Down-Regulation of Key Virulence Factors Makes theSalmonella entericaSerovar TyphimuriumrfaHMutant a Promising Live-Attenuated Vaccine Candidate

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Abstract
Mutants ofSalmonella entericaserovar Typhimurium that lack the transcriptional regulator RfaH are efficient as live oral vaccines against salmonellosis in mice. We show that the attenuation of the vaccine candidate strain is associated with reduced net growth in epithelial and macrophage cells. In order to identify the relevant RfaH-dependent genes, the RfaH regulon was determined withS. entericaserovars Enteritidis and Typhimurium using whole-genomeSalmonellamicroarrays. As well as impacting the expression of genes involved in lipopolysaccharide (LPS) core and O-antigen synthesis, the loss of RfaH results in a marked down-regulation of SPI-4 genes, the flagellum/chemotaxis system, and type III secretion system 1. However, a proportion of these effects could have been the indirect consequence of the altered expression of genes required for LPS biosynthesis. Direct and indirect effects of therfaHmutation were dissociated by genome-wide transcriptional profiling of a structural deep-rough LPS mutant (waaG). We show that truncation of LPS itself is responsible for the decreased intracellular yield observed for ΔrfaHstrains. LPS mutants do not differ in replication ability; rather, they show increased susceptibility to antimicrobial peptides in the intracellular milieu. On the other hand, evidence that deletion ofrfaH, as well as some other genes involved in LPS biosynthesis, results in enhanced invasion of various mammalian cells is shown. Exposure of common minor antigens in the absence of serovar-specific antigens might be responsible for the observed cross-reactive nature of the elicited immune response upon vaccination. Increased invasiveness of theSalmonella rfaHmutant into antigen-presenting cells, combined with increased intracellular killing and the potential for raising a cross-protective immune response, renders therfaHmutant an ideal vaccine candidate.