Inhibitors of autoinducer transporters

Abstract

The present invention relates to the discovery of the lsr operon, the genes therein, and the polypeptides encoded by these genes. The present invention also includes strains with altered expression levels of the polypeptides encoded by the genes and the lsr operon relative to wild type cells. In some embodiments, the strains express a transporter that transports an autoinducer into the cell at a level higher than that of wild type cells. The present invention also includes methods for identifying compounds that modulate the transport of the autoinducer into cells.

1 . (canceled)  
     
     
         2 . (canceled)  
     
     
         3 . (canceled)  
     
     
         4 . (canceled)  
     
     
         5 . (canceled)  
     
     
         6 . (canceled)  
     
     
         7 . (canceled)  
     
     
         8 . (canceled)  
     
     
         9 . (canceled)  
     
     
         10 . (canceled)  
     
     
         11 . (canceled)  
     
     
         12 . (canceled)  
     
     
         13 . (canceled)  
     
     
         14 . (canceled)  
     
     
         15 . (canceled)  
     
     
         16 . (canceled)  
     
     
         17 . (canceled)  
     
     
         18 . (canceled)  
     
     
         19 . (canceled)  
     
     
         20 . (canceled)  
     
     
         21 . (canceled)  
     
     
         22 . (canceled)  
     
     
         23 . (canceled)  
     
     
         24 . (canceled)  
     
     
         25 . (canceled)  
     
     
         26 . (canceled)  
     
     
         27 . (canceled)  
     
     
         28 . A method for identifying a compound that modulates the response to a first autoinducer that is not an acyl-homoserine lactone and that can interact with the  Vibrio harveyi  LuxQ protein, thereby inducing expression of a  Vibrio harveyi  operon comprising the luxCDABE genes, comprising: 
 obtaining a cell having increased expression of a transporter that transports the autoinducer into the cell, wherein the cell produces a detectable signal in response to the first autoinducer;    measuring the response of the cell to the first autoinducer in the presence and absence of a test compound; and    comparing the responses to determine whether the test compound modulates the response to the first autoinducer.    
     
     
         29 . The method of  claim 28 , wherein the cell has been genetically engineered to increase expression of the transporter.  
     
     
         30 . The method of  claim 28 , wherein the transporter comprises at least one polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 37-40 and a sequence having at least 25% identity as determined through use of FASTA version 3.0t78 with the default parameters to one of SEQ ID NOs.: 37-40.  
     
     
         31 . The method of  claim 28 , wherein the transporter comprises a complex comprising each of the amino acid sequences of SEQ ID NOs. 37-40 or amino acid sequences having at least 25% identity as determined through use of FASTA version 3.0t78 with the default parameters to each of the sequences of SEQ ID NOs.: 37-40.  
     
     
         32 . The method of  claim 29 , wherein the cell comprises at least one vector from which one or more polypeptides included in the transporter are expressed.  
     
     
         33 . The method of  claim 29 , wherein the cell comprises a mutation that increases expression of the transporter.  
     
     
         34 . The method of  claim 33 , wherein the mutation is in a gene encoding a repressor that reduces expression of the transporter.  
     
     
         35 . The method of  claim 34  wherein the mutation is in a gene comprising the sequence of SEQ ID NO: 28.  
     
     
         36 . The method of  claim 34 , wherein the mutation is in a gene comprising a sequence having at least 30% identity to SEQ ID NO: 28 as determined through use of BLASTN version 2.0 with the default parameters.  
     
     
         37 . The method of  claim 34 , wherein the mutation is in a gene encoding a polypeptide comprising the sequence of SEQ ID NO: 36  
     
     
         38 . The method of  claim 34 , wherein the mutation is in a nucleic acid comprising a sequence that hybridizes to a probe comprising at least 20 consecutive nucleotides of SEQ ID NO: 28 in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.  
     
     
         39 . The method of  claim 34 , wherein the mutation is in a polypeptide comprising a sequence having at least 25% identity to SEQ ID NO: 36 as determined through use of FASTA version 3.0t78 with the default parameters.  
     
     
         40 . The method of  claim 34  wherein the cell further comprises a mutation in a gene that inhibits the production of the autoinducer.  
     
     
         41 . The method of  claim 40 , wherein the mutation that inhibits the production of the autoinducer is in a luxS gene.  
     
     
         42 . The method of  claim 41 , wherein the mutation that inhibits the production of the autoinducer is in a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9.  
     
     
         43 . The method of  claim 40 , wherein the mutation is in a nucleic acid comprising a sequence having at least 30% nucleotide identity as determined through use of BLASTN version 2.0 with the default parameters to a sequence selected from the group consisting of SEQ ID NOs: 1-9.  
     
     
         44 . The method of  claim 40 , wherein the cell further comprises a mutation that inhibits the detection of a second autoinducer that is an acylhomoserine lactone.  
     
     
         45 . The method of  claim 44 , wherein the second autoinducer is N-(3-hydroxyacyl)-L-homoserine lactone and the acyl group comprises 4-12 carbon atoms.  
     
     
         46 . The method of  claim 45 , wherein the acyl group comprises four carbon atoms.  
     
     
         47 . The method of  claim 44 , wherein the mutation that inhibits the detection of the second autoinducer is in the luxN gene.  
     
     
         48 . The method of  claim 28 , wherein the autoinducer is the autoinducer-2 produced by  Vibrio harveyi.    
     
     
         49 . The method of  claim 28 , wherein the autoinducer is a pentanedione.  
     
     
         50 . The method of  claim 49 , wherein the pentanedione is 4,5-dihydroxy-2,3-pentanedione.  
     
     
         51 . The method of  claim 28 , wherein the cell belongs to a species selected from the group consisting of  S. typhimurium  and  E. coli.    
     
     
         52 . The method of  claim 28 , wherein the cell belongs to a species selected from the group consisting of  Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borrelia burgdorferi  and  Vibrio cholerae.    
     
     
         53 . The method of  claim 1 , wherein the cell is a  Vibrio harveyi  cell.  
     
     
         54 . A method for screening a candidate compound for the ability to bind to a transporter that transports an autoinducer into a cell, wherein the autoinducer is not an acyl-homoserine lactone and can interact with the  Vibrio harveyi  LuxQ protein thereby inducing expression of a  Vibrio harveyi  operon comprising the luxCDABE genes, comprising: 
 contacting the transporter with the candidate compound; and    determining whether the compound specifically binds to the transporter.    
     
     
         55 . The method of  claim 54 , wherein the compound comprises a detectable label.  
     
     
         56 . The method of  claim 54 , wherein the transporter comprises at least one polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs.: 37-40 and a sequence having at least 25% amino acid identity as determined through use of FASTA version 3.0t78 with the default parameters to one of SEQ ID NOs.: 37-40.  
     
     
         57 . The method of  claim 54 , wherein the transporter comprises a complex comprising each of the amino acid sequences of SEQ ID NOs. 37-40 or amino acid sequences having at least 25% amino acid identity as determined through use of FASTA version 3.0t78 with the default parameters to each of the amino acid sequences of SEQ ID NOs.: 37-40.  
     
     
         58 . A method of screening a candidate compound for the ability to modulate the binding of an autoinducer to a transporter, wherein the autoinducer is not an acyl-homoserine lactone and can interact with the  Vibrio harveyi  LuxQ protein thereby inducing expression of a  Vibrio harveyi  operon comprising the luxCDABE genes, comprising: 
 comparing the binding of the autoinducer to the transporter in the presence and absence of the candidate compound; and    determining whether the the extent of binding of the autoinducer to the transporter in the presence of the compound increases or decreases relative to the extent of binding in the absence of the compound.    
     
     
         59 . A method of screening a candidate compound for the ability to bind a polypeptide comprising: 
 contacting a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs. 37-40 and a sequence having at least 25% identity as determined through use of FASTA version 3.0t78 with the default parameters to one of the sequences of SEQ ID NOs.: 37-40 with the compound; and    determining whether the compound specifically binds to the polypeptide.    
     
     
         60 . An isolated bacterial strain comprising a mutation that inhibits the transport of an autoinducer that is not an acyl-homoserine lactone and that can interact with the  Vibrio harveyi  LuxQ protein thereby inducing expression of a  Vibrio harveyi  operon comprising the luxCDABE genes.  
     
     
         61 . The bacterial strain of  claim 60 , wherein the mutation is in a gene selected from the group consisting of the lsrA, lsrB, lsrC, and lsrD genes.  
     
     
         62 . The bacterial strain of  claim 61 , wherein the strain is a Salmonella typhimurium strain.  
     
     
         63 . The bacterial strain of  claim 62 , wherein the mutation is in a sequence selected from the group consisting of SEQ ID NOs.: 29-32.  
     
     
         64 . The bacterial strain of  claim 63 , wherein the mutation is in a gene encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 37-40.  
     
     
         65 . The bacterial strain of  claim 63 , wherein the mutation is in a nucleic acid comprising a sequence selected from the group consisting of a sequence having at least 30% nucleotide identity to one of SEQ ID NOs.: 29-32 as determined through use of BLASTN version 2.0 with the default parameters.  
     
     
         66 . A bacterial strain that overexpresses or underexpresses a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs. 36-43 and a sequence having at least 25% identity to one of SEQ ID NOs: 36-43 relative to a wildtype strain.  
     
     
         67 . A bacterial strain that overexpresses or underexpresses a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs. 36-43 and a sequence having at least 25% identity to one of SEQ ID NOs: 36-43 relative to a wildtype strain.  
     
     
         68 . An isolated or purified nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 28-35 and a fragment comprising at least 20 consecutive nucleotides of one of of SEQ ID NOs.: 28-35.  
     
     
         69 . An isolated or purified nucleic acid comprising a fragment of one of SEQ ID NOs.: 28-35 that encodes a polypeptide that can facilitate the transport of an autoinducer into a cell, wherein the autoinducer is not an acyl-homoserine lactone and can interact with the  Vibrio harveyi  LuxQ protein thereby inducing expression of a  Vibrio harveyi  operon comprising the luxCDABE genes.  
     
     
         70 . A recombinant vector comprising a sequence selected from the group consisting of SEQ ID NOs: 28-35 operably linked to a heterologous promoter.  
     
     
         71 . An isolated or purified protein comprising a sequence selected from the group consisting of SEQ ID NOs.: 36-43 and a fragment comprising at least 10 consecutive amino acids of one of of SEQ ID NOs. 36-43.  
     
     
         72 . An isolated or purified polypeptide comprising a fragment of one of SEQ ID NOs.: 36-43 that encodes a polypeptide that can facilitate the transport of an autoinducer into a cell, wherein the autoinducer is not an acyl-homoserine lactone and can interact with the  Vibrio harveyi  LuxQ protein thereby inducing expression of a  Vibrio harveyi  operon comprising the luxCDABE genes.  
     
     
         73 . An antibody that binds to a polypeptide selected from the group consisting of SEQ ID NOs.: 36-43.