Virulence genes, proteins, and their use

Abstract

A series of genes from Pseudomonas aeruginosa and Klebsiella are shown to encode products that are implicated in virulence. The identification of theses genes therefore allows attenuated microorganisms to be produced. Furthermore, the genes or their encoded products can be used to identify antimicrobial drugs, diagnostic methods for the identification of a pathogen-associated disease, and in the manufacture of vaccines.

1 . An attenuated bacterial mutant derived from a pathogenic bacterial strain, wherein said attenuated mutant has: 
 (i) a mutation of a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14. VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45, and VIR46; and    (ii) reduced inhibition of  Dictyostelium  amoeba growth when compared to the growth observed in the presence of an isogenic bacterial strain.    
     
     
         2 . An attenuated bacterial mutant of  claim 1 , wherein said mutation is insertional inactivation or a gene deletion.  
     
     
         3 . An attenuated bacterial mutant of  claim 1 , wherein said mutant is a gram-negative bacteria.  
     
     
         4 . An attenuated bacterial mutant of  claim 3 , wherein said attenuated gram-negative bacterial mutant is a  Pseudomonas  species.  
     
     
         5 . An attenuated bacterial mutant of  claim 4 , wherein said  Pseudomonas  species is  Pseudomonas aeruginosa.    
     
     
         6 . An attenuated  Pseudomonas  mutant of  claim 5 , wherein said attenuated  Pseudomonas  mutant is selected from the group consisting of: MUT1; MUT2; MUT3; MUT4; MUT5; MUT6; MUT7; MUT8; MUT9; MUT10; MUT11; MUT12; MUT13; MUT14; MUT15; MUT16; MUT17; MUT18; and MUT19.  
     
     
         7 . An attenuated bacterial mutant of  claim 3 , wherein said gram-negative bacterial mutant is a  Klebsiella  species.  
     
     
         8 . An attenuated bacterial mutant of  claim 7 , wherein said  Klebsiella  species is  Klebsiella pneumoniae.    
     
     
         9 . An attenuated  Klebsiella  mutant of  claim 8 , wherein said attenuated  Klebsiella  mutant is selected from the group consisting of: MUT20; MUT21; MUT22; MUT23; MUT24; MUT25; MUT26; MUT27; MUT28; MUT29; MUT30; MUT31; MUT32; MUT33; MUT34; MUT35; MUT36; MUT37; MUT38; MUT39; MUT40; MUT41; MUT42; MUT43; MUT44; MUT45; and MUT46.  
     
     
         10 . A method for identifying an antimicrobial drug, said method comprising: 
 (a) contacting a candidate composition with at least one polypeptide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45 and VIR46; and    (b) comparing the biological activity of said polypeptide in the presence and absence of said candidate composition, wherein alteration of the biological activity of said polypeptide indicates that said candidate composition is an antimicrobial drug.    
     
     
         11 . A method of  claim 10 , wherein said candidate composition contains at least two molecules.  
     
     
         12 . A method of  claim 10 , wherein said candidate composition contains at least one-molecule less than about 500 Daltons.  
     
     
         13 . A method of  claim 10 , wherein said candidate composition contains at least one molecule greater than about 500 Daltons.  
     
     
         14 . A method of  claim 10 , wherein said candidate composition contains at least one molecule selected from a group consisting of a polypeptide, polysaccharide, lipid, nucleic acid, or combination thereof.  
     
     
         15 . A composition of  claim 14 , wherein said polypeptide is an immunoglobulin.  
     
     
         16 . A method for identifying an antimicrobial drug, said method comprising: 
 (a) contacting at a candidate composition with at least one polynucleotide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45, and VIR46; and    (b) comparing the expression of said polynucleotide in the presence and absence of said candidate composition, wherein alteration of the expression of said nucleotide indicates that said candidate composition is an antimicrobial drug.    
     
     
         17 . A method of  claim 16 , wherein said candidate composition contains at least two molecules.  
     
     
         18 . A method of  claim 16 , wherein said candidate composition contains at least one molecule less than about 500 Daltons.  
     
     
         19 . A method of  claim 16 , wherein said candidate composition contains at least one molecule greater than about 500 Daltons.  
     
     
         20 . A method of  claim 16 , wherein said candidate composition contains at least one molecule selected from a group consisting of a polypeptide, polysaccharide, lipid, nucleic acid, or combination thereof.  
     
     
         21 . A composition of  claim 20 , wherein said nucleic acid is a ribonucleic acid.  
     
     
         22 . A nucleic acid of  claim 21 , wherein said nucleic acid is a small interfering ribonucleic acid.  
     
     
         23 . A method for determining the degree of virulence of a pathogen in a subject, said method comprising: 
 (a) measuring the level of expression of at least one polypeptide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45, and VIR46, in a sample from the first subject; and    (b) comparing the amount of said polypeptide in said sample of step (a) to the amount of said polypeptide present in a control sample from a second subject known not to have the presence of said pathogen, wherein an alteration in the expression level of said polypeptide in said first subject as compared to said control sample indicates the degree of virulence of said pathogen.    
     
     
         24 . A method of  claim 23 , wherein said subject is a mammal.  
     
     
         25 . A mammalian subject of  claim 24 , wherein said mammalian subject is a human.  
     
     
         26 . A method for determining the degree of virulence of a pathogen in a subject, said method comprising: 
 (a) measuring the level of expression of at least one polynucleotide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR44, VIR45, and VIR46, in a sample from the first subject; and    (b) comparing the amount of said polynucleotide in said sample of step (a) to the amount of said polynucleotide present in a control sample from a second subject known not to have the presence of said pathogen, wherein an alteration in the expression level of said polynucleotide in said first subject as compared to said control sample indicates the degree of virulence of said pathogen.    
     
     
         27 . A method of  claim 26 , wherein said subject is a mammal.  
     
     
         28 . A mammalian subject of  claim 27 , wherein said mammalian subject is a human.  
     
     
         29 . An attenuated bacterial mutant of  claim 1 , wherein said mutant encodes and expresses a foreign antigen.  
     
     
         30 . An attenuated bacterial mutant of  claim 1 , wherein said mutant contains a plasmid which encodes and expresses, in a eukaryotic cell, a foreign antigen.  
     
     
         31 . A vaccine against a disease caused by a pathogenic microorganism comprising: 
 (a) a pharmaceutically effective dosage of one or more of the attenuated bacterial mutants of  claim 1  and;    (b) a pharmaceutically acceptable diluent or carrier.    
     
     
         32 . An attenuated bacterial mutant derived from a pathogenic bacterial strain, wherein said attenuated mutant has: 
 (i) a mutation of a gene selected from the group consisting of pchE, pchF, pchG, pchH, and pchI; and    (ii) reduced inhibition of  Dictyostelium  amoeba growth when compared to the growth observed in the presence of an isogenic bacterial strain.    
     
     
         33 . A bacterial strain comprising an operon encoding a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR44, VIR45, and VIR46, wherein said bacterial strain includes a mutation that reduces expression of said gene relative to an isogenic bacterial strain lacking said mutation.  
     
     
         34 . A bacterial strain of  claim 33 , wherein said mutation reduces inhibition of  Dictyostelium  amoeba growth when compared to the growth of  Dictyostelium  amoeba in the presence of an isogenic bacterial strain lacking said mutation.