Note: Descriptions are shown in the official language in which they were submitted.
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B&P File No. 2223-92
Title: A SURFACE-EXPOSED LIPOPROTEIN TO Campylobacter jejuni
INVOLVED IN ADHERENCE TO Hep-2 CELLS
FIELD OF THE INVENTION
The invention relates to a novel surface-exposed lipoprotein,
JIpA, specific to Campylobacter jejuni.
BACKGROUND OF THE INVENTION
Campylobacter jejuni is a Gram-negative, spiral,
microaerophilic bacterium that exists as a commensal organism in the
intestinal
tracts of a variety of wild and domestic animals. It is a leading cause of
acute
bacterial enterocolitis in humans worldwide (Altekruse, 1999). Clinical
symptoms of campylobacteriosis range from a mild watery diarrhea to bloody
diarrhea with fever, abdominal cramps, and presence of fecal leukocytes. C.
jejuni infection in human is often associated with undercooked chicken and
turkey, red meat, raw milk, and untreated water. C. jejuni has also been
strongly
implicated in the etiopathogenesis of Guillain-Barre Syndrome (GBS), a
neurological disease that results in respiratory compromise and death.
Pathogenic mechanisms of infection caused by C. jejuni are still not well
understood, but are known to involve colonization, adhesion, and likely also
cellular invasion and toxin production in the human intestinal tract (Ketley,
1997).
Adherence of bacteria to host cells is a critical step in an
infection, which is generally recognized as a multifactoral process and
mediated
by adhesins on the bacterial surface. Bacterial adhesins are either assembled
into
hair-like appendages called pili or directly associated with cell surface
(Soto and
Hultgren, 1999). Although pili have been demonstrated to be adhesins in many
bacterial pathogens, such a structure has not been confirmed in C. jejuni. A
most recent study showed that some environmental stimuli induce a pilus-like
appendage on the cell surface of C. jejuni and C. coli. However, this cell
appendage does not play a role in adherence of C. jejuni to epithelial cells
(Doig,
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et al., 1996). Therefore, the adhesins of C. jejuni might be directly
associated with
the cell surface. Fauchere et al. (1989) reported that several outer membrane
proteins of C. jejuni, including the major cell-binding factor CBF1,
specifically
bind to HeLa cells. Later studies showed that CBF1 is identical to PEB1, one
of
the PEB antigenic proteins (Kervella, et al., 1993). Genetic analysis
indicated that
PEB1 is homologous to a component of the ABC transport system in Gram-
negative bacteria (Pei and Blaser, 1993). The ability of adherence of C.
jejuni to
epithelial cells was reduced 50- to 100-fold but not totally abolished when
the
pebl gene was disrupted, suggesting the possible presence of other adhesins in
C.
jejuni. Konkal et al. (1997) identified a 37 kDa outer membrane protein from
C.
jejuni, named CadF, which specifically binds to fibronectin, a component of
extracellular matrix of mammalian cell. The fibronectin binding activity of
cadF
mutants was reduced 3- to 6-fold. DE Melo and Pechere (1990) discovered four
specifically binding proteins with apparent molecular mass of 28, 32, 36, and
42
kDa when they used the cell surface extracts of C. jejuni to bind HEp-2 cells.
PEB1 (28 kDa) and CadF (37 kDa) possibly correspond to two of these four
proteins. In addition, a 59 kDa outer membrane protein and the 43 kDa major
outer membrane protein (MOMP) were reported to bind to the membrane of
epithelial cells (Schroder and Moser, 1997; Moser et al., 1997).
SUMMARY OF THE INVENTION
The present inventors have isolated a novel cell surface
adhesin of C. jejuni which has homology to a rhoptry protein of the malaria
parasite.
The novel cell surface adhesin, JIpA, plays an important role
in the adhesion of C. jejuni to Hep-2 cells. This polypeptide is released into
the
culture medium during the growth and binds Hep-2 cells. JIpA is the first
protein presently reported in Campylobacter that shares homology with
eukaryotic protein. The glycine-acid extraction and proteinase K digestion
experiments indicate that JIpA is a surface-exposed protein in the said
bacterium.
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Accordingly, the present invention provides a purified and
isolated polypeptide comprising a sequence of 1116-by of the open reading
frame.
The invention also contemplates a jlpA gene encoding a
polypeptide, JIpA, of 372 amino acid residues with a molecular mass of 42.3
kDa.
JIpA contains a typical signal peptide and a lipoprotein processing site at
the N-
terminus. JIpA is a surface exposed lipoprotein. JIpA is loosely associated
with
the cell surface. JIpA binds specifically to Hep-2 cells.
The invention also contemplates truncations of the protein
and analogues, homologs and isoforms of the protein and truncations thereof.
T'he protein may be conjugated to produce other proteins such
as fusion proteins. Such proteins can be generated, for example, by the
synthesis
of N-terminal and C-terminal fusion proteins.
The invention further contemplates the creation of antibodies
to the epitope of the polypeptide of the invention, preferably the creation of
polyclonal antibodies. Furthermore, it is contemplated that such antibodies
can
be used to detect the presence of JIpA protein and like polypeptides under a
variety of conditions and environments.
A kit for the detection of JIpA epitope preferably a
monoclonal antibody and its use is also provided. The kit may also contain
reagents which are needed to bind the antibody to the polypeptide protein in
the
sample.
The nucleic acid sequence for jlpA of the invention allows
those skilled in the art to construct nucleotide probes for the use and
detection
of same, related or analogous polypeptides in a variety of samples including
food, the environment and other biological materials.
The invention further provides for a kit for detection of the
presence of nucleic acid molecules having a sequence encoding a polypeptide
for
JIpA , a related or analogous polypeptide. The kit comprises a nucleotide
probe
which hybridizes with the nucleic acid molecule, reagents required for
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hybridization of the nucleotide probe with the nucleic acid molecule and
directions for its use.
The nucleic acid sequence can be used in the polymerase
chain reaction [PCR] to amplify the nucleic acid molecule of this invention.
Accordingly, the invention relates to a method of determining the presence of
a
nucleic acid molecule having a sequence encoded a JIpA protein or a
predetermined part of JIpA protein in a sample.
The invention further relates to a kit for the determining the
presence of a nucleic acid molecule bearing the sequence of JIpA protein or a
predetermined part of the polypeptide in a sample. The kit comprises of
primers
which are capable of amplifying the nucleic acid molecule in a PCR reaction,
reagents required to amplify the nucleic acid molecule thereof in an
amplification reaction, preferably in a PCR reaction, means for assaying the
amplified sequence and directions for the use of the kit.
The nucleic acid molecules of the invention may also be used
to assay for a substance which inhibit adherence or invasion of C. jejuni. As
such, the invention provides for a method for assaying for a substance that
interferes with a JIpA polypeptide. The method may be used, for example, to
assay for a substance that impacts on the growth or pathogenicity of C.
jejuni.
The substances identified using the method of invention,
antibodies and antisera molecules may be used to inhibit the binding, adhesion
or invasion of C. jejuni . Accordingly, the substances may be used in the
treatment of infectious diseases caused by C. jejuni. There is in this respect
provided a method of inhibiting the binding of C. jejuni comprising
administering to an animal an effective amount of a substance capable of
inhibiting the adherence of a JIpA protein in an animal in need thereof. As
such, these substances may be formulated into pharmaceutical composition for
the administration to subjects.
Other objects, features and advantages of the present
invention will become apparent from the following detailed description. It
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should be understood, however, that the detailed description and the specific
examples while indicating preferred embodiments of the invention are given by
way of illustration only, since various changes and modifications within the
spirit
and scope of the invention will become apparent to those skilled in the art
from this
detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings
in which:
Figure 1 (SEQ. ID. NO.: 1) shows the sequence of 5' nucleotide
sequence and the N-terminus deduced amino acid sequence of the jlpA;
Figure 2 (SEQ. ID. NOS.: 10 and 11) shows the alignment shown
is modified from the alignment obtained from a BlastP database search;
Figure 3 shows the expression of JIpA in E. Coli;
Figure 4 shows surface localization of JIpA in C. jejuni TGH9011;
Figure 5 shows release of JIpA during growth;
Figure 6 shows genetic organization of hip0-jlpA region and
construction of jlpA mutants;
Figure 7 shows specific binding of purified JIpA to Hep-2 cells;
and
Figure 8 shows the presence of jlpA in Campylobacter species.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The following standard abbreviations for the amino acid
residues are used throughout the specifications: A, Ala - alanine; C, Cys -
cysteine;
D, Asp- aspartic acid; E, Glu - glutamic acid; F, Phe - phenylalanine; G, Gly -
glycine;
H, His - histine; I, Ile - isoleucine; K, Lys - lysine; L, Leu - leucine; M,
Met
methionine; N, Asn - asparagine; P, Pro - proline; Q, Gln - glutamine; R, Arg -
arginine; S, Ser - serine; T, Thr - threonine; v, Val - valine; W, Trp -
tryptophan; Y,
Try - tyrosine; and p.Y., P.Tyr - phosphotyrosine.
Administration of an "effective amount" of the compounds of
the present invention is defined as an amount effective, at dosages and for
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periods of time necessary to achieve the desired result. The effective amount
of
a compound of the invention may vary according to factors such as the disease
state, age, sex, and weight of the animal. Dosage regima may be adjusted to
provide the optimum therapeutic response. For example, several divided doses
may be administered daily or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation.
The term "animal" as used herein includes all members of
the animal kingdom, including humans. Preferably, the animal to be treated is
a human.
The description of the invention is divided into three
sections. They are the following sections: [1] novel nucleic acid molecule;
[2]
novel proteins[s]; and [3] application for which the nucleic acid molecules,
proteins and substances identified herein are suitable.
j1] Nucleic acid molecules of this invention:
The present invention relates to an isolated nucleic acid of C.
jejuni encoding a polypeptide associated with adhesion. The term "isolated"
refers to nucleic acid substantially free of cellular material or culture
material
when produced by recombinant DNA technology, or chemical precursors, or
other chemicals when chemically synthesized. The term "nucleic acid" is
intended to include DNA and RNA and can be either double stranded or single
stranded.
In the embodiment of the invention, an isolated nucleic acid
molecule is provided having a sequence which encodes a protein having an
amino acid as shown in Figure 1.
Preferably, the purified and isolated nucleic acid molecule
comprises
[a] a nucleic acid sequence as shown in Figure 1, where T can
also be U;
[b] nucleic acid sequence complementary to [a];
[c] nucleic acid sequences which are homologous to [a] and [b];
._. .___.~..~ ._.....~.~ ~.___. _ _.._ __
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[c] a fragment of [a] to [c] that is at least 15 bases, preferably 20 to
30 bases, and which will hybridize to [a] and [c] under stringent
hybridization
conditions; or
[d] a nucleic acid molecule differing from any of the nucleic
acids of [a] to [c] in codon sequences due to the degeneracy of the genetic
code.
The invention includes nucleic acid molecules comprising
nucleic acid sequences having substantially sequence homology with the nucleic
acid sequence as shown in Figure 1. The term "sequences having substantial
sequence homology" means those nucleic acid sequences which have slight or
inconsequential sequence variations from these sequences, i.e. the sequences
function in substantially the same manner to produce functionally equivalent
proteins. The variations may be attributed to local mutations or structural
modifications.
Another aspect of the invention provides a nucleic acid
molecule, and fragment thereof having at least 15 bases, which hybridizes to
the
nucleic acid molecules of the invention under hybridization conditions,
preferably stringent hybridization conditions. A person skilled in the art is
knowledgable about appropriate stringency conditions which promote DNA
hybridizations and the techniques available thereof.
Isolated and purified nucleic acid molecules having sequences
which differ from the nucleic acid sequences of JIpA due to degeneracy in the
genetic code are also within the scope of this invention.
The determination of whether a particular nucleic acid
molecule encodes a novel protein of the invention may be accomplished by
expressing the cDNA in an appropriate host cell by standard techniques, and
testing the activity of the protein using the methods as described herein. In
addition, regulatory elements may be identified in the DNA through use of
constructs that may identify proteins interacting with the elements by use of
techniques known in the art.
The sequence of a nucleic acid molecule of the invention may
be inverted relative to its normal presentation for transcription to produce
an
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antisense nucleic acid molecule. Preferably, an antisense sequence is
constructed
by inverting a region preceding the initiation codon or unconverted region, In
particular, the nucleic acid sequence contained in the nucleic acid molecule
of
the invention or a fragment thereof, preferably a nucleic acid sequence shown
in
Figure 1 may be inverted relative to its normal presentation for transcription
to
produce antisense nucleic acid molecules.
The antisense nucleic acid molecule of the invention or a
fragment thereof, may be chemically synthesized using naturally occurring
nucleotides or variously modified nucleotides designed to increase the
biological stability of the molecules or to increase the physical stability of
the
duplex formed with mRNA or the native gene, i.e. phosphorothioate
derivatives and acridine substituted nucleotides. The antisense sequences may
be produced biologically using an expression vector induced into cells in the
form of a recombinant plasmid, phagemid or attenuated virus in which
antisense sequences are produced under the control of a high efficiency
regulatory region, the activity of which may be determined by the cell type
into
which the vector is introduced.
The invention also provides nucleic acids encoding fusion
proteins comprising a novel protein of the invention and a selection protein,
or
a selectable marker protein [see below].
j2] Novel proteins of invention
The invention broadly contemplates an isolated protein
characterized in that it has part or all of the primary structural
conformation [i.e.
continuous sequence of amino acid residues] of a novel protein encoded by the
jlpA gene of the invention. In the embodiment of the invention, an isolated
protein is provided which has the amino acid sequence as shown in Figure 1.
Within the context of the present invention, a protein of the
invention may include various structural forms of the primary protein which
retain biological activity. For example, a protein of the invention may be in
the
form of acid or basic salts or in neutral form. In addition, individual amino
acid
residues may be modified by oxidation or reduction.
._..._._______ . _. ___...~~~.~ __.. __..._. . ..._.__~ _...._.
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In addition to the full length amino acid sequence the protein
of the present polypeptide of the invention may also include truncation of the
polypeptide, analogues and homologs of the polypeptide. Truncations may
comprise peptides of at least fifteen amino acid residues.
Analogues of the proteins having the amino acid sequence
shown in Figure 1 and / or truncations thereof as described herein, may
include,
but not be limited to an amino acid sequence containing one or more ammo
acid substitutions, insertions and / or deletions. Amino acid substitutions
may
be of a conserved or non-conserved nature. Conserved amino acid substitutions
involve replacing one or more amino acids of the proteins of the invention
with amino acids of similar charge, size and / or hydrophobic characteristics.
When only conserved substitutions are made the resulting analogue should be
functionally equivalent. Non-conserved substitutions involve replacing one or
more amino acids of the amino acid sequence with one or more amino acids
which possess dissimilar charge, size and / or hydrophobicity characteristics.
One or more amino acid insertions may be introduced into
the amino acid sequences shown in Figure 1. Amino acid insertions may consist
of single amino acid residues or sequential amino acids ranging from 2 to 15
amino acids in length. For example, amino acid insertions may be used to
destroy target sequences so that the protein is no longer active. This
procedure
may be used in vivo to inhibit the activity of a protein of the invention.
Deletions may consist of the removal of one or more amino
acids, or discrete portions from the amino acid sequence shown in Figure 1.
The
deleted amino acids may or may not be contiguous. The lower limit length of
the resulting analog with deletion mutation is about 10 amino acids.
Analogues of a protein of the invention may be prepared by
introducing mutations in the nucleotide sequence encoding the protein.
Mutations in nucleotide sequences constructed for expression of analogue of a
protein of the invention must prevent the reading frame of the coding
sequence. Furthermore, the mutations will preferably not create complementary
regions that could hybridize to produce secondary mRNA structures, such as
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loops or hairpins, which could adversely affect translation of the receptor
mRNA.
Mutations may be introduced at particular loci by synthesizing
oligonucleotides containing a mutant sequence, flanked by restriction sites
enabling ligation to fragments of the native sequence. Following ligation, the
resulting reconstruction sequence encodes an analogue having the desired
amino acid insertion, substitution or deletion.
Alternatively, oligonucleotide-directed site specific
mutagenesis procedures may be employed to provide an altered gene having
particular codons altered according to the substitution, deletion or insertion
required. Deletion or truncation of a protein of the invention may also be
constructed by utilizing convenient restriction endonuclease sites adjacent to
the desired deletion. Subsequent to restriction, overhangs may be filled in,
and
the DNA relegated. Methods of making the alterations are set forth including
by
Sambrook et al. [11J.
The proteins of the invention also include homologs of the
amino acid sequence shown in Figure 1. Such homologs are proteins whose
amino acid sequences are comprised of amino acid sequences that hybridize
under stringent hybridization conditions with a probe used to obtain a protein
on the invention. Homologs of a protein of the invention will have the same
regions which are characteristic of the protein.
In heterologous species of C. jejuni, a homologous protein
includes a protein with an amino acid sequence having at least 30%, preferably
40-50% identity with the amino acid sequence as shown in Figure 1. In
homologous species of C. jejuni, a homologous protein includes a protein with
an amino acid sequence having at least 70% preferably 80-90% identity with the
amino acid sequence as shown in Figure 1.
The invention also contemplates isoforms of the proteins of
the invention. An isoform contains the same number and kinds of amino acids
as a protein of the invention, but the isoform has a different molecular
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structure. The isoforms contemplated by the present invention are those having
the same properties as a protein of the invention as described herein.
The present invention also includes a protein of the
invention conjugated with a selected protein, or a selection maker protein to
produce fusion proteins. Additionally, immunogenic portions of the protein of
the invention are within the scope of the invention.
The protein of the invention [including truncation, analogues
etc.] may be prepared using recombinant DNA methods. Accordingly, the
nucleic acid molecules of the present invention having a sequence which
encodes a protein of the invention may be incorporated in a known manner
into an appropriate expression vector which ensures good expression of the
protein. Possible expression vectors include but are not limited to cosmids,
plasmids or modified viruses [e.g. replication defective retroviruses,
adenoviruses and adeno-associated viruses], so long as the vector is
comparable
with the host cell used. The expression vectors are "suitable for
transformation
of a host", means that the expression vectors contain a nucleic acid molecule
of
the invention and regulatory sequences selected on the basis of the host cells
to
be used for expression, which is operatively linked to the nucleic acid
molecules.
Operatively linked is intended to mean that the nucleic acid is linked to
regulatory sequences in a manner which allows expression of the nucleic acid.
The invention therefore contemplates a recombinant
expression vector of the invention containing a nucleic acid molecule of the
invention, or a fragment thereof, and the necessary regulatory sequences for
the
transcription and translation of the inserted protein-sequence. Suitable
regulatory sequences may be derived from a variety of sources, including
bacterial, fungal or viral genes [12]. Selection of the appropriate regulatory
sequences is dependent on the host cell chosen and may be readily accomplished
by one skilled in the art. Additionally, depending on the host cell chosen and
the vector employed, other sequences, such as an origin of replication,
additional DNA restriction site, enhancers and sequences conferring
inducibility
of transcription may be incorporated into the expression vector. It will also
be
r .....__ .... _.. ... .. _._.....
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appropriate that the necessary regulatory sequences may be supplied by the
native protein and / or its flanking regions.
The invention further provides a recombinant expression
vector comprising a DNA nucleic acid molecule of the invention cloned into
the expression vector in an antisense orientation. That is, the DNA molecule
is
operatively linked to a regulatory sequencer in a manner which allows for
expression, by transcription of the DNA molecule, of an RNA molecule which
is antisense to a nucleotide sequence comprising the nucleotides as shown in
Figure 1. Regulatory sequences operatively linked to the antisense nucleic
acid
can be chosen which direct the continuous expression of the antisense RNA
molecule.
The recombinant expression vector of the invention may also
contain a selection marker gene which facilitates the selection of host cells
transformed or transfected with a recombinant molecule of the invention.
Examples of selectable marker genes are genes encoding a protein such as 6418
and hygromycin which confer resistance to certain drugs, Q-galactosidase,
chloramphenicol acetyltransferase or firefly luciferase. The procedures are
know
to one skilled in the art. It is appreciated that selectable markers can be
introduced on a separate vector from the nucleic acid of interest.
The recombinant expression vector may also contain genes
which encode a fusion moiety which provides increased expression of the
recombinant protein; increased solubility of the recombinant protein; and aid
in
the purification of a target recombinant protein by acting as a ligand in
affinity
purification. For example, a proteolytic cleavage site may be added to the
target
recombinant protein to allow separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Recombinant expression vectors can be introduced into the
host cells to produce a transformant host cell. The term "transformant host
cell"
is intended to include prokaryotic and eukaryotic cells which have been
transformed or transfected with a recombinant expression vector of the
invention. The terms "transformed with", "transfected with", "transformation'
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and "transfection" are intended to encompass introduction of nucleic acid
[e.g. a
vector] into a cell by one of many possible techniques known in the art.
Prokaryotic cells can be transformed with nucleic acid by, for example,
electroporation or calcium-chloride mediated transformation. Nucleic acid can
be introduced into mammalian cells via conventional techniques such as
calcium phosphate or calcium chloride co-precipitation, DEAF-dextran-
mediated transfection, lipofectin, electroporation or microinjection.
Appropriated methods for transforming and transfecting host cells can be found
in a variety of matters including in Sambrook et al.[11].
Suitable host cells including a wide variety of prokaryotic and
eukaryotic host cells. For example, the proteins of the invention may be
expressed in bacterial cells such as E. coli, insect cells [using baclovirus],
yeast
cells or mammalian cells. Other suitable hosts can be found in other matters
including in Goeddell [12].
The proteins of the invention may also be prepared by
chemical synthesis using techniques well known to a person skilled in the art
of
chemistry.
131 Application for which the nucleic acid molecules proteins and substances
identified herein are suitable
[a] detection of nucleic acid molecules, antibodies and diagnostic application
The nucleic acid molecules of the invention, allow those
skilled in the art to construct nucleotide probes for use in the detection of
nucleotide sequences in a sample. A nucleotide probe may be labelled with a
detectable marker such as a radioactive label which provides for an adequate
signal and has sufficient half life such as 32P, 3H, 14C or the like. Other
detectable
markers which may be used include antigens that are recognized by a specific
labelled antibody, fluorescent compound, enzymes, antibodies specific for a
labelled antigen and chemiluminescent compounds. An appropriate label may
be selected having regard to the rate of hybridization and binding of the
probe to
the nucleotide to be detected and the amount of nucleotide available for
hybridization.
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The nucleotide probe may be used to detect genes that encode
proteins related to or analogous to JIpA proteins of the invention.
Accordingly, the present invention also relates to a method of
detecting the presence of the nucleic acid molecule encoding a JIpA protein of
the invention in a sample comprising contacting the sample under
hybridization conditions with one or more of nucleotide probes which hybridize
to the nucleic acid molecule and are labelled with a detectable marker, and
determining the degree of hybridization between the nucleic acid molecule in
the sample and the nucleotide probes.
In an embodiment of the invention, a method is provided for
detecting C. jejuni in a sample comprising contacting the sample with a
nucleic
acid molecule containing a nucleic acid sequence encoding a JIpA protein, or a
fragment thereof, under conditions which permit the nucleic acid molecule to
hybridize with a complementary sequence in the sample to form a hybridization
product, and assaying for hybridization product.
Hybridization conditions which may be used in the methods
of the invention are known in the art and are described for example in
Sambrook[11]. The hybridization product may be assayed using techniques
known in the art. The nucleotide probe may be labelled with a detectable
marker
as described herein and the hybridization product may be assayed by detecting
the detectable marker or the detectable change produced by the detectable
marker.
The nucleic acid molecule of the invention also permits the
identification and isolation, or synthesis of nucleotide sequences which may
be
used as primers to amplify a nucleic acid molecule of the invention, for
example
in PCR. The primers may be used to amplify the genomic DNA of other bacterial
species. The PCR amplification sequence can be examined to determine the
relationship between the various JIpA genes.
It will be appreciated that the primers may contain non-
complementary sequences provided that a sufficient amount of the primer
contains a sequence which is complementary to a nucleic acid molecule of the
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invention or oligonucleotide fragment thereof, which is to be amplified.
Restriction site linkers may also be incorporated into the primers allowing
for
digestion of the amplified products with the appropriate restriction enzymes
facilitating cloning and sequencing of the amplified product.
In an embodiment of the invention a method of determining
the presence of a nucleic acid molecule having a sequence encoding a protein
of
the invention is provided comprising treating the sample with primers which
are capable of amplifying the nucleic acid molecule or predetermining
oligonucleotide fragment thereof in a polymerase chain reaction to form
amplified sequences, under conditions which permit the transformation of
amplified sequences and assaying for amplified sequences.
The amplified products can be isolated and distinguished
based on their respective sizes using techniques known in the art. For
example,
after amplification, the DNA sample can be separated on an agarose gel and
visualized, after staining with ethidium bromide, under ultra violet [UV]
light.
DNA may be amplified to a desired level and a further extension reaction may
be performed to incorporate nucleotide derivatives having detectable markers
such as radioactive labelling or biotin labelled nucleoside triphosphates. The
primers may also be labelled with detectable markers as discussed above. The
detectable markers may be analyzed by restriction and electrophoretic
separation
or other techniques known in the art.
The conditions which may be employed in the methods of the
invention using PCR are those which permit hybridization and amplification
reactions to proceed in the presence of DNA in a sample and appropriate
complementary hybridization primers. Conditions suitable for the polymerase
chain reaction are known in the art. It is appreciated that other techniques
may
be used including Ligase Chain Reaction [LCR] and NASBA.
A JlpA protein of the invention can be used to prepare
antibodies specific for the protein. Antibodies can be prepared which bind a
distinct epitope in an unconserved region of the protein. An unconserved
region of the protein is one which does not have substantial sequence homology
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to other proteins. Alternatively, a region from a well-characterized domain
can
be used to prepare an antibody to a conserved region of a protein of the
invention. Antibodies having specificity for a protein of the invention may
also
be raised from fusion proteins.
Conventional methods can be used to prepare antibodies. For
example, by using a peptide of a protein of the invention, polyclonal antisera
or
monoclonal antibodies can be made using standard methods known to one
skilled in the art.
The term "antibody' as used herein is intended to include
fragments thereof which also specifically react with a protein, of the
invention
or peptide thereof. Antibodies can be fragmented using conventional techniques
and the fragments screened for utility by techniques known to one skilled in
the
art. For example, F[ab']2 fragments can be generated by treating antibody with
pepsin. The resulting F[ab']2 fragment can be treated to reduce disulphide
bridges
to produce Fab' fragments.
Chimeric antibody derivatives, i.e. antibody molecules that
combine a non-human animal variable region and a human constant region are
also contemplated within the scope of the invention. Chimeric antibody
molecules can include, for example, the antigen binding domain from an
antibody of a mouse, rat or other species, with human constant region.
Conventional methods may be used to make chimeric antibodies containing the
immunoglobulin variable region which recognizes a JIpA protein [see for
example [15]].
Monoclonal or chimeric antibodies specifically reactive with a
protein of the invention as described herein can be further humanized by
producing human constant region chimeras, in which parts of the variable
regions, particularly the conserved framework regions of the antigen-binding
domain, are of human origin and only the hypervariable regions are of non
human origin. Such immunoglobulin molecules may be made by techniques
known in the art.
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Specific antibodies or antibody fragments reactive against a
protein of the invention may also be generated by screening expression
libraries
encoding immunoglobulin genes, or portion thereof, expressed in bacteria with
peptides produced from the nucleic acid molecules of the present invention.
For
example, complete Fab fragments, VH regions and FV regions can be expressed
in bacteria using phage expression libraries.
The antibodies may be labelled with a detectable marker
including various enzymes, fluorescent materials, luminescent materials and
radioactive materials. Examples of suitable enzymes include horseradish
peroxidase, biotin, alkaline phosphatase, Q-galactoside or
acetylcholinesterase;
examples of suitable fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride or phycoerythrin; an example of a luminescent material
includes luminol; and examples of suitable radioactive material includes S-35,
Cu-64, Ga-67, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, I-125, I-131, Re-
186,
Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212. the antibodies may also be
labelled or conjugated to one partner of a ligand binding pair. Representative
examples include avidin-biotin and riboflavin-riboflavin binding protein,
methods for conjugating or labelling the antibodies discussed above with the
representative labels set forth above may be readily accomplished using
technique known to one skilled in the art.
The antibodies reactive against proteins of the invention [e.g.
enzyme conjugates or labelled derivatives] may be used to detect a protein of
the
invention in various samples, for example they may be used in any known
immunoassays which rely on the binding interactions between any antigenic
determinant of a protein of the invention and the antibodies. Examples of such
assays are radioimmunoassays, enzyme immunoassays [e.g. ELISA],
immunofluorescence, immunoprecipitation, latex agglutination,
hemagglutination and histochemical tests. Thus, the antibodies may be used to
identify or quantify the amount of a protein of the invention in a sample in
order to diagnose C. jejuni infections.
CA 02296869 2000-O1-24
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A sample may be tested for the presence or absence of a
pathogenic C. jejuni serotype by contacting the sample with an antibody
specific
for an epitope of JIpA protein which antibody is capable of being detected
after it
becomes bound to a JlpA protein in the sample, and assaying for antibody bound
to a JIpA protein in the sample, or unreacted antibody.
In a method of the invention a predetermined amount of a
sample or concentrated sample is mixed with antibody or labelled antibody. The
amount of antibody used in the process is determined upon the labelling agent
chosen. The resulting protein bound to antibody or labelled antibody may be
isolated by conventional isolation techniques, for example, salting out,
chromatography, electrophoresis, gel filtration, fractionation, absorption,
polyacrylamide gel electrophoresis, agglutination, or combination thereof.
For example, one can use labelled and unlabelled or soluble or
insoluble antibody in a given assay mix, e.g. the antibodies can be bound to
suitable carriers such as Sepharose or agarose beads; a labelled antibody can
be
assessed in a sample as it binds to the protein of the invention or one can
assess
the unreactive antibody; and unlabelled antibody can be determined by
measuring the amount of antibody bound to C. jejuni serotype using substances
that interact specifically with the antibody to cause agglutination or
precipitation.
The reagents suitable for applying the methods of the
invention may be packaged into convenient kits providing the necessary
materials, packaged into suitable containers. Such kits may include all the
reagents required to detect a pathogenic C. jejuni serotype in a sample by
means
of the methods described herein, and optionally suitable supports useful in
performing the methods of the invention.
In one embodiment of the invention the kit contains a
nucleotide probe which hybridizes with a nucleic acid molecule of the
invention, reagents required for hybridization of the nucleotide probe with
the
nucleic acid molecule, and directions for its use. In another embodiment of
the
invention the kit includes antibodies of the invention and reagents required
for
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binding of the antibody to a protein specific for a pathogenic C. jejuni
serotype
in a sample. In still another embodiment of the invention, the kit includes
primers which are capable of amplifying a nucleic acid molecule of the
invention or a predetermined oligonucleotide fragment thereof, all the reagent
required to produce the amplified nucleic acid molecule or predetermined
fragment thereof in the polymerase chain reaction, and means for assaying the
amplified sequences.
The methods and kits of the present invention have many
uses. For example, the methods and the kits of the present invention may be
used to detect a pathogenic C. jejuni serotype in any medical or veterinary
sample suspected of containing the said organism. Samples that may be tested
included bodily materials such as blood, urine, serum, tears, saliva, feces,
tissues
and the like. In addition to human samples, samples may be taken from
mammals such as non-human primates, etc. Further, water and food samples
and other environmental samples and industrial wastes may be tested.
Before the samples are tested using the invention described
herein, the samples my be concentrated using the techniques known in the art,
such as centrifugation and filtration. For the hybridization and / or PCR-
based
methods using the invention described herein, nucleic acids may be extracted
from cell extracts of the test sample using techniques known in the art.
Substances that affect adherence and / or invasion of C. jejuni
A JlpA protein of the invention may also be used to assay for
substances which affect adherence and / or invasion of the said organism.
Accordingly, the invention provides a method for assaying for a substance that
affects adherence and / or invasion of C. jejuni comprising mixing a protein
of
the invention with a test substance which is suspected of affecting the
expression or activity of the protein, and determining the effect of the
substance
by comparing to a control.
The reagents suitable for applying the methods of the
invention to identify substances that affect adherence and / or invasion of C.
jejuni may be packaged into convenient kits providing the necessary materials
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packaged into suitable containers. The kits may also include suitable supports
useful in performing the methods of the invention.
Pharmaceutical Compositions and Methods of Treatment
The substances identified by the methods described herein,
antisense nucleic acid molecules, and antibodies, may be used for reducing
adherence and / or invasion of C. jejuni and accordingly may be used in the
treatment of infectious diseases caused by C. Jejuni .
The substances identified using the methods described herein
and antibodies may be formulated into pharmaceutical compositions for
administration to subjects in a biologically compatible form suitable for
administration in vivo. By "biologically compatible form suitable for
administration in vivo" is meant a form of the substance to be administered in
which any toxic effects are outweighed by the therapeutic effect. The
substances
may be administered to living organisms including humans and animals.
Administration of a therapeutically active amount of the pharmaceutical
compositions of the present invention is defined as an amount effective, at
dosages and for periods of time necessary to achieve the desired results. For
example, a therapeutically active amount of a substance may vary according to
factors such as the disease state, age, sex and weight of the individual, and
the
ability of antibody to elicit a desired response in the individual. Dosage
regime
may be administered daily or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation.
The active substance may be administered in a convenient
manner such as by injection [subcutaneous, intravenous, etc.], oral
administration, inhalation, transdermal application or rectal administration.
Depending on the route of administration, the active substance may be coated
in
a material to protect the compound from the action of enzymes, acids and other
natural conditions which may inactivate the compound.
The compositions described herein can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to subjects, such that an effective
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quantity of the active substance is combined in a mixture with a
pharmaceutically acceptable vehicle. Suitable vehicles are described, for
example, in Remington's Pharmaceutical Sciences [Remington's Pharmaceutical
Sciences, Marck Publishing Company, Easton, Pa., USA 1985]. On this basis, the
compositions include, albeit not exclusively, solutions of the substances in
association with one or more pharmaceutically acceptable vehicles or diluents,
and contained in buffered solutions with a suitable pH and iso-osmotic with
the
physiological fluids.
Recombinant molecules comprising an antisense sequence or
oligonucleotide fragment thereof, may be directly introduced into cells or
tissues
in vivo using delivery vehicles such as retroviral vectors, adenoviral vectors
and DNA virus vectors. They may also be introduced into cells in vivo using
physical techniques such as microinjection and electroporation or chemical
methods such as coprecipitation and incorporation of DNA into liposomes.
Recombinant molecules may also be delivered in the form of an aerosol or by
lavage.
The utility of the substances, antibodies, antisense nucleic acid
molecules and compositions of the invitation may be confirmed in animal
experimental model systems.
Vaccines
The present invention relates to a vaccine against an
infectious disease caused by C. jejuni comprising a carrier strain having an
amount of a jlpA protein associated with its surface which is effective to
provide
protection against C. jejuni. "Infectious disease" refers to anv disease or
condition due to the action of C. jejuni. The vaccines may be used for the
prophylaxis or active immunization and treatment of infectious diseases caused
by the said organism.
The carrier strain may be selected so that it is incapable of
multiplying in vivo. Carrier strains are obtained through selection of
variants
which occur naturally, or using conventional means known to those skilled in
CA 02296869 2000-O1-24
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the art. Examples of suitable carrier strains are Shigella species, Salmonella
species, S. Typhimurium species, Vibrio species and Escherichia species.
The invention also relates to a method of preparing a vaccine
against an infectious disease caused by C.jejuni comprising associating with
the
cell surface of a carrier strain a JIpA protein or portion thereof which is
effective
to provide protection against C.jejuni. A JIpA protein or portion thereof may
be
associated with the cell surface of a carrier strain using conventional
methods.
The vaccine may be multivalent vaccine and additionally
contain immunogens related to other infectious diseases in a prophylactically
or
therapeutically effective manner. Multivalent vaccines against infectious
diseases caused by different infectious agents may contain a carrier strain
having
amounts of antigens associated with their surfaces which are effective to
provide protection against the infectious agents.
A multivalent vaccine may comprise at least two carrier
strains each having different immunogens associated with different infectious
agents. A multivalent vaccine may contain a carrier strain having at least two
different immunogens associated with different infectious agents. Thus, a
carrier strain may contain immunogens relating to C.jejuni and other
pathogenic microorganisms.
The vaccine of the invention contains an immunologically
effective amount of the carrier strain[s] with the integrated JIpA protein.
The
optimum amounts of cells per dosage unit depends on the nature of the
infection against which protection is required, the characteristics of the
animals
to be protected and other factors known to persons skilled in the art.
In addition to the carrier strain[s], the vaccine may comprise
an immunologically acceptable carrier such as aqueous diluents, suspending
aids, buffers, excipient and one or more adjuvants known in the art. The
vaccine may also contain preservatives such as sodium azide, thimersol, beta
propiolactone and binary ethyleneimine.
The vaccines of the invention can be intended for
administration to animals, including mammals, avian species and fish;
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preferably humans and various other mammals, including bovines, equines
and swine.
The vaccines of the invention may be administered in
convenient manner, such as intravenously, intramuscularly, subcutaneously,
intraperitoneally, intranasally or orally. The dosage will be dependent on the
nature of the infection, on the desired effect and on the chosen route of
administration and other factors known to persons skilled in the art.
The JIpA proteins and portions thereof of the invention are
also useful for preparing antibodies which may be used as a means of passive
immunization.
While the present invention has been described with
reference to what are presently considered to be the preferred examples, it is
to
be understood that the invention is not limited to the disclosed examples. To
the contrary, the invention is intended to cover various modifications and
equivalent arrangements included within the spirit and scope of the appended
claims.
EXAMPLES
General DNA manipulation and analysis
Plasmid and chromosomal DNA were isolated and
manipulated by using standard techniques (Ausubel, et al., 1989; Sambrook et
al.,
1989). Transformation of plasmids into E. coli strains was performed by the
method of moue et al. (1990). PCR amplification was carried out using
Campylobacter chromosomal DNA as a template for 26 cycles at 95~C, 20 sec;
50~C, 20 sec; and 72~C, 1 min. DNA was sequenced using Sequenase Version
2.0 (United States Biochemical Corp.) and [a-32P] dATP (ICN Biomedicals Inc.)
based on the dideoxy-chain termination method (Sanger, et al., 1977). The
nucleotide sequence was analyzed with the DNAsis program. A database search
for homologous amino acid sequences was performed using the Blast algorithm
at the NCBI via the Internet. The nucleotide sequence has been deposited in
GeneBank under accession number 236940. Campylobacter chromosomal DNA
digested by restriction enzymes were fractionated by electrophoresis in 0.7%
CA 02296869 2001-04-24
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agarose gel (0.8 mg DNA per lane), and DNA was transferred to GeneScreen Plus
nylon membrane (DuPont-NEN) in 0.4 N NaOH solution for Southern
hybridization. A 1.1 kb jlpA DNA fragment, generated by PCR amplification
using
primer P4 (5'-GAGAAACATATGAAAAAAGGTATTTTTCTC-3') (SEQ. ID. NO.: 2)
and primer RP7 (5'-AACTGCCGCCCATTAACATAGAAAAC-3') (SEQ. ID. NO.: 3),
was employed as probe. Southern hybridization analysis was performed using the
DIG (digoxigenin)-High Primer DNA Labeling and Chemiluminescent Detection kit
(Boehringer Mannheim) according to the manufacturer's instructions.
Expression of JIpA in E.coli
Recombinant JIpA was expressed in E.coli using the T7 RNA
polymerase / promoter system [19]. A 1,25 kb jlpA gene product was generated
by
PCR amplification using the primer P4. The PCR product containing artificial
Ndel
and BamHI site at each end was purified, digested with Ndel and BamHI, and
ligated into pT7-7 vector. The resulting recombinant plasmid, pTlp33,
contained a
Ndel in frame fusion of T7 promoter [pT7] with jlpA gene at the start codon.
To
express the recombinant JIpA, pTlp33 was transformed into E.coli BL21[DE3].
The
E.coli BL21 [DE3] [pTlp33] transformant was inoculated in M9 minimal medium
containing 100~,g ml-1 Ap and inoculated at 370. Methionine assay medium was
added at a final concentration of 10% when OD6oo = 0.1, and the culture was
continued to grow to OD6oo=0.2. The expression of JIpA was induced by 1mM
IPTG. The culture was then divided into two aliquots. One of them was
incubated at
370C for 30 min, followed by the incubation at 370C for 60 min with rifampicin
[600
~.g ml-1] for exclusive production of recombinant JIpA. The other one was
incubated
at 370C for 90 min as a control. [35S] methionine [100Ci per ml of culture]
was
added in each aliquot, ad incubated for 5 min. Cells were harvested by
centrifugation and prepared for SDS-PAGE analysis. Radiolabelled proteins were
resolved on a SDS-10% polyacrylamide gel, and visualized by auto radiography.
A 1.2 kb jlpA gene fragment without the first 16 amino acids
coding sequence was PCR amplified and cloned into BamHI site of pGEX-2T.
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The constructed plasmid, pXlpl9, was transformed into E.coli JM101, and GST-
JIpA fusion protein was expressed and purified according to the instructions
provided by the supplier. The purified protein was used for raising
antibodies.
Labelling JIpA with [3H]palmitate
E.coliBL21 was grown in M9 minimal medium containing Ap
at 37°C. Methionine assay medium [10%] and methionine [20~g ml-1] was
added
at OD6oo=0.1. The culture was then grown to OD6oo=0.2, and 1 mM IPTG was
added. After incubation at 37oC for 30 min, [9, 10 [n] - 3H] palmitic acid was
added, and followed by 3h incubation. e.coli cells were harvested and washed
twice in methanol, and the pellet was air dried. The cells were resuspended in
PBS buffer, and lysed with the sample buffer. The released proteins were
separated on a SDS-10% polyacrylamide gel, and radiolabelled lipoproteins were
detected by autoradiography.
Antiserum
Polyclonal antiserum against JlpA was generated by
immunizing a female New Zealand white rabbit. Purified GST-JIpA protein
[500~.g] was homogenized with complete Fread's adjuvant and used to
immunize rabbit by intramuscular injection. Two boosters [250~g of the protein
each] were given at 4 weeks and 6 weeks. Antiserum was harvested at 8 weeks
and stored at -20oC. Antiserum was affinity purified by using protein A
sepharose 6MB as described by Harlow and Lane [20] when needed.
Electrophoresis and Western Blotting
Electrophoresis of SDS-PAGE was performed according to
Laemmli [21]. Protein samples were boiled in SDS sample buffer for 10 min and
separated on SDS- 10% polyacrylamide gel. Proteins were then electroblotted
onto a 0.2~.m nitrocellulose membrane in a transfer buffer containing 25 mM
Tris HCl pH8.3, 0.1% SDS, 0.2M glycine and 20% methanol for 3 hours at 60V.
Membranes were incubated with 1:1000 diluted anti-JIpA antibodies and
followed by anti-rabbit horseradish peroxidase-conjugated secondary antibody.
The immunoreactive proteins were visualized by chemiluminescence.
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Extraction of outer membrane
Outer membrane proteins of C.jejuni TGH9011 were prepared
using Sarcosyl extraction method [22] and Triton X-100 extraction method [23].
Extraction of cell surface proteins
The glycine-acid extraction method has been described to release
surface protein antigens from Campylobacter [8,24,25]. C.jejuni cells
harvested from
five two-day old MH agar plates were washed twice with PBS buffer pH 7.4. The
cell pellet was resuspended in 10 ml of .2 glycine-Hcl buffer pH2.2 and
stirred at
room temperature for 15 min before centrifugation at 10,OOOxg for 15 min. The
supernatant was neutralized with NaOH and then dialyzed against 3x1.5 L of PBS
buffer. The cell pellet was washed once with PBS buffer and resuspended in 10
ml of
PBS buffer.
Construction of isogenic jlpA mutants
An insertion mutant, C. jejuni TGH9011-JA1, and a deletion
mutant, C. jejuni TGH9011-JA2, were constructed .
In order to construct JA1, a plasmid pHDCKlwas constructed by
inserting a 1.45 kb kanamycin (Km) cassette derived from pILL550 (Labigne-
Roussel et al., 1987) in pSK5 (Hani, 1997) into the XbaI site of jlpA gene in
the
plasmid pHDC.
In order to construct JA2, pHDC was digested with XbaI, and
followed by Ba131 digestion at room temperature for 5, 10, 20, 30 min. The
Ba131
digests were pooled, extracted with phenol/chloroform, precipitated with
ethanol,
ligated in the presence of XbaI linker (5'-GTCTAGAC-3') (SEQ. ID. NO.: 4), and
transformed into E. coli DHSa. The transformants were screened for deletions
by
colony PCR using primer P3 (5'-GTGTAAAAATGTAATTAATCACACAC-3') (SEQ.
ID. NO.: 5) located upstream of putative promoter region and PR2 (5'-
TTTGGATCCACTAGGGGAGAAT-3') (SEQ. ID. NO.: 6) located downstream of
jlpA. The PCR products were examined for the presence of XbaI linker by XbaI
digestion. One plasmid (pJLPDS), containing a XbaI linker and the biggest
deletion
(confirmed by sequence analysis), was isolated, digested with XbaI, and
ligated with
Km cassette obtained from pSKS. The ligated DNA was transformed into E. coli
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DHSa. The plasmid pJLPDSK with the insertion of the Km cassette in pJLPD5
was confirmed by digestion with different restriction enzymes, and used to
generate a jlpA deletion mutant.
The plasmids pHDCKI and pJLPDSK were introduced into C.
jejuni TGH9011 by natural transformation (Wang and Taylor, 1990). Km
resistant colonies were isolated from MH agar plates supplemented with Km (25
mg ml-1). The presence of a disrupted jlpA gene with concomitant loss of the
wild-type jlpA due to a double-crossover recombination event was verified by
PCR analysis. One mutant obtained from pHDCKI transformation was
designated as C. jejuni TGH9011-JA1, and another mutant from pJLPDSK
transformation was designated as C. jejuni TGH9011-JA2.
Adherence and invasion assays
HEp-2 cells (ATCC CCL23) were grown at 37~C in Eagle's
minimal essential medium (EMEM) supplemented with 10% fetal bovine
serum (FBS), 100 mg ml-1 streptomycin, and 100 units ml-1 penicillin G (Sigma)
in a humidified 5% C02 incubator. Confluent HEp-2 monolayer was trypsinized,
seeded into 24-well tissue culture plates at about 1105 cells per well in EMEM-
10% FBS without antibiotics, incubated at 37~C for 18 h, and then washed twice
with EMEM. The adherence and invasion assays were performed by co-
incubating jlpA mutants JA1, JA2, and C. jejuni TGH9011 with HEp-2 cells,
respectively, at a HEp-2 cell-bacteria ratio of about 1:100. The medium was
removed after 2 h incubation, and rnonolayers were washed four times with
EMEM, and then lysed with 0.1% (w/v) Triton X-100 at 37~C for 15 min. For the
invasion assay, the monolayers were washed two times with EMEM after 2 h
incubation, and then incubated for an additional 2 h with EMEM containing 100
mg ml-1 gentamicin. The monolayers were washed two times with EMEM, and
lysed with 0.1% (w/v) Triton X-100 for 15 min. The released bacteria were
enumerated by plate counting on MH agar plates. Adherence efficiency was
calculated as percentage of input bacteria adhering after extensive washing
without gentamicin treatment (Yao et al., 1994). Invasion efficiency was
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expressed as percentage of input bacteria surviving after gentamicin treatment
(Oelschlaeger, et al.,1993).
EXAMPLE 1
Identification of the jlpA gene and properties of the gene product
A hip0 gene encoding benzoylglycine amidohydrolase
(hippuricase) was previously identified from C. jejuni TGH9011, and the
nucleotide
sequence of the hip0 franking region revealed the presence of seven other open
reading frames (ORFs) in our laboratory (Hani and Chan, 1995). Analysis of the
deduced amino acid sequences indicated that one of the ORFs, designated orfl4,
encodes a putative lipoprotein. The orfl4 gene was named as jlpA (jejuni
lipoprotein
A).
The 1116 by jlpA gene encodes a polypeptide of 372 amino acids
(aa) with a deduced molecular weight of 42.3kDa. The pSort algorithm analysis
of
the jlpA gene product revealed that the N-terminus of the molecule contained a
18-
amino-acid signal peptide with a central hydrophobic region. The sequence L-F-
S-
A-C (aa 14-18) at the distal end of the signal peptide closely matches the
consensus
processing site for lipid modification (Fig. 1). The G+C content of the jlpA
coding
region as well as flanking regions including hip0 gene is 27.3%, which is
slightly
lower than that of C. jejuni genomic DNA (30-32% G+C) (Owen and Leaper, 1981;
Penner, 1988). A putative s70 promoter, -35(TTTAAA) (SEQ. ID. NO.: 7) and-
10(TATAAT) (SEQ. ID. NO.: 8)(Hawley and McClure,1983), is located 31
nucleotides
upstream of the translational initiation codon. The sequence AGGAGA (SEQ. ID.
NO.: 9), a predicted site for ribosome binding, is located 5 nucleotides
upstream of
the methionine initiator codon (Fig. 1). A potential transcriptional
termination
signal was found immediately downstream of the stop codon of jlpA gene.
Two potential Fur-box sequences, FBSI and FBSII, were
identified upstream of jlpA (Fig. 1). FBSI has 15 of 19 (with 7of the 8 most
conserved
nucleotides) and FBSII has 13 of 19 (with 7 of the 8 most conserved
nucleotides)
nucleotides of the Fur-box consensus sequence (Thomas and Sparling, 1994).
FBSI is
located 80 by upstream of the putative promoter region,
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and FBSII is overlapped 6 by by the -10 promoter element (Fig. 1). The
presence of
two Fur-box sequences on the 5' flanking region of jlpA suggests possible
regulation by Fur.
EXAMPLE 2
Homology of JIpA with a rhoptry protein in malaria parasite
The BLAST search revealed that JIpA shared the highest homology with the
rhoptry protein E8 of Plasmodium yoelii. E8, a huge protein containing 2401
amino
acid residues, is a component of a club-shaped organelle called rhoptry which
plays
an important role in the recognition and invasion of Plasmodium yoelii to the
host
erythrocytes (Sinha et al., 1996). JIpA is homologous to two region of E8.
JlpA
(residues 22-361) shares 19% identity (41% similarity) with as 509 to 877 of
E8 (Fig.
2A) (SEQ. ID. NO.: 10), and JIpA (residues 93-349) has 25% identity (44%
similarity)
with E8 (residues 882-1114) (Fig. 2B) (SEQ. ID. NO. 11). In addition, both
JIpA and E8
are hydrophilic and have similar hydrophobicity profiles at the homologous
regions.
EXAMPLE 3
Expression and post-translational modification of the JIpA protein
The T7 RNA polymerase/promoter expression system (Tabor, 1990) was employed
to determine the expression of JIpA in Escherichia coli. Two prominent
proteins of
42 and 35 kDa, and several minor low molecular weight polypeptides were
observed when E. coli BL21 (DE3) carrying pTlp33 was induced for synthesis of
recombinant proteins in the presence of rifampicin (Fig. 3A). The 42 kDa
polypeptide was close to the predicted size of the prolipoprotein (42.3 kDa)
or
mature JIpA molecule (40.5 kDa). The jlpA gene was the largest and the only
translational ORF in the insert DNA, therefore, the 35 kDa polypeptide might
be a
degradation product of JIpA. The other minor low molecular weight plasmid-
encoded proteins were also likely to be the result of proteolysis of the
recombinant
lipoprotein.
The lipoprotein nature of JIpA was confirmed by [3H]palmitate labeling of the
recombinant protein. E. coli BL21 (DE3)/pTlp33 cells grown in the presence of
[3H]palmitate were harvested and subjected to SDS-PAGE. A band of apparent
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molecular mass of 41 kDa corresponding to recombinant JIpA was observed (Fig.
3B), indicating that the C. jejuni protein was lipid-modified in E. coli. A
radiolabeled high molecular weight complex did not enter the SDS-
polyacrylamide gel, suggesting that an insoluble lipoprotein aggregate present
in
the [3H]palmitate-labeled E. coli BL21 (DE3)/pTlp33 sample.
EXAMPLE 4
Surface localization of JIpA
To localize JIpA in the membrane of C. jejuni cells, sarcosyl extraction
(Amako,
et al., 1996) and Triton X-100 (Skare, et al., 1996) extraction methods were
used
for separating the inner and outer membrane proteins. The results from both
methods showed that JIpA was found predominantly in the inner membrane
fraction (data not shown). However, according to the +2 amino acid rule
(Yamaguchi, et al., 1988), JIpA with a glycine at the +2 position is predicted
to be
located in outer membrane. Glycine-acid extraction method was used to further
investigate the localization of JIpA in the membrane of C. jejuni TGH9011.
Glycine-acid extracts from both C. jejuni TGH9011and JA2 mutant cells were
resolved by SDS-PAGE, and examined by immunoblotting with anti-JIpA
antibodies. JIpA protein was found predominantly in glycine-acid extracts
fraction from C. jejuni TGH9011, while no signal was found in the glycine-acid
extracts from the JA2 mutant, indicating that JIpA was located on the surface
of
C. jejuni (Fig. 4A). However, the surface protein profiles of wild type and
JA2
mutant (data not shown) showed no difference using Coomassie staining.
Similarity of the two protein profiles possibly due to co-migration of JIpA
with
other proteins with similar molecular weight.
To confirm that JIpA is surface exposed, the susceptibility of
JIpA on intact C. jejuni cells to proteinase K was determined. The result
showed
that JIpA of C. jejuni was completely digested when incubated with proteinase
K
(Fig. 4B), while FIgG, the flagellar basal rod protein with molecular weight
of 28
kDa (Chan, et al., 1998), was not affected (Fig. 4C). Anti-FlgG antibodies
also
cross-reacted to a 92 kDa band which is possibly the hook protein FIgE (Fig.
4C).
FIgG has high homology to FIgE (http://www.sanger.ac.uk/Projects/C jejuni/).
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The 92 kDa band was digested by proteinase K, indicating a surface
localization
and consistent with a flagellar hook protein identity. Thus, the results
obtained
from the glycine-acid extraction and proteinase K digestion experiments
indicated that JIpA is localized on the cell surface of C. jejuni. However,
when
sarcosyl and Triton X-100 extraction methods were used, JIpA was found
predominantly in the inner membrane fraction and very little was detected in
the outer membrane fraction. Overall, the above findings suggest that jlpA is
loosely associated with the cell surface.
EXAMPLE 5
Release of jlpA to culture medium during growth
It has been reported that some Gram-negative pathogens
release lipoproteins during bacterial growth, and released lipoproteins play
an
important role in the induction of cytokine production and/or pathology
associated with Gram-negative bacterial infections (Zhang, et al. 1998). The
fact
that JIpA is a surface exposed lipoprotein prompted us to investigate if JIpA
is
released to the culture medium during the bacterial growth. C. jejuni TGH9011
was grown in MH broth at 37~C in C02 incubator with shaking and sampled at
various intervals. Release of JIpA was examined by immunoblotting with anti-
jlpA antibodies. As shown in Fig.S, JIpA was detectable in bacterial culture
supernatant of all stages, and its concentration increased in a time-dependent
fashion.
EXAMPLE 6
Role of jlpA in the adherence to HEp-2 cells
Two isogenic jlpA mutants, C. jejuni TGH9011-JAl (jAl) and
TGH9011-JA2 (jA2) (Fig. 6), were used to examine the role of jlpA in the
adherence and invasion to HEp-2 cells in vitro. C. jejuni JA1 mutant was
constructed by inserting a kanamycin resistant gene cassette (Labigne-Roussel
et
al., 1987) into a unique XbaI site within the jlpA coding sequence. C. jejuni
JA2 is
a deletion mutant, constructed by Bal 3ldigestion followed by insertion with a
kanamycin cassette. Sequence analysis showed that the JIpA N-terminal coding
region including the -35 element of the jlpA promoter was deleted, and only 5
CA 02296869 2000-O1-24
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C-terminal amino acid residues encoding sequence was left intact (Fig. 6).
Immunoblotting analysis using antibodies against JIpA showed that no
immunoreactive JIpA band can be detected in either JA1 or JA2 mutant (data
not shown). No detectable difference in growth was observed among parental C.
jejuni TGH9011 and the two mutant strains when cultured in either MH or
MEM medium (data not shown).
The ability of C. jejuni TGH9011 and two mutants, JA1 and
JA2, to adhere and invade HEp-2 cells was examined. The adherence activity of
JA1 and JA2 to HEp-2 cells was reduced to 19.4% and 18.0% of the wild type
strain, respectively (Table 1). The invasion was also reduced, which might be
due to the reduction of adherence (data not shown).
EXAMPLE 7
Adherence Inhibition of C. jejuni by antibodies against JIpA
To further investigate whether JIpA plays a role in adherence
directly, polyclonal antibodies raised against purified JIpA were used. The
affinity-purified anti-JIpA antibodies inhibited the adherence of C. jejuni
TGH9011 to HEp-2 cells by 68% (Table 2). The lower level of adherence of JA2
was not further reduced by anti-JIpA antibodies (data not shown). Control
preimmunized serum did not inhibit the adherence of TGH9011 to HEp-2 cells
(data not shown).
EXAMPLE 8
Inhibition of C. jejuni adherence by purified recombinant JIpA
Purified JIpA protein was used to examine its effect on
adherence of C. jejuni to HEp-2 cells. The adherence of C. jejuni TGH9011 to
HEp-2 cells was inhibited 83% by co-incubating HEp-2 cells with GST-JIpA
(5mg/ml), while GST (5 mg/ml) only did not affect the adherence of C. jejuni
(Table 2). The binding of JIpA to HEp-2 cells was further confirmed by
immunoblotting analysis with antibodies against GST-JIpA (Fig. 7). A 68 kDa
GST-JIpA band was observed in the GST-JIpA bound HEp-2 cell lysate. No signal
was detected from HEp-2 cell lysate and GST bound HEp-2 cell lysate.
EXAMPLE 9
CA 02296869 2000-O1-24
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Presence of jlpA in C. jejuni strains
To determine whether the jlpA gene is specific to C. jejuni
species, jlpA-specific primers P4 and PR7 were used to amplify the gene from
eight C. jejuni strains and several other Campylobacter species, such as C.
coli, C.
lari, C. sputorum, and C. upsaliensis. A 1.1 kb PCR products was observed in
all
eight C. jejuni strains, isolated from patients or environment and no
detectable
amplicons were observed from the other Campylobacter species (data not
shown). Southern hybridization analysis was used to further confirm the
absence of jlpA homologous sequence in those Campylobacter species. As
shown in Fig. 8A, the jlpA DNA probe which hybridized to a 2.2 kb band of CIaI
digested genomic DNA of C. jejuni TGH9011, did not hybridize with the
genomic DNA digests from the other Campylobacter species. Thus, jlpA gene is
specific to C. jejuni. Expression of JIpA in C. jejuni strains were clarified
by
immunoblotting (Fig. 8B).
DISCUSSION OF EXAMPLES
C. jejuni surface proteins play an essential role in the bacterial
adherence to mammalian cells, and this ligand-receptor interaction are
mediated by adhesins on the bacterial surface. Two adhesins, PEB1 and CadF,
have been characterized in C. jejuni (Pei, et al., 1998; Konkel, et al.,
1997), and
both are outer membrane lipoproteins. PEB1 is a homologue of a component of
ABC transport system in Gram-negative bacteria, and CadF is a peptidoglycan-
associated protein. In this study, we identified a novel C. jejuni-unique
gene,
jlpA, which encodes a surface exposed lipoprotein (JIpA) that binds to HEp-2
cells and is released to the culture medium during growth. JIpA plays an
important role in the adherence of C. jejuni to HEp-2 cells.
Sequence analysis showed that JIpA shares homology with
the E8 rhoptry protein of rodent malaria parasite, P. yoelii (Sinha, et al.,
1996).
The E8 protein, a member of a multigene family proteins with a relative
molecular mass of 235 kDa, is a component of a club-shaped organelle called
rhoptry. These proteins recognize the subset of erythrocytes responsible for
parasite invasion (Holder and Freeman, 1981; Freeman et al., 1980), and are
CA 02296869 2000-O1-24
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associated with generating clonal phenotypic variation in rodent malaria
(Preiser, et al., 1999). JIpA is the first protein reported in Campylobacter
that
shares homology with eukaryotic proteins. The G+C content (27.3%) of the jlpA
coding region is lower than that of the complete genome of C. jejuni (30-32%
G+C) (Owen and Leaper, 1981; Penner, 1988), suggesting a possible acquisition
through horizontal transfer from a eukaryotic genome with a low G+C content,
such as malaria parasite. P. falciparum has a G+C content of 18-20%
(http://www.sanger.ac.uk/Projects/Protozoa). Furthermore, the hip0-jlpA is
located in a 15 kb region specific to C. jejuni (Chap et al., 2000),
indicating that
the region was acquired since the split of C. jejuni and C. coli.
The ability to incorporate [3H]palmitate, the hydrophobic
nature of the N-terminal amino acid sequence, and the presence of a lipobox
sequence L-F-S-A-C (Hayashi and Wu, 1990; Wu, 1996), suggest strongly that
JIpA is a lipoprotein. The low level of [3H]palmitate incorporated may be due
to
the low expression as well as degradation of JIpA in E. coli. Additionally,
the
expression of JIpA may be harmful to E. coli cells since a similar problem has
been reported by Gomez et al. (1994).
In Gram-negative bacteria, prolipoprotein are synthesized in
the cytoplasm. The signal sequence targets the prolipoprotein to the
cytoplasmic
membrane. The lipobox is recognized by the cytoplasmic membrane
prolipoprotein modification and processing enzymes that lead to the formation
of N-acyl-diacylglycerylcystein, a lipid-modified structure at the N-terminus
of
the polypeptide (Hayashi and Wu, 1990; Wu, 1996). The mature lipoprotein is
then transported to its final destination at the outer membrane or retained at
the cytoplasmic membrane. The glycine-acid extraction and proteinase K
digestion experiments indicated that JlpA is a surface-exposed protein in C.
jejuni. We proposed that the identification of JlpA to the inner membrane
fraction by the sarcosyl and Triton X-100 methods may be attributed to a loose
association of JIpA to the outer membrane and thereby released to the inner
membrane fraction by the detergents. Trust and Logan (1984) reported a loosely
associated surface protein of C. jejuni which was also fractionated to the
inner
CA 02296869 2000-O1-24
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membrane fraction by the sarcosyl extraction method. A hydrophobicity
calculation by the method of Kyte & Doolittle identified a potential
hydrophobic
transmembrane domain (aa 353-369) at the C-terminus of JIpA. We proposed
that the lipidated N-terminus and the hydrophobic C-terminus of JIpA are
embedded in the outer membrane while the central hydrophilic region is
exposed on the bacterial surface.
It has been reported that some enteric pathogens, such as E.
coli, Salmonella typhimurium, and Yersinia enterocolitica, release
lipoproteins
during growth (Zhang, et al. 1998). The released lipoproteins may have a role
in
the pathogenesis of bacterial infection because lipoproteins have been shown
to
induce the formation of tumor necrosis factor alpha and the release of
interleukin-6 (IL-6) by macrophages (Zhang, et al., 1997; 1996). JIpA is
released to
the culture medium by C. jejuni. The function of the released JIpA remains to
be established. The mechanism by which lipoproteins are released from bacteria
during growth is still unclear. Gram-negative bacteria are known to release
membrane fragments to the surrounding milieu during growth (Wensink and
Witholt, 1981). C. jejuni has been reported to form blebs consisting of outer
membrane fragments. These blebs are constantly released into the medium
during growing (Pead, 1979; Logan and Trust, 1982). It is possible that the
release
of JIpA is associated with the release of outer membrane fragments and
formation of blebs.
Both insertion mutant JA1 and deletion mutant JA2 of jlpA
showed significant reduction in adherence to HEp-2 cells. The adherence of C.
jejuni to HEp-2 cells was reduced by both affinity-purified anti-JlpA
antibodies
and purified JIpA. In addition, purified JIpA binds to HEp-2 cells. The above
findings suggest that JIpA is an adhesin and has a direct role in the
adherence of
C. jejuni to HEp-2 cells. It also indicates that JIpA is a good potential
candidate
for development of a vaccine against C. jejuni infection.
All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as if each
CA 02296869 2000-O1-24
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individual publication, patent or patent application was specifically and
individually indicated to be incorporated by reference in its entirety.
CA 02296869 2000-O1-24
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FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION
1. Altekruse, SF, Stern, Nj, Felds, PI, Swerdolow, DL (1999) Campylobacter
jejuni
- an emerging foodborn pathogen. Emerg. Infect. Dis. 5: 28-35
2. Ketley, JM (1997) Pathogenesis of enteric infection by Campylobacter.
Microbiology 143:5-21.
3. Soto, GE and Hultgren, SJ (1999) Bacterial adhesins:common themes and
variations in architecture and assembly. J. Bacteriol. 181:1059-1071.
4. Doig, P, Yao, R, Burr, DH, Guerry, P, and Trust, TJ (1996) An
environmentally
regulated pilus-like appendage involved in Campylobacter pathogenesis. Mol.
Microbiol. 20: 885-894.
5. Kervella, M., Pages, JM, Pei, Z, Grollier, G, Blaser, MJ and Fauchere, JL
(1993)
Isolation and characterization of two Campylobacter glycine-extracted proteins
that bind to HeLa cell membrane. Infect. Immun. 61:3440-3448.
6. Pei, Z. And Blaser, MJ (1993) PEB1, the major cell-binding factor of
Campylobacter jejuni, is a homolog of the binding component in Gram-
negative nutrient transport system. J. Biol. Chem. 268: 18717-18725.
7. Konkel, ME, Garvis, SG, Tiptop, SL, Anderson Jr. DE and Cieplak Jr. W
(1997)
Identification and molecular cloning of a gene encoding a fibernectin-binding
protein [CadF] from Campylobacter jejuni. Mol. Microbiol. 24: 953-963.
8. De Melo, MA and Pechere, JC (1990) Identification of Campylobacter jejuni
surface proteins that bind to eukaryotic cells in vitro. Infect. Immun. 64:
1749-
1756.
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9. Schroder, W and Moser, I (1997) Primary structure analysis and adhesion
studies on the major outer membrane protein of Campylobacter jejuni. FEMS
Microbiol. Lett. 150: 141-147.
10. Moser, I, Schroeder, w and Salnikow, J (1997) Campylobacter jejuni major
outer membrane protein and a 59-kDa protein are involved in binding to
fibronectin and INT 407 cell membranes. FEMS Microbiol. Lett. 157: 233-238.
11. Sambrook, J., Fitsch, EF and Maniatis, T. (1989) Molecular Cloning: a
Laboratory Manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring
Harbor Laboratory Press.
12. Goeddel, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, CA (1990).
13. Merrifield, 1964, J. Am. Chem Association. 85: 2149-2154.
14. Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I
and II Thieme, Stuttgart.
l5.Morrison et al. Proc. Natl. Acad. Sci. USA 81; 6851 [1985].
16. Ausubel,FM, Brent R, Kingston, RE, Moore, DD, Seidman, JG, Smith, JA and
Struhl, K (1989) Current Protocols in Molecular Biology. Greene and Wiley-
25 Interscience.
17. moue, H, Nojima, H and Okayama, H. (1990) High efficiency transformation
of Eschericia coli with plasmids. Gene 96: 23-28.
30 18. Sanger, F, Nicklen, S and Coulson, A. (1997) DNA sequencing with chain-
terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.
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19. Tabor, S (1990) Expression using the T7 RNA polymerase / promoter system.
In Current Protocols in Molecular Biology. Ausubel, FA, Breny, R, Kingston,
RE,
Moore, DD, Seidman, JG, Smith JA and Struhl, K [eds] New York: Greene
Publishing and Wiley-Interscience pp16.2.1-16.2.11.
20. Harlow, E and Lane, D (1988) Antibodies - a laboratory manual. Cold Spring
Harbor Laboratory, New York, USA.
21. Laemmli, UK (1970) Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227: 680-685.
22. Amako, K, Wai, SN, Umeda, A, Shigematsu, M and Takade, A (1996)
Electron microscopy of the major outer membrane protein of Campylobacter
jejuni. Microbiol. Immunol. 40: 749-754.
23. Skare, JT, Champion, CI, Mirzabekov, TA, Shang, ES, Blanco, DR,
Erdjument-Bromage, H et al. (1996) Porin activity of the native and
recombinant
outer membrane protein Oms28 of Borrelia burgdorferi. J. Bacteriol. 178: 4909-
4918.
24. Dubreuil, JD, Logan, SM, Cubbage S, Eidhin, DN, McCubbin, WD, Kay, CM et
al. (1988) Structural and biochemical analysis of a surface array protein of
Campylobacter fetus. J. Bacteriol. 170: 4165-4173.
25. Gravis, SG, Puzon, GJ and Konkel, ME (1996) Molecular characterization of
Campylobacter jejuni 29-kilodalton periplasmic binding protein. Infect. Immun.
64: 3537-3534.
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26. Labigne-Roussel, A, Harel, J and Tompkins, L (1987) Gene transfer from
Escherichia coli to Campylobacter species: development of shuttle vectors for
genetic analysis of Campylobacter jejuni. J. Bacteriol. 169: 5320-5323.
27. Hani, EK (1997) Hippurate hydrolase gene of Campylobacter jejuni. Ph.D.
Thesis. University of Toronto, Toronto, Canada.
28. Wang, Y and Taylor, DE (1990) Natural transformation in Campylobacter
species. j. Bacteriol. 172: 949-955.
29. Yao, R., Burr, DH, Doig, P, Trust, TJ, Niu, H and Guerry, P (1994)
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20 DETAILED FIGURE LEGENDS
Figure 1. The 5'nucleotide sequence and the N-terminus deduced amino acid
sequence of the jlpA. Deduced transformational product was shown by using
single-letter amino acid residue codes. A consensus recognition sequence for
signal peptidase II was shown in boldface and the predicted cleavage site was
25 marked by a vertical arrow. The putative ribosome binding site [RBS] and
putative °70 promoter sequence [135 and -10] were underlined. Two
potential
Fur-binding sequences, FBSI and FBSII, were overlined.
Figure 2. The alignment shown is modified from the alignment obtained from a
30 BlastP database search.
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Figure 3. Expression of recombinant JIpA in E.coli. E.coli BL21[DE3] carrying
pT7-
7 vector or pTlp33 was induced to synthesize the recombinant proteins with
IPTG. Radioactive methionine or palmitate was added to label JlpA. Total
cellular proteins were separated on a SDS-10% polyacrylamide gel and
radiolabelled polypeptides were visualized by autoradiography. [A] [35S
methionine-labelled proteins obtained in the absence of presence of
rifampicin.
[B] [3H] palmitate-labelled lipoproteins.
Figure 4. Surface localization of JIpA in C.jejuni TGH9011. [A] Glycine-acid
extraction fraction from wild type C.jejuni TGH9011 and mutant JA2 were
applied to a SDS-10% PAGE, immunoblotted onto a nitrocellulose membrane
and probed with anti-JIpA antibodies. Lanes: 1, 4, untreated whole-cell lysate
[total proteins]; 2,5, glycine-acid treated whole-cell lysate; 3,6, glycine-
acid
extracted proteins. Lanes 1,2,3: proteins from wild type; 4,5,6: proteins from
JA2
mutant. [B] Surface proteolysis of intact C.jejuni TGH9011 cells. C.jejuni
cells
were collected from MH broth, washed with distilled water and resuspended in
water at a concentration of 5x108 cells ml-1. An aliquot of 50 ~,1 cells in
each tube
were exposed to different amount of proteinase K at 37oC for 15 min. Samples
were analyzed by immunoblotting and probed with anti-JIpA antibodies.
Lanes:l, No proteinase K [control]; 2, 0.1 ~,g; 3, 0.5 ~,g; 4, 2.5 ~.g; 5,
10~g of
proteinase K; [C] Same sample as in panel [B] but probed with anti-FIgG
antibodies.
Figure 5. Release of JlpA during growth. C.jejuni TGH9011 was grown in MH
broth medium at 37oC with shaking in the atmosphere of 5% C02 and 95% air.
Samples were withdrawn at the time points indicated. Cells were pelleted and
lysed with the sample buffer. Culture supernatants were filtered through 0.22-
~,m syringe filters and precipitated with TCA. The same OD6oo equivalent of
cell-
associated proteins and the same ten-fold TCA-concentrated OD6oo equivalent of
culture medium proteins were separated on a SDS-10% polyacrylamide gel. Both
CA 02296869 2000-O1-24
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cell proteins and supernatant proteins were transformed onto the same
nitrocellulose membrane followed by immunoblotting with anti-JIpA
antibodies.
Figure 6. Generic organization of hip0-jlpA region and construction of jlpA
mutants. The jlpA insertion mutant, C.jejuni TGH9011-JA1, was constructed by
inserting kanamycin cassette into the Xbal site of the jlpA gene. Deletion
mutant
of jlpA, C.jejuni TGH9011-JA2, was constructed by inserting a kanamycin
cassette into a Ba131 digested jlpA gene.
Figure 7. Specific binding of purified JIpA to HEp-2 cells. Confluent HEp-2
monolayers in a 24-well plate were washed twice with EMEM. One ml EMEM
containing 10 ~,g purified GST-JIpA or GST only as control was added onto the
HEp-2 monolayer. The plate was incubated at 37~C for 2 h in the atmosphere of
5% C02 and 95% air. The HEp-2 monolayers were washed 6 times with EMEM
and then lysed with 150 ~.1 of lOmM Tris.HCl buffer pH 7.4 containing 0.8% SDS
[33]. Protein samples were subjected to SDS-10% polyacrylamide gel, followed
by
immunoblotting with anti-GST-JIpA antibodies. Lanes:l, HEp-2 cells; 2, HEp-2
binded with GST proteins; 3, HEp-2 cells binded with GST-JlpA proteins.
Figure 8. The presence of jlpA in Campylobacter species. [A] Southern
hybridization analysis of Campylobacter DNAs with a jlpA gene probe. A 1.1 kb
DIG-labelled jlpA gene fragment was used to probe Clal-digested genomic
DNAs. Lanes: 1, C.jejuni TGH9011; 2, C.coli ATCC 33559T; 3, C.coli ATCC 43482;
4, C. col i ATCC 43486; 5, C. col i ATCC 49299; 6, C.lari ATCC 35221T; 7, C .
s p a t o r a m
subsp. bubulus ATCC 33562T; 8, C.upsaliensis ATCC 43954T. [B] Immunoblotting
of the whole cell lysates from different C.jejuni strains with anti-JIpA
antibodies. Lanes: 1, TGH9011; 2, 81-176; 3, CEPA-3C; 4, OH4382; 5, OH4384; 6,
LCDC-13267; 7, CA21-106; 8,1-5R.
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Table 1. Adherence of C. jejuni TGH9011 and its jlpA mutants to HEp-2 cells.
Strain % adherence % wild type
TGH9011 0.35 0.14 100
TGH9011-JA1 0.068 0.03 19.4
TGH9011-JA2 0.063 0.02 18.0
Results represent the mean of four independent experiments ~ standard
deviation.
CA 02296869 2000-O1-24
Table 2. Inhibition of adherence by affinity-purified anti-JIpA antibodies (a-
JIpA)
or purified GST-JIpA protein.
Treated cell Addition % inhibition
C. jejuni TGH9011 PBS 0
a-JIpA 68
HEp-2 cells PBS 0
GST 0
GST-JIpA 0
83
C.jejuni TGH9011 cells were incubated with affinity-purified anti-JIpA at 37~C
for 30 min, and then washed with PBS buffer before added to HEp-2 monolayer.
Prior to adding C. jejuni TGH9011 cells, HEp-2 monolayer was incubated with
GST-JIpA or GST only at 37~C for 30 min and washed twice with EMEM.
CA 02296869 2000-O1-24
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Table 3. Strains and plasmids used in this study.
Strain or plasmid
Relevant characteristicsSource
or reference
C. jejuni
TGH9011(ATCC43431)
Serotype 0:3,
clinical isolate
81-176 Clinical isolate
CEPA-3C Clinical isolate from Guillain-Barre Syndrome
patient
OH4382 Clinical isolate from Guillain-Barre Syndrome
patient
OH4384 Clinical isolate from Guillain-Barre Syndrome
patient
LCDC-13267 Environmental isolate
CA21-106 Environmental isolate
1-5R Environmental isolate
J. L. Penner
M. j. Blaser
G. A. Clark
J. L. Penner
J. L. Penner
j.Odumeru
j. Odumeru
j. Odumeru
C. coli
ATCC 33559
ATCC 43482
ATCC 43486
ATCC 49299
Type strain of the species
Serotype reference strain for 0:46
Serotype reference strain for 0:51
Serotype reference strain for 0:61
CA 02296869 2000-O1-24
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J. L. Penner
J. L. Penner
J. L. Penner
J. L. Penner
C.lari
ATCC 35221
Type strain of the species
ATCCa
C. sputorum subsp. Bubulus
ATCC 33562
Type strain of the species
ATCC
C. upsaliensis
ATCC 43954
Type strain of the species
ATCC
E. coli
DHSa
JM101
BL21(DE3)
F- f80dlacZDMl5 recA1 endA1 hsdRl7 gal- gyrA96
supE thi D(lac-proAB) F' [traD36 proAB+ lacIq lacZDMl5]
hsdS gal (lcIts857 indl Sam7 nin5 lacUVS-T7 gene 1)
BRLb
Jenkinson, 1994
Studier, 1990
CA 02296869 2000-O1-24
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Plasmids
pBR322
pBluescriptII KS
pGEX-2T
pILL550
pSK5
pHIP-O
pHDC
pHDCKI
pJLPD5
pJLPDSK
pT7-7
pTlp33
pXlpl9
AmpR, TetR, general cloning vector
AmpR, lacZ, cloning vector
AmpR, glutathione S-transferase (GST) gene fusion vector
AmpR, KmR, E. coli-C. jejuni shuttle vector
AmpR, KmR, 1.45 kb KmR cassette of pILL550 in pUCl9
A pBR322 clone containing hip0-jlpA DNA fragment from C. jejuni DNA
library
pHIP-O deletion derivative carrying jlpA by CIaI digestion
pHDC derivative carrying jlpA::KmR insertion
pHDC derivative carrying Ba131 deleted jlpA and a XbaI linker
pJLPD5 derivative carrying DjlpA::KmR insertion
AmpR, gene expression vector by T7 RNA polymerase
pT7-7 derivative carrying jlpA
CA 02296869 2000-O1-24
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pGEX-2T derivative carrying jlpA
Bolivar, 1977
Stratagene
Pharmacia
Labigne-Roussel, 1990
Hani, 1997
Hani and Chan, 1995
This study
This study
This study
This study
Tabor, 1990
This study
This study
a ATCC, American Type Culture Collection; b BRL, Bethesda Research
Laboratories.
CA 02296869 2001-04-24
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Joe, Angela
Jin, Songmu
Chan, Voon Loong
(ii) TITLE OF INVENTION: A Surface-Exposed Lipoprotein to
Campylobacter Jejuni Involved in Adherence to HEp-2 Cells
(iii) NUMBER OF SEQUENCES: 11
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Bereskin & Parr
(B) STREET: 40 King Street West
(C) CITY: Toronto
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(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
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(A) APPLICATION NUMBER: CA 2,296,869
(B) FILING DATE: 24-JAN-2000
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gravelle, Micheline
(B) REGISTRATION NUMBER: 2800
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(C) STRANDEDNESS: single
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(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AAGCTTAAAC CTGTTGCAAA AATATATAAT AATTGCAAAG TTAATTATAC TTAAAATTAT 60
AAATAATCCA TTTCAAAGAA ACTAAAAAGA TTTTCCTATG TGTAAAAATG TAATTAATCA 120
CACACTCTCT TTTAGTATTG TTTAAATTTT AAGTAAAAAA AGATATAATC TTTTTTTTAA 180
CA 02296869 2001-04-24
-50-
ATTTTTTAAG GAGAAAACTA TGAA.AA.AAGG TATTTTTCTC TCTATTGGAA TAGCTGTTTT 240
GTTTTCAGCT TGCGGAAATT CCATAGATGA AAAAACAGTT AAAA.AATATG AAAATCAACT 300
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
GAGAAACATA TGAAAAAAGG TATTTTTCTC 30
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
AACTGCCGCC CATTAACATA GAAAAC 26
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GTCTAGAC 8
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GTGTAAAAAT GTAATTAATC ACACAC 26
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
TTTGGATCCA CTAGGGGAGA AT 22
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
TTTAAA 6
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
TATAAT 6
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
AGGAGA 6
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 340 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Ile Asp Glu Lys Thr Val Lys Lys Tyr Glu Asn Gln Leu Asn Gln Thr
1 5 10 15
Val Lys Gln Glu Ile Ala Ser Leu Ser Gln Asp Ser Gly Ile Lys Ile
20 25 30
Glu Phe Ser Asp Phe Lys Cys Asn Ala Asp Gly Asp Phe Ile Ala Cys
35 40 45
Leu Ser Pro Asn Phe Lys Thr Leu Ala Lys Asp Asn Asn Asn Glu Tyr
50 55 60
Gln Glu Leu Phe Gln Ala Lys Asn Ile Lys Ile Arg Ser Asn Glu Ile
65 70 75 80
Tyr Lys Gly Glu Ala Asn Ala Ser Ile Ser Ile Lys Glu Tyr Tyr Asn
85 90 95
Asp Leu Phe Lys Asn Gln Lys Ser Ile Gln Ser Asn Leu Val Phe Glu
100 105 110
Asp Phe Lys Leu Gly Glu Lys Val Val Ser Asp Ile Asn Ala Ser Leu
115 120 125
Phe Gln Gln Asp Pro Lys Ile Arg Ser Phe Ile Asn Lys Leu Ser Ser
130 135 140
Asp Ser Tyr Thr Leu Ser Phe Asp Asn Ser Ile Asn Lys Gln Glu Asn
145 150 155 160
Asn Tyr Leu Asp Asn Leu Asp Ile Lys Phe Tyr Asn Ala Lys Leu Asn
165 170 175
Phe Asn Thr Asn Leu Asn Ile Asn Leu Lys Glu Asp Leu Leu Asn Tyr
180 185 190
Leu Asp Ser Lys Gly Ile Lys Phe Asn Thr Gln Thr Leu Ala Met Asp
195 200 205
Glu Gln Ala Ile Asn Glu Leu Leu Asn Ile Ala Asn Tyr Glu Gln Ala
210 215 220
Ser Asp Phe Ser Asn Thr Ile Gln Lys Tyr Ile Ile Leu Asn Asn Phe
225 230 235 240
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Lys Ile Asp Ser Thr Leu Lys Thr Glu Gly Val Phe Ser Ser Tyr Ile
245 250 255
Thr Thr Ala Lys Glu Asn Leu Gln Thr Leu Lys Thr Gln Ser Gln Asn
260 265 270
Glu Glu Gln Ala Leu Ile Phe Asp Lys Ala Leu Ala Ile Leu Asn Asn
275 280 285
Ile Thr Gln Asn Asp Asp Tyr Lys Leu Asn Leu Asp Leu Lys Phe Lys
290 295 300
Asn Ile Pro Val Ser Asp Tyr Ser Thr Gln Gly Ile Asp Ser Ile Glu
305 310 315 320
Lys Leu Ser Ile Asn Asn Gln Asp Ala Thr Glu Val Leu Lys Ile Ile
325 330 335
Leu Pro Phe Ile
340
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 257 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Asn Ile Lys Ile Arg Ser Asn Glu Ile Tyr Lys Gly Glu Ala Asn Ala
1 5 10 15
Ser Ile Ser Ile Lys Glu Tyr Tyr Asn Asp Leu Phe Lys Asn Gln Lys
20 25 30
Ser Ile Gln Ser Asn Leu Val Phe Glu Asp Phe Lys Leu Gly Glu Lys
35 40 45
Val Val Ser Asp Ile Asn Ala Ser Leu Phe Gln Gln Asp Pro Lys Ile
50 55 60
Arg Ser Phe Ile Asn Lys Leu Ser Ser Asp Ser Tyr Thr Leu Ser Phe
65 70 75 80
Asp Asn Ser Ile Asn Lys Gln Glu Asn Asn Tyr Leu Asp Asn Leu Asp
85 90 95
Ile Lys Phe Tyr Asn Ala Lys Leu Asn Phe Asn Thr Asn Leu Asn Ile
100 105 110
Asn Leu Lys Glu Asp Leu Leu Asn Tyr Leu Asp Ser Lys Gly Ile Lys
115 120 125
Phe Asn Thr Gln Thr Leu Ala Met Asp Glu Gln Ala Ile Asn Glu Leu
130 135 140
Leu Asn Ile Ala Asn Tyr Glu Gln Ala Ser Asp Phe Ser Asn Thr Ile
145 150 155 160
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Gln Lys Tyr Ile Ile Leu Asn Asn Phe Lys Ile Asp Ser Thr Leu Lys
165 170 175
Thr Glu Gly Val Phe Ser Ser Tyr Ile Thr Thr Ala Lys Glu Asn Leu
180 185 190
Gln Thr Leu Lys Thr Gln Ser Gln Asn Glu Glu Gln Ala Leu Ile Phe
195 200 205
Asp Lys Ala Leu Ala Ile Leu Asn Asn Ile Thr Gln Asn Asp Asp Tyr
210 215 220
Lys Leu Asn Leu Asp Leu Lys Phe Lys Asn Ile Pro Val Ser Asp Tyr
225 230 235 240
Ser Thr Gln Gly Ile Asp Ser Ile Glu Lys Leu Ser Ile Asn Asn Gln
245 250 255
Asp
