Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2001-05-10
2003-05-06
Weddington, Kevin E. (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C514S418000, C514S438000, C514S441000, C514S461000
Reexamination Certificate
active
06559176
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to bacterial diseases of humans and other mammals. In particular, the invention provides a novel signaling factor involved in regulating bacterial growth and pathogenesis, analogs and derivatives of the signaling factor, and methods for controlling bacterial growth and pathogenesis through use of such analogs and derivatives.
BACKGROUND OF THE INVENTION
Intercellular cooperation confers a considerable advantage on multicellular organisms that was thought to be unavailable to unicellular organisms such as prokaryotes. Research in the last twenty years has revealed, however, that prokaryotes can communicate with each other in a way that modulates gene expression, and thereby can reap benefits that would otherwise be exclusive to eukaryotes. This ability was discovered in luminous marine bacteria such as
Vibrio fischeri
and
Vibrio harveyi,
which activate the expression of genes involved in light production only when their population density exceeds a critical value. This phenomenon, known as quorum-sensing, is now recognized as a general mechanism for gene regulation in many Gram-negative bacteria, and it allows them to perform in unison such activities as bioluminescence, swarming, biofilm formation, production of proteolytic enzymes, synthesis of antibiotics, development of genetic competence, plasmid conjugal transfer, and spoliation.
Quorum-sensing bacteria fall into two classes, depending on how many density-sensing systems they have. Both classes synthesize, release, and respond to signaling molecules called autoinducers to control gene expression as a function of cell density. Bacteria in the larger class use acyl-homoserine lactone signals in a single density-sensing system, with one gene that encodes an autoinducer synthase, and another that encodes a transcriptional activator protein that mediates response to the autoinducer. These genes are homologous to luxI and luxR of
V. fischeri,
respectively (Bassler and Silverman, in
Two component Signal Transduction,
Hoch et al., eds, Am. Soc. Microbiol. Washington D.C., pp 431-435, 1995).
Many bacteria that use the autoinducer-1 signaling factor associate with higher organisms, i.e., plants and animals, at some point during their lifecycles. For example,
Pseudomonas aeruginosa,
an opportunistic pathogen in humans with cystic fibrosis, regulates various virulence determinants with autoinducer-1. Other examples of autoinducer-1-producing bacteria include
Erwinia carotovora, Pseudomonas aureofaciens, Yersinia enterocolitica, Vibrio harveyi,
and
Agrobacterium tumefaciens. E. carotovora
infects certain plants and creates enzymes that degrade the plant's cell walls, resulting in what is called “soft rot disease.”
Yersinia enterocolitica
causes gastrointestinal disease in humans and reportedly produces an autoinducer.
P. aureofaciens
synthesizes antibiotics under autoinducer control that block fungus growth in the roots.
Bacteria of the other class, exemplified by
V. harveyi,
have not one but two independent density-sensing systems.
V. harveyi
apparently uses the more species-specific Signaling System 1 for intra-species communication, and the less species-selective Signaling System 2 for inter-species communication (Bassler et al., J. Bacteriol. 179: 4043-4045, 1997). Each system comprises a sensor-autoinducer pair; Signaling System 1 uses Sensor 1 and autoinducer-1 (AI-1), while Signaling System 2 uses Sensor 2 and autoinducer-2 (AI-2) (Bassler et al., Mol. Microbiol. 13: 273-286, 1994). While autoinducer-1 is N-(3-hydroxy butanoyl)-L-homoserine lactone (HSL) (see Bassler et al., Mol. Microbiol. 9: 773-786, 1993), the structure of autoinducer-2 has not been established, nor have the gene(s) involved in its biosynthesis been identified.
Recent research indicates that quorum-sensing takes place not only among luminous marine bacteria, but also among pathogenic bacteria, where it regulates the production of virulence factors that are critical factors in bacterial pathogenesis. Thus, it would be an advance in the art to identify and characterize compounds with autoinducer-2 activity and the genes encoding the proteins required for production of the naturally-occurring autoinducer-2. Such an advance would provide a way to identify compounds useful for controlling pathogenic bacteria, a way to augment traditional antibiotic treatments, and a new target for the development of new antimicrobial agents.
SUMMARY OF THE INVENTION
The applicants have now discovered that many bacteria, including some non-luminous pathogens, secrete a signaling molecule that mimics
V. harveyi
autoinducer-2 in its function and physical properties. Bacteria that produce the AI-2 signaling factor of the invention include
Vibrio harveyi, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Pseudomonas phosphoreum, Yersinia enterocolitica, Escherichia coli, Salmonella typhimurium, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borrelia burgfdorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Yersinia pestis, Campylobacter jejuni, Deinococcus radiodurans, Mycobacterium tuberculosis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes
and
Staphylococcus aureus.
Free-living bacteria produce this novel signaling molecule only upon shifting to a colonizing, and therefore potentially pathogenic, existence in a host organism. Thus, in addition to stimulating luminescence genes in
V. harveyi,
the signaling molecule is expected to stimulate genes related to pathogenesis in bacteria that produce it. In addition to a purified molecule with autoinducer-2 signaling activity, the invention provides a synthetic form of the molecule and derivatives of it that regulate bacterial growth and pathogenesis.
In another aspect, there is provided a method for regulating the activity of an autoinducer-2 receptor comprising contacting an autoinducer-2 receptor with an AI-2 agonist or antagonist compound.
In another aspect, the invention provides a method of regulating autoinducer-2 activity by contacting a bacterial cell comprising autoinducer-2, or extract thereof, with a compound of structure I, II, III or IV.
In yet another aspect, the invention provides a method for regulating autoinducer-2 receptor activity by contacting an autoinducer-2 receptor with a compound of structure I, II, III or IV.
In another aspect, the invention provides a method for controlling bacterial growth or virulence by identifying a subject infected with an autoinducer-2-producing bacterium and administering to the subject a compound of structure I, II, III or IV.
In yet another aspect, the invention provides a method for inhibiting bacterial growth or virulence in a subject, by identifying a subject in which bacteria are producing autoinducer-2 and administering to the subject an inhibitor of an autoinducer-2 of the present invention.
In another aspect, the invention provides a method for identifying a compound that regulates the activity of autoinducer-2 by comparing the activity of autoinducer-2 obtained in the presence of the compound to that obtained in its absence.
In another aspect, the invention provides a method for identifying an autoinducer analog by contacting a cell, or cell extract, that produces a detectable amount of light in response to an autoinducer with the autoinducer analog and comparing the amount of light produced in the presence and the absence of the autoinducer analog.
In another aspect, the invention provides a method for identifying a compound that regulates the production or activity of autoinducer-2 by contacting with the compound a cell that produces autoinducer-2, and determining whether autoinducer-2 activity is present in the cell.
In another aspect, the invention provides a method for identifying a compound that affects binding of autoinducer-2 to an autoinducer-2 receptor by: (a) contacting autoinducer-2 and the autoinducer-2 receptor with the compound; (b) contacting (a) with a cell, or cell extract, that produces light in response to autoinducer-2
Bassler Bonnie L.
Dammel Carol
Schauder Stephan
Shokat Kevan
Stein Jeffrey
Knobbe Martens Olson & Bear LLP
Princeton University
Weddington Kevin E.
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