Identification of virulence determinants activators in prokaryotic pathogens
OwnerJohn R. Murphy, Li Suninventors
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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.
Claims25
(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.
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.
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.
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.
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.
Dependencies are inferred from the claim text.