Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

Salmonella enterica serovar Typhimurium mutants completely lacking the F(0)F(1) ATPase are novel live attenuated vaccine strains.

The F(0)F(1) ATPase plays a central role in both the generation of ATP and the utilisation of ATP for cellular processes such as rotation of bacterial flagella. We have deleted the entire operon encoding the F(0)F(1) ATPase, as well as genes encoding individual F(0) or F(1) subunits, in Salmonella enteric serovar Typhimurium. These mutants were attenuated for virulence, as assessed by bacterial counts in the livers and spleens of intravenously infected mice. The attenuated in vivo growth of the entire atp operon mutant was complemented by the insertion of the atp operon into the malXY pseudogene region. Following clearance of the attenuated mutants from the organs, mice were protected against challenge with the virulent wild type parent strain. We have shown that the F(0)F(1) ATPase is important for bacterial growth in vivo and that atp mutants are effective live attenuated vaccines against Salmonella infection.

Deletion of tolA in Salmonella Typhimurium generates an attenuated strain with vaccine potential.

The Gram-negative Tol-Pal system of envelope proteins plays a key role in maintaining outer membrane integrity and contributes to the virulence of several pathogens. We have investigated the role of one of these proteins, TolA, in the biology of Salmonella enterica serovar Typhimurium. Deletion of tolA rendered strain SL1344 more susceptible to killing by bile and human serum. In addition the mutant had impaired membrane integrity and displayed alterations in LPS production. The tolA mutant was highly attenuated in mouse infections via the oral and intravenous routes. Importantly, each phenotype displayed by the mutant was complemented by provision of tolA in trans. The tolA gene therefore contributes to virulence, membrane integrity, LPS production and bile and serum resistance in S. enterica serovar Typhimurium SL1344. Finally, immunization with the tolA mutant provided significant protection against subsequent challenge with wild-type SL1344. The Tol-Pal system is therefore a potential target in the development of novel attenuated live vaccines against Salmonella and other Gram-negative pathogens.