Attenuated live bacteria with increased acid resistance and methods of use thereof

Abstract

The present invention relates to inducing acid resistance in a bacterium and methods of increasing the acid resistance of an acid sensitive bacterium.

What is claimed is: 
     
         1 . A recombinant attenuated derivative of a pathogenic enteric bacterium comprising at least one of the following:
 a) a regulatable promoter operably linked to a nucleic acid encoding an arginine decarboxylase and a nucleic acid encoding an arginine agmatine antiporter;   b) a regulatable promoter operably linked to a nucleic acid encoding a glutamate decarboxylase and a nucleic acid encoding a glutamate/γ-aminobutyric acid antiporter; or   c) a regulatable promoter operably linked to a nucleic acid encoding a lysine decarboxylase and a nucleic acid encoding a lysine/cadaverine antiporter.   
     
     
         2 . The recombinant bacterium of  claim 1 , wherein the enteric bacterium is either naturally acid sensitive or becomes more acid sensitive because of attenuating mutations, such that in the absence of induction of the regulatable promoter, the recombinant bacterium is acid sensitive, but upon induction of the regulatable promoter, the recombinant bacterium displays an increase in acid resistance. 
     
     
         3 . The recombinant bacterium of  claim 2 , wherein the bacterium is acid sensitive because of a mutation in a nucleic acid sequence selected from the group consisting of rpoS, fur, phoPQ and guaBA. 
     
     
         4 . The recombinant bacterium of  claim 1 , wherein the regulatable promoter is induced by a sugar. 
     
     
         5 . The recombinant bacterium of  claim 4 , wherein the sugar is selected from the group consisting of arabinose and rhamnose. 
     
     
         6 . The recombinant bacterium of  claim 1 , wherein the bacterium comprises at least one mutation selected from the group consisting of:
 a) ΔP adiA ::TT araC P araBAD  ad/AC mutation;   b) ΔP adiA ::TT rhaSR P rhaBAD  adiAC mutation;   c) rhaSR P rhaBAD  gadBC mutation;   d) araC P araBAD  gadBC mutation;   e) ΔP cadB ::TT rhaSR P rhaBAD  cadBA mutation; and   f) ΔP cadB ::TT araC P araBAD  cadBA mutation.   
     
     
         7 . The recombinant bacterium of  claim 6 , wherein the bacterium further comprises at least one element selected from the group consisting of:
 a) a regulatable promoter operably linked to gadA;   b) the cicA gene from  E. coli  transcribed from its own native promoter, a heterologous constitutive promoter or a heterologous regulatable promoter; and   c) a Ni-dependent urease system from  H. pylori.      
     
     
         8 . The recombinant bacterium of  claim 1 , wherein the bacterium is a  Salmonella  bacterium and:
 a) the nucleic acid encoding the arginine decarboxylase is a  Salmonella  adiA sequence and the nucleic acid encoding the arginine agmatine antiporter is a  Salmonella  adiC sequence; or   b) the nucleic acid encoding the glutamate decarboxylase is an  E. coli  gadB and/or an  E. coli  gadA sequence and the nucleic acid encoding the glutamate γ-aminobutyric acid antiporter is an  E. coli  gadC sequence; or   c) the nucleic acid encoding the lysine decarboxylase is a  Salmonella  cadA sequence and the nucleic acid encoding the lysine/cadaverine antiporter is a cadB sequence.   
     
     
         9 . A vaccine composition, the composition comprising a bacterium of  claim 1 . 
     
     
         10 . A method for increasing the acid resistance of an acid sensitive bacterium, the method comprising introducing into the acid sensitive bacterium a cassette comprising at least one of the following:
 a) a regulatable promoter operably linked to a nucleic acid encoding an arginine decarboxylase and a nucleic acid encoding an arginine agmatine antiporter;   b) a regulatable promoter operably linked to a nucleic acid encoding a glutamate decarboxylase and a nucleic acid encoding a glutamate/γ-aminobutyric acid antiporter; or   c) a regulatable promoter operably linked to a nucleic acid encoding a lysine decarboxylase and a nucleic acid encoding a lysine/cadaverine antiporter,   
       such that in the absence of induction of the regulatable promoter, the recombinant bacterium is acid sensitive, but upon induction of the regulatable promoter, the recombinant bacterium displays an increase in acid resistance. 
     
     
         11 . The method of  claim 9 , wherein the bacterium comprises a mutation in at least one nucleic acid sequence selected from the group consisting of rpoS, fur, phoPQ and guaBA. 
     
     
         12 . The method of  claim 9 , wherein the regulatable promoter is induced by a sugar. 
     
     
         13 . The method of  claim 11 , wherein the sugar is selected from the group consisting of arabinose and rhamnose. 
     
     
         14 . The method of  claim 9 , wherein the bacterium comprises at least one mutation selected from the group consisting of:
 a) ΔP adiA ::TT araC P araBAD  adiAC mutation;   b) AP adiA ::TT rhaSR P rhaBAD adiAC mutation;   c) rhaSR P rhaBAD  gadBC mutation;   d) araC P araBAD  gadBC mutation;   e) AP cadB ::TT rhaSR P rhaBAD  cadBA mutation; and   f) AP cadB ::TT araC P araBAD  cadBA mutation.   
     
     
         15 . The recombinant bacterium of  claim 14 , wherein the bacterium further comprises at least one element selected from the group consisting of:
 a) a regulatable promoter operably linked to gadA;   b) the cicA gene from  E. coli  transcribed from its own native promoter, a heterologous constitutive promoter or a heterologous regulatable promoter; and   c) a Ni-dependent urease system from  H. pylori.      
     
     
         16 . The method of  claim 9 , wherein the bacterium is a  Salmonella  bacterium and:
 a) the nucleic acid encoding the arginine decarboxylase is a  Salmonella  adiA sequence and the nucleic acid encoding the arginine agmatine antiporter is a  Salmonella  adiC sequence; or   b) the nucleic acid encoding the glutamate decarboxylase is an  E. coli  gadB and/or an  E. coli  gadA sequence and the nucleic acid encoding the glutamate γ-aminobutyric acid antiporter is an  E. coli  gadC sequence; or   c) the nucleic acid encoding the lysine decarboxylase is a  Salmonella  cadA sequence and the nucleic acid encoding the lysine/cadaverine antiporter is a cadB sequence.   
     
     
         17 . A recombinant  Salmonella  bacterium, the bacterium comprising a regulatable promoter operably linked to at least one nucleic acid selected from the group consisting of:
 a) adiA and adiC;   b) gadB and gadC; and   c) cadB and cadA.   
     
     
         18 . The recombinant  Salmonella  bacterium of  claim 17 , wherein the bacterium further comprises a mutation in at least one nucleic acid sequence selected from the group consisting of rpoS, fur, phoPQ and guaBA that renders the bacterium acid sensitive. 
     
     
         19 . The recombinant  Salmonella  bacterium of  claim 17 , wherein the bacterium further comprises at least one element selected from the group consisting of:
 a) a regulatable promoter operably linked to gadA;   b) the clcA gene from  E. coli  transcribed from its own native promoter, a heterologous constitutive promoter or a heterologous regulatable promoter; and   c) a Ni-dependent urease system from  H. pylori.      
     
     
         20 . A vaccine composition, the composition comprising a bacterium of  claim 17 .