NovelSalmonella entericaSerovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication

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Abstract
Upon contact with host cells, the intracellular pathogenSalmonella entericaserovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of anS. entericaserovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribedSalmonellagene designatedsspJthat is located between 54.4 and 64 min of theSalmonellachromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104to 107bacteria in C3H/HeN and 101to 104bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carryingsspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, butsspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type bysspJcomplementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.