the presence of any hybridized substantially
complementary nucleic acid complexes that formed.
The presence of a substantially complementary
nucleic acid sequence in a sample, or a significantly
increased level of such a sequence, in comparison to the
levels in a normal or "control" sample, will thus be
indicative of a sample that harbors S. pneumoniae. Here,
substantially complementary nucleic acid sequences are
those that have relatively little sequence divergence and
that are capable of hybridizing to the sequences
disclosed herein under standard conditions.
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Where a substantially complementary nucleic aci~
sequence, or a significantly increased level thereo , is
detected in a clinical sample from a patient suspecred of
having otitis media, pneumococcal pneumonia or
pneumococcal meningitis, for example, this will be
indicative o~ a patient that does have that particular
disease. As used herein, the term "increased levels" is
used to describe a significant increase in the amou = of
the cps loci nucleic acids detected in a given sample in
comparison to that observed in a control sample, e.g., an
equivalent sample from a normal healthy subject.
A variety of hybridization techniques and systems
are known that can be used in connection with the S.
pneumoniae detection aspects of the invention, incl~ing
diagnostic assays such as those described in Falkow
et al ., U. S . Patent 4,358,535.
In general, the "detection" of a cpS locus is
accomplished by attaching or incorporating a detectable
label into the nucleic acid segment used as a probe and
"contacting" a sample with the labeled probe. In such
processes, an effective amount of a nucleic acid seoment
that comprises a detectable label (a probe), is brought
into direct juxtaposition with a composition containing
target nucleic acids. Hybridized nucleic acid complexes
may then be identified by detecting the presence of the
label, for example, by detecting a radio, enzymatic,
fluorescent, or even chemiluminescent label.
Where one simply desires to distinguish S.
pneumoniae DNA from the DNA of other bacteria, it is
contemplated that the non-type specific region sequences
may be employed as probes. However, where one desires
to distinguish among different S. pneumoniae serotypes,
it is contemplated that probes will include both type
specific and non-type specific cps sequences. The type-
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specific sequences, being type specific, will selec~ively
hybridize only to corresponding serotypes. Thus one can
envision a battery of serotype specific cps nucleic acid
hybridization probes can be employed to distinguish and
identify serotype DNA samples. In these instances, it is
not believed to be necessary to employ restriction enzyme
digestion prior to hybridization, but this can be
employed where desired. Alternatively, only one non-type
specific sequences may be employed as a "universal probe"
that allows detection of restriction fragment length
polymorphisms (RFLPs). Typically for RFLP detection, one
will employ the more specific hybridization technique of
Southern analysis wherein restriction digestion of the
unknown or target DNA is carried out using an enzyme that
will cleave either within or surrounding the cps gene
region (FIG. 4 and FIG. 11) show restriction maps for
several of the serotype cps gene regions).
Many suitable variations of hybridization technology
are available for use in the detection of nucleic acids,
as will be known to those of skill in the art. These
include, for example, in situ hybridization, Southern
blotting and Northern blotting. In si tu hybridizatlon
describes the techniques wherein the target nucleic acids
contacted with the probe sequences are those located
within one or more cells, such as cells within a clinical
sample or even cells grown in tissue culture. As is well
known in the art, the cells are prepared for
hybridization by fixation, e.g. chemical fixation, and
placed in conditions that allow for the hybridization of
a detectable probe with nucleic acids located within the
fixed cell.
.,
Alternatively, target nucleic acids may be separated
from a cell or clinical sample prior to contact with a
probe. Any of the wide variety of methods for isolating
target nucleic acids may be employed, such as cesium
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chlorlde gradient centrifugation, chromatography (e.g.,
lon, affinity, magnetic), phenol extraction and the like.
Most often, the isolated nucleic acids will be separated,
e.g., by size, using electrophoretic separation, followed
by immobilization onto a solid matrix, prior to contact
with the labelled probe. ~ These prior separation
techniques are frequently employed in the art and are
generally encompassed by the terms "Southern blotting"
and "Northern blotting~. Although the execution of
various techniques using labeled probes to detect cps
locus DNA or RNA sequences in clinical samples are well
known to those of skill in the art, a particularly
preferred method is described in detail herein, in
Example 4.
PCR Techni~ue~
To detect S. pneumoniae, using a method based upon
PCR technology of U.S. Patent 4,603,102 (incorporated
herein by reference), one may also use one or more probes
with a sequence as set forth in SEQ ID NO:l, SEQ ID NO:2,
SEQ ID NO:3 and SEQ ID NO:4 (including sequence in
between), SEQ ID NO:5 or SEQ ID NO:6, or an equivalent
thereof.
To conduct such a diagnostic method, one would
generally obtain sample nucleic acids from a suitable
source and contact the sample nucleic acids with two
probes or primers corresponding to the cps loci disclosed
herein, under conditions which allow for hybridization
and polymerization to occur. A pair of probes, one
corresponding to the 5' flanking region and the other
corresponding to the 3' flanking region, would be
sufficient to detect the presence of S. pneumoniae in a
sample and may even be used to indicate the amount of
bacteria present. Furthermore the size of the isolated
PCR product, when separated by any of the methods as
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described above, may be sufficient to identify the S.
pneumoniae serotype. Alternatively the PCR produc~ may
be digested with one or more restriction enzymes, whlch
would enable individual serotypes to be distinguished,
using the same principle as RFLP.
In further embodiments, it may be desired to employ
other probes corresponding to type specific or non-type
speciflc regions. A battery of serotype specific probes,
probes corresponding to type specific DNA regions, may be
employed in individual reactions, with a universal probe,
a probe corresponding to non-type specific regions. The
size of PCR products, with or without prior digestion
with restriction enzymes, would distinguish and identify
the S. pneumoniae serotype.
Kits
Kits for use in Southern and Northern blotting or
PCR technology, to identify S. pne71mon;~e and/or
individuals having, or a~ risk ~or developing, otitis
media, pneumococcal pneumoniae or pneumococcal
meningitis, are also contemplated to fall within the
scope of the present invention. Such kits will comprise,
in suitable container means, cps nucleic acid probes;
also, generally, unrelated probes for use as controls;
and optionally, one or more restriction enzymes.
Characterization of Streptococcus pneumoniae Serotypes
In another embodiment of the invention, the non-type
specific DNA may be used, to isolate and characterize the
type-specific DNA sequence for all or any strain of S.
pneumoniae. Consequently, the most suitable probes for
diagnosing S. pneumoniae infection, for use in any
molecular biological technique, could be found.
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The present invention identifies the common flanking
DNA which may be used in hybridizations to target the
location of the type-specific cps genes for any strain of
S. pneumoniae. Once this location has been identified
the cps genes may then be isolated and characterizea by
the use of conventional techniques, such as; chromosome
crawling, PCR technology, cloning and DNA sequencir.g, all
are disclosed herein. As mentioned above, this in turn
would enable suitable probes from each S. pneumoniae
strain to be chosen and then used for, diagnostic and
research purposes. Furthermore, the genetic elements
involved in determining S. pneumoniae serotype may be
elucidated, and an understanding of their effect or
virulence and evolutionary role may be achieved.
In still more particular embodiments, the invention
concerns the preparation and cloning of entire cps gene
regions encoding one or more specific cps genes of a
particular serotype, positioned within a "cassette" for
ease of manipulation, e.g. in plasmid preparation or host
cell introduction, etc. Thus, cps gene cassettes in
accordance with the present invention will typically
include left and right hand flanking regions to allow
homologous recombination in S. pneumoniae host cells.
A preferred method for preparing cps gene cassette
is through the application of PCR technology wherein left
and right hand primer corresponding to left and right
hand flanking region are employed to amplify the cps gene
coding regions. Of course, the primers employed will
typically include at their termini appropriate
restriction enzyme site. Thus, the resulting cassette
will preferably include at each terminus a restriction
enzyme site of choice. The site, of course, will depend
upon the vector that is ultimately employed for
manipulation or transformation but may be the specific
cleavage site for one or more of the restriction enzymes
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shown in FIG. 4 and FIG. 7 or listed in Table l. T~e
list is, of course, exemplary, and any restriction enzyme
could be employed, as is well known to those of skill in
the art.
The present invention also contemplates more
traditional approaches to cps gene regions cloning, such
as by partial fragmentation of S. pneumoniae DNA followed
by cloning into a recombinant cloning host, such as E.
coli, and screening by hybridization and antibiotic
selection (using a selection marker found on the plasmid
or other vector employed for cloning). Of course, f the
cloning host is not a S. pneumonlae host, one may employ
either type specific or non-type specific cps gene
sequences for screening. In these cases, cassettes that
are developed may include enzyme restriction sites
naturally found to occur in the flanking regions, such as
an SphI site.
It is contemplated that virtually any type of host
cell may be employed in connection with the present
invention, depending on the particular application. For
example, where one simply desires to manipulate cps gene
sequences, any acceptable host may be employed, such as
an E. coli, or even an appropriate eukaryotic host where
desired. However, where one contemplated producing
capsule polysaccharides, one will desire to employ a gram
positive host such as bacillus, staphylococcus or
streptococcal hosts. Particularly preferred will be
3 0 S. pneumo~iae host, in that it is contemplated that such
hosts will be more readily ~m~n~hle to manipulation to
increase capsular polysaccharide production.
The inventors contemplate that a particular
application, therefore, will be the use of recombinant
hosts not only for the preparation and manipulation of
cps gene sequences, but also in the large scale
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productlon of capsule saccharides and polysaccharides.
These polysaccharides are useful for the production of
antigenic haptens and epitopes for use in the production
of immunogens. Haptens and epitopes are described herein
as portions of a molecule against which an immune
response is directed. In conjunction with an molecule
that elicits an immune response, that is an immunogen, an
hapten-immunogen complex is able to elicit an immune
response.
Generally speaking, where capsule production is
required, one will employ a S. pneumoniae host cell into
which a selected cps gene region is introduced or
"cassetted in". First, a DNA segment that includes the
selected S. pneumoniae cps gene(s) flanked by sufficient
S. pneumoniae flanking regions to allow homologous
recombination in the S. pneumoniae host is identified. It
is contemplated that flanking regions on the order of 0.1
to 1 kb will be sufficient to allow recombination to
occur. Once an appropriate DNA segment is introduced
into the S. pneumoniae host, either as genomic DNA or as
a recombinant vector (plasmid), transformed host
expressing the introduced cps gene are selected.
DNA may be introduced into a suitable host by a
variety of mechanisms, including natural transformation
of S. pneumoniae, calcium mediated transformation or
electroporation of E. coli. A particularly preferred
method of bacterial transformation includes the steps of
making an S. pneumoniae competent for transformation by
growth in Todd Hewitt broth supplemented with calcium and
bovine serum albumin. Alternatively electroporation may
be employed, one makes the bacterial cells, such as the
E. coli strain LE392, competent in 10~ glycerol in water,
adds the DNA, and electroporates the cells.
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Where the cps gene DNA segment is introduced in the
~orm o~ a plasmid, it would be preferable, at certain
times, to employ a plasmid that is free of a S.
pneumoniae origin of replication. Thus, virtually any
traditional plasmid (which are designed, e.g. for gram
negative hosts such as E. coli) may be employed. The
reason is that where the plasmid is free of a S.
pneumoniae origin o~ replication only those clones that
have successfully undergone homologous recombination with
the recombinant cps gene region will be detected. Stated
another way, in this case, there is no requirement of a
5. pneumoniae origin of replication in order for
homologous recombination to occur, and thus homologous
recombinants are inherently selected for using such a
cloning technique.
Alternatively it may be simpler to introduce the
cassette into S. pneumoniae on a replicating plasmid with
a S. pneumoniae origin of replication. In this way,
higher levels of polysaccharide production as a result of
the elevated copy number of the plasmid (lO to 20) as
opposed to the low copy number of the chromosome (l to 2)
would be achieved and homologous recombination need not
occur.
Additionally, it is contemplated that there will be
some advantage to employing as the starting host a S.
pneumoniae strain that is a high producer of its own
inherent cps gene. These high producers will necessarily
include the genetic environment to support high
production of the newly introduced cps complex, and thus
will likely be ideal hosts ~or such production. To
achieve such recombinants, all that is required is that
~ the heterologous gene containing the flanking regions, or
a genome containing the ~lanking regions, is introduced
into the host cell, and resultant recombinants wherein
the homologous gene has been replaced is selected.
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Although the invention contemplates in particular
embodiments the introductlon of an entire recombinaL,_ cps
gene region where capsule saccharide synthesis is
desired, the invention also contemplates the introdu-tion
of one, two, three or so of the individual CpS genes. It
is contemplated that one or two genes t such as those that
control the biosynthesis of sugar precursors may be
sufficient to, in and of themselves, confer serotype
specific saccharides production on the selected hos., or
can in of themselves, upregulate capsule production by
existing cps genes of the host.
BRIEF DESCRIPTION OF THE DRAWINGS
. The following drawings form part of the presen_
specification and are included to further demonstrate
certain aspects of the present invention. The invention
may be better understood by reference to one or more of
these drawings in combination with the detailed
description of specific embodiments presented hereir~.
FIG. 1. ELISA for the detection of type 3 capsule.
Wells of microtiter dishes were coated with crude
extracts of capsule material. Type 3 capsule was
detected with the monoclonal antibody 16.3 ~Briles et
al., 1981a). The type 2 strain D39 served as a nega_ive
control. Measurements were made in triplicate, and error
bars represent standard errors. Values were standardized
to protein content. Genotypic designations were based on
linkage as determined by transformation mapping.
FIG. 2. Insertion-duplication restoration. The
cloned fragment and the homologous fragment in the
chromosome are represented by the open boxed regions.
The dark block represents the mutation in the chromcsome
of the mutant strain. Insertion of the plasmid clone
into the pneumococcal chromosome results in a duplication
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of the homologous fragment with the plasmid inserte~ in
between. If the recombination occurs to the left or the
- mutation as shown here, a wild type, full-length copy of
the gene is created and function is restored. The
plasmid clone leading to restoration spontaneously
excises at low frequency through homologous recombination
and can therefore be easlly recovered by transformation
to E. coli. pJD330 contains a 2.4 kb Sau3AI fragmen~.
FIG. 3A. Repair of capsule mutations by double
crossover recombination event. Mutants were transformed
with plasmid subclones of pJD330 and no selection for
Er~ was made. The box in the chromosome represents the
region in the mutant chromosome homologous to the
fragment cloned in pJD330. The plasmid represents a
subclone of pJD330 capable of restoring encapsulation in
the mutant strain.
FIG. 3B. Deletion analysis to locate the site of
the cpsAl, capD4, and Rxl mutations. The mutations were
mapped by transformation with plasmid clones cont~'n;ng
the indicated fragments and screening for the mucoid
colony phenotype. Identical fragments repaired the
cpsAl, capD4, and Rxl mutations. No selection was made
for insertion of the plasmids, thus these numbers
represent double crossover events. The actual
fre~uencies of repair, shown for the cpsAl-containing
mutant, are mainly a reflection of the transformation
efficiency of the recipient. No encapsulated
transformants were obtained when pJY4163 (no insert) was
used for transformation. Restriction sites are: M, MunI;
P, PstI; Pv, PvuII; R, RsaI; S3, Sau3A I; Ss, SspI; Xb,
XbaI.
FIG. 4. Physical and yenetic map of the type 3
capsule region of S. pneumoniae WU2. The restriction map
was developed by probing chromosomal digests of WU2 with
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pJD330 and pJD366 and by probing chromosomal digests of
JD770 with pJD330 and pJY4163. The location of primary
clones pJD330 and pJD366 are indicated above the map.
Subcloned fragments used to target insertion-duplica~ion
mutations are indicated below the map. Insertion or
plasmids containing shaded fragments led to loss of
capsule production. + or - at the end of a fragmen~
indicates the presence or absence of transcription
detected at that point in the chromosome. Insertions of
pJD330 and pJD366 are in~the orientation to detect
transcription to the left. Clones pJD351 and pJD364,
which contain the pJD330 and pJD366 fragments,
respectively, in the opposite orientation, were also used
for the transcription studies. The other plasmids used
for insertions were pJD356, pJD337, pJD369, pJD359,
pJD362, pJD361, pJD357, and pJD374, in the order shown.
The genes indicated by genetic data or suggested by
transcription determinations were drawn based on
preliminary sequence information. The cpsDSUM
designations are based on probable functions, as
described in the text. Restriction sites are Bg, BglII;
Ev, EcoRV; H, HindIII; Ha, HaeIII; M, MunI; P, PstI; Pv,
PvuII; R, RsaI; S, SacI; S3, Sau3A I; Sa, SalI; Sp, SphI;
X, XbaI. Restriction sites are not necessarily unique to
the entire region.
FIG. 5. Schematic representation of the capsuie
regions in these strains. Insertions in JD871 and JD872
result from incorporation of pJD366. The insertion in
JD803 is pJD330. The shaded square symbol represents type
2 speci~ic DNA; the open square symbol represents type 3
specific DNA; the hatched square symbol represents
flanking DNA common to both type 2 and type 3 and; the
black square symbol represents pJY4163 or pJY4164. The
locations of the probes used are indicated below the map.
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FIG. 6A. DNA sequence of the 5' flanking region
including partial sequence o~ cps3B and cps3C (SEQ -D
NO:1) encoding for SEQ ID NO:7 and SEQ ID NO:8.
FIG. 6B. DNA sequence o~ the 5' ~lanking regic~
including partial sequence of CpS3C (SEQ ID NO:2),
encoding for SEQ ID NO:9.
FIG. 6C. DNA sequence of the 5' flanking region
including partial sequence of cps3E (SEQ ID NO:3),
encoding for SEQ ID NO:10.
FIG. 6D. DNA sequence of the "repeat" upstream
flanking DNA (SEQ ID NO:4). FIG. 6D consists of two
panels, FIG. 6Di and FIG. 6Dii.
FIG. 6E. DNA sequence of the region containing
cps3D (nucleotides 1 through 1277, SEQ ID NO.5). The -35
and -10 hexamers of potential a-70 type promoters
upstream o~ cps3D are underlined and labeled above the
sequence. Putative ribosome binding sites are
underlined. The precise locations of endpoints of
insertion mutations shown in FIG. 7 are indicated by
triangles below the sequence and are labeled with the
name of the strain containing the given mutation. ~he
locations of spontaneous mutations in cps3D are labeled
with the sequence of the mutation and the name of the
strain containing the mutation. The sequence from the
PvuII-SspI fragment of A66R2 began at nucleotide 1921,
thus it is possible that additional mutations are present
from the PvuII site to this point. Selected restriction
sites are shown. FIG. 6E consists of four panels,
FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii and FIG. 6Eiv.
..
FIG. 6F. DNA sequence of the region containing
cps35 (nucleotides 1277 through 2543, SEQ ID NO:5). The
precise locations of endpoints of insertion mutations
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shown in FIG. 7 are indicated by triangles below t:~e
sequence and are labeled wlth the name of the stra--
containing the given mutation. FIG. 6F consists o four
panels, FIG. 6Fi, FIG. 6Fii, FIG. 6Fiii and FIG. 6-~v.
FIG. 6G. DNA sequence of the region containir_
cps3U (nucleotides 2707 through 3806, SEQ ID NO:5). The
-35 and -10 hexamers of potential a-70 type promote~s
upstream of cps3U are underlined and labeled above _he
sequence. A short region of dyad symmetry just ups~ream
of cps3U is overlined. Putative ribosome binding s-~es
are underlined. The precise locations of endpoints of
insertion mutations shown in FIG. 7 are indicated ~.
triangles below the sequence and are labeled with .~e
name of the strain containing the given mutation.
Selected restriction sites are shown. FIG. 6G cons-sts
of three panels, FIG. 6Gi, FIG. 6Gii and FIG. 6Gii-.
FIG. 6H. DNA sequence of the region containir
cps3M (3746 through 4951, SEQ ID NO:5) with corresp^nding
amino acid sequences. Note that the first line of FIG.
6Hi, FIG. 6Hii and FIG. 6Hiii overlaps the last line of
FIG. 6Gi, FIG. 6Gii and FIG. 6Giii. FIG. 6H consis-s of
three panels, FIG. 6Hi, FIG. 6Hii and FIG. 6Hiii.
FIG. 6I. DNA sequence of the region containir~ the
3' flanking region including 'plpA (SEQ ID NO:6) wi~h
corresponding amino acid sequences (SEQ ID NO:15). FIG.
6I consists of three panels, FIG. 6Ii, FIG. 6Iii and
FIG. 6Iiii.
FIG. 6J. The DNA sequence for a partial trans-osase
A, tnpA, located between 'plpA and overlapping cps~. The
open reading frame is in the opposite orientation
starting at nucleotide 6366 through 5836 of FIG. 6_-,
FIG. 6Iii and FIG. 6Iiii. FIG. 6J consists of two
panels, FIG. 6Ji and FIG. 6Jii.
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FIG. 7. Map of the chromosomal region involvea in
the blosynthesis of S. pneumoniae type 3 capsular
polysaccharide. Triangles indlcate the endpoints of
insertion mutations. Filled triangles represent
insertions which resulted in loss of capsule product_on.
Open triangles represent mutations which did not result
in loss of capsule production. Restriction enzyme s_tes
are: Bg, BglII; Ev, EcoRV; H, HindIII; P, PStI; PV,
PvuII; S, SacI; Sa, SalI; Sp, SphI. Also included is a
diagram showing the position of SEQ ID NO:l, SEQ ID NO:2
and SEQ ID NO:3, including the corresponding amino acid
sequences SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ
ID NO:10.
FIG. 8. Map of the chromosomal region involvea in
the biosynthesis of S. pneumoniae type 3 capsular
polysaccharide showing the relationship between SEQ ID
NO:4, SEQ ID NO: 5 and SEQ ID NO:6, and DNA sequence as
described in FIG. 6Di through FIG. 6Jii.
FIG. 9A and FIG. 9B. Location of insertion
mutations in the type 3-specific region of the S.
pneumoniae WU2 chromosome. Schematic illustration o_ the
insertions. The schematic was derived from Southern blot
analysis such as that shown in FIG. lla and FIG. llb.
The ability of the strains to produce type 3 capsule is
indicated. Restriction sites are: F, FspI; H, HindIII;
K, KpnI; Ms, M5cI; P, PstI; Pv, PvuII, X, XbaI.
FIG. l0. Biosynthetic pathway for type 3 capsular
polysaccharide. The functions of the proteins encoaed by
the type 3-specific genes, based on homologies, genetic,
and biochemical data are shown. Additional functions may
be necessary for capsule transport or attachment.
FIG. ll. Chromosome maps of the capsule regions in
strains of types 2, 3, and 6B. The SacI-HindIII fragment
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(pJD377) from type 3 used for the probe is shown below
the maps. Restriction sites are Bg, BglII; F, FspI; H,
HindIII; S, SalI; Sac, SacI; Sp, SphI.
FIG. 12. Production of type 3 capsule. Buoyant
densities of parents and derivatives expressing the type
3 capsule. Densities were determined by centrifugation
on 0 to 50~ Percoll gradients for 30 min at 8,000 x g.
Samples were grown in duplicate, and the density of each
sample was determined in duplicate gradients. The
results shown were obtained with bacteria grown on solid
medium. Identical results were obtained from growth in
liquid culture.
FIG. 13. Total capsule production. Triplicate
cultures of each strain were grown to an OD600 of ~0.5.
Duplicate wells of polyvinyl plates were coated with
either supernatant fluids or cell sonicates. Total
capsule contents of the type 3 parent and the derivatives
were determined by using a monoclonal antibody to type 3
capsule. See Table 2, footnote d, for explanation of
strain designations.
FIG. 14A. All studies were done in BALB/ByJ mice.
See Table 7, footnote d, for explanation of strain
designations. Virulence of type 2 derivatives. The
parental strains JD770 (3/3) and D39 (2/2) and the
derivatives JD803 (2/3) and JD804 (2/3) had similar LD50s
(50 to 75 CFU). For time-to-death studies, groups of
mice were infected i.p. with doses approximately 5- to
20-fold above the LD50. The observed times to death were
not related to the dose received. Each circle represents
an individual mouse. The median times to death for D39
(2/2) and for derivatives JD803 (2/3) and JD804 (2/3)
were 31.5 and 33.0 h, respectively (not significantly
different). All three values differed significantly (P c
0.005) from that of the type 3 parent JD770 (52h). The
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values for JD803 and JD804 did not differ from each
other.
FIG. 14B. All studies were done in BALB/ByJ mice.
See Table 7, footnote d, for explanation of strain
designations. Virulence of the type 5 derivatives. Mice
were infected i.v. with doses o~ 103 to lo6 CFU (10-fold
increments) of each type 3 derivative. Doses of 10C to
Io2 and 103 to lo6 CFU were used fro the parental strains
DBL5 (5/5) and JD770 (3/3), respectively. The totai
number of mice used per dose is listed beside each datum
point. TK5010* represents the combined data for TK5010
(5/3), TK5011 (5/3), and TK5012 (5/3). The derivatives
did not differ from each other but did differ
significantly from the parental strains ~D770 (P c
0.0005) and DBL5 (P < 0.0001).
FIG. 14C. All studies were done in BALB/ByJ mice.
See Table 7, footnote d, for explanation of strain
designations. Virulence of type 6B derivatives. Mice
were infected i.p. with doses of Iol to lo6 CFU of the
type 6B derivatives. Doses of 10 to 103 CFU and of 103
to Io6 CFU were ~ml ned for the parent strains JD770
(3/3) and DBL1 (6B/6B), respectively. The total number
of mice used per dose is listed beside each datum point.
TK3028 represents the combined data for TK3026 (6B/3)
and TK3028 (6B/3), which did not differ fro each other.
However, these strains did differ significantly from the
parental strains JD770 (3/3) (P < 0.003) and DBL1 (6B/6B)
(P ~ 0.0005).
FIG. 15A. Model for the transfer of type-spec fic
genes. Cassette type-recombination. Replacement of the
recipient's type-specific genes with those of the donor
results from homologous recombination between common
regions that flank the type-specific cassettes. The open
elipsoid symbol represents sequence containing repea~ed
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element; the black oblong symbol represents common DNA
upstream of type-specific cassettes and; the open oblong
symbol represents common DNA (including plpA) downs~ream
of type-specific cassettes.
FIG. 15B. Model for the transfer of type-speci-ic
genes. Binary encapsulation by recombination involvi~g
homology at only one end. Integration at one end o' the
type-specific cassette would occur via homologous
recombination through the repeated element. Integrarion
at the other end would result from an apparent
illegitimate recombination. Linkage of the two type-
specific cassettes would result if the integration
occurred in a repeat element in or closely linked to the
recipient's capsule genes. Symbols represent the same as
in FIG. 15A.
FIG. 15C. Model for the transfer of type-specific
genes. Binary encapsulation via a transposition-like
event. Type-specific cassettes flanked by the repeated
element would resolve out of the chromosome and be
transferred to recipient cells as circular intermediates.
Recombination into the recipient chromosome could occur
at a repeat element unlinked (as shown) or linked to the
recipient's type-specific genes. Transfer of linear DNA
could also yield binary strains as a result of
recombination between the two repeat elements that flank
the type-specific genes and two repeat elements that are
closely linked in the recipient chromosome. Symbols
represent the same as in FIG. 15A.
DET~Tr~n DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present inventors have determined a genetic and
physical map that encompasses the region responsible for
the synthesis of the polysaccharide capsule of S.
pne~moniae. The polysaccharide capsule of S. pneumcniae
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is a potent defense against the immune response of the
host organism and is directly involved in bacterial
virulence. The capsule locus, cps, is basically composed
of two functional regions: a central region that contains
the genes responsible for capsular biosynthesis and is
described herein as the type-specific region, and the
non-type specific regions that flank the central
biosynthetic type-specific region.
S. pneumoniae has evolved a complex 'antigenic
shift' mechanism that allows the bacteria to evade the
host immune system. The antigenic shift of S. pneumoniae
occurs via homologous recombination of a type-specific
cassette that is replaced through natural transformation.
S. pneumoniae is naturally competent allowing for the
acqulsition of chromosomal DNA from exogenous sources,
such as other S. pneumoniae. Disclosed herein is
evidence identifying the non-type specific regions as
being responsible for providing the sequence identity
that allows for homologous recombination cross-over
points.
The present inventors have identified and cloned the
region of the S. pneumoniae chromosome that contains
genes involved in the production of type 3 capsular
polysaccharide, and that is specific to type 3 strains.
They have also cloned approximately 1-3 kb of DNA
flanking both sides of this region and found it to be
common to all capsular serotypes ~m; ned. A genetic and
physical map of the region is presented in FIG. 6A, FIG.
6B, FIG. 6C, FIG. 6Di, FIG 6Dii, FIG. 6Ei, FIG. 6Eii,
FIG. 6Eiii, FIG. 6Eiv, FIG. 6Fi, FIG. 6Fii, FIG. 6Fiii,
~ FIG. 6Fiv, FIG. 6Gi, FIG. 6Gii, FIG. 6Giii, FIG. 6Hi,
FIG. 6Hii, FIG. 6Hiii, FIG. 6Ii, FIG. 6Iii, FIG. 6Iiii,
FIG. 6Ji and FIG. 6Jii. A simplified version of which is
- shown in FIG. 7 and FIG. 8. The sites of insertion
mutations made within the region are shown in FIG. 7.
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The regions found by hybridization studies to be specific
to type 3 or common to all capsule types are also
indicated in FIG. 7. The cloning of the upstream region,
creation of insertion mutations, sequence analysis sf the
region, hybridization analyses using the upstream region,
and an in vi tro assay of type 3 capsule polymerization
are described in the following examples.
The DNA sequence of the region containing the nine
genes; cps3B, cps3C, cps3E, cps3D, cps35, cps3U, cps3M,
'plpA, tnpA and the flanking DNA was determined and is
presented in FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6Di,
FIG. 6Dii, FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii, FIG. 6Eiv,
FIG. 6Fi, FIG. 6Fii, FIG. 6Fiii, FIG. 6Fiv, FIG. 6Gi,
FIG. 6Gii, FIG. 6Giii, FIG. 6Hi, FIG. 6Hii, FIG. 6Hiii,
FIG. 6Ii, FIG. 6Iii, FIG. 6Iiii, FIG. 6Ji and FIG. 6Jii
(SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 and
SEQ ID NO: 6) along with the deduced amino acid sequences
(SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:lC, SEQ
ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15 and SEQ ID NO: 16).
Based on genetic, molecular, and biochemical data
the inventors have been able to assign putative functions
to the type-specific genes in the pathway for type 3
capsular polysaccharide biosynthesis. Two of the genes,
cps3D and cps3S, are required for capsule synthesis.
There is substantial evidence to indicate that cps3D
encodes UDP-glucose dehydrogenase. Described herein is
genetic evidence to indicate that several mutations
causing the capsule-negative phenotype are located in the
gene for UDP-glucose dehydrogenase. The predicted amino
acid sequence has characteristics consistent with this
function. Cps3D shows a high degree of homology to HasB,
which is the UDP-glucose dehydrogenase of S. pyogenes
(Dougherty & van de Rijn, 1993). Within Cps3D are
sequences homologous to the active site and the NAD-
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binding site in known UDP-glucose dehydrogenases. ' is
not possible to perform the standard UDP-glucose
dehydrogenase assay on extracts of S. pneumoniae due to
the presence of a NADH oxidase, which copurifies with the
enzyme (Smith, et al., 1960; Smith, et al., 1958).
However, extracts ~rom cps3D mutants could synthesize
type 3 capsule in vitro if supplied with UDP-glucuronic
acid, i.e., they lacked the ability to convert UDP-
glucose to UDP-glucuronic acid and thus lack UDP-glucose
dehydrogenase activity.
Cps3S is a new member of a family of polysaccharide
synthases. All of these polysaccharide synthases or
which the structures of the polysaccharides are known
produce ~ (1-4) linked polysaccharides. Thus, it is
possible that Cps3S forms the ~ (1-4) linkage in the
disaccharide cellobiuronic acid (glcA ~ (1-4) glc), and
that a second enzyme is required to polymerize (i.e.,
create the ~ 3) linkages) the disaccharides into ~he
full length polysaccharide. However, HasA, the enzyme
most closely related to Cps3S, creates both linkages, a ~
(1-4) and a ~ (1-3), in the production of hyaluronic acid
capsule (DeAngelis, et al ., 1993). HasA has recently
been shown to be sufficient for hyaluronic acid synthesis
in heterologous bacteria, given the nucleotide sugar
substituents (DeAngelis, et al ., 1993a). Because the
inventors did not find another required enzyme in the
type 3-specific region, Cps3S, like HasA, may synthesize
the polysaccharide by monomer addition.
Neither cps3U nor cps3M appears to be required for
type 3 capsule synthesis. Cps3M and Cps3U should
function to convert glucose-6-phosphate into glucose-1-
~ phosphate, and glucose-l-phosphate into UDP-glucose,
respectively (FIG. 10). Since UDP-glucose is necessary
~ for the production of essential cell constituents,
including teichoic acid and lipoteichoic acid (Austrian,
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et al., 1959), the products of other genes may comp;ement
the functions lost in Cps3U and Cps3M mutants. There are
at least two plausible reasons for the retention of ~hese
genes in the type-specific region. One explanation s
that their functions cannot be fully duplicated by the
second enzymes. For example, they may play a role -r.
regulating the amount of polysaccharide produced. vnder
given conditions, such as during infection, increasea
production of capsule could be advantageous. The large
noncoding region upstream of cps3U might be a site c_
which regulation of cps3U and cps3M occurs. It should
also be noted that the reactions carried out by Cps3:I and
Cps3U are each reversible, and the enzymes might be more
active in the reverse reaction. Therefore, Cps3U ar~
Cps3M might function to limit the amount of capsule
produced.
Another possible explanation is that these genes
were obtained along with the necessary type-specific
genes in a horizontal transfer from another organis~. and
have not been lost. This theory is consistent with ~he
hybridization data indicating that none of the type-
specific genes could be detected even at low stringency
in strains of six other pneumococcal types, includir.g
types with related capsular polysaccharide structures
(Dillard, et al., 1994). However, if these genes serve
no necessary function, it is surprising that they have
been maintained in the type 3 cassettes of multiple
strains; i.e., the restriction maps of the type 3 regions
of five non-clonal type 3 strains are identical, and all
have cps3U and cps3M.
There are three requirements for a DNA region
to be considered a gene cassette: 1) more than one copy
of a gene or set of genes must exist, each specifyir.g the
production of a different, but related, product; 2) each
copy must be flanked by DNA which is common to all _he
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copies; and 3) cassettes must undergo recombination
resulting in the replacement of one copy by another.
More than 80 different capsular serotypes of S.
pneumoniae have been identified, and the structures of
more than half o~ the polysaccharides have been
determined (van Dam, et al., 1990).
The presence o~ multiple types implies that as many
different sets of genes exist. The inventors have shown
that all the necessary genes specific for the production
o~ capsules of types 2, 3, and 6B (Example 16) are
closely linked to an approximate 1.2 kb fragment present
in all capsule types examined. This fragment
(corresponding to SEQ ID NO:6 and part of SEQ ID NO:5,
see FIG. 4 and Example 5), cloned from the region
flanking the type 3-specific genes, contains a gene with
a sequence virtually identical to a gene fragment from
type 2 strain, described by Pearce et al., and designated
plpA (Pearce, et al., 1993) . However, the flanking
region from type 3 strain is distinct from the sequence
descri~ed by Pearce et al . (1993) in that it is misslng
about one third of the 5' end of the gene, designated
'plpA. Furthermore Pearce et al. did not identify the
location of the plpA gene nor did they attempt to define
the sequences on either side of the gene.
The mapping studies reported here confirm that the
regions to the right of this fragment are common for at
least 4 kb. The regions map differently to the left of
the fragment (Example 16), implying that these regions
contain the type-specific genes in types 2 and 6B, as
shown herein for type 3.
The upstream left flanking region from type 3, SEQ
ID NO:l, SEQ ID NO:2, SEQ ID NO: 3 and SEQ ID NO: 4 is
common to all capsule types examined ( 2, 3 and 6B, the
Repeat region was also examined in 5, 6A, 8, 9 and 22;
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Example 17). However, the presence of multiple copies of
the Repeat fragment (SEQ ID NO:4) has made the linkage in
other types difficult to determine and it is possible
that the repeat region may not flank the type-speci~ c
genes in other types.
Previous workers have provided biochemical eviaence
of replace~ent of capsule gene cassettes. When a ty~e 1
and a type 2 strain were each transformed to type 14 or
type 23, they no longer produced UDP-glucuronic acid,
implying that the transformants had lost the UDP-glucose
dehydrogenase gene (Austrian, et al., 1959). Similarly,
a type 1 strain transformed to type 3 encapsulation no
longer epimerized UDP-glucuronic acid to UDP-galacturonic
acid. Molecular evidence is presented herein for the
replacement of the type-specific genes for a type 3
strain transformed to type 2 encapsulation and a type 2
strain transformed to type 3 (Example 6). Together with
Example 18, these observations provide strong evidence
for a cassette organization of the type-specific genes.
Since the proposal was put forth that capsule genes
are exchanged through a cassette-type recombination,
there has always been one glaring exception - binary
encapsulation. At low fre~uency, strains of certain
capsule types transformed with DNA from strains of
certain other capsule types were found to produce both
polysaccharides (Austrian, et al., 1959). Evidently,
cassette-type recombination had not occurred in these
transformants since the genes for the original capsule
had been maintained. Bernheimer et al., found that
stable binary strains contained the second set of type-
specific genes at a site unlinked to the recipient's
type-specific genes (Bernheimer, et al., 1967). Unstable
binary strains frequently lost the donor type-specific
genes, which were usually located at a site linked to the
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recipient type-specific genes (Bernheimer, et al ., 1967 ;
Bernheimer and Wermundsen, 1969).
-
Based on the hybridization data described here
concerning the flanking regions and replacement of type-
specific genes, as well as the work of Bernhelmer
concerning transformation to binary capsule types
(Bernheimer, et al ., 1968; Bernheimer, et al ., 1967;
Bernheimer, et al ., 1969 ; Bernheimer and Wermundsen,
1972), the inventors can now propose models for capsule
type change and binary capsule type formation. Cassette-
type recombination would result from crossover events in
the homologous flanking regions, leading to replacement
of the type-specific genes. The left crossover could
take place in the repeated element in strains containing
this region linked to the type-specific genes but would
occur in flanking DNA further upstream in strains that
did not contain the repeat. This type of recombination
is shown in Fig l9A.
The finding of a repeated element upstream of the
type 3 capsule genes (SEQ ID NO:4; Example 17) may
provide an explanation for binary encapsulation. It is
clear that at least one of the copies of the repeated
fragment in types 2 and 6B is unlinked to the capsule
genes, since neither of the type-specific cassettes could
be moved with a marker inserted in this location. In
type 3, a 2.2 kb HindIII fragment containing the repeat
element is linked to the type-specific genes but based on
transformation studies, an 8 kb fragment is not. The
frequency of binary capsule transformants observed by
Bernheimer et al., was significantly lower (10-1000 fold)
than related transformations resulting in replacement,
leading them to suggest that the recombination event
involved strong homology at only one end. Once
- integrated at the "atypical" location (unlinked to the
type-specific cassette), the genes for the second capsule
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type could not be moved to the normal location, exce~t by
transformation of a strain containing the genes of that
type in the normal location, again suggesting that the
non-type specific flanking DNA on at least one end had
been lost (Bernheimer, et al ., 1967; Bernheimer and
Wermundsen, 1972).
Since finding that part of the left flanking D~A
(SEQ ID NO: 4) iS repeated in chromosomes of several
strains, whereas the right flanking region is only
present in one location, it is proposed that the repeated
element of the left flanking region may be involved in
the recombination that results in binary capsule type
formation. The mechanism proposed by Bernheimer et al.,
for stable binary strains could involve homologous
recombination at a repeated element unlinked to the
capsule locus; the recombination at the other end o~ the
capsule genes would occur by an apparent illegitimate
recombination event, as shown in FIG. 15B.
An alternative possibility for the generation of
stable binary strains, shown in FIG. 15C, involves a
transposition-like event that could result if certain
type-specific genes are flanked on both sides by the
repeated element. Unstable binary strains could result
from either type of integration occurring at repeated
elements in, or closely linked to, the recipient's tvpe-
specific genes. Instability could result from
recombination through genes common to both capsule types,
as suggested by Bernheimer et al., for the UDP-glucose
dehydrogenases of types 1 and 3. Results presented here
provide the basis for e~m;n;ng these possibilities.
Binary strains containing the two sets of genes linked
are of particular interest since they might recombine to
form a novel capsule type. ~m; n~tion of strains
producing related capsule structures may help elucidate
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the possible mechanisms involved in novel capsule ty~e
~ormation.
Epidemiological studies have indicated that capsule
type varies independently of other factors, suggestir~g
that a substantial amount o~ genetic exchange has
occurred (Crain et al ., 1990; Coffey et al ., 1991;
Versalovic et al., 1993). Nonetheless, virulence of
clinical isolates appears to correlate with the capsule
type expressed (Briles et al., 1992). Taken together,
these data suggest that the capsule type has a prominent
role in determining virulence. However, epidemiological
studies cannot demonstrate a causal relationship between
capsule type and virulence due to the variability in the
genetic backgrounds o~ the di~erent serotypes. The
characterization of the S. pneumoniae capsule locus
described here has facilitated the construction of
isogenic strains di~fering only in capsule type. These
strains have been used to evaluate the role o~ capsule
type in virulence (Example 18). The cloning of capsule
genes and elucidation of the genetic organization of the
capsule locus is a significant step toward understanding
antigenic variation and virulence in this pathogen.
Cloning of the Type 3 region
There are a number of reasons for first cloning the
type 3 specific genes: the type 3 capsule has a
relatively simple structure that is expected to require a
small number of genes for its synthesis; production of
type 3 capsule is an easily identifiable phenotype; and
finally, the availability of antibodies specific for the
type 3 polysaccharide capsule allowed rapid screening for
the presence of the capsule, of a large number of
isolates, using an ELISA assay as described herein in
~ Example 1. The approach provided and disclosed allowed
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for the first molecular genetic map of the cps gene
locus.
The cloning of additional type-specific genes has
been accomplished using the information derived from the
present invention. Taking advantage of the non-type
specific region one can isolate the DNA encoding other
type-specific genes by simply obtaining a strain of S.
pneumoniae known to have a type-specific capsule.
Polymerase chain reaction using primers specific for
opposite flanking regions and directed toward the
opposite flanking non-type specific region are used to
amplify the type-specific gene cassette. Where the size
of the type-specific region is unknown, restriction
1~ fragment length polymorphism analysis, using probes
specific for either or both of the non-type specific
regions may be used to determine the size.
Antibodies specific for type-specific antigenic
epitopes may be used with the present invention to
distinguish and evaluate the stability of the S.
pneumoniae strain prior to, and after cloning of the
region. It will also be used for verifying the directed
transfer of type-specific genes to a prospective host.
Preferred hosts for polysaccharide capsule production
will be gram positive bacteria, in particular members of
the Streptococcus, Bacillus and even Staphylococcus
specles .
Selection of Hosts for Type-Specific Capsule Production
The present invention provides methods for the
selection, isolation and transformation of Streptococcus
sp. with a type-specific capsule polysaccharide gene
locus. A cps locus can now be isolated and used to
specifically change the capsular phenotype of a selected
host organism. The preferred host organism for use with
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the present invention is a bacteria that produces high
amounts of the capsular polysaccharide.
Once a suitable high producing host is identiried,
it will be used to carry the type-specific genes o~
choice, as shown in Examples 6 and 18. The organisms can
be converted to other serotypes by transforming the nigh
producing recipient bacteria with a gene cassette or with
intact genomic DNA. A gene cassette, as previously
mentioned, is a segment of DNA comprising of one or more
genes flanked by specific DNA sequences which enables
incorporation of the cassette into a recipient's cell
chromosome at a specific site or locus via homologous
recombination. A cassette may contain type-specific
genes, either alone or in combination with non-type
specific genes. Of course, the preferred construct for
transfection will be a cassette containing the non-type
specific flanking regions.
A cassette of the cps locus comprising of the cps
genes and the 5' and 3' flanking regions donated from any
one of the 85 S. pneumoniae serotypes may be transformed
into a recipient S. pneumoniae also belonging to any one
of the 85 serotypes. During transformation, recombination
would occur in the flanking regions, resulting in the
replacement of the recipient's type-specific region by
that of the donor. The capsule type of the recipient
would be expected to change to that of the donor.
The introduction of a gene cassette comprising of
the cps locus or DNA segment or genetic element, into S.
pneumoniae may be performed by a variety of methods. A
particularly preferred embodiment would be to digest the
~ donor S. pneumoniae genomic DNA with one or more
restriction enzymes such as those described in Table 1,
~ for example, and then separate the entire cps locus from
the rest of the genomic DNA by gel purification. This
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specific DNA segment, cps locus or genetic element, .~.ay
then be ligated into a vector such as a plasmid or c~smid
or bacteriophage, and transformed by various methods into
the recipient S. pneumoniae. Alternatively, the donc~ 5.
pneumoniae's entire genomic DNA may be naturally
transformed into the recipient by a suitable method,
e.g., as described in Example 1. Further still, the
donor's genomic DNA may be digested with one or more
restriction enzymes and then ligated into a plasmid,
cosmid or bacteriophage, without selecting specifica ly
for the cps locus. This may then be transformed in~ the
recipient S. pneumoniae.
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TABLE 1
RESTRICTION ENZYMES
Aat II GACGT/C
Acc I GT/MKAC
Acc II CG/CG
Acc III T/CCGGA
Aci I CCGC(2/2)
Acy I GR/CGYC
Afl II C/TTAAG
Afl III A/CRYGT
Age I A/CCGGT
Aha III TTT/A~A
Alu I AG/CT
AlwN I CAGNNN/CTG
Aoc I CC/TNAGG
Apa I GGGCC/C
ApaB I GCANNNNN/TGC
Apah I G/TGCAC
Asc I GG/CGCGCC
Asu I G/GNCC
Asu II TT/CGAA
Ava I C/YCGRG
Ava II G/GWCC
Ava III ATGCAT
Avr III C/CTAGG
Bae I ACNNNNGTAYC
Bal I TGG/CCA
BamH I G/GATCC
Bbv I GCAGC(8/12)
Bbv II GAAGAC(2/6)
Bcc I CCATC
Bcef I ACGGC(12/13)
Bcg I GCANNNNNNCG(12/10)
Bcl I T/GATCA
Bet I W/CCGGW
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Bgl I GCCNNNN/NGGC
Bgl II A/GATCT
Bin I GGATC(4/5)
BpulO I CCTNAGC(-5/2)
Bpu1102 I GC/TNAGC
Bspl286 I GDGCH/C
BsplO6 I AT/CGAT
BspC I CGAT/CG
BsaA I YAC/GTR
BsaB I GATNN/NNATC
BseP I GCGCGC
Bsg I GTGCAG(16/14)
Bsi I CTCGTG(5/1)
BsiY I CCNNNNN/NNGG
Bsm I GA~TGC(1/-1)
BsmA I GTCTC(1/5)
Bsp50 I CG/CG
BspG I CG/CGCTGGAC
BspH I T/CATGA
BspM I ACCTGC(4/8)
BspM II T/CCGGA
Bsr I ACTGG(1/-1)
BsrB I GAGCGG(-3/-3)
BstE II G/GTNACC
BstN I CC/WGG
BstX I CCANNNNN/NTGG
Cac8 I GCN/NGC
Cau II CC/SGG
Cfr I Y/GGCCR
CfrlO I R/CCGGY
Cla I AT/CGAT
CviJ I RG/CY
CviR I TG/CA
Dde I C/TNAG
Dpn I GA/TC
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Dra I TTT/AAA
Dra II TG/GNCCY
Dra III CACNNN/GTG
Drd I GACNNNN/NNGTC
Drd II GAACCA
Dsa I C/CRYGG
EamllO5 I GACNNN/NNGTC
Eci I TCCGCC
Eco3 II GGTCTC(1/5)
Eco47 III AGC/GCT
Eco52 I C/GGCCG
Eco57 I CTGAAG(16/14)
EcoN I CCTNN/NNNAGG
EcoR I G/AATTC
EcoR II /CCWGG
Ecor V GAT/ATC
Esp I GC/TNAGC
Esp3 I CGTCTC(1/5)
Fau I CCCGC(4/6)
Fin I GTCCC
Fnu4H I GC/NGC
FnuD II CG/CG
Fok I GGATG(9/13)
Fse I GGCCGG/CC
Fsi I R/AATTY
Gdi II YGGCCG(-5/-1)
Gsu I CTGGAG(16/14)
Hae I WGG/CCW
Hae II RGCGC/Y
Hae III GG/CC
Hga I GACGC(5/10)
HgiA I GWGCW/C
HgaC I G/GYRCC
HgiE II ACCNNNNNNGGT
HgiJ II GRGCY/C
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Hha I GCG/C
Hind II GTY/RAC
Hind III A/AGCTT
Hinf I G/ANTC
Hinl I GR/CGYC
Hpa I GTT/AAC
Hpa II C/CGG
Hph I GGTCA(8/7)
Kpn I GGTAC/C
Ksp632 I CTCTTC(1/4)
Ksp I CCGC/GG
Mae I C/TAG
Mae II A/CGT
Mae III /GTNAC
Mbo I /GATC
Mbo II GAAGA(8/7)
Mcr I CGRY/CG
Mfe I C/AATTG
Mlu I A/CGCGT
Mly I GACTC(5/5)
Mme I TCCRAC(20/18)
Mnl I CCTC(7/7)
Mse I T/TAA
Msp I C/CGG
Mst I TGC/GCA
Mst II CC/TNAGG
Mwo I G~ 'N N N N ~/NNGC
Nae I GCC/GGC
Nar I GG/CGCC
Nci I CC/SGG
Nco I C/CATGG
Nde I CA/TATG
Nhe I G/CTAGC
Nla III CATG/
Nla IV GGN/NCC
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Not I GC/GGCCGC
Nru I TCG/CGA
Nsi I ATGCA/T
Nsp I RCATG/Y
NSpB II CMG/CKG
Pac I TTAAT/TAA
Pal I GG/CC
PflllO8 I TCGTAG
PflM I CCANNNN/NTGG
Ple I GAGTC(4/5)
PmaC I CAC/GTG
Pme I GTTT/AAAC
PpuM I RG/GWCCY
PshA I GACNN/NNGTC
PspA I C/CCGGG
Pst I CTGCA/G
Pvu I CGAT/CG
Pvu II CAG/CTG
RleA I CCCACA(12/9)
Rsa I GT/AC
Rsr II CG/GWCCG
Sac I GAGCT/C
Sac II CCGC/GG
Sal I G/TCGAC
Sap I GCTCTTC(1/4)
Sau3A I /GATC
Sau96 I G/GNCC
Sau I CC/TNAGG
~ Sca I AGT/ACT
ScrF I CC/NGG
Sdu I GDGCH/C
Sec I C/CNNGG
SfaN I GATC/(5/9)
S~c I CTYRAG
Sfe I C/TYRAG
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Sfi I GGCCNNNN/NGGC
SgrA I CR/CCGGYG
Sma I CCC/GGG
Sna I CTATAC
5 SnaB I TAC/GTA
Spe I A/CTAGT
Sph I GCATG/C
Spl I C/GTACG
Srf I GCCC/GGGC
Sse838 I CCTGCA/GG
Ssp I AAT/ATT
Stu I AGG/CCT
Sty I C/CWWGG
Swa I ATTT/AAAT
15 Taq I T/CGA
Taq II GACCGA(ll/9)
Tfi I GAWTC
Tsp45 I GTSAC
Tsp E I AATT
20 Tthlll I GACN/NNGTC
Tthlll II CAARCA(ll/9)
Vsp I AT/TAAT
Xba I T/CTGAGA
Xcm I CCANNNNN/NNNNTGG
25 Xho I C/TCGAG
Xho II R/GATCY
Xma I C/CCGGG
Xma III C/GGCCG
Xmn I GAANN/NNTTC
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Nucleic Acid Hybridization
The DNA sequences disclosed herein will find u~-lity
as probes or primers in nucleic acid hybridization
embodiments. As such, it is contemplated that
oligonucleotide fragments corresponding to the
sequence(s) of SEQ ID NO:l, SEQ ID NO: 2 and SEQ ID NO:3
(including sequences in between), SEQ ID NO:4, SEQ I_
NO:5 and SEQ ID NO:6 for stretches of between about 10-14
nucleotides to about 20 or to about 30 nucleotides w ll
find particular utility, with even longer sequences,
e.g., 40, 50, 100, 200, 500, and even up to full lenath,
being more preferred for certain embodiments. The
ability of such nucleic acid probes to specifically
hybridize to non-type-specific and to type-specific-
encoding sequences will enable them to be of use in a
variety of embodiments. For example, the probes can be
used in a variety of assays for detecting the preserce of
complementary sequences in a g ven sample, as may be
used, for example to isolate related type-specific aenes.
Alternatively, one may use the non-type-specific regions
to aid in the isolation and cloning of additional ty~e-
specific cassettes. However, other uses are envisioned,
including the use of the sequence information for the
preparation of mutant species primers, or primers fo~ use
in preparing other genetic constructions.
Nucleic acid molecules having stretches of abou_ 10-
14, 20, 30, 50, or even of about 100-200 nucleotides or
so, complementary to SEQ ID NO:1, SEQ ID NO:2 and SEQ ID
NO:3 (including sequences in between), SEQ ID NO:4, SEQ
ID NO:5 and SEQ ID NO:6, will have utility as
hybridization probes. These probes will be useful 1~ a
~ variety of hybridization embodiments, such as Southern
and Northern blotting in connection with analyzing
genomic structure and organization of type-specific genes
or both linked and non-linked regulatory genes in diverse
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strains of S. pneumoniae. The total size of fragmer=, as
well as the size of the complementary stretch(es), ~
ultimately depend on the intended use or application of
the particular nucleic acid segment. Smaller fragmen~s
will generally find use in hybridization embodiments,
wherein the length of the complementary region may be
varied, such as between about 10-14 and about 100
nucleotides, or even up to full length according to she
complementary sequences one wishes to detect.
The use of a hybridization probe of about 10-14
nucleotides in length allows the formation of a duplex
molecule that is both stable and selective. Molecules
having complementary sequences over stretches greater
than 10-14 bases in length are generally preferred,
though, in order to increase stability and selectivity o~
the hybrid, and thereby improve the quality and degree of
specific hybrid molecules obtained. One will generally
prefer to design nucleic acid molecules having gene-
complementary stretches of 15 to 20 nucleotides, or even
longer where desired. Such fragments may be readily
prepared by, for example, directly synthesizing the
fragment by chemical means, by application of nucleic
acid reproduction technology, such as the PCR technology
of U.S. Patent 4, 603,102 (incorporated herein by
reference) or by introducing selected sequences into
recombinant vectors for recombinant production.
Accordingly, the nucleotide sequences of the
invention may be used for their ability to selectively
form duplex molecules with complementary stretches of
non-type- and of type-specific genes. Depending on the
application envisioned, one will desire to employ varying
conditions of hybridization to achieve varying degrees of
selectivity of probe towards target sequence. Such
hybridization conditions are standard in the art and
include low stringency and high stringency. For
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applications re~uiring high selectivity, one will
typically desire to employ relatively stringent
conditions to form the hybrids, e.g., one will select
relatively low salt and\or high temperature conditions,
such as provided by o. 02M-0.15M NaCl at temperatures o~
50C to 70C. One particular example is using the
QuickHyb~ system (Stratagene's Illuminator~
Nonradioactive Detection System) at 68C. Such selective
conditions tolerate little, if any, mismatch between the
probe and the template or target strand, and would be
particularly suitable for isolating other genes encoding
gene products that are involved in the production of
capsule polysaccharides. A preferred embodiment for
hybridization conditions is described in detail in
Example 4. Further standard hybridization conditions can
be found in Sambrook et al., (1989), and are known to
those o~ skill in the art.
Of course, for some applications, for example, where
one desires to prepare mutants employing a mutant primer
strand hybridized to an underlying template or where one
seeks to isolate type-specific-encoding sequences from
related species, functional equivalents, or the like,
less stringent hybridization conditions will typically be
needed in order to allow formation of the heteroduplex.
One may also desire to employ other hybridization
techniques and to change salt conditions such as varying
the amount of salt from between about 0.15M-0.9M. Other
parameters that can be modified may be temperature such
- as those ranging from 20C to 55C to optimize the
signal-to-noise ratio to reduce unwanted background. The
techniques for optimizing hybridization conditions are
well known to those of skill in the art and are generally
also described within the instruction manual for various
reagents and apparatus.
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In any case, it is generally appreciated that
conditions can be rendered more stringent by the addition
of increasing amounts of formamide, which serves to
destabilize the hybrid duplex in the same manner as
increased temperature. Thus, hybridization conditions
can be readily manipulated as is known to those of skill
in the art, and thus will generally be a method of choice
depending on the desired results.
In certain embodiments, it will be advantageous to
employ nucleic acid se~uences of the present invention in
combination with an appropriate means, such as a label,
for determining hybridization. A wide variety of
appropriate indicator means are known in the art,
including fluorescent, radioactive, enzymatic or other
ligands, such as avidin/biotin, which are capable of
giving a detectable signal. In preferred embodiments,
one will likely desire to employ a fluorescent label or
an enzyme tag, such as urease, alkaline phosphatase or
peroxidase, instead of radioactive or other environmental
undesirable reagents. In the case of enzyme tags,
colorimetric indicator substrates are known which can be
employed to provide a means visible to the human eye or
spectrophotometrically, to identify specific
hybridization with complementary nucleic acid-containing
samples.
In general, it is envisioned that the hybridization
probes described herein will be useful both as reagents
in solution hybridization as well as in embodiments
employing a solid phase. In embodiments involving a
solid phase, the test DNA (or RNA) is adsorbed or
otherwise affixed to a selected matrix or surface. This
fixed, single-stranded nucleic acid is then subjected to
specific hybridization with selected probes under desired
conditions. The selected conditions will depend on the
particular circumstances based on the particular criteria
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required (depending, for example, on the G+C conten_s,
type of target nucleic acid, source of nucleic acid, size
of hybridization probe, etc.). Following washing c- the
hybridized surface so as to remove nonspecifically ~und
probe molecules, specific hybridization is detecte~, or
even quantified, by means of the label.
Longer DNA segments will often find particular
utility in the recombinant production of peptides or
proteins. DNA segments which encode peptide antigens
from about 15 to about 50 amino acids in length, or more
preferably, from about 15 to about 30 amino acids lr
length are contemplated to be particularly useful, as are
DNA segments encoding entire cps locus encoded proteins,
such as those of SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO:5 and SEQ ID NO:6. DNA segments encoding
peptides will generally have a minimum coding length in
the order of about 45 to about 150, or to about 90
nucleotides.
The nucleic acid segments of the present inven~ion,
regardless of the length of the coding sequence itself,
may be combined with other DNA sequences, such as
promoters, repressors, attenuators, additional
restriction enzyme sites, multiple cloning sites, other
coding segments, and the like, such that their overall
length may vary considerably. It is contemplated that a
nucleic acid fragment of almost any length may be
employed, with the total length preferably being limited
by the ease of preparation and use in the intended
recombinant DNA protocol. For example, nucleic acid
fragments may be prepared in accordance with the present
invention which are up to about 10,000 base pairs in
length, with segments of about 5,000 or 3,000 being
preferred and segments of about 1,000 base pairs in
length being particularly preferred.
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It will be understood that this invention is not
limited to the particular nucleic acid sequences of SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5 and SEQ ID NO: 6, or to the particular amino ac d
sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ
ID NO:10, SEQ ID NO:11, SEQ ID NO :12, SEQ ID NO :13, SEQ
ID NO:14, SEQ ID NO:15 and SEQ ID NO:16. Therefore, DNA
segments prepared in accordance with the present
invention may also encode biologically functional
equivalent proteins or peptides which have variant amino
acids sequences. Such sequences may arise as a
consequence of codon redundancy and functional
equivalency which are known to occur naturally within
nucleic acid sequences and the proteins thus encoded.
Alternatively, functionally equivalent proteins or
peptides may be created via the application of
recombinant DNA technology, in which changes in the
protein structure may be engineered, based on
considerations of the properties of the amino acids being
exchanged.
DNA segments encoding a gene, including the cpsB,
cpsC, cpsE, cspD, cspS, cspU, cspM, plpA and tnpA genes
may be introduced into recombinant host cells and
employed for expressing and producing the type-specific
proteins for use in producing type-specific capsule
polysaccharides. Alternatively, through the application
of genetic engineering techniques, subportions or
derivatives of selected type-specific gene locus genes
may be employed. Equally, through the application of
site-directed mutagenesis techniques, one may re-engineer
DNA segments of the present invention to alter the coding
sequence, e.g., to introduce improvements to the
antigenicity of the protein or to test mutants in order
to examine the production of capsule polysaccharides at
the molecular level. Where desired, one may also prepare
fusion peptides, e.g., where the type-specific coding
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regions are aligned within the same expression unit with
- other proteins or peptides having desired functions, such
as for immunodetection purposes (e.g., enzyme label
coding regions).
Screening Method Type-Specific Genes
Screening for type-specific genes provides another
utility for the cps loci of the present invention. A
type-specific screening protocol will allow for the
epidemiological identification of S. pneumoniae and its
serotypes at the molecular level. By using one or both
of the non-type specific regions as probes one can
determine the presence of S. pneumoniae from a small
sample by immobilizing DNA from the sample onto a solid
matrix, for example a slot blot using nitrocellulose, and
hybridizing thereto a probe as described in the present
invention.
Using either or both of the non-type specific
regions of the present invention as a probe or probes one
may also screen southern blots. The screening of
southern blots may allow one to determine not only the
presence of S. pneumoniae but also the exact genotype of
S. pneumoniae present in the sample. In conjunction with
densitometric analysis of a southern blot containing
multiple serotypes on may determine not only the relative
frequency of serotypes within a sample, but in addition
one may ~xAm;ne the changing characteristics of the
serotypes by exAm;n;ng samples taken at distinct time
periods.
It also allows the clinician to determine if a
patient is having a recurrence of a particular serotype,
if the patient is susceptible to a particular serotype or
types, or if a new serotype is increasing in the
population.
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Site-Specific Mutagenesis
Site-specific mutagenesis, also known as site-
directed mutagenesis, is a technique useful in the
preparation of changes, directed by the laboratory
technician, that change the characteristics of genes and
their gene products, for the addition of restriction
sites, for modifying the activity of promoters,
repressors, attenuators, and for directed changes
affecting recombination. All of these changes may be
produced through specific mutagenesis of the underlying
non-type- and type-specific DNA of the present invention.
The technique further provides a ready ability to prepare
and test sequence variants, for example, incorporating
one or more of the ~oregoing considerations, by
introducing one or more nucleotide sequence changes into
the DNA. Site-specific mutagenesis allows the production
of mutants through the use of specific oligonucleotide
sequences which encode the DNA sequence of the desired
mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient
size and sequence complexity to form a stable duplex on
both sides of the deletion junction being traversed.
Typically, a primer of about 17 to 25 nucleotides in
length is preferred, with about 5 to 10 residues on both
sides of the junction of the sequence being altered.
In general, the technique of site-specific
mutagenesis is well known in the art, as exemplified by
various publications. As will be appreciated, the
technique typically employs a phage vector which exists
in both a single stranded and double stranded form.
Typical vectors useful in site-directed mutagenesis
include vectors such as the M13 phage. These phage are
readily commercially available and their use is generally
well known to those skilled in the art. Double stranded
plasmids are also routinely employed in site directed
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mutagenesis which eliminates the step of transferrir.g the
gene of interest from a plasmid to a phage.
In general, site-directed mutagenesis in accordance
herewith is performed by first obtaining a single-
stranded vector or melting apart the two strands of a
double stranded vector which includes within its seauence
a DNA sequence which encodes the type-specific protein or
proteins encoded by the type-specific gene locus. An
oligonucleotide primer bearing the desired mutated
sequence is prepared, generally synthetically. This
primer is then annealed with the single-stranded vec~or,
and subjected to DNA polymerizing enzymes such as E. coli
polymerase I Klenow fragment, in order to complete the
synthesis of the mutation-bearing strand. Thus, a
heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears
the desired mutation. This heteroduplex vector is then
used to transform appropriate cells, such as E. coll
cells, and clones are selected which include recombinant
vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of type-
specific genes using site-directed mutagenesis is
provided as a means of producing potentially useful
species, for example a strain having enhanced production
of type-specific capsular polysaccharides, and is not
meant to be limiting as there are other ways in which
sequence variants of other type-specific genes may be
obtained. For example, recombinant vectors encoding
other type-specific genes, as described herein using the
non-type specific regions of the capsule polysaccharide
gene cassette are encompassed.
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siological Functional E~uivalents
Even though the invention has been described with a
certain degree of particularity, it is evident that many
5 alternatives, modifications, and variations will be
apparent to those skilled in the art in light of the
~oregoing disclosure. Accordingly, it is intended ~hat
all such alternatives, modifications, and variations
which fall within the spirit and the scope of the
invention be embraced by the defined claims.
As used in this application, the term "DNA segm.ent"
refers to a DNA molecule that has been isolated free of
total genomic DNA of a particular species. Therefore, a
DNA segment encoding the cps locus refers to a DNA
segment that contains the 5' and/or 3' flanking regions,
or the cpsB, cpsC, cpsE, cpsD, cpsS, cpsU, cpsM, tnpA or
plpA coding sequences, yet is isolated away from, o~
puri~ied free from, total genomic DNA of S. pneumoniae.
Included within the term "DNA segment", are DNA segments
and smaller fragments of such segments, and also
recombinant vectors, including, for example, plasmids,
cosmids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or
purified 5' or 3' flanking region, or cpsB, cpsC, cpsE,
cpsD, cpsS, cpsU, cpsM, plpA or even tnpA gene, refers to
a DNA segment including the coding sequences and, in
certain aspects, regulatory sequences, isolated
substantially away from other naturally occurring genes
or protein encoding sequences. In this respect, the term
"gene" is used for simplicity to refer to a protein,
polypeptide or peptide encoding unit. As will be
understood by those in the art, this term includes both
genomic sequences, cDNA sequences and smaller engineered
gene segments that express, or may be adapted to express,
proteins, polypeptides or peptides.
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"Isolated substantially away from other coding
- sequences'~ means that the locus of interest, in this case
the s~ or 3~ flanking regions, or cps B, cpsC, cps~,
cpsD, cpsS, cpsU, cpsM, tnpA or plpA coding sequences,
S forms the significant part of the DNA segment, and that
the DNA segment does not contain large portions of
naturally-occurring coding DNA, such as large chromosomal
fragments or other functional genes or cDNA coding
regions. Of course, this refers to the DNA segment as
originally isolated, and does not exclude genes or coding
regions later added to the segment by the laboratory
technician.
In particular embodiments, the invention concerns
lS isolated DNA segments and recombinant vectors
incorporating DNA sequences that include the 5' or 3'
flanking regions denoted by SEQ ID NO:l, SEQ ID NO:2, SEQ
ID NO:3 and SEQ ID NO:4 or SEQ ID NO:6, respectively.
DNA segments and vectors that incorporate DNA sequences
that encode CpsB, CpsC, CpsE, CpsD, CpsS, CpsU, CpsM,
PlpA or transposase A proteins that include within their
amino acid sequences an amino acid sequence as set îorth
in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,
SEQ ID NO:15 or SEQ ID NO:16 respectively are also
included.
The term "a sequence as set forth in SEQ ID NO:7-16"
means that the sequence substantially corresponds to a
portion of SEQ ID NO:7-16 and has relatively few amino
acids that are not identical to, or a biologically
functional equivalent of, the amino acids of SEQ ID NO:7-
16. The term "biologically functional equivalent" is
well understood in the art and is further defined in
detail herein. Accordingly, sequences that have between
~ about 75% and about 85~; or more preferably, between
about 86~ and about 95~; or even more preferably, between
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about 96~ and about 99~; of amino acids that are
identical or functionally equivalent to the amino acids
of SEQ ID NO:7-16 will be sequences that are "essentially
as set forth in SEQ ID NO:7-16."
Naturally, it will be understood that for the Cps
proteins, the definition of ~'equivalents" in this sense
does not extend to distinct, but homologous proteins,
such as CpsD and HasB from Streptococcus pyogenes; CpsS
and HasA from S. pyogenes, NodC from Rhizo~ium meliloti;
nor CpsU and GtaB from Bacillus subtilis. Rather, the
scope of equivalents contemplated are such that the
changes made still result in a protein that is
structurally and functionally a Cps protein.
In certain other embodiments, the invention concerns
isolated DNA segments and recombinant vectors that
include within their sequence a nucleic acid sequence
essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4 and SEQ ID NO:6, for the flanking
regions; and SEQ ID NO:5 for the type-speci~ic encoding
regions. The term "as set forth in SEQ ID NO:5" is used
in the same sense as described above and means that the
nucleic acid sequence substantially corresponds to a
portion of SEQ ID NO:5 and has relatively few codons that
are not identical, or functionally equivalent, to the
codons of SEQ ID NO:5. The term "functionally equivalent
codon" is used herein to refer to codons that encode the
same amino acid, such as the six codons for arginine or
serine, and also refers to codons that encode
biologically equivalent amino acids (as in Table 2).
It will be understood that acid and nucleic acid
sequences may include additional residues, such as
additional N- or C-terminal amino acids or 5' or 3'
sequences, and yet still be essentially as set forth in
one of the sequences disclosed herein, so long as the
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sequence meets the criteria set forth above, includl~g
the maintenance of biological protein activlty where
protein expression is concerned. The addition of
terminal sequences particularly applies to coding n~_leic
acid sequences that may, for example, include various
non-coding sequences flanking either of the 5' or 3'
portions of the coding region, such as promoters.
Allowing for the degeneracy of the genetic code,
sequences that have between about 75~ and about 85%; or
more preferably, between about 85~ and about 95~; or even
more preferably, between about 95~ and about 99~ of
nucleotides that are identical to the nucleotides o_ SEQ
ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5 or SEQ ID NO:6 (SEQ ID NO:1-6), will be sequences
that are "as set forth in SEQ ID NO:l-6". Sequences that
are essentially the same as those set forth in SEQ ID
NO:1-6 may also be functionally defined as sequences that
are capable of hybridizing to a nucleic acid segment
containing the complement of SEQ ID NO:l-6 under standard
conditions. Suitable hybridization conditions will be
well known to those of skill in the art and are clearly
set forth herein, e.g., see Example 4.
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~ 68 -
Table 2. Amino Acids and the Corre~po~; n~ Codons.
Amino Acids Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic Asp D GAC GAU
acid
Glutamic Glu E GAA GAG
acid
Phenylal- Phe F UUC W U
anine
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine Ile I AUA AUC A W
~ysine Lys K AAA AAG
~eucine Leu L W A W G CUA CUC CUG C W
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gln Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG G W
Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU
Naturally, the present invention also encompasses
DNA segments that are complementary, or essentially
complementary, to the sequences set forth in SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and
SEQ ID NO:6. Nucleic acid sequences that are
"complementary" are those that are capable o~ base-
pairing according to the standard Watson-Crick
complementarity rules. As used herein, the term
"complementary sequences" means nucleic acid sequences
that are substantially complementary, as may be assessed
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by the same nucleotide comparison set forth above, cr as
defined as being capable o~ hybridizing to the nucleic
acid segment o~ SEQ ID NO:1-6 under relatively stringent
conditions such as those described herein as SEQ ID NO:1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5 and
SEQ ID NO:6.
The DNA segments of the present invention include
those encoding biologically functional equivalent
proteins and peptides. Such sequences may arise as a
consequence of codon redundancy and functional
equivalency that are known to occur naturally within
nucleic acid sequences and the proteins thus encoded.
Alternatively, functionally e~uivalent proteins or
peptides may be created via the application of
recombinant DNA technology, in which changes in the
protein structure may be engineered, based on
considerations of the properties of the amino acids being
exchanged. Changes designed in the laboratory, may be
introduced through the application of site-directed
mutagenesis techniques, e.g., to introduce improvements
to the antigenicity of the protein or to test S.
pneumoniae mutants in order to ~m; ne capsular
productivity at the molecular level.
If desired, one may also prepare fusion proteins and
peptides, e.g., where the cpsB, cpsC, cpsE, cpsD, cpsS,
cpsU, cpsM, plpA and tnpA coding regions are aligned
within the same expression unit with other proteins or
peptides having desired ~unctions, such as for
purification or immunodetection purposes (e.g., proteins
that may be purified by affinity chromatography and
enzyme label coding regions, respectively).
-
AS mentioned above, modification and changes may be
~ made in the structure o~ CpsB, CpsC, CpsE, CpsD, CpsS,
CpsU, CpsM, plpA or tnpA and still obtain a molecule
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having like or otherwise desirable characteristics. For
example, certain amino acids may be substituted for other
amino acids in a protein structure without appreciable
loss of interactive binding capacity with structures such
as, for example, antigen-binding regions of antibodies or
binding sites on substrate molecules, receptors, or
catalytic regulation of capsular polysaccharide
production. Since it is the interactive capacity and
nature of a protein that defines that protein's
biological functional activity, certain amino acid
sequence substitutions can be made in a protein sequence
(or, o~ course, its underlying DNA coding sequence) and
nevertheless obtain a protein with like (agonistic)
properties. Equally, the same considerations may be
employed to create a protein or polypeptide with
countervailing (e.g., antagonistic) properties. It is
thus contemplated by the inventors that various changes
may be made in the sequence of SEQ ID NO: 7, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, SEQ ID NO:15
or SEQ ID NO:16 proteins or peptides (or underlying DNA)
without appreciable loss of their biological utility or
activity.
It is also well understood by the skilled artisan
that, inherent in the definition of a biologically
functional equivalent protein or peptide, is the concept
that there is a limit to the number of changes that may
be made within a defined portion of the molecule and
still result in a molecule with an acceptable level of
equivalent biological activity. Biologically functional
equivalent peptides are thus defined herein as those
peptides in which certain, not most or all, of the amino
acids may be substituted. In particular, the function of
given protein must be retained to be an equivalent. Of
course, a plurality of distinct proteins/peptides with
different substitutions may easily be made and used in
accordance with the invention.
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It is also well understood that where certain
_ residues are shown to be particularly important to ~:~e
biological or structural properties of a protein or
peptide, e.g., residues in active sites, such resiaues
may not generally be exchanged. This is the case i. the
present invention, where CpsD has a putative NAD-binding
site and active site region at residues 2 to 29 and 251-
263 (SEQ ID NO:11) respectively.
Amino acid substitutions are generally based c- the
relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. An analysis
of the size, shape and type of the amino acid side-cnain
substituents reveals that arginine, lysine and histidine
are all positively charged residues; that alanine,
glycine and serine are all a similar size; and that
phenylalanine, tryptophan and tyrosine all have a
generally si~ilar shape. Therefore, based upon these
considerations, arginine, lysine and histidinei alanine,
glycine and serine; and phenylalanine, tryptophan and
tyrosine; are defined herein as biologically functional
equivalents.
In making changes, the hydropathic index of amino
acids may be considered. Each amino acid has been
assigned a hydropathic index on the basis of their
hydrophobicity and charge characteristics, these are:
isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine/cystine (+2.5); methionine
(+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7);
serine (-0.8); tryptophan (-0.9); tyrosine (-1.3);
proline (-1.6); histidine (-3.2); glutamate (-3.5)i
~ glutamine (-3.5); aspartate (-3.5); asparagine (-3.5);
lysine (-3.9); and arginine (-4.5).
.
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The importance of the hydropathic amino acid _..dex
in conferring interactive biological functlon on a
protein is generally understood in the art (Kyte &
Doolittle, 1982, incorporated herein by reference). It
is known that certain amino acids may be substitute~ for
other amino acids having a similar hydropathic index or
score and still retain a similar biological activi~y. In
making changes based upon the hydropathic index, the
substitution of amino acids whose hydropathic indic~s are
within +2 is preferred, those which are within _I ar~
particularly preferred, and those within +0.5 are even
more particularly preferred.
It is also understood in the art that the
substitution of like amino acids can be made effect-~ely
on the basis of hydrophilicity. U.S. Patent 4,554,101,
incorporated herein by reference, states that the
greatest local average hydrophilicity of a protein, as
governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and
antigenicity, i.e. with a biological property of the
protein. It is therefore understood that an amino acid
can be substituted for another having a similar
hydrophilicity value and still obtain a biologically
equivalent, and in particular, an immunologically
equivalent protein.
As detailed in U.S. Patent 4,554,101, the following
hydrophilicity values have been assigned to amino acid
residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0
i l); glutamate (~3.0 + 1); serine (+0.3); asparagine
(+0.2); glutamine (+0.2); glycine (0); threonine (-5.4);
proline (-0.5 _ 1); alanine (-0.5); histidine (-0.5);
cysteine (-1.0); methionine (-1.3); valine (-1.5);
leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
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In making changes based upon similar hydrophilicity
values, the substitution o~ amino acids whose
hydrophilicity values are within +2 is pre~erred, those
which are within +1 are particularly preferred, and those
within +0.5 are even more particularly preerred.
As outlined above, amino acid substitutions are
generally therefore based on the relative similarity of
the amino acid side-chain substituents, for example,
their hydrophobicity, hydrophilicity, charge, size, and
the like. Exemplary substitutions which take various of
the foregoing characteristics into consideration are well
known to those of skill in the art and include: arginine
and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine
and isoleucine.
Antibody Generation
Means for preparing and characterizing antibodies
are well known in the art (See, e.g., Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988;
incorporated herein by reference). This invention thus
contemplates the generation o~ antibodies against the
proteins CpsB, CpsC, CpsE, CpsD, CpsS, CpsU, CpsM, PlpA
and transposase A or peptides derived there~rom. The
CpsB, CpsC, CpsE, CpsD, CpsS, CpsU, CpsM, PlpA and
transposase A proteins or peptides may be obtained using
standard methods of recombinant expression as is
routinely in the art.
..
The methods for generating monoclonal antibodies
(MAbs) generally begin along the same lines as those for
preparing polyclonal antibodies. Briefly, a polyclonal
antibody is prepared by immunizing an animal with an
immunogenic composition in accordance with the present
invention and collecting antisera ~rom that immunized
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~n;m~l. A wlde range of animal species can be used for
the production of antisera. Typically the animal used
for production of anti-antisera is a rabbit, a mouse, a
rat, a hamster, a guinea pig or a goat. Because of the
relatively large blood volume of rabbits, a rabbit is a
preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may
vary in its immunogenicity. It is often necessary
therefore to boost the host immune system, as may be
achieved by coupling a peptide or polypeptide immunogen
to a carrier. Exemplary and preferred carriers are
keyhole limpet hemocyanin (KLH) and bovine serum albumin
(BSA). Other albumins such as ovalbumin, mouse serum
albumin or rabbit serum albumin can also be used as
carriers. Means for conjugating a polypeptide to a
carrier protein are well known in the art and include
glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide
ester, carbodiimyde and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity
of a particular immunogen composition can be enhanced by
the use of non-specific stimulators of the immune
response, known as adjuvants. Exemplary and preferred
adjuvants include complete Freund's adjuvant (a non-
specific stimulator of the immune response containing
killed Mycobacterium tuberculosis), incomplete Freund's
adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the
production of polyclonal antibodies varies upon the
nature of the immunogen as well as the ~n; ~1 used ~or
immunization. A variety of routes can be used to
administer the immunogen (subcutaneous, intramuscular,
intradermal, intravenous and intraperitoneal). The
production of polyclonal antibodies may be monitored by
sampling blood of the immunized animal at various points
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following immunization. A second, booster injection, may
also be given. The process of boosting and titerins is
repeated until a suitable titer is achieved. When a
desired level of immunogenicity is obtained, the
immunized animal can be bled and the serum isolated and
stored, and/or the animal can be used to generate ~bs.
MAbs may be readily prepared through use of well-
known techni~ues, such as those exemplified in u.S.
Patent 4,196,265, incorporated herein by reference.
Typically, this technique involves immunizing a suitable
animal with a selected immunogen composition, e.g., a
purified or partially purified CpsB, CpsC, CpsE, CpsD,
CpsS, CpSU, CpSM, PlpA or transposase A protein,
polypeptide or peptide. The immunizing composition is
administered in a manner effective to stimulate antibody
producing cells. Rodents such as mice and rats are
preferred animals, however, the use of rabbit, sheep frog
cells is also possible. The use of rats may provide
certain advantages tGoding, 1986, pp. 60-61), but mice
are pre~erred, with the BALB/c mouse being most preferred
as this is most routinely used and generally gives a
higher percentage of stable fusions.
Following immunization, somatic cells with the
potential for producing antibodies, specifically B
lymphocytes (B cells), are selected for use in the MAb
generating protocol. These cells may be obtained from
biopsied spleens, tonsils or lymph nodes, or from a
peripheral blood sample. Spleen cells and peripheral
blood cells are preferred, the former because they are a
rich source of antibody-producing cells that are in the
dividing plasmablast stage, and the latter because
peripheral blood is easily accessible. Often, a panel of
animals will have been immunized and the spleen of animal
with the highest antibody titer will be removed and the
spleen lymphocytes obtained by homogenizing the spleen
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with a syrlnge. Typically, a spleen from an immunized
mouse contains approximately 5 X 107 to 2 X 108
lymphocytes.
The antibody-producing B lymphocytes from the
immunized animal are then fused with cells of an immortal
myeloma cell, generally one of the same species as the
animal that was immunized. Myeloma cell lines suited for
use in hybridoma-producing fusion procedures preferably
are non-antibody-producing, have high fusion efficiency,
and enzyme deficiencies that render then incapable o-
growing in certain selective media which support the
growth of only the desired fused cells (hybridomas).
Any one of a number o myeloma cells may be used, as
are known to those of skill in the art (Goding, pp.
65-66, 1986; Campbell, pp. 75-83, 1984). cites). For
example, where the immunized animal is a mouse, one may
use P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4 1, Sp210-Agl4,
FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul;
for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and
4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6
are all useful in connection with human cell fusions.
One preferred murine myeloma cell is the NS-1
myeloma cell line ~also termed P3-NS-1-Ag4-1), which is
readily available from the NIGMS Human Genetic Mutant
Cell Repository by requesting cell line repository number
GM3573. Another mouse myeloma cell line that may be used
is the 8-azaguanine-resistant mouse murine myeloma SP2/0
non-producer cell line.
Methods for generating hybrids of antibody-producing
spleen or lymph node cells and myeloma cells usually
comprise mixing somatic cells with myeloma cells in a 2:1
proportion, though the proportion may vary from about
20:1 to about 1:1, respectively, in the presence o~ an
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agent or agents (chemical or electrical) that promote the
fusion of cell membranes. Fusion methods using Sendai
virus have been described by Kohler and Milstein (1975;
1976), and those using polyethylene glycol (PEG), such as
37~ (v/v) PEG, by Gefter et al. (1977). The use of
electrically induced fusion methods is also appropriate
(Goding pp. 71-74, 1986).
Fusion procedures usually produce viable hybrids at
low frequencies, about 1 X 1o~6 to 1 X 1o~8. However,
this does not pose a problem, as the viable, fused
hybrids are differentiated from the parental, unfused
cells (particularly the unfused myeloma cells that would
normally continue to divide indefinitely) by culturing in
a selective medium. The selective medium is generally
one that contains an agent that blocks the de novo
synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin,
methotrexate, and azaserine. Aminopterin and
methotrexate block de novo synthesis of both purines and
pyrimidines, whereas azaserine blocks only purine
synthesis. Where aminopterin or methotrexate is used,
the media is supplemented with hypoxanthine and thymidine
as a source of nucleotides (HAT medium). Where azaserine
is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells
capable of operating nucleotide salvage pathways are able
to survive in HAT medium. The myeloma cells are
defective in key enzymes of the salvage pathway, e.g.,
- hypoxanthine phosphoribosyl transferase (HPRT), and they
cannot survive. The B cells can operate this pathway,
but they have a limited life span in culture and
generally die within about two weeks. Therefore, the
only cells that can survive in the selective media are
those hybrids formed from myeloma and B cells.
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This culturing provides a population of hybridomas
from which specific hybridomas are selected. Typically,
selection of hybridomas is performed by culturing the
cells by single-clone dilution in microtiter plates,
followed by testing the individual clonal supernatan~s
(after about two to three weeks) for the desired
reactivity. The assay should be sensitive, simple and
rapid, such as radioimmunoassays, enzyme immunoassays,
cytotoxicity assays, plaque assays, dot immunobinding
assays, and the like.
The selected hybridomas would then be serially
diluted and cloned into individual antibody-producing
cell lines, which clones can then be propagated
indefinitely to provide MAbs. The cell lines may be
exploited for MAb production in two basic ways. A sample
of the hybridoma can be injected (often into the
peritoneal cavity) into a histocompatible animal of the
type that was used to provide the somatic and myeloma
cells for the original fusion. The injected animal
develops tumors secreting the specific monoclonal
antibody produced by the fused cell hybrid. The body
fluids of the animal, such as serum or ascites fluid, can
then be tapped to provide MAbs in high concentration.
The individual cell lines could also be cultured in
vitro, where the MAbs are naturally secreted into the
culture medium from which they can be readily obtained in
high concentrations. MAbs produced by either means may
be further purified, if desired, using filtration,
centrifugation and various chromatographic methods such
as HPLC or affinity chromatography.
The following examples are included to demonstrate
preferred embodiments of the invention. It should be
appreciated by those of skill in the art that the
techniques disclosed in the examples which follow
represent techniques discovered by the inventor to
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function well in the practice of the invention, and _hus
can be considered to constitute preferred modes fo~ -ts
practice. However, those of skill in the art shoul-, in
light of the present disclosure, appreciate that many
changes can be made in the specific embodiments whic:~ are
disclosed and still obtain a like or similar result
without departing from the spirit and scope of the
lnvent lon .
EXAMPLE 1
Isolation and Characterization of CaPsule Mutants
A. Methods
1. Bacterial strains, plasmids, and culture conditicns
The bacterial strains and plasmids used are listed
herein in Table 3. Culture conditions for S. pneumcniae
and E. coli were previously described (Dillard and
Yother, 1991). Erythromycin was used at 0.3 ~g/ml and
streptomycin was used at 100 ~g/ml in S. pneumoniae
cultures where indicated. Chloramphenicol was used at 1
~g/ml to detect transcription in S. pneumoniae isolates
carrying pJY4163/4164 chromosomal insertions.
Table 3. Bacterial strains and plasmids.
Strain/ Derivation and Source/Reference
Plasmid properties
Strain
S. pneumoniae
WU2 Type 3 encapsulated Briles et al.
(199lb)
D39 Type 2 encapsulated Avery et al.
(1944)
Rxl Non-encapsulated Ravin (1959)
mutant of R36A-A66 Shoemaker and
hybrid Guild (1974)
L8-2006 Type l encapsulated Dillard and
Yother (1994)
DBL5 Type 5 encapsulated Yother et al.
(1982)
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Strain/ Derivation and Source/Reference
Plasmid properties
BG9273 Type 6A encapsulated Dillard and
Yother (1994)
EP2809 Type 8 encapsulated Dillard and
Yother (1994)
BG5862 Type 9 encapsulated Dillard and
Yother (1994)
LM100 Type 22 encapsulated Dillard and
Yother (1994)
A66R2 Non-encapsulated Muckerman et al.
mutant of (1982)
A66 (Type 3)
661 Non-encapsulated Bernheimer an~
mutant of Wermundsen (1,-72)
A66 (Type 3)
JD531 Non-encapsulated This work
mutant of
WU2, EmR, cpsA1
JD541 Non-encapsulated This work
mutant of
WU2, EMR, cpsA2
JD542 Non-encapsulated This work
mutant of
WU2, EmR, cpsB1
JD551 Non-encapsulated This work
mutant of
WU2, EmR, cpsB2
JD571 Non-encapsulated This work
mutant of
WU2, EmR, cpsB3
JD600 WU2 StrR This work
JD611 JD600 x JD531, EmS, This work
S t rR, cpsA1
JD614 JD600 x JD551, Ems, This work
St rR, cpsB2
JD619 JD600 x JD541, EmS, This work
St rR, cpsA2
JD692 JD600 x JD542, Ems, This work
StrR, cpsB1
JD816 JD600 x JD671, EmS, This work
S t rR, cpsB3
JD636 WIJ2 StrR, RMR, NovR This work
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Strain/ Derivation and Source/Reference
Plasmid properties
JD752 Isolate of This work
trans~ormation pool
W62 x JD811, type 3
encapsulated, EmR
JD770 pJD330 x WU2, t ~ e 3 This work
encapsulated, Em
JD803 JD770 x D39, type 3 This work
encapsulated, Em
JD871 pJD366 x D39, t ~ e 2 This work
encapsulated, Em
JD872 JD871 x WU2, type 2 This work
encapsulated, EmR
JD875 pJD366 x DBL5, t~pe 5 This work
encapsulated, Em
JD908 pJD369 x WU2, non- This work
encapsulated, EmR
E. col i
LE392 F-hsdR514 (Tk Mk Tilghman et al.
) supE44 supF58 ~ (1977)
(faciZY)6 gal K2
galT22 met21 trpR55 A
pJY4163 Lack origin of Yother et al.
and replication for (1992)
pJY4164 S.pneumoniae
Promoterless cat gene
downstream of
multiple cloning site
(opposite
orientations in 4163,
4164), EmR
pJD330 WU2 Sau3Al fragment This work
cloned into p~Y4163
BamHI site, isolated
from JD752
pJD337 pJY4163:: 1.5kb XbaI This work
-P wII fragment of
pJD330
pJD343 pJY4164:- 0.4kb MunI This work
fragment of pJD330
pJD345 pJY4164:: 1.lkb MunI This work
fragment of pJD330
pJD351 pJY4164:: 2.4kb This work
Sau3AI-Sau3AI
fragment of pJD330
orientation opposite
pJD330
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Strain/ Derivation and Source/Reference
Plasmid properties
pJD353 pJY4164:: 1.6kb Thls work
Sau3AI-XbaI fragment
of pJD330
pJD357 pJY4164:: 0.3kb MunI- This work
Sau3AI fragment of
JD330
pJD359 pJY4164:: 0.6kb This work
PvuII-HaeIII fragment
of pJD330
pJD361 pJY4164:: 0.45kb This work
XbaI - Ps tI fragment of
pJD330
pJD362 pJY4164:: 0.4kb This work
HaeI I I -MunI fragment
of pJD330
pJD364 WU2 3.2kb HindIII This work
fragment cloned into
pJY4164 HindII site
pJD366 WU2 3.2kb HindIII This work
fragment cloned into
pJY4164 HindIII site,
orientation opposite
pJD364
pJD368 pJD4164:: O.g5kb ThiS work
RsaI -MunI fragment of
pJD330
pJD369 pJY4164:: 0.55 kb This work
PvuII-MunI fragment
of pJD330
pJD374 WU2 1.2kb Sau3AI This work
fragment cloned in
pJY4163
pJD377 pJY4164:: 1.2kb SacI- This work
HindIII fragment of
pJD364
pJD380 pJY4164:: 0.36kb This work
Sau3AI- SspI fragment
of pJD330
EmR, erythromycin resistant; Ems, erythromycin sensi~ive,
StrR, streptomycin resistant; RifR, rifampicin resis~ant;
NovR, novobiocin resistant.
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2. General DNA techniques
Techniques for DNA fragment isolation, ligatic.s,
and plasmid isolation and purification were performe~ as
previously described (Dillard and Yother, 1991; anc ~s
described by Sambrook et al., 1989, the relevant pc~-ions
incorporated herein by reference). Plasmid screeni~s
were done by scraping colonies from agar plates ana
incubating these in the lysis solution of Kado and ~~u,
3~ SDS, 50mM Tris, pH 12.6 (Kado and Liu, 1981). The
lysates were run on agarose gels to determine plasm-
~sizes.
3. Library construction
A plasmid library of random fragments was
constructed by digesting chromosomal DNA from S.
pneumoniae strain WU2 to completion with Sau3AI and
ligating these fragments into the BamHI site of pJY~163.
The resulting ligation mixture was electroporated i-._o E.
coli LE392, and transformants were selected on L aga~
plates containing 300 ~g erythromycin/ml. Individual
colonies were patched on erythromycin plates. Each ?late
contained 100 colonies and constituted a pool.
Transformants were pooled by scraping the plates.
4. Transformations
Encapsulated strains of S. pneumoniae were induced
to competence as has been described in Yother et al.,
1986, incorporated herein by reference. Non-encaps lated
strains were made competent for transformation by g~owth
in Todd Hewitt broth (Difco, Detroit, MI) supplemen~ed
with 0.01~ CaCl2, 0.5~ BSA, and 0.5~ yeast extract.
S. pneumoniae cells were allowed to express transfcrming
DNA for 2 h. before plating on agar medium.
~ Electroporation of H20 washed E. coli LE392 cells
resuspended in 10~ glycerol was performed in a BTX
Electro Cell Manipulator 600 according to the
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instructions o~ the manufacturer (Biotechnologies a-~
~xperimental Research, Inc., San Diego, CA).
5. Preparation of S. pneumoniae chromosomal DNA
Cultures of S. pneumoniae (100 ml) were grown _o
stationary phase in the presence of 1~ choline chlc~ide
to prevent autolysis (Briese and Hakenbeck, 1983). The
bacteria were centrifuged at 5000 rpm for 10 min ar,~
resuspended in 2.5 ml TE buffer (10 mM TrisHCl, 1 rr~l
EDTA, pX 8.0). SDS was added to 1~, and the cells t;ere
lysed at 65C for 15 min. One fifth volume 5 M KOA^ (pH
8) was added, and incubation was continued at 65C :5
min, followed by incubation on ice for 60 min. Cel_
debris was removed by centrifugation at 10,000 rpm -~r 10
min, the supernatant was added to 2 volumes of etha-.ol,
and the DNA was hooked out with a glass rod. The DNA was
dried, resuspended in TE, and further purified by
CsCl/ethidium bromide buoyant density gradient
centrifugation (Radloff et al., 1967).
6. Recovery of plasmids resolved from S. pneumoniae
chromosomes.
A 10 ml culture of late log phase S. pneumonia2 was
centrifuged at 5000 rpm for 10 min. The supernatan_ was
removed, and the cells were resuspended in 100 ~1 lysis
buEfer (Saunders and Guild, 1980). Following a 5 mln
incubation at 37C, 900 ~l of Birnboim and Doly solution
I was added, and the rest of the alkaline lysis procedure
was carried out as for E. coli (Birnboim and Doly, 1979).
The resulting preparation contained very little plasmid
DNA and was therefore electroporated into E. coli wnere
significant quantities of plasmid could be obtainea and
isolated as described (Birnboim and Doly, 1979).
7. Mapping by chromosomal transformation
The integration frequency was used to determire the
linkage of spontaneous mutations. Chromosomal DNA rom a
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streptomycin resistant derivative of the capsule mutant
was used to transform the other mutants. The number o~
encapsulated transformants obtained divided by the number
of streptomycin resistant trans~ormants obtained is
defined as the integration frequency (Bernhelmer and
Wermundsen, 1972). Integration frequencies of 0.02 to
0.03 resulted ~rom crosses between mutants carrying
mutations in the same locus. Integration frequencies of
about 0.3 indicated the mutations were in different loci.
Linkage o~ plasmid insertions to capsule mutations
was determined by using chromosomal DNA ~rom wild type
strains carrying non-destructive plasmid insertions ~o
transform capsule mutants. Transformants were selected
on erythromycin and screened ~or encapsulation. The
co-transformation frequency determined the degree of
linkage.
20 8. Capsule detection by ELISA
To obtain a crude capsule extract, a lO ml culture
of log phase S. pneumoniae cells was centrifuged at 5000
rpm for 20 min, and the supernatant was removed. The
pellet was resuspended in l ml PBS (50 mM sodium
phosphate pH 7.4, lO0 mM NaCl). Protein content was
determined on lO0 ~l of washed cells using the Bio-Rad
kit (Bio-Rad, Richmond, CA). The rem~1nlng cells were
extracted with an equal volume of 24:l chloroform/isoamyl
alcohol. Following centrifugation, the aqueous phase was
precipitated with two volumes of ethanol. The
precipitate was resuspended in l ml PBS, then treated
with RNase at l mg/ml and DNase at 0.2 mg/ml for 3 h at
37C, and pronase at l mg/ml for 2 h at 37C. The
extract was then re-extracted and precipitated as before.
The precipitate was resuspended in PBS and used to coat
the wells of a microtiter plate for ELISA analysis.
Values were normalized to protein content. ELISAs were
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performed by the standard technique (Ausubel et al.,
1987). Monoclonal antibody 16.3 was used to detect type
3 capsular polysaccharide (Briles et al., 1981a). E~ISA
plates were read at 405 nm in a Biotek model 320 plate
reader (Bio-Tek Instruments, Winooski, VT).
9. Percoll gradient centrifugation
For density determinations, 10 ml of log phase cells
were centrifuged at 4000 rpm for 10 min, washed once with
water, and then resuspended in 1 ml water. A volume of
300 ~l of cells was loaded on top of a 10 ml 0-100 or
25-100~ continuous Percoll gradient. Gradient density
marker beads were loaded on top of the gradients. The
gradients were centrifuged at 10,000 rpm for 15 min with
the brake off. Percoll and density marker beads were
purchased from Pharmacia (Piscataway, NJ).
Non-encapsulated strains of S. pneumoniae exhibit a
higher density in Percoll gradients than encapsulated
strains (Briles et al., 1992). Percoll gradients were
also used to enrich for encapsuiated cells expected to
result from low frequency events. Percoll gradients were
used to obtain binary capsule type transformants and to
enrich for spontaneous revertants to capsule production.
B. Re~ults
To identify the type 3 capsule region of S.
pneumoniae, a Sau3AI library of fragments was cloned from
the type 3 encapsulated strain WU2 to direct insertions
into the chromosome of strain WU2. The library used in
the insertion-duplication mutagenesis procedure was
constructed by cloning random Sau3AI fragments in the
plasmid pJY4163, which carries an erythromycin-resistance
marker. Since this plasmid is unable to replicate in S.
pneumoniae, all erythromycin-resistant transformants
should contain insertions at the chromosomal site cr the
target Sau3AI fragment (Morrison et al., 1984). By
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transforming the library of clones into strain WU2, ~ne
inventors obtained 5 non-mucoid isolates among 491
erythromycin-resistant transformants. However, further
studies involving transformation of the parent strain
with either chromosomal DNA from the mutants or plas~ids
recovered from the mutants showed that the plasmid
insertions were neither linked to nor responsible fcr the
capsule mutations.
To determine if the non-mucoid isolates were truly
deficient in the production o~ type 3 capsular
polysaccharide, several methods were employed. In
slide-agglutination assays, none of the five mutants
reacted with polyclonal antisera specific ~or type 3
polysaccharide. Centrifugation through Percoll density
gradients revealed that the mutant strains were much
denser than the encapsulated parent strain. WU2 cells
had a density cl.O1 g/ml, whereas all five mutants
migrated at 1.09 to 1.10 g/ml. These data suggested that
complete capsules were not produced by the mutants.
However, these tests might not reveal the presence of
short or sparse polysaccharide ch~1n.q on the cell
surfaces or capsular material not translocated to the
surfaces. To determine if such material was presen~,
ELISA analysis was carried out on crude cell extrac~s.
Capsular material in the extracts was detected using a
monoclonal antibody directed against type 3
polysaccharide. The analysis indicated that mutants
JD531 and JD541 (designated mutants A1 and A2
respectively) made no detectable capsular material,
whereas mutants JD542, JD551, and JD571 (designated
mutants B1, B2 and B3 respectively) made significant
levels of reactive material. The common laboratory
strain Rxl was also found to produce significant levels
of type 3 capsular material (FIG. 1). Although Rxl is
generally referred to as a non-encapsulated derivative of
the type 2 strain D39, it was transformed three times
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with chromosomal DNA from derivatives of the type 3
strain A66, chosen twice for type 3 encapsulation, and
chosen finally for non-encapsulation (Ravin, 1959;
Shoemaker and Guild, 1974).
No capsular material could be detected by ELIS~ in
the culture supernatant fluids of mutants JD531 ana
JD541, indicating that these mutants were not merely
defective in attachment of the polysaccharide. Only low
levels of capsular material were detected in supernatants
of Rxl, JD542, JD551, and JD571 cultures.
The five mutations resulting in the
capsule-deficient phenotype were mapped to two loci Dy
chromosomal transformation. Reciprocal crosses between
the mutants yielded encapsulated transformants for each
combination, but not for transformation of a mutant with
DNA ~rom the same strain. The mutations were thereby
determined to be genotypically distinct. The
transformations also revealed that the mutations in JD531
and JD541 were more closely linked to each other than to
the mutations in JD542, JD551, and JD571. Likewise, the
mutations in JD542, JD551, and JD571 were more closely
linked to one another than to the other two mutations.
The integration frequencies for those mutations judged to
be closely linked were similar (0. 02 to 0. 03) and were
ten-fold lower than those judged to be not as closely
linked. The genotypic data thus agreed with the
phenotypic data, i.e., the two mutations leading to total
loss of capsule synthesis mapped together, and the three
mutations causing lack of proper capsular polysaccharide
processing mapped together. The loci containing these
mutations were temporarily designated cpsA and cpsB,
respectively, and the mutations were named as indicated
in FIG. 1.
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Table 4 shows the transformation frequencies o
capsule mutations. S. pneumoniae strains were
transformed with chromosomal DNA from strain JD636, ~
streptomycin resistant WU2 (Table 3), and streptomycin-
resistant transformants were selected. Transformatisnfrequencies are calculated from cultures not induceG to
competence. with optimal induction, strain wU2 may
exhibit a transformation frequency approaching that of
the non-encapsulated mutants. However, during the
lo mutagenesis procedure, sub-optimal transformation
frequencies were observed (0.0003 to 0.006~). JD908
(Table 3) was also included, it contains an insertic~
mutation resulting in loss of capsule expression
(described in FIG. 4). It would appear that the
non-encapsulated mutants are highly transformable,
suggesting that the reason for their over-representation
in the orlginal transformant population was because of
their transformability. The mutagenesis procedure, by
selecting for transformability, has enriched for mutants
already deficient in capsule (Table 4).
Table 4. Transformation frequencies of capsule mutants.
Transfor-
Recipient Mutation Total cfu StrR cfub mation
frequency
(~)
JD531 cpsAl 2.0 x 105 5.0 x 102 0.3
JD541 cpsA2 1. 4 x 108 7.6 x 102 0.6
JD542 cpsBl 2.4 x 105 20 x 102 0.8
JD551 cpsB2 2.0 x 105 4.6 x 102 0.2
JD571 cpsB3 2.4 x 108 5.0 x 102 0.2
WU2 wt 0.O x 105 1 0.0000
JD908 cpsS 10 x 105 10 x 102 0.1
b StrR streptomycin resistant.
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EXAMPLE 2
Identification o~ a Clone Cont~in;nq a Capsule Gene
To identify DNA fragments capable of repairing ~he
cpsA1 mutation, JD611, a derivative of JD531 lackin~ the
pJY4163 insertion, was transformed with pools of pJY4163
clones containing Sau3AI fragments from strain WU2.
Transformations and DNA manipulations were performed as
described in Example 1. In this insertion-duplication
restoration procedure, the plasmid clone is insertea into
the mutant chromosome, leading to duplication of the
homologous target fragment and restoration of one wild
type copy of cpsA (FIG. 2). Erythromycin-resistant
transformants were screened visually for the mucoid
phenotype. One plasmid clone was identified which
restored encapsulation in the cpsAl-containing mutant.
Due to the duplication of the target fragment, the
plasmid insertion could resolve out of the chromosome by
homologous recombination at low frequency. Therefore,
transformation of E. coli with DNA from the encapsulated
transformant and selection for erythromycin-resistance
allowed recovery of the plasmid, designated pJD330, that
had repaired the cpsAl defect.
Transformation of the capsule-deficient mutants with
pJD330 suggested that the clone contained part of cpsA.
When pJD330 was inserted into the chromosome of the
cpsAl-containing mutant, 56~ of the
erythromycin-resistant transformants became encapsulated
(Table 5). The failure of the r~m~;n~er of the
transformants to become encapsulated indicated that the
cloned fragment contained only one end of the gene. The
site of the crossover relative to the site of the
mutation determines whether the mutation will be located
in the incomplete copy of the gene or the full-length
copy. If the recombination occurs on the left, as shown
in FIG. 2, the full-length gene is wild type and capsule
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is restored. However, i~ the crossover occurs on tre
right, the mutation is located in the full-lengtn ccpy,
and no capsule is obtained. This interpretation is
supported by the observation that trans~ormants of the
5 cpsAl mutant which incorporated the plasmid but diG ~ot
become encapsulated, spontaneously gave rise to
encapsulated, erythromycln-resistant colonies, eithe~ by
excision and reinsertion of the plasmid or by gene
conversion. The cpsA2 defect was not repaired by pJ3330,
suggesting that the site of this mutation was eithe~ not
present on the plasmid clone or was located too near the
end of the clone for crossover to repair the defect.
Table 5. Restoration of encapsulation with pJD330.a
Cps+
Recipient Mutation EryR cfub Cps+ cfu frequency
(~)
JD611 cpsAl475 267 56
JD619 cpsA226 0 0
20 JD692 cpsBl13 0 0
JD614 cpsB2124 0 0
Rxl cps~158 49 31
661 capD656 0 0
A66R2 capD44 3 75
a Mutants deficient in capsule production were
transformed with pJD330 DNA. Trans~ormants were
plated on erythromycin to select for those
containing chromosomal insertions of pJD330.
Erythromycin-resistant transformants were screened
for mucoldy. Cps+ frequency is the ratio of
Cps+/EryR cfu.
b EryR. Erythromycin resistant.
Transformation of pJD330 into the cpsB-containing
mutants did not restore any of these to encapsulation,
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suggesting that cpsB is not present on pJD330. However,
strain Rxl was restored to type 3 encapsulation by pJD330
(Table 5).
EXAMPLE 3
c~sA Codes for UDP-Gluco~e Dehydroqenase
Transformation of two prevlously characterizec
mutants suggested that pJD330 contains part of the cene
for UDP-glucose dehydrogenase. UDPG dehydrogenase s the
enzyme which converts UDP-glucose (UDPG) into
UDP-glucuronic acid (UDPGA). UDPG and UDPGA are the two
nucleotide sugars which are required for type 3 capslle
synthesis (Smith et al., 1960). The non-encapsulat_i
mutants A66R2 and 661 were previously shown to be
deficient in the production of UDPG dehydrogenase a_e to
mutations in the locus designated capD ~Bernheimer and
Wermundsen, 1972). Transformation of A66R2 (capD4) with
pJD330 restored encapsulation, whereas transformati_n of
661 (capD6) did not (Table 5).
Transformations with chromosomal DNA (Table 6)
confirmed that the other cpsA and capD mutations we~
closely linked to the region cloned in pJD330.
Transformations and other DNA manipulations were
performed as described in Example 1. Strain JD770 was
obtained by inserting pJD330 into the chromosome o~ the
parental strain WU2. JD770 was found to produce wild
type amounts of type 3 capsule. Chromosomal DNA frcm
JD770 was used to transform those mutants which could not
be restored to encapsulation by pJD330 (Table 6). The
cpsA2 and UDPG dehydrogenase mutation capD6 were fo~nd to
be ~90~ linked to the plasmid insertion.
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Table 6. Restoration o~ encapsulation with chromosomal
DNA linked to pJD330 insertion. a
Cps+
Recipient Mutation EryR cfub Cps+ cfu frequency
(~)
JD692 cpsBl 17 13 70
JD614 CpsB2 79 58 73
JD619 cpsA2 42 39 93
661 capD6 6 6 100
a Transformants were plated on erythromycin to select
for those which had incorporated the region
containing the insertion. Erythromycin-resistant
transformants were screened for mucoldy. Cps+
frequency is the ratio of ~ps+/EryR cfu.
b EryR -- erythromycin resistant.
Deletion analysis was per~ormed to more closely
localize the sites of the mutations cpsAl, capD4, and the
mutation in Rxl (cps-). By transforming with plasmid
subclones and making no selection for insertion of the
plasmids, the inventors were able to observe
recombination events that occurred as a result of double
25 crossovers between the cloned fragment and its homolog in
the chromosome (FIG. 3A). Transformations with several
subclones revealed that the sites of the mutations could
all be localized to a 250 bp PvuII-SspI fragment common
to pJD380 and pJD369 (FIG. 3B). The fact that the same
fragment which restores encapsulation in a UDPG
dehydrogenase mutant also restores encapsulation in the
cpsAl -containing mutant suggests that cpsA encodes UDPG
dehydrogenase. From here on, the cpsA loci is designated
cps3D ( see Example 9).
It is known that transformation of UDPG
dehydrogenase mutants, including 661, with chromosomal
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DNA from a type 1 strain restored type 3 encapsulation by
incorporation of the type 1 specific genes at a site
other than that occupied by the type 3 genes (Bernheimer
et al., 1967, Bernheimer and Wermundsen, 1972). Type 1
capsular polysaccharide contains galacturonic acid;
therefore, type 1 strains are expected to produce UDPG
dehydrogenase (Austrian et al ., 1959). When UDPG
dehydrogenase mutants of a type 3 strain were transformed
with DNA from type 1 strains, the UDPG dehydrogenase from
type 1 complemented the type 3 mutation, allowing the
production of both capsular polysaccharides. The UDPG
dehydrogenase gene from the type 1 strain was never
observed to repair the mutation in the type 3 gene
(Bernheimer and Wermundsen, 1969). When the cpsAl mutant
JD611 was transformed with chromosomal DNA from a type 1
strain, type 3 encapsulated transformants were obtained
at a frequency of 3 X lo~6. This frequency is in
agreement with that observed for transformation of mutant
661 ( capD6 ) to binary encapsulation (Bernheimer and
Wermundsen, 1972) and above the spontaneous reversion
frequency (c8 X 10-9).
EXAMPLE 4
Genetic and PhYsical MaP of the TYpe 3 CaPsule Re~ion
A. Methods
1. Southern Bl o tting
Southern blotting was performed using the vacuum
blotter and chemiluminescent detection system purchased
from Stratagene (La Jolla, CA). The PosiBlot 30-30
pressure blotter is part of an integrated system designed
to transfer DNA or RNA from agarose gels quickly and
efficiently onto solid support matrices, such as
Stratagene~s hybridization membranes including the
Nitrocellulose membranes, Duralose- W membranes
(reinforced nitrocellulose), Duralon- W membranes
(nylon) or Illuminator nylon membranes.
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Following electrophoresis the gels are stainea in 5
~g/ml of ethidium bromide (EtBr) in water, destainea in
water and then photographed~ Prior to Southern trar.srer,
the gels are pretreated by depurination, denaturaticn and
neutralization. Depurination entails treating the cels
with 0.25 N HCl for 5-30 minutes with gentle shakir_.
Denaturation consist of pouring off the HCl and adaing a
0.5 N NaOH and 1.5 M NaCl denaturation solution, erough
to cover the gel. The gels are treated for 5 minutes to
one hour with gentle shaking. Neutralization involves
pouring off the denaturation solution and adding a C.1 M
Tris-HCl (pH 7.5) and 1.5 M NaCl neutralization solution,
enough to cover the gel. They are then treated for -
minutes to one hour with gentle shaking.
Gels are then ready for blotting, which is per~ormed
with gloved hands. The membrane is prewet in distilied
water (dH20) and then in transfer buffer for 5 minu~es.
10x SSC buffer-lOx SSPE buffer or 25 mM sodium phosphate
(pH 6.5) is the transfer buffer for nylon membranes. For
nitrocellulose or Duralose- W membranes, 20x ssc bu~fer
should be used.
The membrane and gel are set up in the Posiblc~ 30-
30 pressure blotter and pressure exerted and adjust ~o 75
mm Hg. Blotting times vary for different gels and depend
on the amount and size of the nucleic acid; size,
thickness and percentage of gel; and depth of gel wells
and volume of sample loaded on the gel; which are
routinely optimized. After the allotted blotting time,
the position of the wells on the membrane is markea and
the gel removed. The gel is generally stained and
destained in ethidium bromide to check the efficiency of
transfer. The membrane is removed from the device and
placed on clean Whatman 3MM paper to allow the excess
buffer to be absorbed. Once the membrane is free of
standing liquid, but still damp, the membrane and the
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Whatman 3MM paper is placed under a W light and
crosslinked. Alternatively, dry the membrane in a 80C
drylng oven for 1-2 hours prior to crosslinking.
Boehringer Mannheim's Genius Nonradioactive
Detection System, a chemlluminescence-based, nucleic acld
detection kit, permits fast, safe and sensitive detection
of DNA and RNA lmmobilized on nylon membranes. As little
as 0.1 pg of target plasmld DNA can be detected in a 30-
minute exposure of the processed blot to X-ray film or,
in a similar exposure time, 1 pg of a single-copy gene
can be detected in less than 1.0 ~g of genomic DNA. The
Nonradioactive Detection System can also be used fcr
rapid Northern-blot analysis of RNA.
After transfer and crosslinking, the membrane is
prehybridized for 1 hour at 42C. The labeled probe is
placed in a mlcrofuge tube contalning 100 ~l of sonicated
salmon sperm DNA (10 mg/ml) stock and heated in a boiling
water bath for 5 minutes. This is pulse-spun to collect
condensation and stored on ice until ready to add to
hybridizatlon. The probe is added to prehybridizatlon
solution and hybridlzed, with shaklng, overnlght at 42C
uslng standard hybrldlzation solutions as described by
the Genius~ protocol. This is washed once for 15 minutes
at room temperature ln O.lx SSC/0.1~ SDS and then washed
twlce for 15 minutes at 60C in O.lx SSC/0.1~ SDS for
each wash. The probe is then ready for detection. The
BRL 1 Kb DNA ladder was used as a molecular weight slze
standard (Bethesda Research haboratorles, Gaithersburg,
MD). Biotin labeled probes for hybridization were
prepared by nlck-translatlon using the BRL BloNlck klt
(Bethesda Research Laboratories). Hlgh stringency
conditions, as described above should result in the
detection of sequences 295~ homologous to the probe.
Reduced strlngency was achleved by lowerlng the wash, or
hybridlzatlon and wash temperatures to room tempera.ure.
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At reduced stringency, sequences with 85~ homology ~~ the
probe should have been detectable.
B. Results
Uslng pJD330 as a probe in Southern hybridizati-ns,
a physical map of the type 3 capsule region of strain WU2
was developed (FIG. 4). Using the information gained
from the chromosomal mapping, the inventors identif~ed
and cloned the HindIII fragment located to the right of
the pJD330 insert. HindIII fragments approximately 3 kb
in size were cloned from the WU2 chromosome into pJY~164.
By using pJD330 to screen for homology, a clone
containing a 3.2 kb insert was identified. This clone,
pJD366, was then used to determine the location of the
15 cpsB mutants.
Transformation mapping using JD770 showed that -he
cpsB mutations were about 74~ linked to the pJD330
insertion (Table 6). This high frequency indicated _hat
cpsB might be adjacent to the fragment contained in
pJD330. When pJD366 was used to transform strains
containing the cpsB mutations, encapsulation was not
restored. Insertion of pJD366 into the WU2 chromosome
did not alter the production of type 3 capsule;
therefore,the inventors were able to ~mlne linkage of
the insertion to the cpsB mutations and determine the
relative location of cpsB. The pJD366 insertion was
found to be only 25~ linked to the cpsB mutations (as
compared to 74~ for the pJD330 insertion), suggesting
that CpSB is located to the left of the pJD330 insert, as
shown in FIG. 4.
In order to localize regions necessary for capsule
production, insertion mutations using subclones of pJD330
and pJD366 were made. Transformation of strain WU2 with
plasmids containing fragments internal to a gene or
operon required for capsule production should result in
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loss of encapsulation. Insertion of the plasmid
containing the Sau3AI - XbaI fragment resulted in loss of
encapsulation, indicating that this entire 1.6 kb
fragment is within a single gene or operon required for
capsule synthesis. Likewise, all insertions within this
region eliminated capsule production (FIG. 4). Insertion
of the plasmid containing the XbaI-PstI fragment did not
disrupt capsule production, indicating that the end of
the gene or operon is contained within this fragment.
None of the other insertions resulted in loss of capsule,
indicating they were not internal to genes or operons
required for capsule synthesis.
Since the plasmids used for the chromosomal
insertions contain a promoterless cat gene, the inventors
were able to establish the directions of transcription at
the insertion sites. All insertions which contained the
cat gene in the orientation to detect transcription
proceeding to the right (as drawn in FIG. 4) resulted in
chloramphenicol resistance. No transcription was
detected in the opposite direction (FIG. 4).
EXAMPLE 5
Homoloqy with Other CaPsule TYPes
If the type-specific genes for capsule production
are contained within a cassette, as has been proposed,
these genes should show little homology to the
type-specific genes from other capsule types. A high
degree of homology should exist in the regions flanking
the type-specific region (Austrian et al ., 1959;
Bernheimer and Wermundsen, 1972). The flanking regions
may contain common genes necessary for production of
capsule of any type or might not be involved in capsule
production.
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To determine if the regions cloned in pJD330 ar~
r pJD366 are specific to type 3 or are present in stra~ns
of other capsuIe types, HindIII digested chromosoma_ DNAs
from strains of types 2, 3, 5, 6A, 8, 9, and 22 were
5 Southern blotted and probed with these plasmids or
fragments thereof. The fragment contained in pJD33C (the
probe used was pJD351, containing 2.4 kb Sau3A1 fracment
from pJD330) hybridized only with DNA from the type 3
strain, detecting the expected bands at 2.2 and 3.2 kb.
10 No hybridization with the chromosomal DNA of the oth~r
six serotypes was detected, nor could the stringency be
sufficiently lowered to detect homology in these strains.
When HindIII digests of chromosomal DNAs from _~ese
15 same strains were probed with the HindIII fragment rom
pJD366, the expected 3.2 kb band was observed in the type
3 strain, but a 1.1 kb band was found in every other
capsule type. Probing with subclones of pJD366
containing the 2.1 kb HindIII- SacI fragment or the 1.2 kb
20 SacI-HindIII fragment revealed that the homology resided
in the more distal 1.2 kb fragment. Therefore, unlike
the remaining 4.2 kb of DNA, which could be detectea only
in the type 3 strains, the 1.2 kb SacI-HindIII fragm~nt
(pJD377) showed a high degree of homology and could be
25 detected at high stringency in all strains (2, 3, 5, 6A,
8, 9 and 22). This result suggests that this regior may
be the highly homologous flanking DNA predicted by t:~e
model to be adjacent to the type-specific genes.
EXAMPLE 6
Transformation of CaPsule TYpe
To determine if all the type-specific genes
necessary for the production of type 3 capsular
polysaccharide were closely linked on the pneumococcal
chromosome, strain JD770 was used as a donor in
transformation of the type 2 strain D39. Laboratory
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techniques were as described in Example 1. Seventy--hree
erythromycin-resistant transformants were obtained, and
all 73 expressed type 3 capsule. No type 2 capsule _ould
be detected by agglutination with type 2 specific
antisera. Using chromosomal DNA from strain JD77C,
succesful trans~ormatlon of strains of type 5 and t-~e 6B
to type 3 encapsulation was also perfomed (Example ~
By transforming the type 2 strain D39 with pJD-~6,
isolates were obtained with the erythromycin-resistance
marker closely linked to the type 2 capsule genes. ~hese
transformants were the result of recombination between
the flanking regions of homologous non-type-specif ~ DNA.
Using DNA from one of these isolates, JD871, to tra sform
the type 3 strain WU2 resulted in 95~ co-transforma~~on
of type 2 encapsulation with erythromycin resistance.
The r~;n;ng 5~ were found to be type 3 encapsulated,
indicating that only the flanking DNA or the plasmi~
alone was transferred to these isolates. Insertion of
pJD366 into the type 5 strain DBL5 also resulted in a
strain - JD875 - with the erythromycin resistance m~rker
linked to the type-specific genes. This strain was
successfully used to transform WU2 to type 5
encapsulation.
EXAMPLE 7
Direct Test of the Cas~ette Model
Transformations and other DNA manipulations werl
performed as described in Example 1. Southern Blot_ing
was performed as described in Example 4.
If capsule type change involves a cassette-type
recombination mechanism, then transformation of capsule
type should result in re~lacement of the recipient's
type-specific genes by those of the donor. In order to
determine if such replacement does occur, DNA was us~d
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from the type 3-specific region to probe HindIII digested
chromosomal DNA ~rom a strain which was originally type 2
and was transformed to type 3 (JD803), and a strain which
was originally type 3 and was transformed to type 2
5 (JD872) (FIG. 5).
Hind III digested chromosomal DNA from strain: 2
(D39 and JD871 from Example 6); 3 (WU2); 3/2 (JD872) and;
2/3 (JD803), were used in Southern blotting. First of
all the Southern blot was probed with pJD343 and pJD368
Together these plasmids contain an 800 bp region
(HaeIII-MunI) specific to type 3 and internal to cpsS
(FIG. 5). The type 3 parent WU2 contained the expected
2.4 kb HindIII ~ragment specific to type 3, whereas
neither the type 2 parent D39 nor its derivative JD871,
which has pJD366 inserted into the chromosome, contained
this fragment. When JD871 (type 2) was used to transform
WU2, the resulting strain JD872 was type 2 encapsula~ed
and had lost the 2.4 kb type 3-specific fragment.
Similarly, when D39 was trans~ormed with DNA ~rom JD770
(type 3), the resulting strain JD803 was type 3
encapsulated and had gained the type 3-specific fragment.
Reprobing of the same blot with the 1.2 kb
SacI-HindIII fragment common to all capsule types
(pJD377), revealed that the 1.1 kb HindIII fragment was
present in each of the strains that now produced the type
2 capsule. Further, JD803 had also gained the 3.2 kb
HindIII fragment present in WU2 (type 3), 2.1 kb of which
is type 3-specific. This fragment was also present in
JD871 and JD872 since it is contained in the plasmia
insert (FIG. 5).
The loss of type 3 genes by the strain converted to
type 2 encapsulation indicated that capsule type change
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does not occur by additlon, but rather by replacemen~ of
the type-specific genes.
EXAMPLE 8
The DNA Seouence and Amino Acid Sequence of cps3D.
A. Methods
1. DNA Sequencing
Templates for sequencing were prepared from double-
stranded plasmid DNA by denaturing with NaOH (2 N) for 5
min at room temperature, and precipitating with 5 M
NH40Ac and ethanol. DNA was sequenced by the Sanger
dideoxy method using the Sequenase 2.0 kit (US
Biochemicals, Cleveland, Ohio) and 35S-dATP. The
oligonucleotide primers 5'-GCCACTATCGACTACGCG-3' (SEQ ID
NO:17) and 5'TCATTTGATATGCCTCCG-3' (SEQ ID NO:18),
corresponding to bases 308 to 325 and 445 to 428 of the
cloning vectors pJY4163 and pJY4164 (Yother, et al.,
1992), respectively, were used routinely. Primers 5'-
GTGAGATAAATAGTAGTGCG-3' (SEQ ID NO:19) and 5'-
TCCAGCTCGTGTCATA~TCT-3' (SEQ ID NO:20), corresponding to
bases 3474 to 3493 and 3596 to 3577, respectively, of the
type 3 capsule locus (FIG. 6Gi, FIG. 6Gii, FIG. 6Giii)
were also used. All oligonucleotide primers were
purchased from Oligos, etc. (Wilsonville, OR). DNA
sequencing of PCR products was performed using the US
Biochem PCR product sequencing kit, according to the
directions of the manufacturer. PCR products were
sequenced at least twice, from separate amplification
reactions. Greater than 97~ of the sequence was obtained
for each strand.
2. Se(luence analysis
The University of Wisconsin Genetics Computer Group
programs (Genetics Computer Group, 1991) were used in the
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analysis of the DNA sequence. Database searches were
performed using the TFASTA program to detect homolo~.~
with the deduced amino acid sequences. Potential s-gma-
70 type promoter sequences were located by using the FIND
program to search for sequences with six or less
mismatches, as compared to the consensus sigma-70
promoter se~uence (Mulligan and McClure, 1986), and
having a spacing of 15 to 20 bp between the -35 anc -10
hexamers. Potential promoter sequences were evaluated
using the equations of Harr et al. (Harr, et al., 1983).
The sequence presented in FIG. 6Di through FIG. 6Jii (SEQ
ID N04, SEQ ID NO:5 and SEQ ID NO:6) has been submi._ed
to GenBank for assignment of an accession number.
15 3. Chromosome crawling and inverse PCR
To isolate the 5' end of cps3D and upstream DNA, S.
pneumoniae WU2 chromosomal DNA was first digested w _h
Ec1136 II (an isoschizomer of SacI that results in blunt
ends) and separated on a 0.6 ~ agarose gel. Fragmen~s
ranging from 6 to 7 kb were excised and puri~ied usi~g
GeneClean (BiolO1, ~a Jolla, CA). A 35 bp XbaI UniAmp
adaptor (Clontech, Palo Alto, CA) was ligated to the
purified fragments. The fragment of interest was then
amplified by using a primer for the adaptor and a prlmer
corresponding to the predicted active site sequence (nt
1802 to 1781 of FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii and FIG.
6Eiv) of cps3D. A 1.8 kb and a 3.8 kb PCR product was
obtained. The 1.8 kb PCR product extended from the
active site to the SacI site upstream of cps3D (FIG. 6Ei,
FIG, 6Eii, FIG. 6Eiii, FIG. 6Eiv). The 3.8 kb PCR
product extended from the active site to the second SacI
site further upstream (FIG. 7). The polymerase chain
reaction (PCR) was performed using AmpliTaq DNA
polymerase (Perkin-Elmer Corp., Norwalk, CT) in a Perkin
Elmer model 480 thermocycler according to the direc~ions
of the manufacturer. In a similar manner, the 0.9 kb
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fragment from the cps3D active site to the EcoRV site
upstream of cps3D was amplified from a 3.5 kb EcoRV
fragment from the WU2 chromosome.
To isolate the region 5' of the repeat sequence, a
SacI-MscI fragment internal to the repeat region
(extending from nucleotide 1 to 257 of SEQ ID N0: 4) was
first cloned into the insertion vector pSF151 (kanamycin
resistant, Kmr), and used to direct an insertion-
duplication event into the type 3 S. Pneumoniae WU2
chromosome. Chromosomal DNA from the resulting Kmr
strain, JD1008, was digested with HindIII, self-ligated,
and transformed into the E. coli. The resulting Kmr
plasmid, pRS111, contained in the pSF151 vector and DNA
flanking the insertion, i.e., DNA extending from the
HindIII site in cps3B to the HindIII site in the repeat
sequence (~2.3 kb of S. pneumoniae DNA).
B. Results
The cps3D nucleotide sequence is shown in FIG. 6Ei,
FIG. 6Eii, FIG. 6Eiii and FIG. 6Eiv (SEQ ID NO:5). The
Cps3D amino acid sequence :(SEQ ID N0:11) is highly
homologous (56~ identity, 73~ similarity) to that of the
UDP-glucose dehydrogenase (HasB) from Streptococcus
pyogenes (Dougherty and van de Rijn, 1993). Two other
sequences were detected in the GenBank which shared a
high degree of homology with Cps3D. These open reading
frames from the Escherichia coli and Salmonella
enteri tica rfb clusters have not been shown biochemically
or genetically to be UDP-glucose dehydrogenases (Bastin,
et al ., 1993), but they share a high degree of homology
with HasB and Cps3D.
Cps3D (SEQ ID NO:11) has several characteristics
consistent with it being UDP-glucose dehydrogenase. The
N-terminal amino acid residues 2 to 29 have all the
characteristics of an NAD-binding site (Wierenga, e t al .,
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1986), and this sequence is very homologous to regions
~rom HasB, AlgD (the GDP-mannose dehydrogenase of
Pseudomonas aeruginosa [Deretic, et al., 1987]), and the
two potential UDP-glucose dehydrogenases from E. coli and
S. enteritica. The homology with AlgD was previously
noted by Garcia et al., in the deduced amino acid
sequence of the S. pneumoniae gene cap3-1 (Garcla, et
al., 1993). They suggested that Cap3-1 was the type 3
UDP-glucose dehydrogenase. Sequence ID NO:1 and SEQ ID
NO:5 is in complete agreement with that of Garcla et al.,
from the EcoRV site to the ScaI site (nucleotide 883 to
1377 FIG. 6Di, FIG. 6Dii, FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii
and FIG. 6Eiv, containing amino acids 1 to 117, SEQ ID
NO:11). However, no other homology was seen, suggesting
that these investigators had cloned only the 5' end of
the gene.
The Cps3D sequence at amino acid residues 251 to 263
(SEQ ID NO:11) is consistent with this being the active
site of the enzyme. This region is identical at the
amino acid level with that of HasB and the putative E.
coli and S. enteri tica UDP-glucose dehydrogenases. The
homology of the active site region of HasB with that of
bovine UDP-glucose dehydrogenase and AlgD has been fully
described (Dougherty and van de Rijn, 1993). The
cysteine at residue 259 (SEQ ID NO:11) of Cps3D contains
the essential thiol group of the reactive site (Ridley,
et al., 1975). The predicted size of Cps3D (45 kDa) is
also similar to the size of the E. coli enzyme (47 kDa)
(Schiller, et al., 1976).
EXAMPLE 9
Identification of Ca~sule Mutants
DNA sequencing and manipulations were performed as
described in Example 8.
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To determine the nature of the two cpsA mutations,
identified in Example 1, the regions were amplified rom
the chromosomes of the mutant strains and sequenced.
Each mutant (JD611 and JD619) contained a single base
pair transversion resulting in a premature stop codon in
the cps3D sequence. The locations of the mutations are
indicated in FIG. 6Ei, FIG 6Eii, FIG. 6Eiii and FIG.
6Eiv.
To localize the three cpsB mutations, also
identified in Example 1, located upstream of the UDP-
glucose dehydrogenase mutations ( cpsA), standard PC~ or
chromosome crawling was used to amplify fragments from
the parent type 3 chromosome that contained either the
5' end coding sequence of cps3D (nucleotide 1027 to 1802,
FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii, FIG. 6Eiv), the promoter
and the 5~ end of cps3D (nucleotide 885 to 1802, FIG.
6Di, FIG. 6Dii, FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii and FIG.
6Eiv), or the 5' end of cps3D plus approximately 1 kb of
upstream DNA (nucleotide 1 to 1802). Each of these
fragments was used to transform the capsule-deficient
mutants JD614 and JD692. JD692 could be transformed to
encapsulation using the 5' end coding sequence of cFs3D,
whereas JD614 was not restored to encapsulation by this
fragment but was restored by the fragment containing the
5' end plus 141 bp of upstream DNA, including the
promoter. Both of the mutants were restored by the 1.8
kb fragment containing the 5' end of cps3D and the
upstream DNA, and neither was restored with a fragment
containing the 3' end of cps3D (nucleotide 1759 to 2385,
FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii and FIG. 6Eiv). Thus,
these upstream mutations are not located in a separate
gene but are in either the cps3D structural gene or its
promoter. Since some capsule material is produced by
these mutants, a mutation within the coding region (as in
JD692) must be a missense mutation or an in-frame
deletion or insertion which reduces the activity of the
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enzyme. The mutation in JD614 may be in the promote~,
and thus, a promoter down mutation, or it may be ir ~he
structural gene but too close to the beglnning of t~e
gene for recombination and repair to occur with the
fragment used.
Amplification and sequencing of the 250 bp Pvu _-
SspI fragment from the mutant strains A66R2 and Rxl
showed that each contained a missense mutation in t~e
cps3D coding se~uence (FIG. 6Ei, FIG. 6Eii, FIG. 6E ii,
FIG. 6Eiv).
EXAMPLE 10
DNA Sequences of CPs35 and CPS3U
DNA sequencing and analysis was performed as
described in Example 8.
The region just downstream of cps3D contains a
second gene, cps3S, that is required for type 3 capsular
polysaccharide biosynthesis. An open-reading frame, l248
bp in length, is transcribed in the same direction as
cps3D and is in the same reading frame (SEQ ID NO:5,.
The direction of transcription is in agreement with ~hat
determined using cat insertions as described in Exam~le
4. Only 15 bp separate a potential start codon for cps3S
from the stop codon of cps3D. The sequence AGGGG just
upstream of the putative start codon may serve as a
ribosome binding site (FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii,
FIG. 6Eiv), or due to the close proximity of cps3D, no
ribosome binding site may be necessary. The deduced
amino acid sequence of Cps3S predicts a protein of ~8 kDa
(SEQ ID NO:12), if the first start codon at nucleotide l
(nucleotide 2227 in FIG. 6Fi, FIG. 6Fii, FIG. 6Fiii, FIG.
6Fiv) is utilized. Other potential start codons are
located at nucleotide l plus l9 and +61 (nucleotide 2245
and 2287 FIG. 6Fi, FIG. 6Fii, FIG. 6Fiii, FIG. 6Fiv,
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contains both ~(1-3) and ~(1-4) linkages, however the
linkage to glucuronic acid is ~ 4) in hyaluronic acid
but ~(1-3) in type 3 capsule (Reeves and Goebel, 1941).
Homology was also seen between Cps3S and NodC from
Rhizobium meliloti (21~ identity, 47~ similarity). NodC
is necessary for the synthesis of nodulation factor, a
substituted oligosaccharide consisting of ~ 4) linked
N-acetyl glucosamine residues (Lerouge, et al., 1990).
It has previously been noted that HasA and NodC are
homologous to polysaccharide synthases, including FBF15
of Stigmatella aurantiaca, pDG42 of Xenopus laevis, and
chitin synthases from both Saccharomyces cerevisiae and
Candida albicans (DeAngelis, et al., 1993b; Dougherty and
van de Rijn, 1994; Atkinson and Long, 1992; Debellé, et
al., 1992). Cps3S is also homologous to these proteins.
These results suggest that Cps3S is the type 3 capsular
polysaccharide synthase.
The PILEUP program was used to align the amino acid
sequences of the bacterial polysaccharide synthases Cps3S
(SEQ ID NO:12), HasA, NodC, and FBF15. Only a few
clusters of amino acids are found to be conserved in all
four proteins. A few of these, GKR (residues 131 to 133
SEQ ID NO:12), an acidic region VDSD (153 to 156 SEQ ID
25 NO:12), DRXLT (256 to 260 SEQ ID NO:12), QQXRW (292 to
296 SEQ ID NO:12), and WXTR (418 to 421 SEQ ID NO:12),
are also found in the eukaryotic polysaccharide
synthases.
Since all four proteins contain highly hydrophobic
stretches, hydrophobic amino acids are found conserved at
several locations throughout the proteins. Four
hydrophobic stretches identified in Cps3S are found in
~ all four proteins. These regions may span the cell
membrane. This hypothesis has been supported for NodC.
Immunogold labeling revealed a surface location for NodC,
and the C-terminal hydrophobic region was shown to direct
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the insertion of an alkaline phosphatase fuslon protein
to the cell membrane (Johnson, et al., 1989; John, et
al ., 1988 ) . Earlier studies indicated that the type 3
capsule synthesizing activity also has a membrane
location (Smith, et al., 1961). The last hydrophobic
stretch may be required for the function of Cps3S slnce
the insertion in JD897 which elimlnated this reglon (the
last 45 amlno aclds of the protein) resulted in loss of
capsule production (FIG. 6Fi, FIG. 6Fii, FIG. 6Fiii, FIG.
6Fiv). Expression of Cps3S ln E. coli was, llke that of
NodC, lethal to the host.
A. Method
1. Expression of Cps3S
A 2.1 kb Sau3AI-PstI fragment containing the 3' end
of cps3D and the entire cps35 gene was cloned from
pJD351 into the expression vector pKK223-3 (Brosius and
Holy, 1984) at the polylinker BamHI-PstI sites to yleld
pJD424. Cultures of E. coli TG-l (Sambrook, et al.,
1989) or TG-l transformants were grown to exponentlal
phase, at whlch tlme lsopropyl-b-D-thiogalactoside (IPTG)
was added to a concentration of 1 mM to induce expression
from the tac promoter of pKK223-3.
Transformations and other DNA manipulations were
performed as described in Example 1.
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B. Results
The sequence ~rom resldues 211 to 233 ln NodC was
noted for the large number of cysteine residues. _ has
been suggested that this region participates in the
blnding of divalent cations which are necessary fc~ the
production of chitin and chitin-like molecules (Atkinson
and Long, 1992). Type 3 capsule synthesis requires Mg+~
(Smith, et al., 1960). Although this region in Cps3S
contains only one cysteine, the region is highly
conserved between all four proteins.
The GenBank search also revealed that Cps3S has
homology over short stretches to the rhamnosyl
transferase RfbN from Salmonella enteritica, which is
necessary for the production o 0-antigen in type B
strains. This enzyme creates an ~(1-4) linkage to
mannose in the O-antigen repeat unit. The homologous
regions are a subset of those conserved regions common to
HasA, NodC, and Cps3S, but the best homology is seen in
the region 229 to 278 (SEQ ID NO:12).
In the production of Group B type III capsular
polysaccharide, the galactosyl trans~erase CpsD transfers
a galactose to a molecule located in the cell membrane.
2S Rubens et al . ( 1993), suggested that the acceptor may be
dolichol or a rela~ed molecule, and identified a region
of CpsD with homology to putative dolichol binding
regions of several proteins. Although it is not clear
that such sequences are actually involved in dolichol
recognition or binding (Schutzbach, et al., 1993),
several similar regions (e.g., at residues 7 to 20, 21 to
38, and 388 to 401, as numbered in SEQ ID NO:12) are
present in CpsS. Since the putative dolichol binding
motif [FL(F/I)VXFXXI(P/L)FXFY] (Albright, et al., 1989;
Kelleher, et al., 1992) is a highly hydrophobic sequence
that is rich in phenylalanines, the sites in Cps3S may
actually reflect the hydrophobicity of the molecule and
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the A-T rich bias in the DNA sequence rather than
indicating a specificity for dolichol-like molecules. It
is not known whether S. pneumoniae utilizes an
intermediate acceptor in capsule synthesis, however -he
capsular polysaccharides of several serotypes have been
found to be covalently linked to the cell wall. Type 3
capsule, by contrast, is not covalently linked to the
cell and is generally considered an exopolysacchariae
(Sorensen, et al., 1990). Therefore, if Cps3S does use a
membrane bound acceptor, it is likely not the final
acceptor.
EXAMPLE 12
Cps3U is Homologous to Glucose-1-Phosphate
Uridylyltransfera~es and Cps3M is Homologou~
to PhosPhomutases
The gene downstream of cps3S is designated as cps3U
(SEQ ID NO:5) based on its probable function. The amino
acid sequence of Cps3U (SEQ ID NO:13) showed a high
degree of homology with glucose-l-phosphate
uridylyltransferases from several other bacterial
species. The highest degree of homology was found with
GtaB from Bacillus subtili~ (55~ identity, 73~
similarity). The active site of glucose-l-phosphate
uridylyltransferase has not been characterized from any
of the bacterial enzymes, however, the active site in the
enzyme from potato tuber (Solanum tuberosum) has been
investigated. Kazuta et al., recognlzed 5 lysine
residues present at the active site (Kazuta, et al.,
1991), and by mutational studies Katsube et al., showed
that one of these residues was important for function,
and a second was absolutely required (Katsube, et al .,
1991). Cps3U contains 24 lysines, six of which are
absolutely conserved among the six bacterial glucose-l-
phosphate uridylyltransferases in the database. Only one
region from Cps3U containing a conserved lysine can be
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aligned well with the potato tuber enzyme sequence. It
is homologous to the region containing the required
lysine.
The Iinal gene in SEQ ID NO:5, is cpsM with a
deduced amino acid sequence (SEQ ID NO:14~. The CpsM
amino acid sequence revealed significant homology tc both
phosphoglucomutases (PGM) and phosphomannomutases (~
from a diverse group of microorganisms. Contained with
CpsM is a phosphoserine signature sequence
(GIMVTASHTPAPFNG) conserved within the reported active
sites of both PGMs and PMMs. However, approximately 15
of the C terminus present in other phosphomutases, and
apparently more important ~or their function, is absent
from CpsM. Phosphomutase activity from a recombinan~
CpsM was not detected in E. coli, suggesting that cpsM
may encode a non-functional protein.
EXAMPLE 13
2 0 cps3S and cps3D are Transcribed as an Operon
A. Methods
Southern blotting was performed as described in
Example 4, all other DNA manipulations, including
insertion deletion mutations, were performed as described
in Example 1. The locations of mutations can be seen in
FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii, FIG. 6Eiv, FIG. 6Fi,
FIG. 6Fii, FIG. 6Fiii and FIG. 6Fiv.
B. Results
Use of fragments subcloned from the cps3DSU region
to direct insertion-duplication mutations in the parent
type 3 chromosome resulted in several mutants that
~ produced no detectable capsule (FIG. 9A and FIG. 9B) and
exhibited the extremely rough phenotype described by
Taylor (1949). The colonies were very small and rough,
and the cells clumped when grown in liquid culture. DNA
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sequencing revealed that the sites of the mutations are
within cps3S (FIG. 6Fi, FIG. 6Fii, FIG. 6Fiii, FIG.
6Fiv). The lack of capsule production in these mutants
must be due to loss of cps3S expression, rather than to a
polar effect on downstream genes, since insertions within
cps3U or cps3M, the next genes downstream, had no
apparent effect on capsule production.
Molecular and genetic evidence suggest cps35 is in
an operon with cps3D. Sequence analysis revealed no
potential promoter sequences in the region upstream of
cps3S (FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii and FIG. 6Eiv, SEQ
ID NO:5). The phenotypes of several insertion mutants
also suggest that no promoter is located in the 3' end of
15 cps3D and that cps3S is transcribed from the cps3D
promoter. The sites of these insertions are shown in
FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii, FIG. 6Eiv, FIG. 6Fi,
FIG. 6Fii, FIG. 6Fiii, FIG. 6Fiv and FIG. 7, however, the
structures of the mutations are more fully illustrated in
FIG. 9A and FIG. 9B. To insure that the plasmids had
inserted as expected for insertion-duplication mutations,
chromosomal DNA from the mutant strains was subjected to
Southern blot analysis.
Insertion mutants were digested with MscI/FspI for
JD982, MscI/SalI for JD983, and MscI/KpnI for JD908,
JD902, and JD900 and run on agarose gels and blotted as
described in Example 4. The blots were probed with
vector pJY4164. Increasing distance from the MscI site
to the end of the vector was demonstrated by an increase
in the size of the upper band. A faint band in the JD982
lane was observed, likely a result of partial digestion.
The 4. 7 kb and 4.8 kb bands in JD982 and JD908,
respectively, indicate that these mutants contain a
duplication of the inserted plasmid.
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Insertion of the plasmids results in a duplica=ion
t of the cloned fragment. Therefore, mutant strains slch
as JD908, in which the duplicated fragment contains ~oth
the 5' end of cps3S and the 3' end of cps3D, have G -ull-
5 length copy of cps3S downstream of the plasmid inse~_ion.
In addition, the full-length copy is contiguous wit~ the
3' end of cps3D. Therefore, if cps35 had its own
promoter, or if one were located in the 3' end of c~s3D,
these insertions should not result in a loss of cps35
lO expression. However, four such insertions have bee- made
in the WU2 chromosome (JD846, JD897, JD898, and JD5r8),
and even with a duplication of up to 450 bp of the ' end
of cps3D, a loss of capsule production was observed.
Two more internal insertions in cps3D were creared.
As expected these insertions eliminated capsule
production (FIG. 9A and FIG. 9B). However, since c_s3D
and cps35 are transcribed as an operon, this result ~oes
not prove that cps3D is required for capsule synthesis.
20 That fact is demonstrated by the lack of capsule
production seen in strains containing non-polar poi-.~
mutations in cps3D (Example 14).
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EX~iMPLE 14
In Vi tro Polymerization A~say
To evaluate the competence of the mutants to
synthesize type 3 capsule, an in vitro polymerizati~.
assay was used.
A. Methods
1. In vitro polysaccharide synthesis
Type 3 capsular polysaccharide was synthesized and
quantitated in vitro using a modification of the me_hod
of Smith, et al., 1961. Crude extracts containing cell
membranes and cytoplasm were prepared from 200 ml o- S.
pneumoniae cultures harvested at an 0600 of 0.25 as
described (Yother and White, 1994), except that cell
material was concentrated 200-fold, and all steps were
performed using a thioglycolate buffer (10 mM sodium
thioglycolate, 5 mM MgSO4, 100 mM Tris-HCl pH 8.3) to
stabilize the enzymes (Smith, et al., 1960). The
digestion of cell wall material by mutanolysin treatment
was performed in this buffer and 20~ sucrose.
Protoplasts were sonicated three times for 15 s at 35
power at 0C.
Polysaccharide synthesis was carried out at 34 C for
2 h in a 1 ml reaction containing 100 ml of extract, 5 mM
UDP-glucose, 5 mM UDP-glucuronic acid (where indicated),
and 1 mM NAD, in the thioglycolate buffer. The reaction
was boiled 1 min then quickly cooled to 25 C in H2O.
Following centrifugation for 30 s at 8160 x g, the type 3
specific monoclonal antibody 16.3 (Briles, et al., :981a)
was added in excess to the supernatant and incubaticn was
continued at 37C for 30 min.
The specific antigen-antibody complexes were
measured at 650 nm in a spectrophotometer, and the amount
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of capsule was determined by comparison with a stanaard
, curve prepared using purified type 3 polysaccharide
- purchased ~rom ATCC (Rockville, MD) (sernheimer, 1953).
Reactions were done in triplicate and were standardized
to protein content of the crude extract, as determined in
duplicate using the sio-Rad Laboratories (Hercules, CA)
protein assay kit.
B. Results
The spontaneous mutants JD611 and JD619 ( cpsAl and
cpsA2), which contain stop mutations in cps3D, produce no
detectable capsular materlal. However, both synthesized
high molecular weight type 3 polysaccharide in a cell-
free system in vitro when provided with the nucleotide
sugar precursors, i.e., UDP-glucose and UDP-glucuronic
acid (Table 7). No capsule was produced by these mutants
when UDP-glucuronic acid was omitted from the reaction.
These results indicate that these mutants produce no
capsule due to the lack of UDP-glucuronic acid and
support the conclusion that Cps3D is the UDP-glucose
dehydrogenase. They also confirm that stop mutations in
cps3D are not polar on cps35. The increased amount of
polysaccharide produced by the WU2 extract (as compared
to that produced by that of JD611 or JD619) may be
explained by the observation of Smith et al. (1961), that
increased amounts of type 3 capsule are produced in vitro
when a small amount of unpurified polysaccharide is
already present in the reaction.
The mutants which contain insertions within cps3S
(JD902), or between the full-length copies of cps3D and
cps35 (JD908, JD897) were unable to synthesize
significant amounts of capsule even with both precursors
~ present. These results emphasize the role of Cps3S in
capsule synthesis and support the conclusion that cps3D
and cps3S are transcribed as an operon.
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Table 7. In vi tro cap~ule ~ynthesis assay.
strain Cps phenotype a UDPGAb CPS (~g/mg
protein)
JD611 Cps3D-S+ + 9.8 iO.6
_ o.g +0.2
~D619 Cps3D-S+ + 5-7 +0-3
- 0.2 +0.1
JD614 Cps3D*S* NAb 5.4 +0.4 (to)C
+ 5.9 +0.5 (0.5)c
JD692 Cps3D*S* NA 4.8 +0.3 (to)
t 7.0 + 1.0 (2.2)
JD902 Cps3D+S- + 1.7 +0.3
JD908 Cps3D+S- + 1.5 +0.1
JD897 Cps3D+S~ t 1.1 +0.1
WU2 Cps3D+S+ NA 3.8 +0.2 (to)
+ 16.6 +0.3 (12.8)
- 16.3 ~0.8
D39 Cps2+ + 0.5 +0.3
a Capsule phenotypes are based on the cps3D and cps3S
genotypes. - indicates either a stop or insertion
mutation (see FIG. 6Ei, FIG. 6Eii, FIG. 6Eiii, FIG.
6Elv, FIG. 6Fi, FIG. 6Fii, FIG. 6Fiii, FIG. 6Fiv,
FIG. 9A and FIG. 9B for locations of mutations).
*indicates either a missense or in-frame deletion or
insertion in cps3D that apparently also affects
cps3S .
30 b NA, not applicable.
c For strains which produce capsule in vivo, the
amount of polysaccharide present at the start of the
assay ~to) is given, and the amount Of
polysaccharide produced during the assay is
~ 35 indicated in parentheses.
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EX~MPLE 15
Biochemical PathwaY
Based on the genetic analysis, the homology of _he
amino acid sequences of the type-specific genes to t:~e
sequences of enzymes of known function, the behavior of
the mutants in biochemical and immunochemical assays, and
previous biochemical characteriæations of type 3 strains
(Austrian, et al ., 1959; Dillard and Yother, 1994; Smith,
et al ., 1960; Smith, et al ., 1961; Bernheimer, 1953), a
pathway for the biosynthesis of type 3 capsular
polysaccharide is proposed (FIG. lO). The last of the
type-specific genes, cps3M, is homologous with
phospxhoglucomutases from several bacterial species. Even
though maintained in the type 3-specific region, Cps3U
and Cps3M may not be required for capsule synthesis,
since an insertion internal to cps3U (which has a polar
e~ect on cps3M) does not result in loss of capsule
production (FIG. 7, FIG. 9A and FIG. 9B), as judged by
colony morphology on blood agar medium.
EXAMPLE 16
The Downstream Non Type-specific
Flankina Reqion and Mappinq Other Capsule Types
Southern blots of digested chromosomal DNA from
strains 2, 3 and 6B and probed with pJD377 was performed
as described in Example 4. Faint bands in addition to
the band of interest was observed on Southern blots.
This was probably due to the detection of fragments
containing the amiA-like genes which have homology to
plpA. DNA was either digested with BglII, SacI of Hind
III. Other laboratory techniques were as described in
Example 1 or Example 8.
Sequence analysis of the 1.2 Kb SacI-HindIII
fragment (from plasmid JD377) employed in Example 5
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contained the 3' end of cps3M and the 5' half of a gene
with 50~ identity to the s. pneumoniae amiA (SEQ ID
NO:6). The amiA-like sequence has recently also been
identified by Pearce et al. and named expl (Pearce, et
al., 1993), and subsequently renamed plpA (Pearce, et
al., 1994). Further Southern hybridizations performed as
described in Example 4 showed that the non-type-specific
homologous DNA in the 1.2 kb SacI-HindIII fragment is
plpA .
A partial copy of a transposase gene was also
identified immediately adjacent to and between cpsM and
plpA . Previous findings of repetitive elements linked to
the capsule locus suggest that the deletions in this
region may be the result of a transposition event,
possibly one which introduced the type 3-specific
cassette.
If, as in type 3, the homologous region is directly
adjacent to the type-specific genes in other serotypes,
it should be possible to map other type-æpecific genes
using this fragment. This was found to be the case, and
the chromosome maps of the capsule regions in strains of
types 2, 3, and 6B, from Southern blots, are shown in
FIG. 11. It can be seen in FIG. 11 that restriction
sites located to the right of the plpA fragment are
highly conserved in all three strains. The type 3 strain
differs slightly in this region due to a deletion of the
5' end of plpA. The sites located to the left of plpA
are divergent among the capsule types. The close linkage
of the region to all the necessary type-specific genes
for each type, combined with the different restriction
maps and the fact that the type 3-specific genes are
~ located directly adjacent to this fragment, suggests that
this region contains the type-specific genes in all three
capsule types.
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EX~MPLE 17
The Upstream Non Type-s~ecific Flankinq Reqion
In order to isolate DNA 5' of the biosynthetic
genes, a 1. 8 kb fragment extending from the upstrear SacI
sites to just before the PvuII site in cps3D (nucleo~ide
1 through 1802 of FIG. 6Di, FIG. 6Dii, FIG. 6Ei, FIC-.
6Eii, FIG. 6Eiii and FIG. 6Eiv, nucleotide 1 throush 934
of SEQ ID NO:4 and nucleotide 1 through 868 of SEQ ID
NO:5) was amplified from the type 3 WU2 chromosome using
inverse PCR as described in Example 8. All other
materials and methods were as described in Example _, 4
and 8.
The 1.8 kb fragment was then used to probe HindIII-
digested chromosomal DNA from seven S. pneumoniae
serotypes (2, 3, 5, 6, 8, 9 and 22). The fragment
hybridized strongly with the expected fragments at 2.2
and 2. 3 kb in the type 3 strain. However, hybridization
was also observed with fragments of 2. 6 and 8 kb, aiong
with weak hybridization with several other fragments
(3.0, 3.1, and 4.4 kb). Likewise, each of the strains
representing other capsule types contained two strongly
homologous ~ragments (4. 8 and 8.0 kb for types 2, 6B, 8,
9; 2.2 and 4.8 or 12 for types 5 and 22, respectively)
and at least one weakly homologous fragment (4.4 kb).
When chromosomal DNAs of types 2, 3, and 6B were digested
with Ps tI, PvuII, or SacI/HindIII, and probed with the
604 bp SacI-HindIII fragment (pJD392) upstream of cps3D
(within nucleotides 1 through 610; SEQ ID NO: 4, FIG. 6Di
and FIG. 6Dii), 4 to 10 bands were detected in each.
Transformation studies were performed to examine
linkage of the repeat upstream region to the type-
specific capsule genes. The plasmid (pJD392) containing
the 604 bp SacI-HindIII i~ragment was introduced into the
chromosome of the type 3 strain. The insert, located in
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the 2.2 kb HindIII ~ragment (FIG. 7) adjacent to tr~ type
3-specific genes, did not affect capsule production.
- When the resulting strain was used to transform
recipients o~ types 2 and 6s, greater than 95~ of thQ
erythromycin-resistant transformants expressed type 3
capsule. However, when pJD392 was transformed into
strains of types 2 and 6B, the plasmid inserted intc the
8 kb HindIII fragment, and the type specific genes ~ould
not be moved to strains of heterologous types (i.e., 2,
3, or 6B) by transformation and selection for linkac~ to
the erythromycin marker in the insertions. This suagests
that this region may be found in more than one loca~~on
and not necessarily located, adjacent to the cps lccus in
all other serotypes.
To isolate DNA 5~ of the SacI site in the repea_
region (SEQ ID NO:4), a 257 nucleotide SacI-MscI fragment
internal to the repeat region was used to direct an
insertion-duplication event in the type 3 S. pneumcniae
WU2 chromosome. The flanking 2.3 kb HindIII fragmen~ was
then closed from the chromosome, as described in Example
8. The 1.4 kb SacI fragment from within this HindIII
fragment was then used to probe HindIII-digested
chromosomal DNA from three 5. p~eumoniae serotypes (2, 3,
and 6B). The fragment hybridized strongly with the
expected 2.3 kb band in the type 3 strain. Less intense
bands were also observed at 0.8, 3.0 and 8.0 kb. The
type 2 and 6B strains each contained a strongly
homologous band at 0.8 kb and a strong but less intense
band at 9.0 kb. Digestion of types 2, 3, and 6B with
other enzymes followed by hybridization with the SacI
fragment yielded the results shown in Table 8.
The results show that this region is found in only one
location in all serotypes examined. The weak bands
observed for serotype 3 probably represent hybridization
c of the 5' end of the repeat region 132 nucleotides
upstream of the SacI site (FIG. 7).
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TA8LE 8. BA~DS DETECTED BY SOU1~ BLGlll~
Str~ striction Enzyme used to digest chromosomal DNA.
Bgl II SacI SphI
type 2 ~12* 4.0, >12 12,~12
type 3 ~12* 1.4, (weak at 10 (weak at
3.5,8.5,>12) 12.5,13)
type 6B ~12* 4.0,12 12,~12
* The BglII fragments were not identical in size.
Numbers represent size of bands in kb as observed on
southern blots carried out as described in Example ~.
DNA in the 1.4 kb SacI fragment was sequenced using
techniques as described in previous Examples, and can be
seen, alongside the predicted amino acid sequences, in
FIG. 6A, FIG. 6B and FIG. 6C (SEQ ID N0:1, SEQ ID N0:2,
SEQ ID N0:3, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9 and
SEQ ID N0:10).
EXAMPLE 18
Capsule Type Expression and Virulence in ~. Pneumoniae
In these studies, isogenic strains expressing the
type 3 capsule were constructed and the effect on
virulence was determined. Strains of types 2, 5, and 6B
were used as recipients. The type 2 and 5 strains dlffer
in virulence from the type 3 strain in terms of time
required to cause death (shorter with type 2) and LD50
(lower with type 5). The type 6B strain is of low
virulence in mice. The results showed that expression of
the type 3 capsule attenuated the virulence of the type 5
strain, caused the type 6B strain to become highly
virulent, and had no effect on the type 2 strain. Thus,
in general, the expression of virulence was correlated
with the type of capsule expressed.
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A. Methods
l. Transformatlons, serotyping, ELISAs and restriction
enzyme fragment patterns.
Transformations ELISAs and DNA manipulations were
performed as described previously in Example 1. All
trans~ormants and parental strains were serotyped with
capsule type-specific antisera (Statens Seruminstitut,
Copenhagen, Denmark) in slide agglutination assays.
Genomic DNA, was digested with HindIII for 4 h at 37C
and electrophoresed overnight through 0.7~ agarose ln
Tris-borate-EDTA buffer.
2. Analysis of PspA.
Bacteria were grown in CDM containing 2~ choline, a
condition that causes release o~ PspA into the culture
medium. Filtered, unconcentrated supernatant fluids (20
~1) were electrophoresed in sodium dodecyl sulfate (SDS)-
12~ polyacrylamide gels. Western blotting (immuno-
blotting) was performed by using a semidry electroblotter
(Bio-Rad Laboratories, Richmond, Calif.), and the blots
were processed as described previously (Yother et al.,
1992). The PspA-specific monoclonal antibodies XiR278,
Xil26, and 2A4 were kindly provided by Larry McDaniel
(University of Alabama at Birmingham). Silver staining
was performed by using the Silver Stain Kit from
Stratagene Cloning Systems, Inc. (La Jolla, Calif.).
3. Characterization of morphology and capsule production.
For average chain length determinations, bacteria
were grown in THY to an optical density at 600 nm (OD600)
of ~0.3. Chain lengths were determined microscopically
by using a Petroff-Hauser counting chamber (Auther C.
Thomas Co., Philadelphia, PA). An average of five
squares was counted ~or each strain. Comparisons o~
average chain lengths were determined by using the two-
sample rank test (Zar, 1984).
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The number of cells per colony was determined by
using bacteria grown on blood agar medium for 18 h at
37OC in 5~ CO2. A plug containing a single colony was
obtained with a sterile Pasteur pipette and then
resuspended in 50 ~l of THY. Tenfold serial dilutions
were performed in THY and plated on blood agar medium.
Plates were incubated overnight at 37C in 5~ CO2, and
the number of CFU per colony was calculated.
Buoyant density determinations were performed by
using bacteria grown on blood agar medium or in THY.
Bacteria grown on solid medium were harvested by washing
each plate with water, centrifuging the suspension, and
then resuspending the pellet to an OD600 of ~0.4 with
water. Ten-milliliter liquid cultures, grown to an OD600
of -a . 5, were harvested by centrifugation for 10 to 15
min at 8,000 to 16,000 x g. Bacteria were washed twice
with water prior to being loaded onto 10-ml, continuous,
0 to 50~ Percoll (Pharmacia, Piscataway, N.J.) gradients.
As standards, 5 ~l of density marker beads (Pharmacia)
ranging in size from 1.033 to 1.076 g/ml, were also
loaded. Gradients were centrifuged for 30 min at 8,000 x
g with the brake off. A standard curve based on the
migration of the marker beads was generated, and the
density of the bacteria was determined by extrapolation.
For determinations of total capsule content, l.S-ml
cultures grown in CDM containing 0.0005~ choline were
harvested by centrifugation at 8,000 to 16,000 x g for 10
min. Culture supernatant fluids were filtered and saved,
and the cells were resuspended in 500 ~l of protoplast
buffer (20~ sucrose, 0.005 M Tris [pH 7.4], 0.0025 M
MgSO4). Cell sonicates were produced by three cycles of
a 10-s pulse, followed by a 10-s incubation on ice, with
a Fisher Sonic Dismembrator model 300 (Fisher
Biotechnology, St. Louis, Mo.) with the intensity control
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set at 30. Culture supernatant fluids and cell soni_ates
were stored at ~20C.
For surface localization assays, 1.5-ml culture_
grown to an OD600 of ~0 5 were heat kllled by incuba-_on
at 65C for 30 min. Bacteria were harvested by
centrifugation, and culture supernatant ~luids were
filtered and saved. After the pellets were washed t..ice
with phosphate-bu~ered saline (PBS; 137 nM NaCl, 2. mM
KC1, 4.3 mM Na2HPO4 7H20, 1.4 mM KH2PO4), the pelle_s
were resuspended in 1.5 ml of THY. Samples were stored
at 4C.
4 . Virul ence assays .
The virulence o~ the type 3 derivatives was com~ared
with that of the parental strains in BALB/ByJ female ~ice
(Jackson Laboratory, Bar Harbor, Maine). Bacteria were
grown to the mid-log phase in THY. Samples were dilu=ed
serially in sterile lactated Ringer's solution, and r . 2
ml was used to infect mice intraperitoneally (i.p.) cr
intravenously (i.v.), as indicated. Fi~ty percent l_thal
doses (LD50s) were determined by the method of Reed and
Muench (1938) and compared by Fisher's exact test (Zar
1984). Median times to death were analyzed by using the
two-sample rank test (Zar 1984). The P values were
determined by using a two-tailed table.
B. Results
As described in Table 3, strain JD770 contains a
non-destructive insertion in the type 3 capsule locus.
The amount and cellular localization o~ the capsular
material produced by JD770 is identical to that of i-s
parent strain WU2. Trans~ormation of JD770 chromosc~al
~ DNA, and selection ~or erythromycin resistance, resu_ts
in isolates that express the type 3 capsule of the a_nor
but not the capsule of the recipient strain (Example 5).
Based on this result, JD770 DNA was used to trans~o-~.
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type 2, 5, and 6B recipients and selected for
erythromycin-resistant isolates (see Table 9). All of
the type 2 Eryr transformants expressed the type 3
capsule but not the type 2 capsule. ~95~ of the type 5
and type 6B Eryr transformants expressed the type 3
capsule but not the capsule of the recipient parent. The
remainder of the type 5 and 6B transformants expressed
the capsular type of the recipient parent only.
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CA 02201772 1997-04-03
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To produce essentially isogenic strains, twoindependent transformants from each cross were ~
backcrossed at least three times to their respective
parent recipient strains. The final isolates were
examined for restriction enzyme fragment patterns,
pneumococcal surface protein A (PspA) expression, capsule
expresslon, and morphological characteristics prior to
testing in a mouse virulence model.
1. Res tri c ti on enzyme fragmen t pa t t erns .
The HindIII restriction patterns of the strains used
in these studies can be easily distinguished. In all
cases, the type 3 derivatives constructed here were ~ound
to have the HindIII pattern of the recipient strain,
indicating that gross alterations in the genomic DNA
content had not occurred and that the parent donor strain
JD770 had not been inadvertently re-isolated.
2 . PspA expressi on .
PspA varies with respect to molecular weight,
antigenic determinants, and strain distribution. PspA
serotypes and capsular serotypes do not correlate. The
strains used in these studies expressed PspAs that had
different molecular weights and reacted with different
PspA-specific monoclonal antibodies. In all cases, the
PspAs of the type 3 derivatives constructed here were
found to have the molecular weight and antibody
reactivities of the parent recipient strains.
30 3. Morphologic characterization and capsule production.
Microscopic ex~m;n~tion revealed that alteration of
capsular type had no e~fect on the chain length of the
~ type 2 and type 6B derivative strains. However, the
chain lengths of the type 5 derivatives di~fered
significantly from that of the type 5 parental strain and
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were almost identical to that of the type 3 parent
(Table 9).
Morphologically, type 3 strains exhibit large mucoid
capsules when grown on blood agar plates, whereas ty?e 2,
5, and 6B strains have small mucoid capsules. The .vpe 3
derivatives of the type 2, 5, and 6B strains had a
similar appearance to the type 3 parent on blood aga~
plates. The increase in colony size compared with that
of the recipient parents did not appear to be due to cell
number since similar numbers of cells per colony were
observed for all of the parent and derivative type 3
strains (data not shown). To examine capsule produc_ion,
Percoll density gradients and ELISAs were performed.
Percoll density gradient centrifugation has been shown
previously to differentiate capsular serotypes and
amounts by density (Briles et al ., 1992). In this assay,
all of the derivatives had densities similar to that of
the parent type 3 strain and distinct from that of the
recipient parent strains (FIG. 12). Thus, all of the
derivatives produced cell-associated, surface-localized
type 3 capsule in amounts similar to that of the type 3
parent. The total amounts of capsule material produced,
i.e., both cell associated and released, were determ ned
in ELISAs to be similar for both the type 3 parent and
each of the derivatives (FIG. 18). ELISAs were also used
to confirm that the amounts of surface-accessible ca~sule
were similar in the type 3 parent and the derivatives.
~. Virulence of type 3 derivatives.
To assess the effect of alteration of capsule type
on virulence, BALB/ByJ female mice were infected i.p. or
i.v. with the type 3 derivatives and parent strains.
Strain JD770, which contains the nondestructive
erythromycin resistance marker in the type 3 capsule
locus, did not differ from its parent type 3 strain r,~2
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in terms of median time to death=(52 versus 49.5 h, i.p.)
or LD50 (75 versus 50 CFU, i.p.; 1 x 105 versus 2 x 105
CFU, i.v.). Thus JD770 was used in subsequent studies
for comparisons with the type 3 derivatives. As
expected, the recipient parent strains were significantly
different from JD770 with respect to time to death or
LD50s (FIG. 14). Expression of the type 3 capsule had no
apparent effect on the virulence of the type 2 recipient
strain; i.e., the time required to cause death was not
significantly different from that of the type 2 parent
but was significantly different fro~ that of the type 3
parent (FIG. 14A). However, alteration of capsular type
had dramatic effects on the virulence of the type 5 and
6B strains. In contrast to the highly virulent type 5
lS parental strain (LD50, ~10 CFU) and the virulent type 3
parental strain (LD50, -105), the type 3 derivatives were
not virulent even at doses of lo6 CFU (FIG. 143).
Switching of the type 6B capsule to type 3 resulted in a
reduction of the LD50 from >1 x 1o6 to ~6 x 103 CFU, a
value that was similar to but still greater than the 7.5
x 102 value observed for the type 3 parent strain (FIG.
14C).
These results may be indicative of the role other
factors play in pneumococcal virulence. For example, the
type 5 capsule may represent one that results in high
virulence with few other factors required, whereas the
type 3 capsule may require the presence of other factors
to be highly virulent. The introduction of the type 3
capsule into the type 5 genetic background may thus
result in the expression of a virulent capsule but, in
the absence of other necessary factors, in an avirulent
- strain. The increase in virulence of the type 6B strain
suggests that the type 3 capsule is probably more
virulent than the type 6B. However, its failure to
become as virulent as the type 3 parent is suggestive of
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a lack of other virulence factors in the 6B background.
The type 2 reciplent was only slightly more virulent than
the type 3 donor and no significant change was noted in
the virulence of its derivatives (FIG. 12). This result
may suggest that the type 2 and type 3 capsules are of
equal virulence and that the "accessory factors"
necessary for full virulence are present in both strains.
Whether the decrease in virulence of type 5
derivatives is related to the alteration of cell chain
length is not known. Clearly, the parent type 3 strain
is highly virulent with a similar chain length. The
alteration in chain length may reflect a general change
in the surface structure of the type 5 strains possibly
resulting from the change in capsule expression. Because
the strains constructed were transformed with chromosomal
DNA the inventors cannot rule out the possibility that
determinants closely linked to the capsule locus are
affecting the outcome of these studies. However, because
several backcrosses were performed, and because
independent isolates exhibited identical characteristics,
it is unlikely that unlinked determinants are responsible
for the results.
EXAMPLE 19
Increa~ed Virulence of S. pneumo~iae
tYpe 6B bY Inactivation of ~l~A.
In Example 18, the introduction of the type 3-
specific cassette and linked genes into an avirulent type
6B strain resulted in expression of the type 3 capsule
and an increase in virulence. To more clearly define the
contribution of the capsular serotype to the virulence of
5. pneumonlae, insertion-duplication mutagenesis was used
to insert an erythromycin marker adjacent to the type 6B-
specific capsule cassette in the 3' flanking region.
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Surprisingly, introduction o~ this insertion resulted in
an increase in virulence of the type 6B strain
(derivative LD50 of 103 versus parental LD50 of ~106,
intraperitoneal). This enhancement of virulence c~_ld be
attributed to the l.2 kb SacI-HindIII fragment fro~. ~he
type 3 strain WU2 (pJD377) that was used to direct ~he
erythromycin marker into the type 6B chromosome.
Transformation of the wild type 6B strain with the SacI-
HindIII fragment alone, followed by intraperitonea
infection of the transformation mixture into mice
resulted in death in less than 24 hours. Identical
results were obtained using multiple smaller fragme-rs of
the SacI-HindIII ~ragment. JD377 (Example 5) compr ses
the 3' end of cps3M and part of the gene plpA. The
fragments used contained mutations that, like the
original insertion, resulted in a defective plpA ir the
type 6B strain. These data suggest that avirulence of
type 6B observed via the intraperitoneal route is aue to
expression o~ plpA, and that the increase in virulence of
the type 6B strain expressing the type 3 capsule is the
result of inactivation of the linked plpA and not
expression of the type 3 capsule.
* * *
All of the compositions and methods disclosed and
claimed herein can be made and executed without unaue
experimentation in light of the present disclosure.
While the compositions and methods of this invention have
been described in terms of preferred embodiments, i_ will
be apparent to those of skill in the art that varia=ions
may be applied to the composition, methods and in t;~e
~ steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and
scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically
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and physiologically related may be substituted for the
agents described herein while the same or similar results
would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are
deemed to be within the spirit, scope and concept o_ the
invention as defined by the appended claims.
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SU~STITUTE SI~EET ~RUI E ~)
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Yother et al., ~Transformation of encapsulated
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SEQUENCE LISTING
-
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: UAB RESEARCH FOUNDATION
(B) STREET: 701 SOUTH 20TH STREET
. SUITE 112OG
ADMINISTRATION BUILDING
(C) CITY: BIRMINGHAM
(D) STATE: ALABAMA
(E) COUNTRY: UNITED STATES OF AMERICA
(F) POSTAL (ZIP) CODE: 35294-0111
(ii) INVENTORS: YOTHER, Janet
DILLARD, Joseph P.
(iii) TITLE OF INVENTION: STREPTOCOCCUS PNEUMONIAE
CAPSULAR POLYSACCHARIDE
GENES AND FLANKING REGIONS
(iv) NUMBER OF SEQUENCES: 20
(v) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Arnold, White & Durkee
(B) STREET: P.O. Box 4433
(C) CITY: Houston
(D) STATE: TX
(E) COUNTRY: United States of America
(F) ZIP: 77210
- (vi) COM~Ul~:~ READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COM~U~l~: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS/ASCII
SU~SrITUTE S~ET ~ l)LE 2~
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(D) SOFTWARE: PatentIn Release #1.0, Version
#1.30
(vii) CURRENT APPLICATION DATA:
(A) APPLICATICN NUMBER: UNKNOWN
(B) FILING DATE: CONCURRENTLY HEREWITH
(C) CLASSIFICATION: UNKNOWN
(viii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/243,546
(B) FILING DATE: 16-MAY- 1994
(C) CLASSIFICATION: UNKNOWN
(ix) ATTORNEY/AGENT INFORMATION:
(A) NAME: Parker, David L.
(B) REGISTRATION NUMBER: 32,165
(C) REFERENCE/DOCKET NUMBER: AMCY018P--
(x) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (512) 418-3000
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(2) INFORMATION FOR SEQ ID NO:l:
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(C) STRANDEDNESS: single
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(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
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CA 02201772 1997-04-03
W~ gS13154g
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WO95/31548 CA 02201772 1997-04-03
PCTrUS95/06119
-176-
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W O9S/31548 CA 02201772 1997-04-03
PCTnUS9SI06119
-177-
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W O95/31548 CA 02201772 1997-04-03
PCTAUS95/06119
-178-
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WO 95~31548
PCTrUS9S1~6119
- -179-
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-180-
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CA 02201772 1997-04-03
W Og~/31~48 PCTnUS9~106119
-181-
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CA 02201772 1997-04-03
WO 95/31548 PCTrUS95/06119
-182-
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CA 02201772 1997-04-03
PCTnVS90~61~9
W O9513~548
-183-.
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CA 02201772 1997-04-03 PCTnUS95/06119
W O9~/31~48
. -184-
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CA 02201772 1997-04-03
PCTn~S9SJ06~19
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CA 02201772 1997-04-03
W O95/31548 PCTrUS95/06119
-186-
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SUBSTITUTE SHEET (RllEE 26)
CA 02201772 1997-04-03
~CTn~S95J06119`
W O95131548
-187-
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CA 02201772 1997-04-03
W O95/31548 PCTrUS95/06119
-188-
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CA 02201772 1997-04-03
WO 95/31548 PCT/I~S95/06119
--189--
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CA 02201772 1997-04-03
W O95/31548 PCTrUS95/06119
-190 -
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CA 02201772 1997-04-03
WO 951315~8 PC'rNS95~6119
-191-
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S~JBSTITIJTE SHEET (RULE 26)
CA 02201772 1997-04-03
W O9S/31548 PCTAUS95/06119
- 192 -
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l inear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
TCATTTGATA TGCCTCCG 18
(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l inear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
30 GTGAGATAAA TAGTAGTGCG 20
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
SlJBSTITl~TE SHEET (~LE 26
CA 02201772 1997-04-03
WO ~5/31548 PCT~US9~/0611s
- 193 -
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
10 TCCAGCTCGT GTCATAATCT 20
SUE~TlllJlE SHEET (RlJLE 26)
