Identification of inhibitors of a bacterial stress response

Abstract

A system, a composition, a method and a kit for identifying anti-bacterial agents are provided. The invention described herein is useful in identifying inhibitors of any bacterial stress response. Moreover, the invention can be applied to any sRNA and its target, any transcription factor and its target, and any transcription factor/sRNA pair (i.e., a transcription factor that regulates a sRNA). In particular, the present invention provides a system, a composition, a method and a kit for the identification of cyclic peptides that block the σ E pathway in Escherichia coli.

1 .- 8 . (canceled) 
     
     
         9 . A composition for identifying inhibitors of a bacterial stress response comprising:
 A) at least one candidate inhibitor of a bacterial stress response;   B) a bacterial strain including:
 a) a nucleotide sequence comprising:
 i) a first nucleic acid encoding σ E ; 
 ii) a second nucleic acid encoding RybB, wherein the second nucleic acid is operably linked to the first nucleic acid, 
 iii) at least one transcriptional terminator sequence disposed between the first and second nucleic acids; 
 
 b) a nucleotide sequence comprising a third nucleic acid encoding a reporter protein, wherein the third nucleic acid is operably linked to a porin regulatory sequence; 
   C) a culture medium that is used for the bacterial strain.   
     
     
         10 . The composition of  claim 9  further comprising a means for detecting, measuring and comparing expression of the reporter protein. 
     
     
         11 . The composition of  claim 9  further comprising a means for isolating and identifying the inhibitors of the bacterial stress response. 
     
     
         12 . The composition of  claim 9  wherein the reporter protein comprises a luminescent or fluorescent protein. 
     
     
         13 . The composition of  claim 10  wherein the means for detecting and measuring expression of the reported protein comprises a luminometer, a fluorometer, or a fluorescence activated cell sorter. 
     
     
         14 . The composition of  claim 9  wherein the at least one candidate inhibitor of a bacterial stress response comprises a cyclic peptide. 
     
     
         15 . The composition of  claim 9  wherein the at least one candidate inhibitor of a bacterial stress response comprises a library of cyclic peptides. 
     
     
         16 . The composition of  claim 9  wherein the first nucleic acid includes rpoE, the second nucleic acid includes rybB, and the porin regulator sequence comprises one or more ompC regulatory-sequences. 
     
     
         17 .- 25 . (canceled) 
     
     
         26 . A kit for screening for an antibiotic comprising:
 A) at least one candidate antibiotic;   B) a first bacterial strain including;
 a) a nucleotide sequence comprising;
 i) a first nucleic acid encoding σ E ; 
 ii) a second nucleic acid encoding RybB, wherein the second nucleic acid is operably linked to the first nucleic acid; 
 iii) at least one transcriptional terminator sequence disposed between the first and second nucleic acids; 
 
 b) a nucleotide sequence comprising a third nucleic acid encoding a reporter protein, wherein the third nucleic acid is operably linked to a porin regulatory sequence; 
   C) a second bacterial strain including a nucleotide sequence comprising the third nucleic acid encoding a reporter protein and at least one mutation that allows the growth of the bacterial strain in the absence of σ E  activity, wherein the third nucleic acid is operably linked to the porin regulatory sequence; and   D) instructions for use.   
     
     
         27 . A composition comprising a cyclic peptide inhibitor of the σ E  pathway in an amount effective for inhibiting growth of pathogenic bacterial cells and/or blocking the ability of pathogenic bacterial cells to cause disease in a mammal, and a pharmaceutically acceptable carrier. 
     
     
         28 . A composition comprising a cyclic peptide inhibitor of an Hfq activity in an amount effective for inhibiting growth of pathogenic bacterial cells and/or blocking the ability of pathogenic bacterial cells to cause disease in a mammal, and a pharmaceutically acceptable carrier.