Note: Descriptions are shown in the official language in which they were submitted.
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ANTIMICROBIAL COMPOUNDS
Baclc:=round of the Invention
Triclosan is a trichlorinated biphenyl broad spectrum antibacterial/fungal
agent
S (Furia, T.E., et al. Soap & Chemical Specialties 44, 47-50, 116-122 (1968);
Regos, J., et
al. Dermatologica 158, 72-79 (1979)). Because of its general biocidal
activity, triclosan
has been used as a topical disinfectant in soaps, cosmetics, and lotions
(Regos, J., et al.
Dermatologica 158, 72-79 (1979)), and more recently has been added to
toothpastes
(Cummins, D. J. Clin. Periodont. 18, 455-461 (1991), to fabrics for use in
bedding and
clothing, and to plastics for use in toys, cutting boards, and flooring.
The mechanism of action of triclosan has been uncertain; biochemical and
physical assays have shown inhibition of uptake of nutrients (Regos, J., et
al. Zbl. Bakt.
Hyg.,1. Abt. Orig. A 226, 390-401 ( 1974)), inhibition of proteases (Cummins,
D. J. Clin.
Periodont. 18, 455-461 (1991)), and cell lysis (Regos, J., et al. Zbl. Bakt.
Hyg., I. Abt.
Orig. A 226, 390-401 (1974)); Cummins, D. J. Clin. Periodont. 18, 455-461
(1991)). A
plasmid-mediated triclosan resistance has been reported in Staphylococcus
aureus but
the mechanism is unknown. (Cookson, B.D., et al. The Lancet 337, 1548-1549
(1991)).
Summary of the Invention
The present invention is based, at least in part, on the discovery that
triclosan (an
antimicrobial compound commonly used in consumer products, e.g., soaps and
detergents), has a genomic target which is involved in its ability to impart
antimicrobial
activity. The present invention further includes the identification of the
genomic target
for triclosan in Escherichia coli and in Mycobacterium smegmatis, as FabI and
InhA,
respectively, and provides for methods of identifying antimicrobial compounds
based
upon this identification (hereinafter screening assays will be used
interchangeably for
such methods for discussion purposes).
The present invention also is based, at least in part, on the discovery of
triclosan-
resistant microbial cells and the identification of mutant enoyl ACP-reductase
(ER)
polypeptides, e.g., E. coli FabI polypeptides or M. smegmatis InhA
polypeptides, which
confer the triclosan-resistance to the microbial cells. (For discussion
purposes below,
the term ER will be used to refer to these reductase enzymes, and it should be
understood that the descriptions apply to the ER polypeptide as well as to the
FabI and
InhA polypeptide embodiments.) The present invention includes the development
of
screening assays using these mutant polypeptides and triclosan-resistant
microbial cells
for antimicrobial compounds which can be used against triclosan-resistant
microbial
cells, e.g., in lieu of triclosan or in addition to triclosan.
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It should be appreciated that the present invention is the first time that a
non-
specific antimicrobial agent (hereinafter NSAM) was shown to be target
specific on a
genomic level, i.e., have a genomic target which is involved in its ability to
impart
antimicrobial activity. NSAMs for the purpose of this invention is intended to
include
the broad class of antimicrobial compounds, e.g., found in consumer products,
that (prior
to the present discovery) were not believed to be target specific on the
genomic level by
those of ordinary skill in the art. NSAMs are not intended to include
antibiotics or other
antimicrobials which one of ordinary skill in the art would have expected to
be target
specific on a genomic level prior to the discovery of the present invention.
The present
invention includes the identification of genomic targets involved in an NSAM's
ability to
impart antimicrobial activity and the development of screening assays for
antimicrobial
compounds based upon these genomic targets.
The invention features identification of a second genomic target that
influences
cell sensitivity to triclosan, the efflux pump which is the product of the
acrAB gene. In
1 S one embodiment, the invention describes double mutants altered both in er
and acrAB,
such that inactivation of the efflux pump renders both er+ (wild type) and er
mutant
cells more sensitive to triclosan. This embodiment provides that, in the
presence of an
inhibitor of the AcrAB efflux pump, a lower effective dose of an inhibitor of
an ER
protein is required to effectively inhibit the ER protein and achieve
biocidal,
antimicrobial, or antibiotic activity.
Other aspects of the invention include the reagents used in the aforementioned
screening assays, antimicrobial compounds identified using the screening
assays, and
methods of using the identified compounds in combination products, e.g.,
consumer
products and in therapeutic methods.
Brief Description of the Drawing
Figure 1 is a schematic of a restriction map of the pLYT8 region encoding
triclosan resistance, and deletion mutants. The thick (gray or black)
horizontal region
represents chromosomal DNA inserted into the tet gene of the pBR322 vector
(thin,
white). The deleted regions of the mutants are represented by interruptions of
the black
horizontal line; pLYTl 1 was created using BsmI and pLYTl2 using SspI. The
response
to triclosan (MIC, ~g ml-1 ) encoded by the plasmids in hypersusceptible host
strain
AG100A are the numbers shown in parentheses.
Figure 2 is a diagram illustrating an exemplary alignment of the protein
sequences of Fabl and InhA.
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Detailed Description of the Invention
The present invention pertains to a method for identifying an antimicrobial
compound which interacts with an ER polypeptide, e.g., a FabI or InhA
polypeptide.
(For discussion purposes below, the term ER will be understood to the ER
polypeptide
and to the FabI and InhA polypeptide embodiments.) The method involves
contacting
the ER polypeptide with a compound under conditions which allow interaction of
the
compound with the ER polypeptide to occur. The method further includes
detecting the
interaction of the compound with the ER polypeptide as an indication of
whether the
compound is an antimicrobial compound.
The language "antimicrobial compound" is art-recognized and is intended to
include a compound which inhibits the proliferation or viability of a microbe
which is
undesirable and/or which disrupts a microbial cell. The language further
includes
significant diminishment of a biological activity which is undesirable and
associated
with the microbe, such that a subject would not be detrimentally affected by
the microbe.
Examples include antibiotics, biocides, antibacterial compounds.
The language "ER polypeptide" is intended to include a polypeptides having
enoyl-acyl can:ier protein reductase activity. The ER polypeptides of the
present
invention include full length ER polypeptides and/or biologically active
fragments
thereof. The preferred fragments contain the reducing agent binding cleft
and/or the
triclosan binding portion and/or the substrate binding site, and are of a size
which allows
for their use in the screening methods of the present invention. An example of
such a
polypeptide is a FabI or InhA polypeptide. In addition to these two exemplary
ER
polypeptides, the term ER polypeptide is also meant to cover ER polypeptides
from
other microorganisms, e.g., from species other than E. coli or M. smegmatis.
The language "Fabl" and "InhA" is art-recognized and is intended to exemplify
ER polypeptides having enoyl-acyl carrier protein reductase activity. The Fabl
and
InhA polypeptides of the present invention include the full length
polypeptides and/or
biologically active fragments thereof. The preferred fragments contain the
reducing
agent binding cleft and/or the triclosan binding portion and/or the substrate
binding site,
and are of a size which allows for their use in the screening methods of the
present
invention. ER polypeptides of the present invention are discussed in further
detail
below.
The term "compound" is art-recognized and includes compounds being tested for
antimicrobial activity. The compound can be designed to incorporate a moiety
known to
interact with a ER polypeptide or can be selected from a library of diverse
compounds,
e.g., based on a desired activity, e.g., random drug screening based on a
desired activity.
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Preferably, the compound of the present invention is a small molecule.
Examples of
compounds of the present invention include NSAMs and triclosan compounds.
"NSAM" for the purpose of this invention is as defined above. An NSAM
compound includes functional and structural analogs of a parent NSAM compound.
The
analogs can be selected or designed either using the genomic target involved
in its ability
to impart antimicrobial activity and/or based upon knowledge derived from
studying the
interaction between the NSAM and the genomic target.
The language "triclosan compound" includes functional and structural analogs
of
triclosan. The analogs can be selected or designed either using the genomic
target and/
or based upon knowledge derived from studying the interaction between
triclosan and
the genomic target.
In one embodiment the compound is not an antibiotic. In another embodiment,
the compound is not isoniazid, diazaborine, or ethionamide.
The compound can be a single compound or can be a member of a test library.
Exemplary test libraries that can be used include combinatorial libraries or
libraries of
natural products.
The synthesis of combinatorial libraries is well known in the art and has been
reviewed (see, e.g., E.M. Gordon et al., J. Med Chem. (1994) 37:1385-1401 ;
DeWitt, S.
H.; Czarnik, A. W. Acc. Chem. Res. (1996) 29:114; Armstrong, R. W.; Combs, A.
P.;
Tempest, P. A.; Brown, S. D.; Keating, T. A. Acc. Chem. Res. (1996) 29:123;
Ellman, J.
A. Acc. Chem. Res. (1996) 29:132; Gordon, E. M.; Gallop, M. A.; Patel, D. V.
Acc.
Chem. Res. (1996) 29:144; Lowe, G. Chem. Soc. Rev. (1995) 309, Blondelle et
al.
Trends Anal. Chem. (1995) 14:83; Chen et al. J. Am. Chem. Soc. (1994)
116:2661; U.S.
Patents 5,359,115, 5,362,899, and 5,288,514; PCT Publication Nos. W092/10092,
W093/09668, W091/07087, W093/20242, W094/08051).
In another illustrative synthesis, a "diversomer library" is created by the
method
of Hobbs DeWitt et al. (Proc. Natl. Acad. Sci. U.S.A. 90:6909 (1993)). Other
synthesis
methods, including the "tea-bag" technique of Houghten (see, e.g., Houghten et
al.,
Nature 354:84-86 ( 1991 )) can also be used to synthesize libraries of
compounds
according to the subject invention.
The language "interacts with an ER polypeptide" include interactions with the
polypeptide which result in the identification of a compound having
antimicrobial
activity. Such interactions include binding of the compound to the
polypeptide, e.g.,
direct or indirect binding, which allows for identification of a compound
having
antimicrobial activity. In one embodiment, the interaction occurs with the
reducing
agent binding cleft of the ER polypeptide. In another embodiment, the
interaction
occurs with the triclosan binding portion of the ER polypeptide.
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The language "reducing agent binding cleft" is intended to include that
portion of
the ER polypeptide which interacts with, e.g., binds with, a reducing agent.
An example
of a reducing agent cleft is the NAD (or NADH+)/NADP (or NADPH+) binding cleft
of
the ER polypeptide.
The language "triclosan binding portion" is that portion of the ER polypeptide
which binds, e.g. directly or indirectly, triclosan. In one embodiment the
triclosan
binding portion is within the reducing agent binding cleft.
The language "detecting the interaction of the compound with the ER
polypeptide" includes means of detection which result in the identification of
a
compound having antimicrobial activity. For example, the interaction can be
detected
based on the presence or absence of enzyme activity, e.g., using art-
recognized
techniques.
The present invention further pertains to a method for identifying an
antimicrobial compound by contacting an enoyl reductase molecule with a
compound
under conditions which allows enzyme activity to occur. In this method, the
presence or
absence of enzyme activity is detected as an indication of whether the
compound is an
antimicrobial compound.
The language and terms of this method are as defined above and/or below . The
language "enoyl reductase molecule" (ER) is art recognized and is a
cytoplaslnic enzyme
involved in the synthesis of fatty acids. The enzymatic activity can be
measured using
art-recognized techniques some of which are discussed below.
The present invention further pertains to a method for identifying an
antimicrobial compound by exposing or contacting a microorganism to a compound
under conditions which allow fatty acid biosynthesis to occur. In this method,
the
inhibition of fatty acid biosynthesis is detected as an indication of whether
the
compound is an antimicrobial compound. The language and terms of this method
are as
defined above and/or below.
The language " inhibition of fatty acid biosynthesis" is art recognized and
includes the inhibition of the synthesis of at least one fatty acid in the
microorganism .
The inhibition of fatty acid biosynthesis can be measured as discussed below.
The term
"microorganism" is art- recognized and for purposes of this invention is used
interchangeably with "microbe or microbial cell".
The present invention further pertains to a method for identifying an
antimicrobial compound which interacts with a mutant ER polypeptide by
contacting the
mutant ER polypeptide with a compound under conditions which allow interaction
of the
compound with the mutant ER polypeptide to occur. In this method, the presence
or
absence of interaction of the compound with the mutant ER polypeptide is
detected as an
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indication of whether the compound is an antimicrobial compound. The language
and
terms of this method are as defined above and below.
The language "mutant of an ER polypeptide" is intended to include polypeptides
which differ from the ER polypeptide in an alteration of at least one amino
acid residue
but retain their ability to be useful within the screening assays of the
present invention.
The mutant ER polypeptides of the present invention include the full length
mutant ER
polypeptide and/ or biologically active fragments thereof. The preferred
fragments
contain the reducing agent binding cleft and/ or the triclosan binding portion
and are of a
size which allows for their use in the screening methods of the present
invention.
In one embodiment, the protein product of the mutant gene is capable of
conferring resistance to triclosan in a microbial cell. In another embodiment,
the protein
product of the mutant gene is capable of conferring resistance to an NSAM in a
microbial cell. In another embodiment, the mutant has a gly93val substitution.
(The
convention used here to describe the substitution mutation lists the wild-type
amino acid
followed by the position of the residue in the protein followed by the
substituted mutant
amino acid.) In another embodiment, the mutant has a met159thr or phe2031eu
substitution. In another embodiment, the mutant has an alteration of at least
one amino
acid in the reducing agent, e.g., NAD/NADP binding cleft of the ER molecule or
an
alteration of at least one amino acid residue in the triclosan binding
portion. In even
more specific embodiments the mutant ER protein is a mutant Fabl polypeptide
having
an amino acid sequence as shown in SEQ ID NO: 3 except that it comprises an
amino
acid substitution at a position selected from the group consisting of G 13, S
16, S 19, I20,
A21, S91, I92, G93, F94, A95, L 100, L 144, S 145, Y 156, M 159, K 163, G 190,
P 191,
I192, 8193, T194, L195, A196, I200, K201, D202, F203, 8204 and K205. Exemplary
residues for substitution underlined in Figure 2. One of ordinary skill in the
art would
understand that the numbering system is based on the E. coli FabI polypeptide.
Based
on this finding, one of ordinary skill in the art would further be able to
select comparable
residues which are applicable to another microorganism. For example, an
alignment of
FabI can be made with other, related ER molecules. An exemplary alignment of
FabI
and InhA, made using the BLAST algorithm, is shown in Figure 2. Using such an
alignment, it is possible to determine mutations in other ER polypeptides that
would
correspond to mutations in a FabI or InhA polypeptide which have been shown to
confer
resistance to triclosan. As used herein, the language "corresponds to" is
meant to
include an approximate correspondence when the sequence are aligned in a
biologically
meaningful manner by one of ordinary skill in the art. The language
"corresponds to"
also includes residues which spatially correspond, e.g., are in the same
functional
position upon crystallography, but which may not correspond when aligned using
an
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alignment program. The language "corresponds to" also includes residues which
perform the same function, e.g., mediate an enzymatic activity or bind the
same cofactor.
Other exemplary mutant ER proteins include, e.g., InhA mutants Ser94Ala
(corresponding to FabI S91); Met103Thr (corresponding to FabI L100); A1a124Va1
(corresponding to Fab 1 S 121 ); Met 161 Val (corresponding to FabI M 159).
Mutant ER
polypeptides are discussed in further detail below.
The present invention further pertains to a method for identifying an
antimicrobial compound capable of inhibiting proliferation or viability of a
triclosan-
resistant microbial cell. The method involves contacting a triclosan-resistant
microbial
cell with a compound under conditions which allow a triclosan-resistant
microbial cell to
proliferate or remain viable. The method further includes determining whether
the
compound is capable of inhibiting proliferation or viability of the cell
thereby
identifying an antimicrobial compound capable of inhibiting proliferation or
viability of
a triclosan-resistant microbial cell. The language and terms of this method
are as defined
above and/or below.
The language "triclosan-resistant microbial cell" is intended to include a
microbial cell which has become resistant to the antimicrobial effects) of
triclosan, e.g.,
triclosan no longer inhibits the proliferation of the microbial cell or the
cell remains
viable when exposed to triclosan, at a concentration of triclosan sufficient
to kill the
parent sensitive cell. Sensitivity is measured by a parameter known as
"minimum
inhibitory concentration" (MIC), such that a triclosan-resistant microbial
cell has a MIC
that is at least 1.5-fold greater than the sensitive parent, at least 2-fold
greater than the
sensitive parent, preferably at least 4-fold greater than the sensitive
parent, even more
preferably at least 10-fold greater than the sensitive parent. Examples of
triclosan-
resistant microbial cell include the cell lines described in the examples
below such as
AGT11, AGT23, and AGT25. The triclosan-resistant microbial cell also can be
acrAB+, i.e., it possesses at least the efflux pump protein of the acrAB+
gene, such that
triclosan sensitivity is enhanced by genetic loss of this gene, or by chemical
inhibition of
its activity.
The inhibition of proliferation or viability of the cell can be determined or
can be
detected using art-recognized techniques, e.g., optical detection. For
example, the
presence of lysis of the triclosan-resistant microbial cell can be used to
identify an
antimicrobial compound capable of inhibiting proliferation or viability,
and/or
disrupting, a triclosan-resistant microbial cell.
The present invention further pertains to a method for identifying an
antimicrobial compound capable of inhibiting proliferation or viability of a
triclosan-
resistant microbial cell by contacting a polypeptide capable of conferring
resistance to
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triclosan with a compound under conditions which allow interaction of the
compound to
the polypeptide to occur. In this method, the presence or absence of
interaction of the
compound with the polypeptide is detected as an indication of whether the
compound is
an antimicrobial compound capable of inhibiting proliferation or viability of
a triclosan-
S resistant microbial cell. The language and terms of this method are as
defined above
and/or below.
The language "polypeptide capable of conferring resistance to triclosan" is
intended to include a polypeptide which when present in the microbial cell
under
appropriate conditions confers resistance to triclosan to the microbial cell,
e.g., the
microbial cell can proliferate and remain viable in the presence of triclosan.
The invention further pertains to a method for identifying an antimicrobial
compound capable of inhibiting proliferation or viability of a NSAM-resistant
microbial
cell. The method involves contacting a polypeptide capable of conferring
resistance to a
NSAM with a compound under conditions which allow interaction of the compound
1 S with the polypeptide to occur. The method further involves detecting the
presence or
absence of interaction with the polypeptide as an indication of whether the
compound is
an antimicrobial compound capable of inhibiting proliferation or viability of
a NSAM-
resistant microbial cell. The language and terms of this method are as defined
above
and/or below.
The language "polypeptide capable of conferring resistance to a NSAM" is
intended to include a polypeptide which when present in the microbial cell
under
appropriate conditions confers resistance to a NSAM to the microbial cell,
e.g., the
microbial cell can proliferate and remain viable in the presence of the NSAM.
Other aspects of this invention include antimicrobial compounds identified
using
2S any of the aforementioned methods or screening assays and the use of these
compounds
in combination products or in therapy as an active agent in a pharmaceutical
composition.
The "combination product" includes an antimicrobial compound identified using
a screening method of the invention and a product forming the combination
product.
The term "product" is intended to include consumer products such as
detergents, soaps,
deodorant mouthwash, toothpaste, and lotions.
The present invention further pertains to a combination product containing a
structural analog of triclosan and a product forming a combination product. In
a
preferred embodiment, the combination product containing the structural analog
of
3S triclosan is effective against a triclosan-resistant microbial cell.
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The present invention further pertains to a combination product containing a
structural analog of an NSAM and a product forming a combination product. In a
preferred embodiment, the combination product containing the structural analog
of the
NSAM is effective against a triclosan-resistant microbial cell or an NSAM-
resistant
microbial cell.
The present invention further pertains to a method for inhibiting the growth
of an
unwanted microorganism with a NSAM or an NSAM compound by administering to the
subject an effective amount of the NSAM or the NSAM compound such that the
growth
of the unwanted microorganism is inhibited. The present invention even further
pertains
to a method for inhibiting the growth of an unwanted microorganism with a
triclosan
compound or with the parent triclosan compound by contacting a surface, e.g.,
the
surface of an instrument, the surface of the skin of a subject, the surface of
a room, or the
surface of a container, with an effective amount of the NSAM such that the
growth of
the unwanted microorganism is inhibited.
The present invention further pertains to a method for treating a subject
having
growth of an unwanted microorganism with a NSAM or an NSAM compound by
administering to the subject an effective amount of the NSAM or the NSAM
compound
such that the subject is treated for the unwanted microorganism. The present
invention
even further pertains to a method for treating a subject having growth of an
unwanted
microorganism with a triclosan compound or with the parent triclosan compound
by
administering to the subject an effective amount of the NSAM such that the
subject is
treated for the unwanted microorganism.
The term "subject" refers to a living animal or human in need of treatment
for, or
susceptible to, a condition involving an unwanted or undesirable
microorganism, e.g., a
particular treatment for having an unwanted pathogenic cell as defined below.
In
preferred embodiments, the subject is a mammal, including humans and non-human
mammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice.
In the
most preferred embodiment, the subject is a human. The term "subject" does not
preclude individuals that are entirely normal with respect to having an
unwanted
pathogen or normal in all respects. The subject may fonmerly have been treated
with
antibiotic or antimicrobial therapy, and may be under treatment, or have been
treated by
antibiotic or antimicrobial therapy in the past.
The term "patient," as used herein, refers to a human subject who has
presented
at a clinical setting with a particular symptom or symptoms suggesting one or
more
diagnoses of having an infectious disease, or having the presence of an
unwanted
microbial cell. A patient's diagnosis can alter during the course of disease
progression,
such as development of further disease symptoms, or remission of the disease,
either
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spontaneously or during the course of a therapeutic regimen or treatment.
Thus, the term
"diagnosis" does not preclude different earlier or later diagnoses for any
particular
patient or subject. The term "prognosis" refers to assessment for a subject or
patient of a
probability of developing a condition associated with or otherwise indicated
by presence
of one or more unwanted pathogenic cells in the patient.
Methods and Uses
The environment contains a variety of microbes which are pathogenic disease
organisms. These include viruses, bacteria, fungi, and protozoans, which can
cause
pathological damage to the subject organism if present as an unwanted cell.
The term "infectious disease" is meant to include disorders caused by one or
more species of bacteria, viruses, fungi, and protozoans, species of which
that are
disease-producing organisms collectively referred to as "pathogens." The term
"fungi" is
meant to include the yeasts. In this invention, pathogens are exemplified, but
not limited
to, Gram-positive bacteria such as Actinomyces bovis, Enterococcus fecalis,
Hemophilus
pneumoniae, Listeria monocytogenes, Mycobacterium tuberculosis, M. leprae, M.
smegmatis, Proprionibacterium acnes, Sarcina ventriculi, Staphylococcus
aureus, S.
epidermis, S. intermedias, Streptococcus hemolyticus, S. pneumoniae; Gram-
negative
bacteria such as Campylobacter fetus, Erwinia carotovora, Flavobacterium
meningosepticum, Helicobacter pylori, Hemophilus pneumoniae, H. influenzae,
Klebsiella pneumonia, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Shigella
dysenteria, Salmonella typhi, S. paratyphi, Yersinia pestis, Escherichia coli
serotype
0157, and Chlamydia species, Helicobacter species; viruses such as HIV-1, 2,
and -3,
HSV-I and -II, non-A non-B non-C hepatitis virus, pox viruses, rabies viruses,
and
Newcastle disease virus; fungi such as Candida albicans, C. tropicalis, C.
krusei, C.
pseudotropicalis, C. parapsilosis, C. guillermondii, C. stellatoidea,
Aspergillus
fumigatus, A. niger, A. nidulans, A. flavus, A. terreus, Absidia corymbifera,
A. ramosa,
Cryptococcus neoforms, Histoplasma capsulatum, Coccidioides immitis,
Pneumocystis
carinii, Rhizopus arrhizus, R. oryzae, Mucor pusillus and other fungi; and
protozoa such
as Entamoeba histolytica, Entamoeba coli, Giardia lamblia, G. intestinalis,
Eimeria
sp., Toxoplasma sp., Cryptosporidium parvum, C. muris, C. baileyi, C.
meleagridis, C.
wrairi, and C. nosarum. Obtaining unique epitopes from these organisms by
screening
proteins and by assaying peptides in vitro are commonly known to those skilled
in the
art.
In preferred embodiments compounds of the invention can be used to inhibit the
growth of an unwanted organism, e.g., an infectious, pathogenic organism or an
organism that causes spoilage or biofouling, by contacting the organism with
the
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compound. The compound can be applied prior infection by the organism to
prevent a
subject from becoming infected. For example, the compounds can be used for
cleaning
surfaces, e.g., counter tops, instruments, or the skin of the subject, to
inhibit the growth
of the organism and reduce the possibility of the subject actually becoming
infected with
one of the organisms.
Treating or treatment of a state characterized by the presence of an unwanted
cell, e.g., an unwanted pathogenic cell, e.g., an unwanted bacterium, is
intended to
include the alleviation of or diminishment of at least one symptom, for
example, fever or
inflammation, typically associated with the state. The treatment also includes
alleviation
or diminishment of more than one symptom. Preferably, the treatment cures,
e.g.,
substantially eliminates, the symptoms associated with the state.
The language "therapeutically effective dose" or "therapeutically effective
amount" of a compound described herein, is that amount necessary or sufficient
to
perform its intended function, e.g., on a surface or on or within a subject,
e.g., to
eradicate or inhibit growth of an unwanted pathogen, e.g., microorganism. The
therapeutically effective amount can vary depending on such factors as the
species or
strain of the pathogen, the amount of the pathogen to be inhibited ant the
manner in
which the compound is to be used. One of ordinary skill in the art would be
able to
study the aforementioned factors and make a determination regarding the
effective
amount of the compound required without undue experimentation. For
administration,
one of ordinary skill in the art would be able to determine such amounts based
on such
factors as the subject's size, the severity of the subject's symptoms, and the
particular
composition or route of administration selected. An in vitro or in vivo assay
can be here
used to determine an "effective amount" of the compounds described herein to
achieve
inhibition of growth or proliferation of the cell by binding and inhibiting
the specific
target.
A "therapeutically effective dosage" is a dosage of a compound that preferably
inhibits growth of an unwanted pathogenic cell, or destroys cell viability, by
at least
about 50%, more preferably by at least about 80%, even more preferably by at
least
about 90%, and still more preferably by at least about 95% relative to the
absence of the
compound. The ability of a compound to inhibit or kill infectious disease
cells can be
evaluated in an in vitro inhibitory concentration assay, or, e.g., an animal
model system
predictive of efficacy in infectious diseases. Alternatively, this property of
a compound
can be evaluated by examining the ability of the compound to inhibit in vitro
by using
assays well-known to the skilled practitioner. Assays include the of effect on
viability of
the test pathogenic cell, by assay of quantity of "colony forming units"
(cfu), in the
presence and absence of the compound; assay of capability to carry out a
physiological
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process, such as cellular uptake of a metabolite; assay of uptake and
incorporation of a
metabolite into a macromolecule, such as a nucleic acid or protein; each assay
conducted
in the presence of a range of concentrations and in the absence of the
compound. For
compounds having a known specific target, the effective dosage to inhibit the
activity of
that target, such as an enzyme, can be assessed using isolated target
material.
The present invention also pertains to antimicrobial soap or detergent
preparations containing triclosan in amounts which are much lower than the
amounts
contained in the commercially available antimicrobial soap or detergent
preparations.
The commercially available antimicrobial soap or detergent preparations
contain
triclosan in higher amounts and part of the present invention includes the
realization that
higher amounts, e.g., than 0.3 % triclosan found in Total~ toothpaste, or 3 mg
ml-1, are
not necessary for the triclosan to interact with its genomic target. The
antimicrobial soap
or detergent preparations contain triclosan at a concentration of less than
about 500 pg
per milliliter of soap or detergent preparation forming an antimicrobial soap
or detergent
preparation. In other embodiments, the antimicrobial soap or detergent
preparations
contain triclosan at a concentration of less than about, e.g., 500 pg ml-1
(one ml being
roughly equivalent to one gram of solid, which can be corrected by the density
of the
solid), less than about 100 ~g ml-1 , less than about 50 ~g ml-1, e.g., less
than about 10
pg ml-1, less than about 5 p,g ml-1~ less than about 1 p.g ml-land e.g., less
than about
0.5 p,g ml-1.
In addition to the above uses for the antimicrobial agents and compounds of
the
invention, the following uses are included: ( 1 ) a skin antiseptic: a safe,
nonirritating,
antimicrobial-containing preparation that prevents overt skin infection; (2) a
patient
preoperative skin preparation: a safe, fast-acting, broad-spectrum,
antimicrobial-
containing preparation that significantly reduces the number of micro-
organisms on
intact skin; (3) a surgical hand scrub: a safe, nonirritating, antimicrobial-
containing
preparation that significantly reduces the number of microorganisms on the
intact skin.
A surgical hand scrub should be broad-spectrum, fast-acting and persistent;
(4) a health-care personnel hand wash: a safe, nonirritating preparation
designed for
frequent use that reduces the number of transient microorganisms on intact
skin to an
initial baseline level after adequate washing, rinsing and drying. If the
preparation
contains an antimicrobial agent, it should be broad-spectrum, fast-acting,
and, if
possible, persistent; (5) a skin wound cleanser: a safe, nonirritating, liquid
preparation
(or product to be used with water) that assists in the removal of foreign
material from
small, superficial wounds and does not delay wound healing; (6) a skin wound
protectant: a safe, nonirritating preparation applied to small cleansed wounds
that
provides a protective barrier (physical, chemical, or both) and neither delays
healing nor
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favors the growth of microorganisms; and (7) an antimicrobial soap: a soap
containing
an active ingredient with in vitro and in vivo activity against skin
microorganisms.
The present invention also pertains to antimicrobial soap or detergent
preparations containing triclosan compounds, e.g., structural analogs of
triclosan, in a
S soap or detergent preparation. In a preferred embodiment, the structural
analog of
triclosan is a compound capable of inhibiting the proliferation and viability
of a
triclosan-resistant microbial cell.
Antimicrobial Compounds
The language "antimicrobial compound" is art-recognized and is intended to
include a compound which inhibits the proliferation or viability of a microbe
which is
undesirable and/or which disrupts a microbial cell. The language further
includes
diminishment of an activity which is undesirable and associated with the
microbe.
Examples include antibiotics, biocides, antibacterial compounds.
The term "antibiotics" is art recognized and includes antimicrobial agents
synthesized by an organism in nature and isolated from this natural source,
and
chemically synthesized antibiotics. The term includes but is not limited to:
poIyether
ionophore such as monensin and nigericin; macrolide antibiotics such as
erythromycin
and tylosin; aminoglycoside antibiotics such as streptomycin and kanamycin; (3-
lactam
antibiotics such as penicillin and cephalosporin; and polypeptide antibiotics
such as
subtilisin and neosporin. Semi-synthetic derivatives of antibiotics, and
antibiotics
produced by chemical methods are also encompassed by this term.
Chemically-derived antimicrobial agents such as isoniazid, trimethoprim,
quinolines, and sulfa drugs are considered antibacterial drugs, although the
term
antibiotic has been applied to these. These agents and antibiotics have
specific cellular
targets for which binding and inhibition by the agent or antibiotic can be
measured. For
example, erythromycin, streptomycin and kanamycin inhibit specific proteins
involved
in bacterial ribosomal activity; penicillin and cephalosporin inhibit enzymes
of cell wall
synthesis; and rifampicin inhibits the (3 subunit of bacterial RNA polymerase.
It is
within the scope of the screens of the present invention to include compounds
derived
from natural products and compounds that are chemically synthesized.
The term "biocidal" is art recognized and includes an agent that those
ordinarily
skilled in the art prior to the present invention believed would kill a cell
"non-
specifically," or a broad spectrum agent whose mechanism of action is unknown,
e.g.,
prior to the present invention, one of ordinary skill in the art would not
have expected
the agent to be target-specific. Examples of biocidal agents include paraben,
chlorbutanol, phenol, alkylating agents such as ethylene oxide and
formaldehyde,
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halides, mercurials and other heavy metals, detergents, acids, alkalis, and
chlorhexidine.
The term "bactericidal" refers to an agent that can kill a bacterium;
"bacteriostatic" refers
to an agent that inhibits the growth of a bacterium.
In contrast to the term "biocidal," an antibiotic or an "anti-microbial drug
approved for human use" is considered to have a specific molecular target in a
microbial
cell. Preferably a microbial target of a therapeutic agent is sufficiently
different from its
physiological counterpart in a subject in need of treatment that the
antibiotic or drug has
minimal adverse effects on the subject.
A specific target for drug or antibiotic therapy can be ribosomal protein (S
12 of
the 30s ribosome); an RNA polymerise subunit (~i of bacterial RNA polymerise);
a cell
wall (a cross-linking enzyme of a bacterial cell wall); or a DNA polymerise-
associated
proteins (e.g., a gyrase). In the invention here, an enzymatic component of
fatty acid
biosynthesis, enoyl-ACP reductase, is determined to be a specific target of an
effective
dose of an agent which was previously classified as a non-specific biocidal
agent when
used at significantly higher concentrations than the effective dose.
The term "enzyme" includes polymorphic variants that are silent mutations
naturally found within the microorganism population of a strain or species.
The
enzymes in the preferred embodiment of the invention are fatty acid
biosynthesis
enzymes, preferably enoyl-ACP reductase (enoyl reductase) enzymes,.however,
there is
no intent to limit the invention to these enzymes. The term fatty acid
biosynthesis
enzymes (and its equivalent term fatty acid biosynthetases) is intended to
include those
components of a proteins or polypeptides capable of synthesizing fatty acids
via the
three-carbon intermediate, malonyl CoA. The proteins include acyl Garner
protein
(ACP), acetyl CoA-ACP transacetylase, malonyl CoA-transferase (3-ketoacyl-ACP
synthase, (3-ketoacyl-ACP reductase, ~i-hydroxyacyl-ACP dehydratase, and enoyl-
ACP
reductase (Lehninger, A., et al. Principles of Biochemistry, 2nd Ed., 1993
Worth, New
York, p. 642-653). The ACP of E. coli and of other organisms contains the
prosthetic
group 4'-phosphopantetheine, to which the growing fatty acid chain is
covalently linked
by a thioester bond. The term "enzymes" is art recognized for purposes of this
invention
and can refer to whole intact enzyme or portions or fragments thereof.
The terms "protein," "polypeptide" and "peptide" are used interchangeably
herein.
The term "variant" as used herein refers to a protein or nucleic acid molecule
that
is substantially similar in structure and biological activity and may
substitute for the
molecule of which it is a variant. Thus, provided that two molecules possess a
common
activity and may substitute for each other, they are considered variants as
that term is
used herein even if the composition or secondary, tertiary or quaternary
structure of one
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of the molecules is not identical to that found in the other, or if the amino
acid or
nucleotide sequence is not identical. Variants of the ER polypeptides are
intended to be
included as part of this invention.
The term "fragment," as used herein with respect to a molecule such as ER or
antibody protein or a nucleic acid encoding ER, refers to a portion of a
native or variant
amino acid residue or nucleotide sequence. The team "fragment" includes a
chemically
synthesized protein fragment.
The term "antibody" as used herein is intended to include fragments thereof
which are also specifically reactive with one of the components in the methods
and kits
of the invention. Antibodies can be fragmented using conventional techniques
and the
fragments screened for utility in the same manner as described above for whole
antibodies. For example, F(ab)2 fragments can be generated by treating an
antibody
with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide
bridges to
produce Flab) fragments. The term "antibody" is further intended to include
single
chain, bispecific and chimeric molecules. The term "antibody" includes
possible use
both of monoclonal and polyclonal antibodies (Ab) directed against a target,
according
to the requirements of the application.
Polyclonal antibodies can be obtained by immunizing animals, for example
rabbits or goats, with a purified form of the antigen of interest e.g., wild-
type or mutant
ER protein, or a fragment of the antigen containing at least one antigenic
site.
Conditions for obtaining optimal immunization of the animal, such as use of a
particular
immunization schedule, and using adjuvants e.g. Freund's adjuvant, or
immunigenic
substituents covalently attached to the antigen, e.g. keyhole limpet
hemocyanin, to
enhance the yield of antibody titers in serum, are well-known to those in the
art.
Monoclonal antibodies are prepared by procedures well-known to the skilled
artisan,
involving obtaining clones of antibody-producing lymphocyte, i.e. cell lines
derived
from single cell line isolates, from an animal, e.g. a mouse, immunized with
an antigen
or antigen fragment containing a minimal number of antigenic determinants, and
fusing
said clone with a myeloma cell Iine to produce an immortalized high-yielding
cell line.
Many monoclonal and polyclonal antibody preparations are commercially
available, and
commercial service companies that offer expertise in purifying antigens,
immunizing
animals, maintaining and bleeding the animals, purifying sera and IgG
fractions, or for
selecting and fusing monoclonal antibody producing cell lines, are available.
Specific high affinity binding proteins or peptides, that can be used in place
of
antibodies, can be made according to methods known to those in the art. For
example,
proteins that bind specific DNA sequences may be engineered (Ladner, R.C.,et.
al., U.S.
Patent 5,096,815), and proteins, polypeptides, or oligopeptides
("miniproteins") that bind
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a variety of other targets, especially protein targets (Ladner, R.C., et. al.,
U.S. Patent
5,233,409; Ladner, R.C., et.al., U.S. Patent 5,403,484) may be engineered and
used in
the present invention for covalent linkage of a genetically replicating unit,
such as a
bacteriophage, displaying a library of variant peptides, to select amino acid
sequences
S that are capable of binding to an immobilized wild-type or a mutant ER
protein. The
consensus of amino acid sequences of such obtained engineered binding peptides
can be
used as a probe of the structure of the target ER protein, and can serve as
the basis of
design of a peptidomimetic drug.
Antibodies and binding proteins can be incorporated into large scale
diagnostic
or assay protocols that require immobilizing the compositions of the present
invention
onto surfaces, for example in mufti-well plate assays, or on beads for column
purification.
Immunoassays
1 S General techniques to be used in performing various immunoassays are known
to
those of ordinary skill in the art. Moreover, a general description of these
procedures is
provided in U.S. Patent No. 5,051,361 which is incorporated herein by
reference, and by
procedures known to the skilled artisan, and described in manuals of the art
(Ishikawa,
E., et. al. (1988), Enzyme Immunoassay Igaku-shoin, Tokyo, NY; Harlow, E. and
D.
Lane, Antibodies.' A Laboratory Manual, CSH Press, NY). Examples of several
immunoassays are given discussed here.
Radioimmunoassays (RIA) utilizing radioactively labeled ligands, for example,
antigen directly labeled with 3H, or 14C, or 1251, measure presence of ER as
antigenic
material. A fixed quantity of labeled mutant ER, for example, competes with
unlabeled
2S antigen from the sample for a limited number of antibody binding sites.
After the bound
complex of labeled antigen-antibody is separated from the unbound (free)
antigen, the
radioactivity in the bound fraction, or free fraction, or both, is determined
in an
appropriate radiation counter. The concentration of bound labeled antigen is
inversely
proportional to the concentration of unlabeled antigen present in the sample.
The
antibody to ER can be in solution, and separation of free and bound antigen ER
can be
accomplished using agents such as protein A, or a second antibody specific for
the
animal species whose immunoglobuIin contains the antibody to ER.
Alternatively,
antibody to ER can be attached to the surface of an insoluble material, which
in this
case, separation of bound and free ER is performed by appropriate washing.
3S Other preferred immunoassay techniques use enzyme labels such as
horseradish
peroxidase, alkaline phosphatase, luciferase, urease, and 13-galactosidase.
For example,
ER conjugated to horseradish peroxidase can compete with free sample ER for a
limited
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number of antibody combining sites present on antibodies to ER attached to a
solid
surface such as a microtiter plate. The anti-ER antibodies may be attached to
the
microtiter plate directly, or indirectly, by first coating the microtiter
plate with
multivalent ER conjugates (coating antigens) prepared for example by
conjugating ER
with serum proteins such as rabbit serum albumin (RSA). After separation of
the bound
labeled ER from the unbound labeled ER, the enzyme activity in the bound
fraction is
determined colorimetrically, for example by a mufti-well microtiter plate
reader, at a
fixed period of time after the addition of horseradish peroxidase chromogenic
substrate.
The above examples of preferred immunoassays describe the use of radioactively
and enzymatically labeled tracers. Assays also may include use of fluorescent
materials
such as fluorescein and analogs thereof, 5-dimethylaminonaphthalene-1-sulfonyl
derivatives, rhodamine and analogs thereof, coumarin analogs, and
phycobiliproteins
such as allophycocyanin and R-phycoerythrin; phosphorescent materials such as
erythrosin and europium; luminescent materials such as luminol and luciferin;
and sols
such as gold and organic dyes. In one embodiment of the present invention, the
biological sample is treated to remove low molecular weight contaminants.
The term "substantially pure" or "isolated" with respect to a population of
genetically modified cells means that the cells contain fewer than about 20%,
more
preferably fewer than about 10%, most preferably fewer than about 1 %, non-
modified
cells. The term "genetically modified" refers to mutation, including without
limitation
point mutation, substitution, transition, transversion, deletion, insertion,
inversion and
translocation mutation of nucleic acid. It includes manipulation of a
recipient cell by
introduction of recombinant or genetically engineered nucleic acid such as
transformation and transfection.
The term "substantially pure" or "isolated" with respect to a nucleic acid or
a
protein means that the nucleic acid or protein is at least about 75%,
preferably at least
about 85%, more preferably at least about 90%, even more preferably at least
about
95%, and most preferably at least about 99% free of other nucleic acids or
proteins.
The term "culture medium" refers generally to any preparation suitable for
cultivating living cells, preferably microorganisms. A "cell culture" refers
to a cell
population sustained in vitro using sterilized culture medium.
Bacterial Enovl-ACP Reductase Mutants. Structure, and Assay
A mutation of E. coli known as envM was characterized as having a temperature-
sensitive osmotic fragility phenotype (Egan, A. et al. Genet. Res. Cambr. 21:
139-152
(1973)), and was subsequently shown to be the gene for enoyl reductase and for
resistance to diazoborine in this species and in Salmonella typhimurium
(Turnowsky, F.,
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et al. J. Bacteriol. 171, 6555-6565 ( 1989)). The envM gene had been
characterized as
encoding a protein involved in biosynthesis of lipopolysaccharide (Hogenauer,
G. et al,
Nature 293: 662-664 (1981)), and this mutation was shown to reduce virulence
in E. coli
clinical isolates 0111:B4 and O1:K1. The envM'~ gene was then shown to encode
the
FabI enoyl ACP reductase ((Turnowsky, F., et al. J. Bacteriol. 171, 6555-6565
(1989);
Bergler, H., et al. J. Biol. Chem. 269, 5493-5496 {1994)).
FabI wild type and mutant proteins were expressed on plasmids in E. coli cells
(Bergler, H., et al. Eur. J. Biochem. 242, 689-694 ( 1996)), and the proteins
were
overproduced, facilitating purification and assay. FabI has been engineered as
an N-
terminal insertion of six histidine residues, enabling purification using a
Nip-agarose
column (Qiagen, Hilden, Germany) for use in reconstitution of purified fatty
acid
biosynthesis components for synthesis and assay in vitro (Heath, R. et al., J.
Biol. Chem.
270: 26538-26542 (1995)).
InhA of Mycobacterium smegmatis, a species susceptible to triclosan(Vischer,
W.A. et al. 1974. Zbl. Bakt. Hyg., I. Abt. Orig. A 226:376-389), is 35%
identical to E.
coli FabI (GAP program of Genetics computer Group, Inc.[GCG]) and has enoyl
reductase activity (Dessen, A. et al. 1995. Science 267:1638-1641) (Quemard,
A. et al.
1995. Biochemistry 34:8235-8241). The inhA locus was originally identified by
a
mutation replacing serine 94 with alanine (S94A) in the gene product which
caused
resistance to the antitubercular drug isoniazid (Banerjee, A. et al. 1994.
Science
263:227-230). Mutations conferred resistance to isoniazid, and to another anti-
tuberculosis drug, ethioamide, in Mycobacterium smegmatis, M. tuberculosis, M.
bovis,
and Mavium (Banerjee, A., et al. Science 263, 227-230 (1994)). It is 87%
identical to
M. tuberculosis InhA, the three dimensional structure of which has been
determined by
X-ray crystallography (Dessen, A. et al. 1995. Science 267:1638-1641) in the
presence
of modified isoniazid (Rozwarski, D.A. et al. 1998. Science 279:98-102). X-ray
crystallography of E. coli FabI (Baldock, C. et al. 1996. Science 274:2107-
2110)
demonstrates its structural similarity to InhA.
Structural studies involving crystallization of enoyl reductase from E coli
and X-
ray crystallography of the enzyme alone and co-crystallized with diazaborine
derivatives
(Baldock, C., et al. Acta Cryst. D52: 1181-1184 ( 1996); Science 274, 2107-
2110 ( 1996))
revealed that a covalent bond is formed between cofactor NAD and
benzodiazaborine (or
thienodiazaborine) through the boron atom. These analyses reveal that the drug
enters
the NAD cleft, and the analyses identify the residues of the cleft. Similar
studies of the
InhA ser94ala mutant protein reveal that isoniazid resistance is due to a
decreased
affinity of the mutant protein for NAD (Dessen, A., et al. Science 267, 1638-
1641
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(1995)). Further, covalent attachment of isoniazid to NAD in the NAD cleft can
be
observed (Rozwarski, D. et al. Science 279: 98-102 (1998)).
The complete fatty acid biosynthesis set of reactions can be measured in vivo
using incorporation into E. coli cells of [i-[3-3H]alanine into medium and
long chain
acyl-ACPs, which are analyzed by conformationally sensitive gel
electrophoresis in 13%
polyacrylamide containing 0.5 M urea (Heath, R. et al., J. Biol. Chem. 270:
26538-
26542 (1995)). This in vivo assay is useful herein for screens of drug
candidates among
natural products and synthetic chemicals for use as antimicrobial agents, for
activity that
inhibits fatty acid biosynthesis, by performing the assay in the presence and
absence of
each compound or extract. Fatty acid synthesis can be assayed in an entirely
pure in
vitro system, using purified components for each reaction ((Heath, R. et al.,
J. Biol.
Chem. 270: 26538-26542 (1995)).
Enoyl reductase activity can be measured using crude cell extracts or
substantially purified enzymes by following NADH oxidation at 340 nm with a
Uvikon
93310 spectrophotometer {Kontron Instruments), with 2-traps-octenoyl-ACP as a
substrate (Dessen, A., et al. Science 267, 1638-1641 (1995)). This reaction
can be
carried out in small volumes in 96-well or 384-well mufti-well plastic dishes,
and can be
automated for use in large-scale screens of antimicrobial agents using FabI or
InhA as
the specific target.
Enoyl reductase activity can also be measured in whole cells by growth with
32p1 ~d measurement of incorporation into phospholipids. Following this
procedure,
cells are extracted with chloroform-methanol (2:1), which is then mixed with
0.25
volumes of water, and the chloroform layer is removed and analyzed for
phospholipids
by two-dimensional thin-layer chromatography on silica plates (Turnowsky, F.,
et al. J.
Bacteriol. 171, 6555-6565 (1989)). Reactions can be performed in vivo in the
presence
and absence of drug candidates, to determine the effect on distribution of
radioactivity
into the spectrum of phospholipid intermediates.
Potential drug candidates can be assayed by ability to bind to an ER protein
which has been immobilized on a bead or on a plastic surface, for example, the
plastic of
mufti-well plastic dishes. A large variety of techniques for immobilization to
beads and
to surfaces of target proteins are described in U.S.P.N. 5,233,409. Candidate
agents can
be incubated with immobilized ER protein under appropriate conditions, for
example, in
the presence of NAD or NADP, and under conditions of different temperature and
pH,
using the known inhibitors and mutants of the invention to optimize the assay.
Following incubation with the potential candidates, the immobilized ER is
separated
from unbound compounds, washed to remove non-specifically bound materials, and
then
bound materials are eluted, for example, with solutions of decreased pH, or
increased
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detergent concentration, to obtain and analyze the specifically bound
materials. Agents
that are found to bind immobilized ER in this primary screen can be tested for
ability to
inhibit the enoyl reductase activity, and for antimicrobial activity using
whole cells.
Viability assays, and assays of cell lysis can also be performed in mufti-well
plastic
dishes, in which viability is measured by cfu content following incubation in
the
presence and absence of drug, of dilutions of the contents of each well. Lysis
can be
measured by loss of optical density at, e.g., 540 nm, using an automated plate
reader.
Genes, Nucleic Acids. Hybridization to Clone Homologs of ER, and Vectors
Homologs of ER proteins can be generated by mutagenesis, such as by at least
one of a discrete point mutation which can give rise to a substitution, or by
at least one
of deletion or insertion. The present invention also is intended to encompass
homologs
of the ER polypeptide and mutant ER polypeptides described above. These
fragments
and homologs, which are biologically active in a manner which is the same or
similar to
the parent ER polypeptide. For example, a polypeptide or protein has ER
biological
activity if it can bind and reduce the double bond of an enoyl such as an
octenoyl which
is linked to ACP.
As used herein, the term "nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
The
term should also be understood to include, as equivalents, analogs of either
RNA or
DNA made from nucleotide analogs, and, as applicable to the embodiment being
described, single-stranded (such as sense or antisense) and double-stranded
polynucleotides.
As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid
comprising an open reading frame encoding a ER of the present invention. A
"recombinant gene" refers to nucleic acid encoding a ER protein encoded by a
gene that
has been engineered by recombinant techniques. The nucleotide sequence
encoding
Fab 1 is shown in SEQ ID NO: 2.
The term "vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. The term "expression vector"
includes
any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene
construct
in a form suitable for expression by a cell (e.g., linked to a promoter). In
the present
specification, "plasmid" and "vector" are used interchangeably, as a plasmid
is a
commonly used form of vector. Moreover, the invention is intended to include
other
vectors which serve equivalent functions.
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The terms "transformation" and "transfection" mean the introduction of a
nucleic acid, e.g., an expression vector, into a recipient or "host" cell. The
term
"transduction" means transfer of a nucleic acid sequence, preferably DNA, from
a donor
to a recipient cell, by means of infection with a virus previously grown in
the donor,
preferably a bacteriophage, preferably phage P 1.
The term "gene product" includes an RNA molecule transcribed from a gene, or
a protein translated from the RNA transcribed from the gene.
Vectors capable of directing the expression of genes to which they are
operatively linked are referred to herein as "expression vectors". Expression
vectors for
expression of the er gene and capable of replication in a cell of a bacterium,
such as an
Escherichia, a Bacillus, a Streptomyces, a Streptococcus, or in a cell of a
simple
eukaryotic fungus such as a Saccharomyces or, a Pichia, or in a cell of a
eukaryotic
organism such as an insect, a bird, a mammal, or a plant, are within the
present
invention. Such vectors may carry functional replication-specifying sequences
(replicons) both for a host for expression, for example a Streptomyces, and
for a host, for
example, E. coli, for genetic manipulations and vector construction. See e.g.
U.S.P.N
4,745,056. Suitable vectors for a variety of organisms are described in
Ausubel, F. et
al., Short Protocols in Molecular Biology, Wiley, New York (1995), and for
example,
for Pichia, can be obtained from Invitrogen (Carlsbad, CA).
"Transcriptional regulatory sequence" is a generic term to refer to DNA
sequences,
such as initiation signals, enhancers, and promoters, which induce or control
transcription of protein coding sequences with which they are operably linked.
In
preferred embodiments, transcription of a recombinant ER gene, a marRAB
sequence or
acrAB sequence, is under the control of a promoter sequence (or other
transcriptional
regulatory sequence) which controls the expression of the recombinant gene in
a cell-
type in which expression is intended. It will also be understood that the
recombinant
gene can be under the control of transcriptional regulatory sequences which
are the same
or which are different from those sequences which control transcription of the
naturally-
occurring form of the ER protein. Exemplary regulatory sequences are described
in
Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic
Press,
San Diego, CA ( I 990). For instance, any of a wide variety of expression
control
sequences, that control the expression of a DNA sequence when operatively
linked to it,
may be used in these vectors to express DNA sequences encoding the ER proteins
of this
invention.
"Homology" refers to sequence similarity between two peptides or between two
nucleic acid molecules. Homology can be determined by comparing a position in
each
sequence which may be aligned for purposes of comparison. When a position in
the
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compared sequence is occupied by the same base or amino acid, then the
molecules are
homologous or identical at that position. A degree of homology between
sequences is a
function of the number of matching or identical positions shared by the
sequences.
"Cells," "host cells," "recipient cells," or "sensitive recipient cells," are
terms
used interchangeably herein. It is understood that such terms refer not only
to the
particular subject cell but to the progeny or potential progeny of such a
cell. Recipient
cells are "sensitive" for the drug that is used to select for the particular
drug-resistant
trait of interest encoded by the transducing or transforming nucleic acid,
e.g., in the
invention, the cell can be sensitive to one or more of ampicillin, kanamycin,
or triclosan.
In one embodiment, the invention includes a nucleic acid which encodes a
peptide having enoyl reductase enzyme activity, e.g., Fabl or InhA.
Preferably, the
nucleic acid is a PCR product molecule comprising at least a portion of the
nucleotide
sequence represented in SEQ ID NO: 1 or SEQ ID NO: 2 from nucleotide (nt) 404
to
1189, or a homolog or variant thereof.
Preferred nucleic acids encode a bacterial FabI protein comprising an amino
acid
sequence at least 50% homologous, more preferably 75% homologous and most
preferably 80%, 90%, or 95% homologous with an amino acid sequence shown in
one of
SEQ ID NO: 3. Nucleic acids which encode polypeptides having an activity of a
FabI
protein and having at least about 90%, more preferably at least about 95%, and
most
preferably at least about 98-99% homology with a sequence shown in SEQ ID NO:
2 are
within the scope of the invention.
Preferred nucleic acids encode a bacterial InhA protein comprising an amino
acid
sequence at least 50% homologous, more preferably 75% homologous and most
preferably 80%, 90%, or 95% homologous with an amino acid sequence shown in
one of
SEQ ID NO: 12. Nucleic acids which encode polypeptides having an activity of a
InhA
protein and having at least about 90%, more preferably at least about 95%, and
most
preferably at least about 98-99% homology with a sequence shown in SEQ ID NO:
11
are within the scope of the invention.
Another aspect of the invention provides a nucleic acid which hybridizes under
high stringency conditions to a "probe", which is a nucleic acid molecule
which binds
specifically to a nucleic acid molecule encoding an ER enzyme. A suitable
probe is at
least 12 nucleotides in length, is single-stranded, and is labeled, for
example,
radiolabeled or fluorescently labeled. Appropriate moderate stringency
conditions
which promote DNA hybridization, for example, 6.0 x sodium chloride/sodium
citrate
(SSC) at about 45°C, are followed by successive washes of increased
stringency, e.g.,
2.0 x SSC at 50°C, and are known to those skilled in the art or can be
found in Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
Other
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suitable stringency conditions include selecting the salt concentration in the
wash step
from a low stringency of about 2.0 x SSC at 50°C, and then using a wash
of a high
stringency condition, of about 0.2 x SSC at 50°C. In addition, the
temperature in the
wash step can be increased from low stringency conditions at room temperature,
about
22°C, to high stringency conditions at about 65°C. Exemplary
probes for DNA
sequencing and for PCR analysis of FabI are shown in SEQ ID NOs: 4-10.
Conditions for hybridizations are largely dependent on the melting temperature
that is observed for half of the molecules of a substantially pure population
of a double-
stranded nucleic acid, a parameter known as the Tm which is the temperature in
°C at
which half the molecules of a given sequence are melted or single-stranded.
For nucleic
acids of sequence 11 to 23 bases, the Tm can be estimated in degrees C as
2(number of
A+T residues) + 4(number of C+G residues). Hybridization or annealing of the
probe to
the nucleic acid being probed should be conducted at a temperature lower than
the Tm,
e.g., 15°C, 20°C, 25°C or 30°C lower than the Tm.
The effect of salt concentration (in
M of NaCI) can also be calculated, see for example, Brown, A., "Hybridization"
pp. 503-
506, in The Encyclopedia ofMolec. Biol., J. Kendrew, Ed., Blackwell, Oxford
(1994).
Fragments of the nucleic acids encoding ER proteins are within the scope of
the
invention. As used herein, a fragment of the nucleic acid encoding a portion
of a ER
protein refers to a nucleic acid molecule having fewer nucleotides than the
nucleotide
sequence encoding the entire amino acid sequence of ER protein but which
nevertheless
encodes a peptide having the biological activity, e.g., enoyl-ACP reductase
activity.
Nucleic acid fragments within the scope of the present invention include those
capable
of hybridizing under high stringency conditions with nucleic acids from other
species for
use in screening protocols to detect ER homologs and naturally occurring
polymorphic
alleles.
Useful expression control sequences, include, for example, the early and late
promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the
lac
system, the trp system, the TAC or TRC system, T7 promoter whose expression is
directed by T7 RNA polymerase, the major operator and promoter regions of
phage
lambda , the control regions for fd coat protein, the promoter for 3-
phosphoglycerate
kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g.,
PhoS, the
promoters of the yeast a-mating factors, the polyhedron promoter of the
baculovirus
system and other sequences known to control the expression of genes of
prokaryotic or
eukaryotic cells or their viruses, and various combinations thereof. A useful
translational enhancer sequence is described in U.S.P.N. 4,820,639.
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It should be understood that the design of the expression vector may depend on
such factors as the choice of the host cell to be transformed and/or the type
of protein
desired to be expressed. In one embodiment, the expression vector includes a
recombinant gene encoding a peptide having an activity of a ER protein. Such
expression vectors can be used to transfect cells and thereby produce proteins
or
peptides, including fusion proteins or peptides, encoded by nucleic acids as
described
herein.
As used herein, a "derivative" or "analog" of an antimicrobial compound (e.g.,
a
peptide) refers to a form of that compound in which one or more reaction
groups on the
compound have been derivatized with a substituent group (e.g., alkylated or
acylated
peptides). As used herein an "analog" of a compound refers to a compound that
retains
chemical structures necessary for functional activity yet that also contains
certain
chemical structures that differ. An example of an analog of a naturally-
occurring peptide
is a peptide that includes one or more non-naturally-occurring amino acids. As
used
herein, a "mimetic" of a compound refers to a compound in which chemical
structures
necessary for functional activity have been replaced with other chemical
structures that
mimic the conformation. Examples of peptidomimetics include peptidic compounds
in
which the peptide backbone is substituted with one or more benzodiazapine
molecules
{see e.g., James, G.L. et al., (1993) Science 260:1937-1942) and "retro-
inverso" peptides
(see U.S. Patent No. 4,522,752 by Sisto), described further below. A "residue"
refers to
an amino acid in a position in a peptide, or an amino acid mimetic
incorporated in the
peptide compound by an amide bond or amide bond mimetic. Approaches to
designing
peptide derivatives, analogs and mimetics are known in the art. For example,
see
Farmer, P.S. in Dru;~ Design (E.J. Ariens, ed.) Academic Press, New York,
1980, vol.
10, pp. 119-143; Ball. J.B. and Alewood, P.F. (1990) J. Mol. Recognition 3:55;
Morgan,
B.A. and Gainor, J.A. (1989) Ann. Rep. Med. Ckem. 24:243; and Freidinger, R.M.
( 1989) Trends Pharmacol. Sci. 10:270.
An "amino acid mimetic" refers to a moiety, other than a naturally occurring
amino acid, that conformationally and functionally serves as a substitute for
a particular
amino acid in a peptide-like compound without adversely interfering to a
significant
extent with the function of the compound (e.g., inhibition of ER). In some
circumstances, substitution with an amino acid mimetic may actually enhance
properties
of the inhibitor (e.g., interaction of the inhibitor with ER). Examples of
amino acid
mimetics include D-amino acids. Peptides substituted with one or more D-amino
acids
may be made using well known peptide synthesis procedures. The effect of amino
acid
substitutions with D-amino acids and other peptidomimetics can be tested using
assays
as described herein.
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The peptide analogs or mimetics of the invention include isosteres. The term
"isostere" as used herein refers to a sequence of two or more residues that
can be
substituted for a second sequence because the steric conformation of the first
sequence
fits a binding site specific for the second sequence. The term specifically
includes
peptide backbone modifications (i.e., amide bond mimetics) well known to those
skilled
in the art. Such modifications include modifications of the amide nitrogen,
the a-
carbon, amide carbonyl, complete replacement of the amide bond, extensions,
deletions
or backbone crosslinks. Several peptide backbone modifications are known,
including yr
[CHZS], yr[CH2NH], ~r[C(S)NH2], yr[NHCO], yr[C{O)CH2], and yr[CH=CH]. In the
nomenclature used above, yr indicates the absence of an amide bond. The
structure that
replaces the amide group is specified within the brackets. Other examples of
isosteres
include peptides substituted with one or more benzodiazapine molecules (see
e.g.,
James, G.L. et al. {1993) Science 260:1937-1942)
Other possible modifications include an N-alkyl (or aryl) substitution (~
[CONR]), backbone crosslinking to construct lactams and other cyclic
structures, or
retro-inverso amino acid incorporation (y[NHCO]). By "inverso" is meant
replacing L-
amino acids of a sequence with D-amino acids, and by "retro-inverso" or
"enantio-retro"
is meant reversing the sequence of the amino acids ("retro") and replacing the
L-amino
acids with D-amino acids. For example, if a parent peptide is Thr-Ala-Tyr, the
retro
modified form is Tyr-Ala-Thr, the inverso form is thr-ala-tyr, and the retro-
inverso form
is tyr-ala-thr using lower case letters to refer to D-amino acids. Compared to
the parent
peptide, a retro-inverso peptide has a reversed backbone while retaining
substantially the
original spatial conformation of the side chains, resulting in a retro-inverso
isomer with a
topology that closely resembles the parent peptide and is able to bind the
selected
cysteine protease. See Goodman et al. "Perspectives in Peptide Chemistry" pp.
283-294
( 1981 ). See also U.S. Patent No. 4,522,752 by Sisto for further description
of "retro-
inverso" peptides.
Pharmaceutical Compositions
The invention provides pharmaceutically acceptable compositions which include
a therapeutically-effective amount or dose of an antimicrobial compound, e.g.,
triclosan,
and one or more pharmaceutically acceptable carriers (additives) and/or
diluents. A
composition can also include a second antimicrobial agent, e.g., an inhibitor
of an efflux
Pump.
As described in detail below, the pharmaceutical compositions can be
formulated
for administration in solid or liquid form, including those adapted for the
following: (1)
oral administration, for example, drenches (aqueous or non-aqueous solutions
or
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suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral
administration,
for example, by subcutaneous, intramuscular or intravenous injection as, for
example, a
sterile solution or suspension; (3) topical application, for example, as a
cream, ointment
or spray applied to the skin; (4) intravaginally or intrarectally, for
example, as a pessary,
cream, foam, or suppository; or (5) aerosol, for example, as an aqueous
aerosol,
liposomal preparation or solid particles containing the compound.
The phrase "pharmaceutically-acceptable carrier" as used herein means a
pharmaceutically-acceptable material, composition or vehicle, such as a liquid
or solid
filler, diluent, excipient, solvent or encapsulating material, involved in
canying or
transporting the antimicrobial agents or compounds of the invention from one
organ, or
portion of the body, to another organ, or portion of the body without
affecting its
biological effect. Each carrier should be "acceptable" in the sense of being
compatible
with the other ingredients of the composition and not injurious to the
subject. Some
examples of materials which can serve as pharmaceutically-acceptable carriers
include:
(1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn
starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose,
ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6)
gelatin; (7)
talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut
oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil; (10)
glycols, such as propylene glycol; ( 11 ) polyols, such as glycerin, sorbitol,
mannitol and
polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13)
agar; (14)
buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15)
alginic
acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl
alcohol; (20) phosphate buffer solutions; and (21 ) other non-toxic compatible
substances
employed in pharmaceutical compositions. Proper fluidity can be maintained,
for
example, by the use of coating materials, such as lecithin, by the maintenance
of the
required particle size in the case of dispersions, and by the use of
surfactants.
These compositions may also contain adjuvants such as preservatives, wetting
agents, emulsifying agents and dispersing agents. Prevention of the action of
microorganisms may be ensured by the inclusion of various antibacterial and
antifungal
agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like.
It may also
be desirable to include isotonic agents, such as sugars, sodium chloride, and
the like into
the compositions. In addition, prolonged absorption of the injectable
pharmaceutical
form may be brought about by the inclusion of agents which delay absorption
such as
aluminum monostearate and gelatin.
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In some cases, in order to prolong the effect of a drug, it is desirable to
slow the
absorption of the drug from subcutaneous or intramuscular injection. This may
be
accomplished by the use of a liquid suspension of crystalline or amorphous
material
having poor water solubility. The rate of absorption of the drug then depends
upon its
rate of dissolution which, in turn, may depend upon crystal size and
crystalline form.
Alternatively, delayed absorption of a parenterally-administered drug form is
accomplished by dissolving or suspending the drug in an oil vehicle.
Pharmaceutical compositions of the present invention may be administered to
epithelial surfaces of the body orally, parenterally, topically, rectally,
nasally,
intravaginally, intracisternally. They are of course given by forms suitable
for each
administration route. For example, they are administered in tablets or capsule
form, by
injection, inhalation, eye lotion, ointment, etc., administration by
injection, infusion or
inhalation; topical by lotion or ointment; and rectal or vaginal
suppositories.
The phrases "parenteral administration" and "administered parenterally" as
used
herein means modes of administration other than enteral and topical
administration,
usually by injection, and includes, without limitation, intravenous,
intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,
intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular,
subarachnoid, intraspinal and intrasternal injection and infusion.
The phrases "systemic administration," "administered systemically,"
"peripheral
administration" and "administered peripherally" as used herein mean the
administration
of a sucrose octasulfate and/or an antibacterial or a contraceptive agent,
drug or other
material other than directly into the central nervous system, such that it
enters the
subject's system and, thus, is subject to metabolism and other like processes,
for
example, subcutaneous administration.
In some methods, the compositions of the invention can be topically
administered
to any epithelial surface. An "epithelial surface" according to this invention
is defined as
an area of tissue that covers external surfaces of a body, or which and lines
hollow
structures including, but not limited to, cutaneous and mucosal surfaces. Such
epithelial
surfaces include oral, pharyngeal, esophageal, pulmonary, ocular, aural,
nasal, buccal,
lingual, vaginal, cervical, genitourinary, alimentary, and anorectal surfaces.
Compositions can be formulated in a variety of conventional forms employed for
topical administration. These include, for example, semi-solid and liquid
dosage forms,
such as liquid solutions or suspensions, suppositories, douches, enemas, gels,
creams,
emulsions, lotions, slurries, powders, sprays, lipsticks, foams, pastes,
toothpastes,
ointments, salves, balms, douches, drops, troches, chewing gums, lozenges,
mouthwashes, rinses.
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Conventionally used carriers for topical applications include pectin, gelatin
and
derivatives thereof, polylactic acid or polyglycolic acid polymers or
copolymers thereof,
cellulose derivatives such as methyl cellulose, carboxymethyl cellulose, or
oxidized
cellulose, guar gum, acacia gum, karaya gum, tragacanth gum, bentonite, agar,
carbomer,
bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum,
hectorite, ispaghula
husk, polyvinypyrrolidone, silica and derivatives thereof, xanthan gum,
kaolin, talc,
starch and derivatives thereof, paraffin, water, vegetable and animal oils,
polyethylene,
polyethylene oxide, polyethylene glycol, polypropylene glycol, glycerol,
ethanol,
propanol, propylene glycol (glycols, alcohols), fixed oils, sodium, potassium,
aluminum,
magnesium or calcium salts (such as chloride, carbonate, bicarbonate, citrate,
gluconate,
lactate, acetate, gluceptate or tartrate).
Such compositions can be particularly useful, for example, for treatment or
prevention of an unwanted cell, e.g., vaginal Neisseria gonorrhea, or
infections of the
oral cavity, including cold sores, infections of eye, the skin, or the lower
intestinal tract.
I 5 Standard composition strategies for topical agents can be applied to the
antimicrobial
compounds, e.g., triclosan or a pharmaceutically acceptable salt thereof in
order to
enhance the persistence and residence time of the drug, and to improve the
prophylactic
efficacy achieved.
For topical application to be used in the lower intestinal tract or vaginally,
a
rectal suppository, a suitable enema, a gel, an ointment, a solution, a
suspension or an
insert can be used. Topical transdermal patches may also be used. Transdermal
patches
have the added advantage of providing controlled delivery of the compositions
of the
invention to the body. Such dosage forms can be made by dissolving or
dispersing the
agent in the proper medium.
Compositions of the invention can be administered in the form of suppositories
for rectal or vaginal administration. These can be prepared by mixing the
agent with a
suitable non-irritating carrier which is solid at room temperature but liquid
at rectal
temperature and therefore will melt in the rectum or vagina to release the
drug. Such
materials include cocoa butter, beeswax, polyethylene glycols, a suppository
wax or a
salicylate, and which is solid at room temperature, but liquid at body
temperature and,
therefore, will melt in the rectum or vaginal cavity and release the active
agent.
Compositions which are suitable for vaginal administration also include
pessaries, tampons, creams, gels, pastes, foams, films, or spray compositions
containing
such carriers as are known in the art to be appropriate. The carrier employed
in the
sucrose octasulfate /contraceptive agent should be compatible with vaginal
administration and/or coating of contraceptive devices. Combinations can be in
solid,
semi-solid and liquid dosage forms, such as diaphragm, jelly, douches, foams,
films,
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ointments, creams, balms, gels, salves, pastes, slunries, vaginal
suppositories, sexual
lubricants, and coatings for devices, such as condoms, contraceptive sponges,
cervical
caps and diaphragms.
For ophthalmic applications, the pharmaceutical compositions can be formulated
as micronized suspensions in isotonic, pH adjusted sterile saline, or,
preferably, as
solutions in isotonic, pH adjusted sterile saline, either with or without a
preservative
such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the
compositions
can be formulated in an ointment such as petrolatum. Exemplary ophthalmic
compositions include eye ointments, powders, solutions and the like.
Powders and sprays can contain, in addition to sucrose octasulfate and/or
antibiotic or contraceptive agent(s), carriers such as lactose, talc, silicic
acid, aluminum
hydroxide, calcium silicates and polyamide powder, or mixtures of these
substances.
Sprays can additionally contain customary propellants, such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as
butane and
propane.
Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or
suspension of the agent together with conventional pharmaceutically acceptable
carriers
and stabilizers. The Garners and stabilizers vary with the requirements of the
particular
compound, but typically include nonionic surfactants ('Tweens, Pluronics, or
polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters,
oleic acid,
lecithin, amino acids such as glycine, buffers, salts, sugars or sugar
alcohols. Aerosols
generally are prepared from isotonic solutions.
Compositions of the invention can also be orally administered in any orally
acceptable dosage form including, but not limited to, capsules, cachets,
pills, tablets,
lozenges (using a flavored basis, usually sucrose and acacia or tragacanth),
powders,
granules, or as a solution or a suspension in an aqueous or non-aqueous
liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as
pastilles (using
an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as
mouth
washes and the like, each containing a predetermined amount of sucrose
octasulfate
and/or antibiotic or contraceptive agents) as an active ingredient. A compound
may also
be administered as a bolus, electuary or paste. In the case of tablets for
oral use, Garners
which are commonly used include lactose and corn starch. Lubricating agents,
such as
magnesium stearate, are also typically added. For oral administration in a
capsule form,
useful diluents include lactose and dried corn starch. When aqueous
suspensions are
required for oral use, the active ingredient is combined with emulsifying and
suspending
agents. If desired, certain sweetening, flavoring or coloring agents may also
be added.
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Tablets, and other solid dosage forms, such as dragees, capsules, pills and
granules, may be scored or prepared with coatings and shells, such as enteric
coatings
and other coatings well known in the pharmaceutical-formulating art. They may
also be
formulated so as to provide slow or controlled release of the active
ingredient therein
S using, for example, hydroxypropylmethyl cellulose in varying proportions to
provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They
may be sterilized by, for example, filtration through a bacteria-retaining
filter, or by
incorporating sterilizing agents in the form of sterile solid compositions
which can be
dissolved in sterile water, or some other sterile injectable medium
immediately before
use. These compositions may also optionally contain opacifying agents and may
be of a
composition that they release the active ingredients) only, or preferentially,
in a certain
portion of the gastrointestinal tract, optionally, in a delayed manner.
Examples of
embedding compositions which can be used include polymeric substances and
waxes.
The active ingredient can also be in micro-encapsulated form, if appropriate,
with one or
more of the above-described excipients.
Liquid dosage forms for oral administration include pharmaceutically
acceptable
emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the
active ingredient, the liquid dosage forms may contain inert diluents commonly
used in
the art, such as, for example, water or other solvents, solubilizing agents
and emulsifiers,
such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,
benzyl alcohol,
benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed,
groundnut, corn, germ, olive, castor and sesame oils), glycerol,
tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and mixtwes thereof.
Besides inert diluents, the oral compositions can also include adjuvants such
as
wetting agents, emulsifying and suspending agents, sweetening, flavoring,
coloring,
perfuming and preservative agents.
Suspensions, in addition to the antimicrobial agents) may contain suspending
agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene
sorbitol and
sorbitan esters, microcrystalline cellulose, aluminum metahydroxide,
bentonite, agar
agar and tragacanth, and mixtwes thereof.
Sterile injectable forms of the compositions of this invention can be aqueous
or
oleaginous suspension. These suspensions may be formulated according to
techniques
known in the art using suitable dispersing or wetting agents and suspending
agents.
Wetting agents, emulsifiers and lubricants, such as sodium lawyl sulfate and
magnesium
stearate, as well as coloring agents, release agents, coating agents,
sweetening, flavoring
and perfuming agents, preservatives and antioxidants can also be present in
the
compositions.
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The sterile injectable preparation may also be a sterile injectable solution
or
suspension in a nontoxic parenterally-acceptable diluent or solvent, for
example as a
solution in 1,3-butanediol. Among the acceptable vehicles and solvents that
may be
employed are water, Ringer's solution and isotonic sodium chloride solution.
In
addition, sterile, fixed oils are conventionally employed as a solvent or
suspending
medium. For this purpose, any bland fixed oil may be employed including
synthetic
mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceride
derivatives are
useful in the preparation of injectables, as are natural pharmaceutically-
acceptable oils,
such as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil
solutions or suspensions may also contain a long-chain alcohol diluent or
dispersant,
such as Ph. Helv or similar alcohol.
The antimicrobial agent or a pharmaceutically acceptable salt thereof will
represent some percentage of the total dose in other dosage forms in a
material forming a
combination product, including liquid solutions or suspensions, suppositories,
douches,
enemas, gels, creams, emulsions, lotions slurries, soaps, shampoos,
detergents, powders,
sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms,
douches, drops,
troches, lozenges, mouthwashes, rinses and others. Creams and gels for
example, are
typically limited by the physical chemical properties of the delivery medium
to
concentrations less than 20% (e.g., 200 mg/gm). For special uses, far less
concentrated
preparations can be prepared, (e.g., lower percent formulations for pediatric
applications). For example, the pharmaceutical composition of the invention
can
comprise sucrose octasulfate in an amount of 0.001-99%, typically 0.01-75%,
more
typically 0.1-20%, especially 1-10% by weight of the total preparation. In
particular, a
preferred concentration thereof in the preparation is 0.5-50%, especially 0.5-
25%, such
as 1-10%. It can be suitably applied 1-10 times a day, depending on the type
and
severity of the condition to be treated or prevented.
Given the low toxicity of an antimicrobial agent or a pharmaceutically
acceptable
salt thereof over many decades of use as a biocide [W.R. Garnett, Clin. Pharm.
1:307-
314 (I982); R.N. Brogden et al., Drugs 27:194-209 (1984); D.M. McCartlly, New
EngJ
Med., 325:1017-1025 (1991), an upper limit for the therapeutically effective
dose is not a
critical issue. For most forms of triclosan the minimum amount present in the
materials
forming combinations of this invention that is effective in treating or
preventing bacterial
disease due to direct interaction with the organism should produce be less
than 0.1 pg
ml-1, less than 0.5 p,g ml-I, preferably less than 1 pg ml-1, even more
preferably less
than less than 5 p,g ml-I, and most preferably less than 10 p.g ml-1.
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For prophylactic applications, the pharmaceutical composition of the invention
can be applied prior to physical contact. The timing of application prior to
physical
contact can be optimized to maximize the prophylactic effectiveness of the
compound.
The timing of application will vary depending on the mode of administration,
the
epithelial surface to which it is applied, the surface area, doses, the
stability and
effectiveness of composition under the pH of the epithelial surface, the
frequency of
application, e.g., single application or multiple applications. Preferably,
the timing of
application can be determined such that a single application of composition is
sufficient.
One skilled in the art will be able to determine the most appropriate time
interval
required to maximize prophylactive effectiveness of the compound.
One of ordinary skill in the art can determine and prescribe the effective
amount
of the pharmaceutical composition required. For example, one could start doses
at levels
lower than that required in order to achieve the desired therapeutic effect
and gradually
increase the dosage until the desired effect is achieved. In general, a
suitable daily dose
of a composition of the invention will be that amount of the composition which
is the
lowest dose effective to produce a therapeutic effect. Such an effective dose
will
generally depend upon the factors described above. It is preferred that
administration be
intravenous, intracoronary, intramuscular, intraperitoneal, or subcutaneous.
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of cell biology, cell culture, molecular biology,
microbiology,
recombinant DNA, and immunology, which are within the skill of the art. Such
techniques are explained fully in the literature. See, for example, Molecular
Cloning A
Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al. (Cold Spring Harbor
Laboratory
Press (1989)); Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel,
F. et al.
(Wiley, NY (1995)); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);
Oligonucleotide Synthesis (M. J. Gait ed. (1984)); Mullis et al. U.S. Patent
No:
4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.
(1984)); the
treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical
Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,
London (1987)); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir
and C. C. Blackwell, eds. (1986)); and Miller, J. Experiments in Molecular
Genetics
(Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1972)).
The invention is further illustrated by the following examples, which should
not
be construed as further limiting. The contents of all references, pending
patent
applications and published patents, cited throughout this application are
hereby
expressly incorporated by reference.
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EXAMPLES
The following methodology described in the Materials and Methods section was
used throughout Examples 1-9.
Materials and Methods
Isolation of triclosan resistant mutants
All experiments were performed at 37oC using LB broth or agar (Ausubel et al.
supra) (Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel, F. et
al. (Wiley,
NY (1995)). Independent cultures of an E. coli K12 strain, AG100 (George, A.M.
&
Levy, S.B. J. Bacteriol. 155, 531-540 (1983)) were grown overnight to
stationary phase
at 37oC and 108 colony-forming-units (cfu) from each culture were plated onto
agar
containing 0.2 or 0.3 ~g ml-1 triclosan having the structure 2,4,4'-trichlor-
2'-
hydroxydiphenyl ether, CAS # 3380-34-5 (trade name Irgasan~ DP300, Ciba
CH3565,
available from Ciba Specialty Chemicals Corp., Greensboro, NC; stock solutions
dissolved in ethanol).
After incubation for 24-48 h, one resistant colony from each of six cultures
was
purified on agar containing triclosan. Resistance to triclosan was quantitated
using serial
dilution plates with 2.0 fold steps of increasing concentrations of triclosan.
Five ~,1 of
log phase cells containing approximately 4 x 104 cfu was applied as a spot to
the
dilution plates. The lowest triclosan concentration which inhibited growth
after 20 h
defined the minimal inhibitory concentration (MIC). Inhibition of growth rate
was
determined in broth culture by adding triclosan at various concentrations to
log phase
cells which had reached an absorbance (A530) of 0.1 and determining the effect
on the
rate of change of absorbance 1 h later; lysis was identified by a loss of
absorbance (about
50%) accompanied by a 4-5 log loss in viable cfu per A530 umt.
A chromosomal library was prepared from mutant AGT11 by cloning 1-7kb
Sau3aI partial digestion fragments into the BamHI site of the tet gene in
pBR322,
transforming into strain DHSa (GibcoBRL, Bethesda MD), and selecting on
ampicillin
(Sigma, St. Louis, MO). Approximately 16,000 transformants were pooled to form
the
library, and the clones encoding triclosan resistance were found by plating
about 80,000
cfu from the library on 0.3 pg ml-1 triclosan.
Other methods and strains.
Chromosomal DNA was prepared using a puregene kit (Gentra Systems,
Minneapolis, MN). PCR products of AGT23 and AGT25 were generated for
sequencing
using Taq DNA polymerise (Gibco) and oligonucleotide pairs LMO11, SEQ ID No:
4,
and LMO10, SEQ ID No. 5 (respectively, nt 160-179 and 1168-1149). The
numbering
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system for fabl of Bergler, H., et al. (J. Gen. Microbiol. 138, 2093-2100 (
1992)), in
which the fabl gene is nt 404-1189; see SEQ ID No. 1) was used. Other
sequencing
primer pairs were LM019, SEQ ID No: 6, and LM020, SEQ ID No: 7 (respectively,
nt
1291-1275 and 74S-762). The same oligonucleotides were used for sequencing the
products. Junctional DNA in pLYT6 and pLYT8 was sequenced using
oligonucleotide
BR346, SEQ ID No: 9 (nt 346-357 in pBR322, in which the BamHI site is at nt
375 (see
the catalog of New England Biolabs, Beverly, MA). The fabl gene in pLYT8 was
sequenced using LMO10, LM019, and LMO11. pLYT27 was sequenced using LMO15
(nt 875-856; see SEQ ID No: 8) and LM021, SEQ ID No: 10 (in pBR322, nt 4068-
4086).
Strain JZM 120 (~arcrAB: : kan; Ma, D., et al. Molec. Microbiol. 16, 45-55 (
1995))
(from H. Nikaido) served as the donor strain for bacteriophage P1-mediated
transduction
(Provence, D.L. & Curtiss, R.I. in Methods for General and Molecular
Bacteriology eds.
Gerhardt, P., Murray, R.G.E., Wood, W.A. & Kreig, N.R. 317-347 American
Society
for Microbiology, Washington, D. C., (1994); Miller, J. Experiments in
Molecular
Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1972)) in the
inactivation of the acrAB locus in other strains. Strain AG 100/kan
(OmarCORAB;
Maneewannakul, K., et al. Antimicrobial Agents Chemother. 40, 1695-1698
(1996))
similarly was used to inactivate the mar locus. Because of the reported low
aqueous
solubility of triclosan (10 mg ml-1; Ciba-Geigy, Irgasan~ DP300 material
safety data
sheet No. 235 (1996)), some MIC experiments were performed in hypersusceptible
host
AG100A (which is AG100 DacrAB::kan) to reduce the triclosan concentration
required.
Strain AGT 11 K is AGT 11 ~arcrAB: : kan.
EXAMPLE 1
Isolation of Mutants Resistant to Triclosan
A genetic approach was used to find the mechanism of triclosan action in
Escherichia toll. Mutants resistant to triclosan were isolated and then the
resistance
locus was cloned and identified. The roles of the AcrAB multidrug ei~lux pump
and of
its positive regulator MarA in the susceptibility of strains to triclosan were
then
investigated.
Six independent triclosan resistant mutants of E. toll K12 strain AG100 were
isolated as described in Methods. The MICs ranged from 1.7 to 145 times the
0.28 ~,g
ml-1 MIC of the parental strain (Table 1, MIC column 1). Further, triclosan-
resistant E.
toll strain AGT11 had several times the isoniazid resistance of the isogenic
parent
AG100 (determined in the presence of 250 ~,M hydrogen peroxide to reduce the
inherently high resistance of E. toll to isoniazid).
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EXAMPLE 2
The role of Two Loci acrAB and marRAB in Triclosan Resistance
The acrAB operon in Escherichia coli encodes a multidrug efflux pump which
provides intrinsic resistance to many diverse compounds including antibiotics
and
disinfectants (Nikaido, H. J. Bacteriol. 178, 5853-5859 (1996)). This operon
can be up-
regulated by MarA (Ma, D., et al. Molec. Microbiol. 16, 45-55 (1995), a
transcriptional
activator encoded by the marRAB operon involved in multiple antibiotic
resistance
(Alekshun, M.N., et al. Antimicrob. Agents Chemother. 41, 2067-2075 (1997)).
Mar mutants overexpressing the mar operon were twice as resistant to triclosan
as the parental strain AG100. In the mutants selected on triclosan,
inactivation of
marRAB had little effect upon triclosan resistance (Table 1, MIC column 2) in
comparison to these mutants having marRAB+ activity (Table 1, MIC column 1).
Table 1
Strain Mutation MIC of strain
in divided
fabl by MIC of
AG1000*
none* * mar* * acrAB*
AG 100 none 1 0.7 i 0.063
AGT7 NI 1.7 1.7 0.071
AGT8 NI 4 3.4 0.25
AGT9 NI 2.3 2.3 0.32
AGTll G93V 145 145 11.4
AGT23 M159T 11.4 ND 1.7
AGT25 F203L 4.6 ND 0.57
nC~anve mclosan resistance of mutants selected upon triclosan and effect of
inactivation
of the marRAB and acrAB loci. Minimal inhibitory concentrations (MICs) were
determined in duplicate on the complete set of strains by the agar dilution
technique as
described in Methods, and the mean values are presented as ratios to the MIC
of wild
type strain AG100. The greatest average deviation from the mean, seen for one
strain,
was 33%. NI, not identified, mutation in fabl based on P1 transduction
experiments
*The MIC of AG100 was 0.28 ~ 0.04 ~g ml-1
**Inactivated locus
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Inactivation of acrAB increased the susceptibility of all strains ( including
that of
the triclosan susceptible parent AG100) approximately 7 - 24 fold (Table 1,
MIC column
3). Increased triclosan resistance of fabl acrAB mutants was observed compared
to
acrAB inactivated in the fabl+ AG 100. The AcrAB multidrug pump was an
effective
exporter of triclosan but was not the basis of the enhanced resistance in the
fabl mutants.
Loss of the AcrAB multidrug efflux pump presumably permits a greater
concentration of triclosan within the cytoplasm of the cell, where FabI is
located
(Cronan, J.E., Jr. & Rock, C.O. in Escherichia coli and Salmonella: Cellular
and
Molecular Biology (ed. Neidhardt, F.C.) 612-636 (ASM Press, Washington, DC,
1996)),
resulting in the observed increase in susceptibility of cells to the drug.
EXAMPLE 3
Transduction and Cloning of Triclosan Resistance
The triclosan resistance phenotype of mutant AGT11 could be transduced to
recipient strain AG100A using P1 phage (Provence, D.L., et al. Methods for
General
and Molecular Bacteriology, eds. Gerhardt, P., et al. pp. 317-347, American
Society for
Microbiology, Washington, D. C., 1994); Miller, J. Experiments in Molecular
Genetics
(Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1972)), indicating that
the
mutation conferring the resistance phenotype might lie in one clonable locus.
A genomic Sau3AI library from strain AGT11 was prepared in plasmid
pBR322, and transformed into strain DHSa (see Methods). Clones mediating
triclosan
resistance were obtained in the library at a frequency of about 1 in 2500
transformants.
Ten clones, named pLYTI through pLYTlO, were isolated and screened. The
plasmids
isolated from these clones bore inserts of various sizes. Digestion of
plasmids with
HindIII and SphI revealed that all plasmid clones had a fragment of
approximately 1530
bp. All clones gave the same MIC (about 4 p,g ml-1, measured in hypersensitive
strain
AG100A), compared to 0.005 - 0.02 pg ml-1 for the vector alone in the same
host. The
level of resistance by the mutation present as a single copy on the chromosome
(for
example, the MIC of strain AGT11K) was 2 to 4 ~,g ml-1.
EXAMPLE 4
Identification of the Triclosan Resistance Gene by Sequence
The functional DNA sequences present in two clones with inserts of different
sizes, pLYT6 and pLYTB, were obtained using a pBR322 primer. The sequences
were
compared to those deposited in the E.coli genomic database. The sequence data
and the
sizes of the inserts showed that each insert bore the fabl gene together with
an upstream
putative open reading frame ycjD (Fig. 1, pLYTB). The 1530 by fragment
possessed by
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all tested clones proved to be a HindIII fragment extending from the HindIII
site in the
vector to a HindIII site in the middle of fabl. The inserts in these clones
may all have
had the same orientation (that of the tet gene in the vector).
To see which gene, ycjD or fabl, was able to confer triclosan resistance, a
BsmI
fragment containing half of fabl was deleted from pLYTB, producing plasmid
pLYT 11
(Fig. 1 ). This deletion produced loss of triclosan resistance (Fig. 1 ).
Further, an SspI
fragment which included the tet promoter and all of ycjD was deleted from
pLYT8,
producing plasmid pLYTl2 (Fig. 1 ). This deletion had no effect on triclosan
resistance
(Fig 1 ). These results show that triclosan resistance was conferred by fabl
gene.
Further, transcription from the tet promoter was not required for expression
of triclosan
resistance.
EXAMPLE 5
Substitution Mutation in fabl cause Triclosan Resistance
The fabl gene encodes enoyl ACP reductase, an enzyme involved in the
synthesis of fatty acids (Cronan, J.E., Jr., et al. in Escherichia coli and
Salmonella:
Cellular and Molecular Biology, ed. Neidhardt, F.C., 612-636 ASM Press,
Washington,
DC, (1996)) which reduces a double bond using NADH or NADPH (Bergler, H., et
al.
Eur. J. Biochem. 242, 689-694 (1996)). To determine if mutations were present
in fabl,
the entire fabl gene of pLYT8 from residues 190 to 1260 was sequenced
{residues are
identified using the numbering system of Bergler (Bergler, H., et al. J. Gen.
Microbiol.
138, 2093-2100 (1992)), including the upstream "BoxC" region (Bergler, H., et
al. J.
Gen. Microbiol. 13 8, 2093-2100 ( 1992)).
The sequence obtained was compared to that of fabl in the database (shown in
SEQ ID NO: l, from nt 404-1189). Codon 93 in fabl was found to have mutated
from
ggt to gtt, thereby substituting the wild type glycine at residue 93 of the
fabl protein
(SEQ ID N0:2) with valine in the mutant enzyme.
EXAMPLE 6
Demonstration of sly 93va1 Mutation Role in Triclosan Resistance by Backcross
To determine whether the mutation is the cause of resistance, or whether it is
a
mere sequence variant unique to strain AG100 and that pLYT8 resistance was due
to the
presence of a wild-type fabl gene in multicopies on the plasmid, a "backcross"
of wild-
type DNA into the mutant was performed. This cross tests whether the real
chromosomal mutation leading to triclosan resistance in mutant strain AGT11
had been
identified. The mutation gly93va1 affects the same residue as the gly93ser
mutation in
the FabI protein which was shown to cause resistance to the heterocyclic
inhibitor
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diazaborine (Bergler, H., et al. J. Gen. Microbiol. 138, 2093-2100 (1992);
Turnowsky,
F., et al. J. Bacteriol. 171, 6555-6565 (1989)).
The mutation-bearing 606 by SspI-HindIII fragment of pLYTl2 was replaced
with the wild type Sspl HindIII counterpart from a PCR product of chromosomal
DNA
S from parental strain AG100. The 606 by region of the resulting plasmid,
pLYT27, was
sequenced to confirm that the DNA derived from AG100 in fact carried the wild
type
sequence identical to that in the database. The triclosan MIC measured for
pLYT27 in
host AGI OOA was 20-30 fold greater than that for vector pBR322 itself,
showing a clear
multicopy effect. However, this increase in resistance was notably less than
the 280-340
fold increased MIC measured for pLYT8 and pLYTI2, the plasmids bearing the
gly93val mutation {Fig. 1 ). Therefore the gly93val mutation was responsible
for
triclosan resistance in the original mutant AGTI I, as its replacement with
the wild-type
allele confered triclosan sensitivity.
How triclosan might inhibit FabI is informed by studies on diazaborine, a
boron-
1 S containing, heterocyclic inhibitor of E. coli and Salmonella typhimurium
FabI.
Diazaborine resistance results from a gly93ser mutation (Bergler, H., et al.
J. Gen.
Microbiol. 138, 2093-2100 (1992); Turnowsky, F., et al. J. Bacteriol. 171,
6555-6565
(1989)), similar to the gly93val mutation shown here to cause a high level of
triclosan
resistance. In the wild type FabI enzyme, binding of diazaborine is dependent
upon the
presence of the cofactor NAD(H) (Kater, M.M., et al. Plant Molec. Biol. 25,
771-790
(1994); Bergler, H., et al. J. Biol. Chem. 269, 5493-5496 (1994)). The
gly93ser
mutation reduces the binding of diazaborine to the enzyme (Bergler, H., et al.
J. Biol.
Clrem. 269, 5493-5496 (1994)) and also results in lowered specific activity of
the
enzyme (Bergler, H., et al. Eur. J. Biochem. 242, 689-694 (1996)).
Of the triclosan-resistant fabl mutants isolated here, the growth rate in
broth of
mutant AGT11 was about 40% less, and that of mutant AGT23 about 15% less, than
that
of the wild type parent. These data show that the FabI enzyme in the mutants
is a less
active enzyme than that of the fabl+ parent.
EXAMPLE 7
Seauences of fabl in Other Triclosan Resistant Mutants
PCR products of the entire fabl gene of two other triclosan resistant mutants,
AGT23 and AGT25, were synthesized using chromosomal DNA as template. The
sequence of the PCR product of strain AGT23 revealed a single point mutation
(atg
became acg), leading to replacement of methionine 159 by threonine. Strain
AGT25 had
a single point mutation (ttc became ctc), leading to replacement of
phenylalanine 203 by
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leucine. Thus, mutations in fabl were responsible also for the triclosan
resistance
phenotype of strains AGT 11, AGT23, and AGT25.
Mutations at residues 93, 159, and 203 led to triclosan resistance,
correlating
with the recently-determined crystal structure of wild type E. coli FabI
protein (Baldock,
C., et al. Science 274, 2107-2110 ( 1996)). This structure shows that these
three residues
line the cleft of FabI in which NAD+ (and diazaborine) bind. The structure
also shows
NAD+ and diazaborine covalently linked to each other via the boron of the
latter.
Triclosan, diazaborine and isoniazid can interact in a related manner with
enoyl-
ACP reductases as indicated by the following facts. InhA, the gene encoding
enoyl-ACP
reductase of Mycobacterium tuberculosis, has 40% sequence identity with E.
coli FabI
(Banerjee, A., et al. Science 263, 227-230 (1994)). A mutation of serine 94 to
alanine is
associated with isoniazid resistance in both M. smegmatis and M. tuberculosis
(Banerjee,
A., et al. Science 263, 227-230 (1994)). In this organism the crystal
structures of both
the wild type and mutant InhA proteins were determined, showing that they have
different conformations in the NAD binding site near amino acid residue 94,
leading in
the mutant to decreased affinity for NAD, and thus for the inhibitor (Dessen,
A., et al.
Science 267, 1638-1641 (1995)).
Further, triclosan-resistant E. coli strain AGT11 had several times the
isoniazid
resistance of the isogenic parent AG100 (determined in the presence of 250 pM
hydrogen peroxide to reduce the inherently high resistance of E. coli to
isoniazid).
Although M. smegmatis is susceptible to triclosan, M. tuberculosis is not
sensitive
(Vischer, W.A., et al. Zbl. Bakt. Hyg., 1. Abt. Orig. A 226, 376-389 (1974)).
The related crystal structure for another homologous enoyl reductase, that of
the
rape seed oil plant, Brassica napus, has also been determined (Rafferty, J.B.,
et a1
Structure 3, 927-938 (1995)). Diazaborine and triclosan both have two
unsaturated rings
but otherwise are structrualIy different, and isoniazid has a single ring. Two
of these
structures can covalently bind with NADH when present together in the ER site
for
reducing agents.
EXAMPLE 8
Chromosomal Manning of Triclosan Resistance to Min 28 5
Linkage of the triclosan resistance locus in three unsequenced mutants AGT7,
AGT8, and AGT9 (and in the sequenced mutants AGT1 l and AGT23 provided as
controls) was used to map this gene to min 28.5, the location of fabl. This
was done
using P1 transduction ofzci-3118::TnlOkan at approximately min 28.5 (Singer,
M., et
al. Microbiol. Rev. 53, 1-24 (1989)), from a wild type donor strain into each
of the
mutants. Of 10 kanamycin resistant transductants analysed for each mutant, 3
to 6 had
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acquired triclosan sensitivity. These data support triclosan resistance being
due to
mutations in the fabl gene.
These findings together suggested that triclosan most likely acts upon wild
type
FabI, thereby inhibiting synthesis of fatty acids and consequently of lipids,
lipopolysaccharides, and membranes, leading to decreased growth.
EXAMPLE 9
Lysis of Cells by Triclosan Occurs at Higher Concentrations than Inhibition of
the
Fabl Primarx Target
Triclosan lyses cells of E. coli (Regos, J., et al. Zbl. Bakt. Hyg.,1. Abt.
Orig. A
226, 390-401 (1974)) and Porphyromonas gingivalis (Cummins, D. J. Clin.
Periodont.
18, 455-461 ( 1991 )).
Triclosan here was found to cause a loss of absorbance in broth cultures of
growing susceptible E. coli, accompanied by a decrease in recoverable viable
cells due
to cell lysis, at concentrations of triclosan higher than those which affected
the growth
rate. To inhibit the growth rate 50%, about 0.15 p,g ml-1 triclosan was
required for wild
type strain AG100, about 0.02 p,g ml'1 triclosan for AGl00A (deleted of
acrAB), and
about 1 p,g ml-1 for triclosan-resistant mutant derivative AGT11K (gly93va1,
otherwise
isogenic to AG 1 OOA). On the other hand, the amount of triclosan required to
give lysis
was 2-8 p.g ml-1 for these strains. However, strain AGT11 fabl gly93val,
otherwise
isogenic to AG100, did not display lysis even when triclosan up to a level of
256 pg
ml-1 was added. This indicates that FabI is involved in protection from cell
lysis even at
high concentrations of triclosan. Therefore mutations to triclosan resistance
in fabl
affect both the FabI inhibitory activity of this agent and also its lysis
activity in bacteria.
These data indicate that cells that are currently resistant to triclosan can
be made
susceptible to lysis by use of one or more additional agents specific for an
efflux pump.
Further, as the data in Table 1 show that acrAB deletions can restore
triclosan
sensitivity and lysis to the fabl mutants, cells that are currently resistant
to triclosan can
be made susceptible to lysis by use of one or more agents that can inactivate
acrAB.
EXAMPLE 10
Isolation and characterization of mutants of M smegmmatis selected for
resistance to
triclosan or to isoniazid.
Three Mycobacterium smegmatis mutants were selected for resistance to
triclosan and were found to have different mutations in InhA, an enoyl
reductase
involved in fatty acid synthesis. Isoniazid resistance accompanied triclosan
resistance
for the Metl61 Val mutation and to a lesser extent for A1a124Va1, but not for
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Met103Thr. A Ser94Ala mutation originally selected on isoniazid also mediated
triclosan resistance, as did the wild type inhA eliminated resistance. These
results
suggest that M. smegmatis InhA, like its Escherichia toll homolog FabI, is a
target for
triclosan.
M. smegmatis strain mc2155 was grown in LB broth or 7H9 medium (see legend
to Table 1 ) to stationary phase and approximately 108 colony-forming units
were plated
onto LB agar (without Tween 80 or glycerol) containing 0.8-1.6 ~,gml-1
triclosan (a
trichlorinated diphenyl ether, from Ciba-Geigy Corp., Greensboro, NC). After a
3 day
incubation, the largest of the 20-200 colonies of various sizes which appeared
per plate
were selected. Three independent mutants, MT 1, MT9, and MT 17, were chosen
for
study. Each was 4-6 times more resistant to triclosan than was the parental
strain (Table
2). Mutant MT1 manifested considerable resistance to isoniazid, MT17 less, and
MT9
none (Table I ). Mutant mc2651 (from W.R. Jacobs, Jr.), which has the S94A
substitution in InhA (Banerjee, A. et al. 1994. Science 263:227-230), as
expected
showed isoniazid resistance. In addition, it had a 4-6 fold triclosan
resistance (Table 2).
The wild type M. smegmatis inhA gene on multicopy plasmid pMD31::inhA+ (an
unpublished KanR E. toll-mycobacterial shuttle plasmid derived by subcloning a
3kb
BamHI fragment including orfl-inhA-orf3 into pMD31 (Donnelly-Wu et al. 1993.
Mol.
Microbiol. 7:407-417); gift of L. Miesel) caused resistance to triclosan and
isoniazid
(Table 1 ), likely related to target overexpression. These data suggested that
M.
smegmatis InhA is a target for triclosan.
EXAMPLE 11
Substitution of wild type inhA for mutant inhA
If a mutation in inhA were responsible for both the triclosan and isoniazid
resistance, homologous replacement of the mutant inhA chromosomal gene with a
wild
type inhA gene would eliminate the resistances. The method employed pYUB325
(Miesel, L. et al. 1998. J. Bacteriol. 180-2459-2467), from W.R. Jacobs,
Jr.,), a shuttle
cosmid containing a large PacI restriction fragment from the mc2155 genome.
Within
this fragment are the wild type inhA+ gene and a nearby kanamycin resistance
gene
insert. pYUB325 (prepared from E. toll host STBL-2 [Gibco/BRL]) was digested
with
PacI and extracted with phenol/chloroform. Cells in logarithmic phase in LB
broth/0.2%
Tween 80 were chilled on ice for 1.Shr and pelleted at 4°C. The pellets
were resupended
gently in 0.2 vol of cold 10% glycerol/ 0.1 % Tween 80, and then I 0% glycerol
was
added up to 1 vol. Cells were pelleted and the resuspension and washing
process
repeated once, with final resuspension in 0.01 vol of glycerol/Tween 80.
Electroporation
was performed using 0.1 ml cell suspension with 0.2 pgDNA in 0.2 cm chilled
cuvettes
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at 2.5 kV, 25 p.F, 100052. Then 1 ml LB broth/0.5% Tween 80 was added, the
cells
grown fro 4-l6hr, plated on LB agar containing 15 ~g ml-1 kanamycin, and
incubated 4-
6 days.
Four kanamycin resistant transformants of each mutant were assayed for drug
susceptibility by agar dilution. All four transformants of mutant MT9, three
of both MTI
and mc2651, and one of MT17 had lost both triclosan and any isoniazid
resistance. The
rest retained the parental resistance phenotype. These results are compatible
with the
expected frequency of 30-70% for coinheritance of inhA+ and KanR (Miesel, L.
et al.
1998. J. Bacteriol. 180:2459-2467). Therefore the mutant inhA gene, or a gene
very
closely linked to it, had been responsible for both resistances in each
mutant.
EXAMPLE 12
DNA sequence in inhA gene from mutants
The inhA gene in each of the three triclosan-selected mutants was sequenced.
Chromosomal DNA was prepared as described (Ausubel, F.M. et al. 1996. Current
Protocols in Molecular Biology, vol 1 John Wiley Sons, p. 2.4.1.) using a 2 hr
preliminary incubation at 37°C of cells with 4 mg ml-1 lysozyme.
Polymerise chain
reaction (PCR) of the entire inhA gene was performed for each mutant using Taq
DNA
polymerise (Gibco/BRL) at 2 mM Mgr in EasyStart reaction tubes (Molecular Bio-
Products). Primers LM026 (forward): S'-AAAGCCCGGACACACAAGA-3') (SEQ ID
NO: 13) and LM027 (reverse): 5'-CGAACGACAGCAGTAGCAAG-3' (SEQ ID
N0:14) were chosen from sequences bracketing inhA (see GenBank accession
number
(I73544) using the PRIME program of GCG and were annealed at 52°C. Both
strands of
the resulting 890 by PCR product were sequenced (Tufts Core Facility) using
the same
two primers.
The inhA structural gene of each mutant differed by a single nucleotide from
the
wild type sequence (GenBank accession number U02530). Together with the other
results, this finding proved that a mutated inhA gene was responsible for the
triclosan
resistance in each mutant. Mutant MT1 had replacement of methionine 161 (ATG)
by
valine (GTG), mutant MT9 had replacement of methionine 103 (ATG) by threonine
(ACG), and mutant MT17 had replacement of alanine 124 (GCG) by valine (GTG).
EXAMPLE 13
InhA Mediates Triclosan Resistance in M smegmatis
All three of the M. smegmatis InhA residues mutated in the present study, like
those in FabI of triclosan-resistant E. coli (McMurry, L.M. et al. 1998.
Nature 394:531-
532), lie close to the NADH cofactor and putative acyl substrate binding sites
(observed
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using the program STING (Neshich, G.R. et al. 1998. Submitted to Protein Data
Bank
Quarterly Newsletter 84.) with M. tuberculosis InhA [Protein Data Base lENY].
Triclosan might, like isoniazid (Rozwarski, D.A. 1998. Science 279:98-102) and
diazaborine (Baldock, C. et al. 1996. Science 274:2107-2110), bind covalently
to
NADH. Resistance then might be explained, as for isoniazid (Basso, L. A. et
al. 1998. J.
Infect. Dis. 178:769-775) (Dessen, A. et al. 1995. Science 267:1638-1641)
(Rozwarski,
D.A. et al. 1998. Science 278:98-102), by reduced binding of NADH to the
enzyme. In
this regard, replacement of methionine 161, near the amino terminus of helix
A5, by
valine in M. smegmatis InhA leads to triclosan/isoniazid resistance.
Replacement of the
equivalent diazaborine-interacting (Baldock, C. et al. 1996. Science 274:2107-
2110)
methionine 159 of E. coli FabI by threonine led to triclosan, but not
diazaborine,
resistance (McMurry, L.M. et al. 1998. Nature 394:531-532). These
substitutions may
interfere with the hydrogen bond to NADH formed by the conserved lysine 165
one
helical turn away (Baldock, C. et al. 1996. Science 274:2107-2110; Dessen, A.
et al.
1995. Science 267:1638-1641; Rafferty, J.B. et al. 1995. Structure 3:927-938).
Near
methionine 161 in InhA is methionine 103, located in the loop connecting
strand B4 to
helix A4. Its replacement by threonine conferred only triclosan resistance.
The third
altered residue, alanine 134, is in the middle of helix A4, near but facing
away from
NADH. Since this residue seems to lie outside the putative active site, the
resistance
caused by substitution of a more bulky valine may occur by an indirect
allosteric effect.
Steric interference with binding of diazaborine to the putative fatty acyl
substrate
binding site of E. coli FabI has been suggested as the resistance mechanism
for the G93 S
mutation (Baldock, C. et al. 1996. Science 274:2107-2110). Whether or not
triclosan
binds to NADH, this hydrophobic molecule might block fatty acyl substrate
binding.
M. smegmatis is suseptible to triclosan whereas M. tuberculosis is not
(Vischer,
W.A. et al. 1974. Zbl. Bakt. Hyg., I. Abt. Orig. A 226:376-389). The four
residues in M.
smegmatis InhA which influence triclosan resitance, S94, M103, A124, and M161,
are
conserved in M. tuberculosis. They would not, therefore, identify any residues
unique to
M. tuberculosis InhA which might account for the intrinsic resistance. On the
other
hand, that resistance may be due to mechanisms unrelated to InhA, such as the
activity
of endogenous efflux pumps) analogous to those which operate on triclosan in
other
organisms (McMurry, L.M. et al. FEMS Microbiol. Lett.; Schweizer, H.P. 1998.
Antimicrob. Agents Chemother. 42:394-398).
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Table 2. Characteristics of strains of Mycobacterium smegmatis
Strain Characteristics (reference) Inha relative MIC (S.D~
mutation triclosan triclosan isoniazid
LB 7H9 7H9
mc2155 wild type (see Meise) et al none 1.0 1.0 1.0
1998. 3. Bacteriol. 180:2459)
MTI mc2155 selected on triclosan M161V 4.9(0.9) 6.3(2.0) 8.5(2.5)
(this work)
MT9 mc2155 selected on triclosan M103T 4.4(1.1) 6.3(2.0) 1.2(0.5)
(this work)
MT17 mc2155 selected on triclosan A124V 4.0(1.2) 5.8(1.7) 2.0(0.7)
(this work)
mc2651 mc2155 selected on isoniazid S94A 4.4(1.3) 6.3(2.0) 22(12)
(See Banerjee et al. 1994. Science. 263:227).
mc2155/ mc2155 bearing multicopy none 4.6(0.6) 6.3(2.0) >64
pMD3l::inhA+ inhA+(see text)
Table 2. Minimal inhibitory concerntrations (MICs) are expressed as ratios to
the MIC
of M. smegmatis mc2155. All MICs wre determined on agar plates by 2-fold
serial
dilutions using logarithmic phase cells as described (McMurry, L.M. et al.
1998. Nature
394:531-532). Cells were grown with 0.05% Tween 80 either in LB broth or in
7H9
medium supplemented with ADC plus 0.2% glycerol and were tested on the
corresponding solid media without Tween 80. All plates with triclosan also
contained
0.1 % ethanol. Less clumping of cells during growth was seen in 7H9 than in
LB, but the
MIC for mutants in 7H9 agar approached the solubility limit of triclosan in
this medium
(50-100 p,g ml'1, observed visually). Results are means (+/- standard
deviation [S.D.])
of 4-5 experiments. The MICs for mc2155 (in pg ml' 1 ) were: triclosan in LB,
0.61 (+/-
0.15); triclosan in 7H9, 14 (+/-5); isoniazid in 7H9, 7 (+/-2).
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EXAMPLE 14
Overexoression of the multidrug efflux pump locus acrAB, or of mar A or soxS
both encodine uositive regulators of acrAB, decreased susceutibility to
triclosan 2
fold.
Deletion of the acrAB locus increased the susceptibility to triclosan
approximately 10-fold. Four of five clinical E. coli strains which
overexpressed mar A
or soxS also showed enhanced triclosan resistance. The acrAB Iocus was
involved in the
effects of triclosan upon both cell growth rate and cell lysis.
Triclosan inhibits the synthesis of lipids in Escherichia coli, presumably by
action upon FabI, an enoyl reductase required for the synthesis of fatty acids
(McMurry
et al. (I998) Triclosan targets lipid synthesis. Nature 394, 531-532). At
higher
concentrations, triclosan also causes cell lysis (McMurry et al. (1998)
Triclosan targets
lipid synthesis. Nature 394, 531-532; Regos et al. (1974) Investigations on
the mode of
action of triclosan, a broad spectrum antimicrobial agent. Zbl. Bakt. Hyg., 1.
Abt. Orig. A
226, 390-401). AcrAB is a multidrug efflux pump in E. coli (Nikaido, H. (1996)
Multidrug efflux pumps of Gram-negative bacteria. J. Bacteriol. 178, 5853-
5859;
Okusu et al. (1996) AcrAB efflux pump plays a major role in the antibiotic
resistance
phenotype of Esherichia coli multiple antibiotic-resistance(Mar) mutants. J.
Bacteriol.
178, 306-308)whose normal physiological role is unknown, although it may
assist in
protection of cells against bile salts in the mammalian small intestine
(Thanassi et al.
(1997) Active efflux of bile salts by Escherichia coli. J. Bacteriol. 179,
2512-2518).
AcrAB confers intrinsic resistance to many diverse, mostly lipophilic,
compounds
including antibiotics and disinfectants (Nikaido, H. ( 1996) Multidrug efflux
pumps of
Gram-negative bacteria. J. Bacteriol. 178, 5853-5859; Okusu et al. (1996)
AcrAB efflux
pump plays a major role in the antibiotic resistance phenotype of Esherichia
coli
multiple antibiotic-resistance(Mar) mutants. J. Bacteriol. 178, 306-308; Moken
et al.
(1997) Selection of multiple-antibiotic-resistant (Mar) mutants of Escherichia
coli by
using the disinfectant pine oiI: roles of the mar and acrAB loci. Antimicrob.
Chemother.
41, 2770-2772). The acrAB operon is upregulated by MarA (Ma et al. (1995)
Genes
acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol.
Microbiol. 16, 45-55), a transcriptional activator encoded by the marRAB
operon
involved in multiple antibiotic resistance (Alekshun et al. ( 1997) Regulation
of
chromosomally mediated multiple antibiotic resistance: the mar regulation.
Antimicrob.
Agents Chemother. 41, 2067-2075). Mutations in the repressor gene marR lead to
overexpression of marA (Alekshun et al. (1997) Regulation of chromosomally
mediated
multiple antibiotic resistance: the mar regulation. Antimicrob. Agents
Chemother. 41,
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2067-2075; Cohen et al. (1993) Genetic and functional analysis of the multiple
antibiotic
resistance {mar) locus in Escherichia coli. J. Bacteriol. 175, 1484-
492);Seoane et al.
(1995) Characterization of MarR, the repressor of the multiple antibiotic
resistance
(mar) operon of Escherichia coli. J. Bacteriol. 177, 3414-3419). The soxS gene
encodes a MarA homolog (Alekshun et al. ( 1997) Regulation of chromosomally
mediated multiple antibiotic resistance: the mar regulation. Antimicrob.
Agents
Chemother. 41, 2067-2075; Li et al. (1996) Sequence specificity for DNA
binding by
Escherichia coli SoxS and Rob proteins. Mol. Microbiol. 20, 937-945; Miller et
al.
( 1996) Overlaps and parallels in the regulation of intrinsic multiple-
antibiotic resistance
in Escherichia coli. Mol. Microbiol. 21, 441-448) which also positively
regulates acrAB
(Ma et al. (1996) The local repressor AcrR plays a modulating role in the
regulation of
acrAB genes of Escherichia coli by global stress signals. Mol. Mlcrobiol. 19,
101-112).
Table 3
15 Triclosan susceptibility of strains overexpressing marA (mutations in marR
), soxS (mutation in soxR), or
acrAB (mutation in acrR)
Strain (plasmid)/reference Characteristics Relative M1C of triclosana
HH180 (Cohen et al. (1993) Genetic Wild-type Mearb I.0
and functional analysis of the multiple
antibiotic resistance (mar) locus in
Fscherichia coli. J. Bacteriol. 175,
1484-492)
HH180(pHHM184) (Cohen et al. Wild-type Amarb (mar+
1.1
(1993) Genetic and functional analysis
of the multiple antibiotic resistance
(mar) locus in Escherichia coli. J.
Bacteriol. 175, 1484-492)
HH180(pHHM191) (Cohen et al. Wild-type Amarb (marR2) 3.0
(1993) Genetic and functional analysis
of the multiple antibiotic resistance
(mar) locus in Escherichia coli. J.
Bacteriol 175, 1484-492)
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HH180(pHHM193) (Cohen et al. Wild-type Amarb (marRS) 4.6
(1993) Genetic and functional analysis
of the multiple antibiotic resistance
(mar) locus in Escherichia coli. J.
Bacteriol. 175, 1484-492)
GC4488 (Greenberg et al. (1991) Wild-type I.0
Activation of oxidative stress genes by
mutation at the soxQllcfxBllmarA
locus of Escherichia coli. J. Bacteriol.
173,4433-4439)
JTG 1078 (Greenberg et al. ( 1991 ) GC4488 saxRl05 zjc-2204:: Tn IOkan 2.1
Activation of oxidative stress genes by
mutation at the saxQllcfxBllmarA
locus of Escherichia coli. J. Bacteriol.
173,4433-4439)
AG100 (George et al. (1983) Wild-type 1.0
Amplifiable resistance to tetracycline,
chloramphenicol, and other antibiotics
in Escherichia coli: involvement of a
non-plasmid-determined efflux of
tetracycline. J. Bacteriol. 155, 531-
540)
AG 100B (Okusu et al. (1996) AcrAB AG100 acrR:: kan 1.9
efflux pump plays a major role in the
antibiotic resistance phenotype of
Esherichia coli multiple antibiotic-
resistance(Mar) mutants. J. Bacteriol.
178, 306-308)
aMIC of strain divided by MIC of corresponding wild-type strain. MIC for AGI00
was 0.17 ug ml'1, for HH180,
0.07 pg ml-l, and for GC4488, 0.08 pg ml-1. MIC values are means from two to
five determinations.
bHas a 39 kb chromosomal deletion encompassing the mar locus.
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Materials and methods
All strains except those designated as'clinicaf were E. coli K-12 derivatives.
Cells were grown in LB broth or on LB agar at 37°C. Minimal inhibitory
concentration
(MIC) was determined using serial dilution LB agar plates with steps of 1.2-
1.5-fold
increasing concentrations of triclosan (also called Irgasan DP300; a gift from
Ciba-
Geigy). A 5 p,l amount of exponential phase cells at OD530-0.01 (about 3x10
colony-
forming units) was applied to the agar and the MIC was defined as the lowest
concentration which allowed no visible growth after 20 h at 37°C.
Results
Overexpression of the mar, sox, or acrAB locus decreased susceptibility to
triclosan
Defined mutations in marR within the marRAB operon cloned on low copy
plasmids (pHHM191, pHHM193) lead to overexpression of marA (Alekshun et al.
1 S (1997) Regulation of chromosomally mediated multiple antibiotic
resistance: the mar
regulation. Antimicrob. Agents Chemother. 41, 2067-2075; Cohen et al. (1993)
Genetic
and functional analysis of the multiple antibiotic resistance (mar) locus in
Escherichia
coli. J. Bacteriol. 175, 1484-492; Seoane et al. (1995) Characterization of
MarR, the
repressor of the multiple antibiotic resistance (mar) operon of Escherichia
coli. J.
Bacteriol. 177, 3414-3419). These mutations caused a 2.8-4.2-fold reduction in
the
susceptibility to triclosan as compared to the wild-type strain HH180/pHHMl84
(deleted
for the chromosomal mar locus and bearing the wild-type mar+ locus on a low
copy
plasmid) (Table 1 ). Chromosomal Mar mutants (overexpressing marA) showed a 2-
fold
lower susceptibility to triclosan (Table 2, strains AG102 and APS). Strain
JTG1078,
overexpressing soxS, had a triclosan MIC twice that of its parental strain
GC4488 (Table
1 ). Overexpression of acrAB resulting from a mutation in acrR doubled the
triclosan
MIC (strain AG100B, Table 3).
Effect of deletion of the mar or acrAB locus on susceptibility to triclosan
Deletion of the marCORAB locus from wild-type strain AG100 had little effect
on susceptibility to triclosan, while deletion from Mar mutants AG102 and APS
eliminated their resistance to triclosan (Table 4). Deletion of the acrAB
locus increased
the triclosan susceptibility about 10-fold in parental strain AG100 and some
20-fold in
the Mar mutants AG102 and APS, thereby equalizing the susceptibility of the
two
classes of strains (Table 4). Evidently, the amount of MarA in a Mar mutant,
but not in
the wild type strain, was sufficient to up-regulate acrAB.
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Table 4
Effect of deletion of the marCORAB acrAB locus upon susceptibility to
triclosan
Parental strain/reference Characteristics Relative MIC of triclosana
Control AmarCORABb DacrABb
AG 100 (George et al. (1983) Amplifiable Wild-type 1.0 0.87 0.11
resistance to tetracycline,
chloramphenicol, and other antibiotics in
Escherichia coli: involvement of a non-
plasmid-determined efflux of tetracycline.
J. Bacteriol. 155, 531-540)
AG102 (Cohen et al. (1993) Genetic and AG100 marRl 2.0 0.86 0.092
functional analysis of the multiple
antibiotic resistance (mar) locus in
Escherichia coli. J. Bacteriol. 175, 1484-
492)
APS (Nikaido, H. (1996) Multi-drug AG100 mar 2.0 0.95 0.092
efflux pumps of Gram-negative bacteria.
J. Bacteriol. 178, 5853-5859)
Strains AG 102 and APS are chromosomal Mar mutants and overexpress marA.
aMIC of strain divided by MIC of AG100 control strain (with no deletion). The
MIC for AG 100 was 0.17
ug ml-I.
bParental strain with this additional deletion; construction of these
inactivated strains has been described
(Moken et al. ( 1997) Selection of multiple-antibiotic-resistant (Mar) mutants
of Fscherichia coli by using
the disinfectant pine oil: roles of the mar and acrAB loci. Antimicrob.
Chemother. 41, 2770-2772).
Deletion of acrAB decreased the concentration of triclosan reauired for cell
lvsis
Use of liquid cultures permitted both growth rate and cell lysis to be
monitored.
Lysis was defined by loss of absorbance together with loss of viability. AG100
in liquid
culture required 0.6 ~.g ml-1 triclosan to inhibit the growth rate 90% but 8
~,g ml-I for
lysis (Table 5). Since the MIC (determined on agar) was 0.17-0.28 ~g ml-I for
AG100
15 (Tables 3-S), the MIC values almost surely reflected growth inhibition
rather than cell
lysis. That deletion of the acrAB locus decreased the MIC for triclosan 10-
fold (Table 4)
suggested that the AcrAB efflux pump lowers the internal concentration of
triclosan
affecting enoyl reductase, a cytoplasmic enzyme which is the putative target
of triclosan
(McMurry et al. (1998) Triclosan targets lipid synthesis. Nature 394, 531-532)
and
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which is essential for cell growth (Cronan et al. (1996) Biosynthesis of
membrane lipids.
In: Escherichia coli and Salmonella: Cellular and Molecular Biology
(Neidhardt, F.C.,
Ed.), pp. 612-636, ASM Press, Washington, D.C.).
AcrAB also influenced the effect of triclosan upon cell lysis. The
susceptibility
of wild-type cells to lysis by triclosan was increased about 2-fold by loss of
the efflux
pump (Table 5). The mechanism of triclosan-induced lysis is not known.
However, the
G93V mutation in enoyl reductase in triclosan-resistant mutant AGT11 (isogenic
with
AG100; (McMurry et al. (1998) Triclosan targets lipid synthesis. Nature 394,
531-532))
led to resistance of cells both to growth rate inhibition and to lysis (Table
5; (McMuny
et al. (1998) Triclosan targets lipid synthesis. Nature 394, 531-532)),
suggesting that
synthesis of fatty acids/lipids might not only be needed for growth but also
to prevent
lysis. On the other hand, when the acrAB locus was deleted from AGT1 l,
notable
protection by the G93V mutation remained against growth rate inhibition but
not against
lysis (Table 5, AG 1 OOA vs. AGT11 K). If the AcrAB pump were to remove drugs,
such
as triclosan, directly from the membrane (Nikaido, H. (1996) Multidrug efflux
pumps of
Gram-negative bacteria. J. Bacteriol. 178, 5853-5859), loss of this pump might
allow
the hydrophobic triclosan to accumulate in the membrane bilayer to a critical
level
leading to lysis regardless of the rate of fatty acid synthesis.
Table 5
Concentration of triclosanliquid culture to inhibit growth and
required in to cause lysis in strains deleted
for acrAB and/or bearing
a fabl mutation mediating
triclosan resistance
Strain/reference CharacteristicsMICa (fig ml-1) Concentration (fig
ml-I) of triclosan which
Inhibited growth rate 50% (90%) Caused
lysis
AG100 (George et al. Wild-type0.28 0.15 (0.6) g
(1983) Amplifiable
resistance to tetracycline,
chloramphenicol, and other
antibiotics in Fscherichia
coli: involvement of a
non-
plasmid-determined efflux
oftetracyeline. J.
Bacteriol. 155, 531-540)
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AG100A (Okusu et al. AG100 DacrAB::kan 0.018 0.02 (0.05) 3.4
(1996) AcrAB efflux pump
plays a major role in the
antibiotic resistance
phenotype of Esherichia
coli multiple antibiotic-
resistance(Mar) mutants. J.
Bacteriol. 178, 306-308)
AGT11 (McMurry et al. AG100fabl (G93V) 41 13 (>32)b >32b
(1998) Triclosan targets
lipid synthesis. Nature
394,531-532)
AGTIIKc (this work) AGTI I DacrAB::kan 3.2 1.3 (2.1) 3~
The concentration of triclosan required to slow the growth rate by 50% (or
90%) an hour after addition was
determined using OD530 to monitor growth. 'Lysis' was defined in such cultures
by a 30-50% loss of OD530 within 2
h of triclosan addition accompanied by a 4-6 log loss in viability (as
indicated by colony-forming units).
aDetennined using agar dilution plates.
$ b'friciosan formed an insoluble precipitate above 32 ug ml-I ~ no lysis of
cells was seen even at nominal triclosan
concentrations of 256 pg ml-I.
cAF"T11K was constructed by Pl transduction (Provence et al. (1994) Gene
transfer in Gram-negative bacteria. In:
Methods for General and Molecular Bacteriology (Gerhardt et al.), pp. 317-347.
American Society for Microbiology,
Washington, D.C.) of AacrAB:: kan from strain )ZM120 (Okusu et al. (1996)
AcrAB efflux pump plays a major role
in the antibiotic resistance phenotype of Esherichia coli multiple antibiotic-
resistance(Mar) mutants. J. Bacteriol.
178, 306-308; Ma et al. (1995) Genes acrA and acrB encode a stress-induced
efflux system of Fscherichia coli. Mol.
Microbiol. 16, 45-55) into AGTI 1.
Relationship of triclosan susceptibility to overexpression of marA or soxS in
clinical
strains
Triclosan susceptibility of clinical strains of E. coli from blood samples
taken in
hematology-oncology hospital wards in Europe (Oethinger et al. (1997)
Association of
organic solvent tolerance and fluoroquinolone resistance in clinical isolates
of
Escherichia coli. J. Antimicrob. Chemother. 41, 111-114). All strains chosen
from
Series S were susceptible to fluoroquinolones, tetracycline, ampicillin, and
chloramphenicol, while all chosen from series HO and E were resistant to all
four
antibiotics. Of 15 susceptible strains, 14 had a mean triclosan MIC of 0.090
p,g ml-I
(S.D. 0.014). The remaining susceptible strain, S20, was exceptional in
overexpressing
CA 02319115 2000-07-24
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marA (Oethinger et al. (1998) Overexpression of to regulatory marA or soxS
gene in
cliniciai topoisomerase mutants of Escherichia coli. Antimicrob. Agents
Chemother. 42,
2089-2094) and had a correspondingly higher triclosan MIC, 0.27 p,g ml-1. Of
31
multiply resistant strains, three (E3, E19, H099) overexpressed either marA or
soxS
(Oethinger et al. (1998) Overexpression of to regulatory marA or soxS gene in
clinicial
topoisomerase mutants of Escherichia coli. Antimicrob. Agents Chemother. 42,
2089-
2094), which correlated with a higher mean triclosan MIC of 0.33 ~,g ml-~
(S.D. 0.03);
the fourth strain (H017) also overexpressed marA (Oethinger et al. (1998)
Overexpression of to regulatory marA or soxS gene in clinicial topoisomerase
mutants of
Escherichia coli. Antimicrob. Agents Chemother. 42, 2089-2094) but had a
triclosan
MIC of only 0.15 p,g ml-~. Multiply resistant strain E10 had a triclosan MIC
of 0.38 ~.g
ml-i, but overexpressed neither marA or soxS, nor was it tolerant to
cyclohexane
(Oethinger et al. (1998) Overexpression of the regulatory marA or soxS gene in
clinicial
topoisomerase mutants of Escherichia coli. Antimicrob. Agents Chemother. 42,
2089-
2094), a hallmark of strains overexpressing marA, soxS, robA, or acrAB (White
et al.
(1997) Role of the acrAB locus in organic solvent tolerance mediated by
expression of
marA, soxS, or robA in Escherichia coli. J. Bacteriol. 179, 6122-6126). This
strain
probably has mutations(s) at other loci, possibly including fabl. The
remaining 26
multiply resistant strains overexpressing neither marA or soxS had a mean
triclosan MIC
of 0.13 ~,g ml-~ (S.D. 0.04). In summary, regardless of the multiple
antibiotic resistance
phenotypes, four of the five clinical strains which overexpressed marA or soxS
had a
triclosan MIC more than twice that of strains which did not overexpress either
gene.
This effect was consistent with the findings in the laboratory K-12 strains.
Discussion
The deletion of AcrAB multidrug efflux pump increases the susceptibility of E.
coli strains to triclosan, both at the level of growth inhibition and of
lysis. Triclosan can
now be added to the list (Nikaido, H. ( 1996) Multidrug efflux pumps of Gram-
negative
bacteria. J. Bacteriol. 178, 5853-5859) of presumed AcrAB substrates. In
Pseudomonas
aeruginosa, a recent study indicates that triclosan is also a substrate for
the MexAB-
OprM multidrug efflux pump (Schweizer, H.P. (1998) Intrinsic resistance to
inhibitors
of fatty acid biosynthesis in Psuedomonas aeruginosa is due to e$lux:
application of a
novel technique for generation of unmarked chromosomal mutations for the study
of
efflux systems. Antimicrob. Agents Chemother. 42, 394-398). Mutations at the
secondary loci acr, mar, and sox in E. coli conferred only a 2-fold resistance
to triclosan,
presumably via a small up-regulation of acrAB. A mutation at any one of these
three
loci might not by itself threaten triclosan efficacy, but might act
synergistically with
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mutations at other loci such as fabl, where mutations can increase triclosan
resistance
90-140-fold (Table 5). Finally, low levels of the very stable triclosan in the
environment
might encourage preferential survival of acrlmarlsox mutants resistant to
multiple
antibiotics.
S The contents of all references, pending patent applications and published
patents,
cited throughout this application are hereby expressly incorporated by
reference. In
addition, the contents of McMurry et al. 1998. FEMS Microbiology Letters
166:305 are
also expressly incorporated by this reference.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following
claims.
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SEQUENCE LISTING
<110> Trustees of Tufts College
<120> Antimicrobial Compounds
<130> pkz-OOicppc
<140>
<141>
<150> 09/027,130
<151> 1998-02-20
1$ <160> 14
<170> PatentIn Ver. 2.0
<210> 1
<211> 1301
<212> DNA
<213> Escherichia coli
<220>
<221> CDS
<222> (14) . . (232)
<220>
<221> CDS
<222> (236)..(343)
<220>
<221> CDS
<222> (347)..(1189)
<220>
<221> CDS
<222> (1193)..(1204)
<220>
<221> CDS
<222> (1208) . . (1300)
<400> 1
ctgcaggaac tga acc gcc ggt cac cct ctc cct gaa aga gcg agg ggg 49
Thr Ala Gly His Pro Leu Pro Glu Arg Ala Arg Gly
1 5 10
cag acc gag ccg aat agc tgt tgt ggt gaa aac atg gag acg gtg ctg 97
Gln Thr Glu Pro Asn Ser Cys Cys Gly Glu Asn Met Glu Thr Val Leu
15 20 25
gag aat att cgg caa ggt ctg aac cgt ccc agc cat cgc cat gaa agg 145
Glu Asn Ile Arg Gln Gly Leu Asn Arg Pro Ser His Arg His Glu Arg
30 35 40
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gtt agg ggc tgt atg agc ctg ttt gtt get ggg gta aca ata ttt gca 193
Val Arg Gly Cys Met Ser Leu Phe Val Ala Gly Val Thr Ile Phe Ala
45 50 55 60
S
caa tac ggt ccc ctc gcc cct ctg ggg aga ggg tta ggg tga ggg gaa 241
Gln Tyr Gly Pro Leu Ala Pro Leu Gly Arg Gly Leu Gly Gly Glu
65 70 75
aag cgc ccc ccc tgc cgc agc ctg ctc cgg tcg gac ctg gca act ata 289
Lys Arg Pro Pro Cys Arg Ser Leu Leu Arg Ser Asp Leu Ala Thr Ile
80 85 90
get act cac agc cag gtt gat tat aat aac cgt tta tct gtt cgt act 337
Ala Thr His Ser Gln Val Asp Tyr Asn Asn Arg Leu Ser Val Arg Thr
95 100 105
gtt tac taa aac gac gaa tcg cct gat ttt cag gca caa caa gca tca 385
Val Tyr Asn Asp Glu Ser Pro Asp Phe Gln Ala Gln Gln Ala Ser
110 115 120
aca ata agg att aaa get atg ggt ttt ctt tcc ggt aag cgc att ctg 433
Thr Ile Arg Ile Lys Ala Met Gly Phe Leu Ser Gly Lys Arg Ile Leu
125 130 135
gta acc ggt gtt gcc agc aaa cta tcc atc gcc tac ggt atc get cag 481
Val Thr Gly Val Ala Ser Lys Leu Ser Ile Ala Tyr Gly Ile Ala Gln
140 145 150
gcg atg cac cgc gaa gga get gaa ctg gca ttc acc tac cag aac gac 529
Ala Met His Arg Glu Gly Ala Glu Leu Ala Phe Thr Tyr Gln Asn Asp
155 160 165 170
aaa ctg aaa ggc cgc gta gaa gaa ttt gcc get caa ttg ggt tct gac 577
Lys Leu Lys Gly Arg Val Glu Glu Phe Ala Ala Gln Leu Gly Ser Asp
175 180 185
atc gtt ctg cag tgc gat gtt gca gaa gat gcc agc atc gac acc atg 625
Ile Val Leu Gln Cys Asp Val Ala Glu Asp Ala Ser Ile Asp Thr Met
190 195 200
ttc get gaa ctg ggg aaa gtt tgg ccg aaa ttt gac ggt ttc gta cac 673
Phe Ala Glu Leu Gly Lys Val Trp Pro Lys Phe Asp Gly Phe Val His
205 210 215
tct att ggt ttt gca cct ggc gat cag ctg gat ggt gac tat gtt aac 721
Ser Ile Gly Phe Ala Pro Gly Asp Gln Leu Asp Gly Asp Tyr Val Asn
220 225 230
SO gcc gtt acc cgt gaa ggc ttc aaa att gcc cac gac atc agc tcc tac 769
Ala Val Thr Arg Glu Gly Phe Lys Ile Ala His Asp Ile Ser Ser Tyr
235 240 245 250
agc ttc gtt gca atg gca aaa get tgc cgc tcc atg ctg aat ccg ggt 817
Ser Phe Val Ala Met Ala Lys Ala Cys Arg Ser Met Leu Asn Pro Gly
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255 260 265
tct gcc ctg ctg acc ctt tcc tac ctt ggc get gag cgc get atc ccg 865
Ser Ala Leu Leu Thr Leu Ser Tyr Leu Gly Ala Glu Arg Ala Ile Pro
270 275 280
aac tac aac gtt atg ggt ctg gca aaa gcg tct ctg gaa gcg aac gtg 913
Asn Tyr Asn Val Met Gly Leu Ala Lys Ala Ser Leu Glu Ala Asn Val
285 290 295
cgc tat atg gcg aac gcg atg ggt ccg gaa ggt gtg cgt gtt aac gcc 961
Arg Tyr Met Ala Asn Ala Met Gly Pro Glu Gly Val Arg Val Asn Ala
300 305 310
atc tct get ggt ccg atc cgt act ctg gcg gcc tcc ggt atc aaa gac 1009
Ile Ser Ala Gly Pro Ile Arg Thr Leu Ala Aia Ser Gly Ile Lys Asp
315 320 325 330
ttc cgc aaa atg ctg get cat tgc gaa gcc gtt acc ccg att cgc cgt 1057
Phe Arg Lys Met Leu Ala His Cys Glu Ala Val Thr Pro Ile Arg Arg
335 340 345
acc gtt act att gaa gat gtg ggt aac tct gcg gca ttc ctg tgc tcc 1105
Thr VaI Thr Ile Glu Asp Val Gly Asn Ser Ala Ala Phe Leu Cys Ser
350 355 360
gat etc tct gcc ggt atc tcc ggt gaa gtg gtc cac gtt gac ggc ggt 1153
Asp Leu Ser Ala Gly Ile Ser Gly Glu Val Val His Val Asp Gly Gly
365 370 375
ttc agc att get gca atg aac gaa ctc gaa ctg aaa taa tcg ttc tgt 1201
Phe Ser Ile Ala Ala Met Asn Glu Leu Glu Leu Lys Ser Phe Cys
380 385 390
tgg taa aga tgg gcg gcg ttc tgc cgc ccg tta tct ctg tta tac ctt 1249
Trp Arg Trp Ala Ala Phe Cys Arg Pro Leu Ser Leu Leu Tyr Leu
395 400 405
tct gat att tgt tat cgc cga tcc gtc ttt ctc ccc ttc ccg cct tgc 1297
Ser Asp Ile Cys Tyr Arg Arg Ser Val Phe Leu Pro Phe Pro Pro Cys
410 415 420
gtc a 1301
Val
425
<210> 2
<211> 786
<212> DNA
<213> Escherichia coli
<220>
<221> CDS
<222> (1)..(786)
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<400> 2
atg ggt ttt ctt tcc ggt aag cgc att ctg gta acc ggt gtt gcc agc 48
Met Gly Phe Leu Ser Gly Lys Arg Ile Leu Val Thr Gly Val Ala Ser
1 5 10 15
aaa cta tcc atc gcc tac ggt atc get cag gcg atg cac cgc gaa gga 96
Lys Leu Ser Ile Ala Tyr Gly Ile Ala Gln Ala Met His Arg Glu Gly
20 25 30
get gaa ctg gca ttc acc tac cag aac gac aaa ctg aaa ggc cgc gta 144
Ala Glu Leu Ala Phe Thr Tyr Gln Asn Asp Lys Leu Lys Gly Arg Val
35 4p 45
gaa gaa ttt gcc get caa,ttg ggt tct gac atc gtt ctg cag tgc gat 192
Glu Glu Phe Ala Ala Gln Leu Gly Ser Asp Ile Val Leu Gln Cys Asp
50 55 60
gtt gca gaa gat gcc agc atc gac acc atg ttc get gaa ctg ggg aaa 240
Val Ala Glu Asp Ala Ser Ile Asp Thr Met Phe Ala Glu Leu Gly Lys
65 70 75 80
gtt tgg ccg aaa ttt gac ggt ttc gta cac tct att ggt ttt gca cct 288
Val Trp Pro Lys Phe Asp Gly Phe Val His Ser Ile Gly Phe Ala Pro
2S 85 90 95
ggc gat cag ctg gat ggt gac tat gtt aac gcc gtt acc cgt gaa ggc 336
Gly Asp Gln Leu Asp Gly Asp Tyr Val Asn Ala Val Thr Arg Glu Gly
100 105 110
ttc aaa attgcccac gacatc agctcctac agcttcgtt gcaatggca 384
Phe Lys IleAlaHis AspIle SerSerTyr SerPheVal AlaMetAla
115 120 125
35aaa get tgccgctcc atgctg aatccgggt tctgccctg ctgaccctt 432
Lys Ala CysArgSer MetLeu AsnProGly SerAlaLeu LeuThrLeu
130 135 140
tcc tac cttggcget gagcgc getatcccg aactacaac gttatgggt 480
40Ser Tyr LeuGlyAla GluArg AlaIlePro AsnTyrAsn ValMetGly
145 150 155 160
ctg gca aaagcgtct ctggaa gcgaacgtg cgctatatg gcgaacgcg 528
Leu Ala LysAlaSer LeuGlu AlaAsnVal ArgTyrMet AlaAsnAla
45 165 170 175
atg ggt ccggaaggt gtgcgt gttaacgcc atctctget ggtccgatc 576
Met Gly ProGluGly ValArg ValAsnAla IleSerAla GlyProIle
180 185 190
50
cgt act ctggcggcc tccggt atcaaagac ttccgcaaa atgctgget 624
Arg Thr LeuAlaAla SerGly IleLysAsp PheArgLys MetLeuAla
195 200 205
55cat tgc gaagccgtt accccg attcgccgt accgttact attgaagat 672
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His Cys Glu Ala Val Thr Pro Ile Arg Arg Thr Val Thr Ile Glu Asp
210 215 220
gtg ggt aac tct gcg gca ttc ctg tgc tcc gat ctc tct gcc ggt atc 720
Val Gly Asn Ser Ala Ala Phe Leu Cys Ser Asp Leu Ser Ala Gly Ile
225 230 235 240
tcc ggt gaa gtg gtc cac gtt gac ggc ggt ttc agc att get gca atg 768
Ser Gly Glu Val Val His Val Asp.Gly Gly Phe Ser Ile Ala Ala Met
245 250 255
aac gaa ctc gaa ctg aaa 786
Asn Glu Leu Glu Leu Lys
260
<210> 3
<211> 262
<212> PRT
<213> Escherichia coli
<400> 3
Met Gly Phe Leu Ser Gly Lys Arg Ile Leu Val Thr Gly Val Ala Ser
1 5 10 15
Lys Leu Ser Ile Ala Tyr Gly Ile Ala Gln Ala Met His Arg Glu Gly
20 25 30
Ala Glu Leu Ala Phe Thr Tyr Gln Asn Asp Lys Leu Lys Gly Arg Val
40 45
Glu Glu Phe Ala Ala Gln Leu Gly Ser Asp Ile Val Leu Gln Cys Asp
50 55 60
35 Val Ala Glu Asp Ala Ser Ile Asp Thr Met Phe Ala Glu Leu Gly Lys
65 70 75 BO
Val Trp Pro Lys Phe Asp Gly Phe Val His Ser Ile Gly Phe Ala Pro
85 90 95
Gly Asp Gln Leu Asp Gly Asp Tyr Val Asn Ala Val Thr Arg Glu Gly
100 105 110
Phe Lys Ile Ala His/Asp Ile Ser Ser Tyr Ser Phe Val Ala Met Ala
115 120 125
Lys Ala Cys Arg Ser Met Leu Asn Pro Gly Ser Ala Leu Leu Thr Leu
130 135 140
Ser Tyr Leu Gly Ala Glu Arg Ala Ile Pro Asn Tyr Asn Val Met Gly
145 150 155 160
Leu Ala Lys Ala Ser Leu Glu Ala Asn Val Arg Tyr Met Ala Asn Ala
165 170 175
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Met Gly Pro Glu Gly Val Arg Val Asn Ala Ile Ser Ala Gly Pro Ile
180 185 190
Arg Thr Leu Ala Ala Ser Gly Ile Lys Asp Phe Arg Lys Met Leu Ala
195 200 205
His Cys Glu Ala Val Thr Pro Ile Arg Arg Thr Val Thr Ile Glu Asp
210 215 220
Val Gly Asn Ser Ala Ala Phe Leu Cys Ser Asp Leu Ser Ala Gly Ile
225 230 235 240
Ser Gly Glu Val Val His Val Asp Gly Gly Phe Ser Ile Ala Ala Met
245 250 255
Asn Glu Leu Glu Leu Lys
260
<210> 4
<211> 20
<212> DNA
<213> synthetic construct
<400> 4
gagcctgttt gttgctgggg 20
<210> 5
<211> 20
<212> DNA
<213> synthetic construct
<400> 5
tgcagcaatg ctgaaaccgc 20
<210> 6
<211> 17
<212> DNA
<213> synthetic construct
<400> 6
cgggaagggg agaaaga 17
<210> 7
<211> 18
<212> DNA
<213> synthetic construct
<400> 7
aattgcccac gacatcag 18
CA 02319115 2000-07-24
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_ 'j _
<210> 8
<211> 20
<212> DNA
<213> synthetic construct
<400> 8
cgttgtagtt cgggatagcg 20
<210> 9
<211> 16
<212> DNA
<213> synthetic
construct
<400> 9
cgcgatcatg gcgacc 16
<210> 10
<211> 19
<212> DNA
<213> synthetic struct
con
<400> 10
accagcgttt ctgggtgag 19
<210> 11
<211> 850
<212> DNA
<213> Mycobacteriumsmegmatis
<220>
<221> CDS
<222> (40)..(849)
<400> 11
ccggacacac aagatttctc atgacaggc ctactc 54
gctcacaagg agtcaccaa
MetThrGly LeuLeu
1 5
gaa ggc aag cgc ctc acggggatc atcaccgattcg tcgatc 102
atc gtc
Glu Gly Lys Arg Leu ThrGlyIle IleThrAspSer SerIle
Ile Val
10 15 20
gcg ttc cac atc aag gcccaggag gccggcgccgaa ctggtg 150
gcc gtc
Ala Phe His Ile Lys AlaGlnGlu AlaGlyAlaGlu LeuVal
Ala Val
25 30 35
ctg acc ggt ttc cgc aagttggtc aagcgcatcgcc gaccgc 198
gac ctg
Leu Thr Gly Phe Arg LysLeuVal LysArgIleAla AspArg
Asp Leu
40 45 50
etg ccc aag ccg ccg ctggaaetc gacgtgcagaac gaggag 246
gcc ctg
Leu Pro Lys Pro Pro LeuGluLeu AspValGlnAsn GluGlu
Ala Leu
CA 02319115 2000-07-24
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_g_
55 60 65
cac ctg tcg act ctg gcc gac cgg atc acc gcc gag atc ggt gag ggc 294
His Leu Ser Thr Leu Ala Asp Arg Ile Thr Ala Glu Ile Gly Glu Gly
S 70 75 80 85
aac aag atc gac ggt gtg gtg cac tcg atc ggg ttc atg ccg cag agc 342
Asn Lys Ile Asp Gly Val Val His Ser Ile Gly Phe Met Pro Gln Ser
90 95 100
ggt atg ggc atc aac ccg ttc ttc gac gcg ccg tac gag gat gtg tcc 390
Gly Met Gly Ile Asn Pro Phe Phe Asp Ala Pro Tyr Glu Asp Val Ser
105 110 115
aag ggc atc cac atc tcg gcg tac tcg tac gcc tcg ctc gcc aaa gcc 438
Lys Gly Ile His Ile Ser Ala Tyr Ser Tyr Ala Ser Leu Ala Lys Ala
120 125 130
gtt ctg ccg atc atg aat ccg ggc ggc ggc atc gtc ggc atg gac ttc 486
Val Leu Pro Ile Met Asn Pro Gly Gly Gly Ile Val Gly Met Asp Phe
135 140 145
gac ccc acg cgc gcg atg ccg gcc tac aac tgg atg acc gtc gcc aag 534
Asp Pro Thr Arg Ala Met Pro Ala Tyr Asn Trp Met Thr Val Ala Lys
2$ 150 155 160 165
agc gcg ctc gaa tcg gtc aac cgg ttc gtc gcg cgt gag gcg ggc aag 582
Ser Ala Leu Glu Ser Val Asn Arg Phe Val Ala Arg Glu Ala Gly Lys
170 175 180
gtg ggc gtg cgc tcg aat ctc gtt gcg gca gga ccg atc cgc acg ctg 630
Val Gly Val Arg Ser Asn Leu Val Ala Ala Gly Pro Ile Arg Thr Leu
185 190 195
gcg atg agc gca atc gtg ggc ggt gcg ctg ggc gac gag gcc ggc cag 678
Ala Met Ser Ala Ile Val Gly Gly Ala Leu Gly Asp Glu Ala Gly Gln
200 205 210
cag atg cag ctg ctc gaa gag ggc tgg gat cag cgc gcg ccg ctg ggc 726
Gln Met Gln Leu Leu Glu Glu Gly Trp Asp Gln Arg Ala Pro Leu Gly
215 220 225
tgg aac atg aag gac ccg acg ccc gtc gcc aag acc gtg tgc gca ctg 774
Trp Asn Met Lys Asp Pro Thr Pro Val Ala Lys Thr Val Cys Ala Leu
230 235 240 245
ctg tcg gac tgg ctg ccg gcc acc acc ggc acc gtg atc tac gcc gac B22
Leu Ser Asp Trp Leu Pro Ala Thr Thr Gly Thr Val Ile Tyr Ala Asp
250 255 260
ggc ggc gcc agc acg cag ctg ttg tga t 850
Gly Gly Ala Ser Thr Gln Leu Leu
265 270
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<210> lz
<211> 269
<212> PRT
<213> Mycobacterium smegmatis
<400> 12
Met Thr Gly Leu Leu Glu Gly Lys Arg Ile Leu Val Thr Gly Ile Ile
1 5 10 15
Thr Asp Ser Ser Ile Ala Phe His Ile Ala Lys Val Ala Gln Glu Ala
25 30
Gly Ala Glu Leu Val Leu Thr Gly Phe Asp Arg Leu Lys Leu Val Lys
35 40 45
Arg Ile Ala Asp Arg Leu Pro Lys Pro Ala Pro Leu Leu Glu Leu Asp
50 55 60
Val Gln Asn Glu Glu His Leu Ser Thr Leu Ala Asp Arg Ile Thr Ala
65 70 75 80
Glu Ile Gly Glu Gly Asn Lys Ile Asp Gly Val Val His Ser Ile Gly
85 90 95
2$ Phe Met Pro Gln Ser Gly Met Gly Ile Asn Pro Phe Phe Asp Ala Pro
100 105 110
Tyr Glu Asp Val Ser Lys Gly Ile His Ile Ser Ala Tyr Ser Tyr Ala
115 120 125
Ser Leu Ala Lys Ala Val Leu Pro Ile Met Asn Pro Gly Gly Gly Ile
130 135 140
Val Gly Met Asp Phe Asp Pro Thr Arg Ala Met Pro Ala Tyr Asn Trp
3$ 145 150 155 160
Met Thr Val Ala Lys Ser Ala Leu Glu Ser Val Asn Arg Phe Val Ala
165 170 175
Arg Glu Ala Gly Lys Val Gly Val Arg Ser Asn Leu Val Ala Ala Gly
180 185 190
Pro Ile Arg Thr Leu Ala Met Ser Ala Ile Val Gly Gly Ala Leu Gly
195 200 205
Asp Glu Ala Gly Gln Gln Met Gln Leu Leu Glu Glu Gly Trp Asp Gln
210 215 220
Arg Ala Pro Leu Gly Trp Asn Met Lys Asp Pro Thr Pro Val Ala Lys
SO 225 230 235 240
Thr Val Cys Ala Leu Leu Ser Asp Trp Leu Pro Ala Thr Thr Gly Thr
245 250 255
Val Ile Tyr Ala Asp Gly Gly Ala Ser Thr Gln Leu Leu
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260 265
<210> 13
S <211> 19
<212> DNA
<213> synthetic construct
<400> 13
aaagcccgga cacacaaga 19
<210> 14
<211> 20
<212> DNA
<213> synthetic construct
<400> 14
cgaacgacag cagtagcaag 20
