Compositions and methods for regulating bacterial pathogenesis
AssigneeJoseph J. Mallom
InventorsBONNIE L. BASSLER, MICHAEL G. SURETTE
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Abstract
This invention provides a purified extracellular bacterial autoinducer-2 signaling molecule, the production of which is regulated by changes in environmental conditions associated with a shift from a free-living existence to a colonizing or pathogenic existence in a host organism. The signaling molecule stimulates LuxQ luminescence genes, and is believed also to stimulate a variety of pathogenesis related genes in the bacterial species that produce it. This invention also provides is a new class of bacterial genes involved in the biosynthesis of the signaling molecule.
Claims117
An isolated bacterial extracellular signaling factor comprising at least one molecule that is polar and uncharged, and having an approximate molecular weight of less than 1,000 kDa, wherein the factor interacts with LuxQ protein thereby inducing expression of a Vibrio harveyi operon comprising luminescence genes luxCDABE.
An isolated bacterial signaling factor comprising a factor of the formula:
- 5
An isolated bacterial signaling factor comprising a factor having the formula:
- 6
- wherein R1, R2, R3 and R4 are independently selected from the group consisting of hydrido, halo, alkyl, haloalkyl, cycloalkyl, cycloalkenyl, heterocyclyl, methyl, cyano, alkoxycarbonyl, amino, carboxyl, hydroxyl, formyl, nitro, fluoro, chloro, bromo, methyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkylsulfonyl, haloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, hydroxyalkyl, mercaptoalkyl, alkoxyalkyl, aryloxyalkyl, heteroaryloxyalkyl, aralkyloxyalkyl, heteroarylalkyloxyalkyl, alkylthioalkyl, arylthioalkyl, heteroarylthioalkyl, aralkylthioalkyl, heteroarylalkylthioalkyl, haloalkylcarbonyl, haloalkyl(hydroxy)alkyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl, carboxyalkyl, alkoxycarbonylalkyl, alkylcarbonyloxyalkyl, aminoalkyl, alkylaminoalkyl, arylaminoalkyl, aralkylaminoalkyl, heteroarylaminoalkyl, heteroarylalkylaminoalkyl, alkoxy, and aryloxy;
- phenyl, cyclohexyl, cyclohexenyl, benzofuryl, benzodioxolyl, furyl, imidazolyl, thienyl, thiazolyl, pyrrolyl, oxazolyl, isoxazolyl, triazolyl, pyrimidinyl, isoquinolyl, quinolinyl, benzimidazolyl, indolyl, pyrazolyl and pyridyl, aminosulfonyl, fluoro, chloro, bromo, methylthio, methyl, ethyl, isopropyl, tert-butyl, isobutyl, pentyl, hexyl, cyano, methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, tert-butoxycarbonyl, propoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, pentoxycarbonyl, methylcarbonyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, dichloropropyl, methoxy, methylenedioxy, ethoxy, propoxy, n-butoxy, hydroxymethyl, hydroxyethyl, methoxymethyl, ethoxymethyl, trifluoromethoxy, methylamino, N,N-dimethylamino, phenylamino, ethoxycarbonylethyl, and methoxycarbonylmethyl, methyl, ethyl, fluoromethyl, difluoromethyl, trifluoromethyl, cyano, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, benzyl, phenylethyl, phenylpropyl, methylsulfonyl, phenylsulfonyl, trifluoromethylsulfonyl, hydroxymethyl, hydroxyethyl, methoxymethyl, ethoxymethyl, methylcarbonyl, ethylcarbonyl, trifluoromethylcarbonyl, trifluoro(hydroxy)ethyl, phenylcarbonyl, benzylcarbonyl, methoxycarbonylmethyl, ethoxycarbonylethyl, carboxymethyl, carboxypropyl, methylcarbonyloxymethyl, phenyloxy, phenyloxymethyl, thienyl, furyl, and pyridyl, wherein the thienyl, furyl, pyridyl, methylthio, methylsulfinyl, methyl, ethyl, isopropyl, tert-butyl, isobutyl, pentyl, hexyl, cyano, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, dichloropropyl, methoxy, methylenedioxy, ethoxy, propoxy, n-butoxy, hydroxymethyl, hydroxyethyl and trifluoromethoxy.
A method for identifying a compound that regulates the activity of a signaling factor comprising:
- a) contacting the signaling factor with the compound;
- b) measuring the activity of the signaling factor in the presence of the compound and comparing the activity of the signaling factor obtained in the presence of the compound to the activity of the signaling factor obtained in the absence of the compound;
- and
- c) identifying a compound that regulates the activity of the signaling factor.
A method for detecting an autoinducer molecule in a sample comprising:
- a) contacting the sample with a bacterial cell, or extract thereof, comprising biosynthetic pathways that produce a detectable amount of light in response to an exogenous autoinducer, the bacterial cell having at least two distinct alterations in gene loci that participate in autoinducer pathways, wherein a first alteration in a gene locus comprises an alteration that inhibits detection of a first autoinducer and wherein a second alteration in a gene locus comprises an alteration that inhibits production of a second autoinducer;
- and
- b) measuring light produced by the bacterial cell, or extract thereof, of a).
A bacterial cell comprising at least two distinct alterations in gene loci that participate in autoinducer pathways, wherein a first alteration in a gene locus comprises an alteration that inhibits detection of a first autoinducer and wherein a second alteration in a gene locus comprises an alteration that inhibits production of a second autoinducer and wherein the cell is bioluminescent when contacted with an autoinducer.
A method for identifying an autoinducer analog that regulates the activity of an autoinducer, comprising:
- a) contacting a bacterial cell, or extract thereof, comprising biosynthetic pathways which will produce a detectable amount of light in response to an autoinducer with an autoinducer analog;
- b) comparing the amount of light produced by the bacterial cell, or extract thereof, in the presence of an autoinducer with the amount produced in the presence of the autoinducer analog, wherein a change in the production of light is indicative of an autoinducer analog that regulates the activity of an autoinducer.
A method for producing autoinducer-2 comprising contacting S-adenosylhomocysteine (SAH) with a LuxS protein under conditions and for such time as to promote the conversion of S-adenosylhomocysteine to autoinducer-2.
A method for producing autoinducer-2 comprising contacting S-ribosylhomocysteine (SRH) with a LuxS polypeptide under conditions and for such time as to promote the conversion of S-ribosylhomocysteine to autoinducer-2.
A method for producing autoinducer-2 comprising:
- a) contacting S-adenosylhomocysteine (SAH) with a 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (pfs) protein under conditions and for such time as to promote the conversion of S-adenosylhomocysteine to S-ribosylhomocysteine;
- b) contacting the S-ribosylhomocysteine from a) with a LuxS protein under conditions and for such time as to promote the conversion of S-ribosylhomocysteine to autoinducer-2.
A method for detecting an autoinducer-associated bacterial biomarker comprising;
- a) contacting at least one bacterial cell with an autoinducer molecule under conditions and for such time as to promote induction of a bacterial biomarker;
- and
- b) detecting the bacterial biomarker.
A method for detecting a target compound that binds to a LuxP protein comprising contacting the LuxP protein with the target compound and detecting binding of the compound to LuxP.
A method for regulating bacterial biofilm formation comprising contacting a bacterium capable of biofilm formation with a compound capable of regulating biofilm formation wherein the compound regulates autoinducer-2 activity.
An isolated nucleic acid molecule that encodes a protein necessary for biosynthesis of a bacterial extracellular signaling factor, wherein said factor interacts with LuxQ protein thereby inducing expression of a Vibrio harveyi operon comprising luminescence genes luxCDABE.
An isolated nucleic acid molecule having a sequence selected from the group consisting of:
- a) SEQ ID NO:1;
- b) a variant of SEQ ID NO:1;
- c) a natural mutant of SEQ ID NO:1;
- d) a sequence hybridizing with SEQ ID NO:1 or its complement and encoding a polypeptide substantially the same as the polypeptide encoded by SEQ ID NO:1;
- and
- e) a sequence encoding part or all of a polypeptide having amino acid SEQ ID NO:10.
An isolated nucleic acid molecule having a sequence selected from the group consisting of:
- a) SEQ ID NO:2;
- b) a variant of SEQ ID NO:2;
- c) a natural mutant of SEQ ID NO:2;
- d) a sequence hybridizing with SEQ ID NO:2 or its complement and encoding a polypeptide substantially the same as the polypeptide encoded by SEQ ID NO:2;
- and
- e) a sequence encoding part or all of a polypeptide having amino acid SEQ ID NO:11.
An isolated nucleic acid molecule having a sequence selected from the group consisting of:
- a) SEQ ID NO:4;
- b) a variant of SEQ ID NO:4;
- c) a natural mutant of SEQ ID NO:4;
- d) a sequence hybridizing with SEQ ID NO:4 or its complement and encoding a polypeptide substantially the same as the polypeptide encoded by SEQ ID NO:4;
- and
- e) a sequence encoding part or all of a polypeptide having amino acid SEQ ID NO:12.
A method of purifying a bacterial extracellular signaling factor wherein said factor interacts with LuxQ protein thereby inducing expression of a Vibrio harveyi operon comprising luminescence genes luxCDABE, comprising:
- a) growing, in a culture medium, bacterial cells that produce the signaling molecule;
- b) separating the bacterial cells from the culture medium;
- c) incubating the bacterial cells in a solution having high osmolarity, under conditions that permit production and secretion of the signaling molecule from the bacterial cells;
- d) separating the bacterial cells from the high osmolarity solution;
- and
- e) purifying the factor from the high osmolarity solution.
A kit comprising a carrier means being compartmentalized to receive in close confinement one or more container means containing a strain of bacteria, or extract thereof, comprising biosynthetic pathways that produce a detectable amount of light in response to an exogenous autoinducer, the bacterial cell having at least two distinct alterations in gene loci that participate in autoinducer pathways, wherein a first alteration in a gene locus comprises an alteration that inhibits detection of a first autoinducer and wherein a second alteration in a gene locus comprises an alteration that inhibits production of a second autoinducer.
Dependencies are inferred from the claim text.