Attenuated live bacteria with increased acid resistance and methods of use thereof
The present invention relates to inducing acid resistance in a bacterium and methods of increasing the acid resistance of an acid sensitive bacterium.
1. A recombinant attenuated derivative of a pathogenic enteric Salmonella bacterium comprising a nucleic acid encoding an antigen and at least one of the following:
a) a regulatable promoter operably linked to a nucleic acid encoding a Salmonella AdiA arginine decarboxylase and a nucleic acid encoding a Salmonella AdiC arginine agmatine antiporter;
b) a regulatable promoter operably linked to a nucleic acid encoding an E. coli GadB and/or an E. coli GadA glutamate decarboxylase and a nucleic acid encoding an E. coli GadC glutamate/γ-aminobutyric acid antiporter; or
c) a regulatable promoter operably linked to a nucleic acid encoding a Salmonella CadA lysine decarboxylase and a nucleic acid encoding a Salmonella CadB lysine/cadaverine anti porter.
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 adiAC 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. A vaccine composition, the composition comprising a bacterium of claim 1 .