Identification of virulence determinants activators in prokaryotic pathogens

Abstract

Disclosed is a method for identifying activators of a transition metal-dependent repressor of virulence gene expression in infectious prokaryotic pathogens. The method utilizes genetic circuitry that represents the response of a given prokaryote to nutritional stress and the expression of genes that contribute to the establishment of the infectious process. The exposure of recombinant cells or a cell-free system containing the genetic circuitry to a non-metal ion test substance that activates the repressor produces a detectable response. The method is applicable for any prokaryote employing metal ion-dependent repressors to regulate specific gene expression, specifically as it pertains to virulence determinant expression.

We claim:  
     
         1 . (Amended) A method for identifying a non-metal ion activator of a transition metal-dependent repressor of gene expression in a prokaryote, comprising: 
 (a) providing recombinant cells comprising a first recombinant DNA segment containing a first promoter operably linked to a first regulatory gene encoding a first repressor native to or functional in a given prokaryote, a second DNA segment containing a second promoter operably linked to a first operator that binds said first repressor and a second regulatory gene encoding a second repressor, and a third recombinant DNA segment comprising a third promoter operably linked to a second operator that binds the second repressor, and a reporter gene;    (b) culturing said recombinant cells in medium substantially free of metal ion activators of said first repressor and which contains a selection agent that directly or indirectly causes a detectable response upon expression or lack of expression of the reporter gene;    (c) adding a non-metal ion test substance to said medium; and    (d) determining whether the response occurs as an indication of whether said test substance activates said first repressor.    
     
     
         2 . The method of  claim 1  wherein said first regulatory gene encodes a diphtheria tox repressor (DtxR) protein and said first operator binds said DtxR protein.  
     
     
         3 . The method of  claim 2  wherein said first regulatory gene encodes DtxR and said first operator comprises native tox operator, a functional fragment of said operator or a variant of a DtxR consensus binding sequence.  
     
     
         4 . The method of  claim 1  wherein said first regulatory gene encodes a diphtheria tox repressor (DtxR) homologue and said first operator binds said DtxR homologue.  
     
     
         5 . The method of  claim 4 , wherein said DtxR homologue is an iron dependent regulator (IdeR) and said first operator binds said IdeR.  
     
     
         6 . The method of  claim 1 , wherein said first repressor encodes ferric uptake regulator (Fur).  
     
     
         7 . The method of  claim 1  wherein said second regulatory gene encodes TetR and said second operator comprises tetO.  
     
     
         8 . The method of  claim 1  wherein said reporter gene encodes chloramphenicol acetyltransferase and said selection agent is chloramphenicol.  
     
     
         9 . The method of  claim 1  wherein said medium comprises a chelating agent that binds metal ion activators of said first repressor.  
     
     
         10 . The method of  claim 9  wherein said chelating agent is 2,2′-dipyridyl.  
     
     
         11 . The method of  claim 1  wherein said first and second recombinant DNA segments are contained in a first vector and said third recombinant DNA segment is contained in a second vector.  
     
     
         12 . The method of  claim 11  wherein said second vector is a lambda phage.  
     
     
         13 . The method of  claim 1  wherein said cells are  E. coli  cells.  
     
     
         14 . A method for identifying a non-metal ion activator of a diphtheria tox repressor (DtxR) protein in a prokaryote, comprising: 
 (a) providing recombinant cells comprising a recombinant vector, wherein said vector comprises a first DNA segment containing a first promoter operably linked to a first regulatory gene encoding a DtxR protein, a second DNA segment comprising a second promoter operably linked to an operator that binds said DtxR protein and a second regulatory gene encoding a tetracycline repressor (TetR), and a third DNA segment comprising a third promoter operably linked to a tetracycline operator (tetO) and a reporter gene encoding chloramphenicol acetyltransferase;    (b) culturing said recombinant cells in medium substantially free of metal ion activators of said DtxR protein and which comprises chloramphenicol;    (c) adding a test substance to said medium; and    (d) determining the extent of growth of said cells as an indication of whether said test substance activates said DtxR protein.    
     
     
         15 . A method for identifying a non-metal ion activator of a metal-dependent repressor of gene expression in a prokaryote, comprising: 
 providing a solution containing (a) purified repressor native to or functional in a given prokaryote; (b) a DNA construct comprising in operable association, a promoter, an operator and a reporter gene; (c) a coupled transcriptional and translational system that allows expression of said reporter gene; (d) a chelating agent that binds metal activators of said repressor; and (e) a non-metal test substance to allow a reaction to occur; and    detecting expression or lack of expression of said reporter gene as an indication of whether the test substance activates said repressor.    
     
     
         16 . The method of  claim 15  wherein the coupled transcriptional and translational system comprises bacterial extract.  
     
     
         17 . The method of  claim 15  wherein said reporter gene encodes β-galactosidase or luciferase.  
     
     
         18 . A composition of matter, comprising: a recombinant vector comprising a first DNA segment containing a first promoter operably linked to a first regulatory gene encoding a first repressor native to or functional in a given procaryote, and a second DNA segment containing a second promoter operably linked to a first operator that binds said first repressor, and a second regulatory gene encoding a second repressor.  
     
     
         19 . The composition of matter of  claim 18  wherein said recombinant vector further comprises a third DNA segment comprising a third promoter operably linked to a second operator that binds the second repressor, and a reporter gene.  
     
     
         20 . The composition of matter of  claim 18  wherein said recombinant vector is a first recombinant vector and said composition further comprises a second recombinant vector comprising a third DNA segment comprising a third promoter operably linked to a second operator that binds the second repressor, and a reporter gene.  
     
     
         21 . The composition of matter of  claim 18  which is an  E. coli  cell.  
     
     
         22 . The composition of matter of  claim 20  which is an  E. coli  cell.  
     
     
         23 . A composition of matter comprising: (a) purified repressor protein native to or functional in a given procaryote, a, (b) a DNA construct comprising in operable association, a promoter, an operator that binds said repressor protein and a reporter gene, (c) a transcriptional and translational system that allows expression of said reporter gene and (d) a chelating agent that binds metal ion activators of said repressor protein.  
     
     
         24 . The composition of matter of  claim 23  further comprising a non-metal ion test substance.  
     
     
         25 . The composition of matter of  claim 23  wherein said system comprises bacterial extract.