Recombinant staphylococcus thioredoxin reductase, and inhibitors thereof useful as antimicrobial agents

Abstract

Isolated and purified Staphylococcus thioredoxin reductases (TrxB) are provided. Polynucleotides encoding the TrxBs, vectors and host cells containing such polynucleotides are also provided. In addition, antibodies reactive with the TrxBs are provided, as are methods of isolating the TrxBs, as well as methods for producing recombinant TrxBs, using TrxBs for screening compounds for TrxB-modulating activity, and detecting Staphylococcus in a test sample.

We claim:  
     
         1 . A method for identifying a compound that modulates Staphylococcus thioredoxin reductase (TrxB) activity, comprising: 
 (a) providing a Staphylococcus TrxB polypeptide comprising an amino acid sequence having Staphylococcus thioredoxin reductase activity capable of catalyzing a specific reduction of thioredoxin with a concomitant oxidation of NADPH to NADP + ;    (b) contacting a test compound with the TrxB polypeptide in the presence of thioredoxin, NADPH and a disulfide-containing substrate, under conditions that favor reduction of the disulfide-containing substrate; and    (c) detecting the presence of free sulfhydryl groups formed by the reduction of disulfides of the disulfide-containing substrate, as a measure of TrxB activity, thereby identifying a compound that modulates Staphylococcus TrxB activity.    
     
     
         2 . The method of  claim 1 , wherein the amino acid sequence comprises residues 6-21, 134-137, 146-161 and 267-277 of SEQ ID NO:2 or SEQ ID NO:10; or residues 6-21, 134-137, 146-161 and 267-277 of SEQ ID NO:2 or SEQ ID NO:10, with conservative amino acid substitutions.  
     
     
         3 . The method of  claim 1 , wherein the amino acid sequence comprises residues 1-277 of SEQ ID NO:2 or SEQ ID NO:10; or residues 1-277 of SEQ ID NO:2 or SEQ ID NO:10, with conservative amino acid substitutions.  
     
     
         4 . The method of  claim 1 , wherein the amino acid sequence is SEQ ID NO:2, SEQ. ID NO:10, or an amino acid sequence having 90% identity to SEQ ID NO:2 or SEQ ID NO:10.  
     
     
         5 . The method of  claim 2 , wherein the amino acid sequence comprises SEQ ID NO:2.  
     
     
         6 . The method of  claim 2 , wherein the amino acid sequence comprises SEQ ID NO:10.  
     
     
         7 . The method of  claim 1 , wherein the disulfide-containing substrate is insulin.  
     
     
         8 . The method of  claim 1 , wherein the disulfide-containing substrate is 5,5′-dithio-bis-2-nitrobenzoic acid.  
     
     
         9 . A compound identified by the method of  claim 1 , wherein the compound modulates Staphylococcus thioredoxin reductase (TrxB) activity.  
     
     
         10 . A composition comprising the compound of  claim 9  and a pharmaceutically acceptable excipient.  
     
     
         11 . A method of treating a Staphylococcus infection in an infected subject, comprising administering to the subject an effective antibacterial amount of the composition of  claim 10 .  
     
     
         12 . The method of  claim 11 , wherein the infection comprises a  Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus albus, Staphylococcus hyicus, Staphylococcus hyos, Staphylococcus intermedius  or  Staphylococcus simulans  infection.  
     
     
         13 . The method of  claim 12 , wherein the infection is a  Staphylococcus aureus  infection.