Virulence-associated nucleic acid sequences and uses thereof

Abstract

Disclosed are bacterial virulence polypeptides and nucleic acid sequences (e.g., DNA) encoding such polypeptides, and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing such polypeptides to screen for antibacterial or bacteriostatic compounds.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule comprising a sequence substantially identical to SEQ ID NO:252.  
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein said nucleic acid molecule comprises the sequence shown in SEQ ID NO:252  
     
     
         3 . An isolated nucleic acid molecule comprising a sequence substantially identical to SEQ ID NO:105.  
     
     
         4 . The isolated nucleic acid molecule of  claim 3 , wherein said nucleic acid molecule comprises the sequence shown in SEQ ID NO:105.  
     
     
         5 . An isolated nucleic acid molecule comprising a sequence substantially identical to SEQ ID NO:106.  
     
     
         6 . The isolated nucleic acid molecule of  claim 5 , wherein said nucleic acid molecule comprises the sequence shown in SEQ ID NO:106.  
     
     
         7 . A substantially pure polypeptide comprising an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO:253.  
     
     
         8 . The substantially pure polypeptide of  claim 7 , wherein said amino acid sequence comprises the sequence shown in SEQ ID NO:253.  
     
     
         9 . A substantially pure polypeptide comprising an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO:107.  
     
     
         10 . The substantially pure polypeptide of  claim 9 , wherein said amino acid sequence comprises the sequence shown in SEQ ID NO:107.  
     
     
         11 . A substantially pure polypeptide comprising an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO:108.  
     
     
         12 . The substantially pure polypeptide of  claim 11 , wherein said amino acid sequence comprises the sequence shown in SEQ ID NO:108.  
     
     
         13 . A method for identifying a compound which is capable of decreasing the expression of a pathogenic virulence factor, said method comprising the steps of: 
 (a) providing a pathogenic cell expressing a nucleic acid molecule of  claim 1;  and    (b) contacting said pathogenic cell with a candidate compound, a decrease in expression of said nucleic acid molecule following contact with said candidate compound identifying a compound which decreases the expression of a pathogenic virulence factor.    
     
     
         14 . The method of  claim 13 , wherein said pathogenic cell infects a mammal.  
     
     
         15 . The method of  claim 13 , wherein said pathogenic cell infects a plant.  
     
     
         16 . A method for identifying a compound which is capable of decreasing the expression of a pathogenic virulence factor, said method comprising the steps of: 
 (a) providing a pathogenic cell expressing a nucleic acid molecule of  claim 3;  and    (b) contacting said pathogenic cell with a candidate compound, a decrease in expression of said nucleic acid molecule following contact with said candidate compound identifying a compound which decreases the expression of a pathogenic virulence factor.    
     
     
         17 . The method of  claim 16 , wherein said pathogenic cell infects a mammal.  
     
     
         18 . The method of  claim 16 , wherein said pathogenic cell infects a plant.  
     
     
         19 . A method for identifying a compound which is capable of decreasing the expression of a pathogenic virulence factor, said method comprising the steps of: 
 (a) providing a pathogenic cell expressing a nucleic acid molecule of  claim 5;  and    (b) contacting said pathogenic cell with a candidate compound, a decrease in expression of said nucleic acid molecule following contact with said candidate compound identifying a compound which decreases the expression of a pathogenic virulence factor.    
     
     
         20 . The method of  claim 19 , wherein said pathogenic cell infects a mammal.  
     
     
         21 . The method of  claim 19 , wherein said pathogenic cell infects a plant.  
     
     
         22 . A method for identifying a compound which binds a polypeptide, said method comprising the steps of: 
 (a) contacting a candidate compound with a substantially pure polypeptide comprising an amino acid sequence of  claim 7  under conditions that allow binding; and    (b) detecting binding of the candidate compound to the polypeptide.    
     
     
         23 . A method for identifying a compound which binds a polypeptide, said method comprising the steps of: 
 (a) contacting a candidate compound with a substantially pure polypeptide comprising an amino acid sequence of  claim 9  under conditions that allow binding; and    (b) detecting binding of the candidate compound to the polypeptide.    
     
     
         24 . A method for identifying a compound which binds a polypeptide, said method comprising the steps of: 
 (a) contacting a candidate compound with a substantially pure polypeptide comprising an amino acid sequence of  claim 11  under conditions that allow binding; and    (b) detecting binding of the candidate compound to the polypeptide.    
     
     
         25 . A method of treating a pathogenic infection in mammal, said method comprising the steps of: 
 (a) identifying a mammal having a pathogenic infection; and    (b) administering to said mammal a therapeutically effective amount of a composition which inhibits the expression or activity of a polypeptide encoded by a nucleic acid molecule of  claim 1  in said pathogen.    
     
     
         26 . A method of treating a pathogenic infection in mammal, said method comprising the steps of: 
 (a) identifying a mammal having a pathogenic infection; and    (b) administering to said mammal a therapeutically effective amount of a composition which inhibits the expression or activity of a polypeptide encoded by a nucleic acid molecule of  claim 3  in said pathogen.    
     
     
         27 . The method of  claim 26 , wherein said pathogen is  Pseudomonas aeruginosa.    
     
     
         28 . A method of treating a pathogenic infection in mammal, said method comprising the steps of: 
 (a) identifying a mammal having a pathogenic infection; and    (b) administering to said mammal a therapeutically effective amount of a composition which inhibits the expression or activity of a polypeptide encoded by a nucleic acid molecule of  claim 5  in said pathogen.    
     
     
         29 . The method of  claim 28 , wherein said pathogen is  Pseudomonas aeruginosa.    
     
     
         30 . A method of treating a pathogenic infection in a mammal, said method comprising the steps of: 
 (a) identifying a mammal having a pathogenic infection; and    (b) administering to said mammal a therapeutically effective amount of a composition which binds and inhibits a polypeptide encoded by an amino acid sequence of  claim 5 .    
     
     
         31 . The method of  claim 30 , wherein said pathogen is  Pseudomonas aeruginosa.    
     
     
         32 . A method of treating a pathogenic infection in a mammal, said method comprising the steps of: 
 (a) identifying a mammal having a pathogenic infection; and    (b) administering to said mammal a therapeutically effective amount of a composition which binds and inhibits a polypeptide encoded by an amino acid sequence of  claim 7 .    
     
     
         33 . The method of  claim 32 , wherein said pathogen infection is caused by  Pseudomonas aeruginosa.    
     
     
         34 . A method of treating a pathogenic infection in a mammal, said method comprising the steps of: 
 (a) identifying a mammal having a pathogenic infection; and    (b) administering to said mammal a therapeutically effective amount of a composition which binds and inhibits a polypeptide encoded by an amino acid sequence of  claim 9 .    
     
     
         35 . The method of  claim 34 , wherein said pathogen infection is caused by  Pseudomonas aeruginosa.    
     
     
         36 . A method of treating a pathogenic infection in mammal, said method comprising the steps of: 
 (a) identifying a mammal having a pathogenic infection; and    (b) administering to said mammal a therapeutically effective amount of a composition which binds and inhibits a substantially pure polypeptide comprising an amino acid sequence of  claim 11 .    
     
     
         37 . The method of  claim 36 , wherein said pathogen infection is caused by  Pseudomonas aeruginosa.    
     
     
         38 . A method of identifying a compound which inhibits the virulence of a Pseudomonas cell, said method comprising the steps of: 
 (a) providing a Pseudomonas cell;    (b) contacting said cell with a candidate compound; and    (c) detecting the presence of a phenazine, wherein a decrease in said phenazine relative to an untreated control cell is an indication that the compound inhibits the virulence of said Pseudomonas cell.    
     
     
         39 . The method of  claim 38 , wherein said cell is  Pseudomonas aeruginosa.    
     
     
         40 . The method of  claim 38 , wherein said phenazine is detected by spectroscopy.  
     
     
         41 . The method of  claim 38 , wherein said phenazine is a pyocyanin.  
     
     
         42 . The method of  claim 41 , wherein said pyocyanin is detected by measuring the absorbance at 520 nm  
     
     
         43 . The method of  claim 38 , wherein said cell is present in a cell culture.