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.

1 - 17 . (canceled).  
     
     
         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 - 22 . (canceled)  
     
     
         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.  
     
     
         26 . The composition of  claim 18  wherein said first regulatory gene encodes a diphtheria tox repressor (DtxR) protein.  
     
     
         27 . The composition of  claim 18  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.  
     
     
         28 . The composition of  claim 18  wherein said first regulatory gene encodes a diphtheria tox repressor (DtxR) homologue.  
     
     
         29 . The composition of  claim 18  wherein said DtxR homologue is an iron dependent regulator (IdeR).  
     
     
         30 . The composition of  claim 18  wherein said first regulatory gene encodes ferric uptake regulator (Fur).  
     
     
         31 . The composition of  claim 18  wherein said second regulatory gene encodes TetR.  
     
     
         32 . The composition of  claim 19  wherein said second regulatory gene encodes TetR and said second operator comprises tetO.  
     
     
         33 . The composition of  claim 19  wherein said reporter gene encodes chloramphenicol acetyltransferase.  
     
     
         34 . The composition of  claim 20  wherein said second vector is a lambda phage.  
     
     
         35 . The composition of  claim 19 , wherein said first regulatory gene encodes a diptheria tox repressor (DTXR) protein or a homologue thereof, said first operator binds said DtxR protein or homologue, said second regulatory gene encodes TetR, said second operator comprises tetO, and said reporter gene encodes chloramphenicol acetyltransferase.  
     
     
         36 . The composition of  claim 20 , wherein said first regulatory gene encodes a diptheria tox repressor (DTXR) protein or a homologue thereof, said first operator binds said DtxR protein or homologue, said second regulatory gene encodes TetR, said second operator comprises tetO, and said reporter gene encodes chloramphenicol acetyltransferase.  
     
     
         37 . The composition of  claim 23  wherein said chelating agent is 2,2′-dipyridyl.  
     
     
         38 . A recombinant host cell comprising the composition of matter of  claim 18 .  
     
     
         39 . The recombinant host cell of  claim 36  which is an  E. coli  cell.  
     
     
         40 . A recombinant host cell comprising the composition of matter of  claim 20 .  
     
     
         41 . The recombinant host cell of  claim 38  which is a  E. coli  cell.  
     
     
         42 . A composition comprising the recombinant host cell of  claim 39  and a chelating agent.  
     
     
         43 . A composition comprising the recombinant host cell of  claim 41  and a chelating agent.