Nagy Gábor, Danino Vittoria, Dobrindt Ulrich, Pallen Mark, Chaudhuri Roy, Emödy Levente, Hinton Jay C, Hacker Jörg
Department of Medical Microbiology and Immunology, University of Pécs, Szigeti út 12, 7624 Pécs, Hungary.
Infect Immun. 2006 Oct;74(10):5914-25. doi: 10.1128/IAI.00619-06.
Mutants of Salmonella enterica serovar 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 with S. enterica serovars Enteritidis and Typhimurium using whole-genome Salmonella microarrays. 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 the rfaH mutation 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 DeltarfaH strains. 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 of rfaH, 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 the Salmonella rfaH mutant into antigen-presenting cells, combined with increased intracellular killing and the potential for raising a cross-protective immune response, renders the rfaH mutant an ideal vaccine candidate.
缺乏转录调节因子RfaH的鼠伤寒沙门氏菌突变体作为抗小鼠沙门氏菌病的口服活疫苗效果显著。我们发现，候选疫苗菌株的减毒与上皮细胞和巨噬细胞中净生长的减少有关。为了鉴定相关的RfaH依赖性基因，利用全基因组沙门氏菌微阵列对肠炎沙门氏菌和鼠伤寒沙门氏菌血清型确定了RfaH调控子。除了影响参与脂多糖（LPS）核心和O抗原合成的基因表达外，RfaH的缺失还导致SPI-4基因、鞭毛/趋化系统和III型分泌系统1的显著下调。然而，这些影响中的一部分可能是LPS生物合成所需基因表达改变的间接后果。通过对结构性深粗糙LPS突变体（waaG）进行全基因组转录谱分析，分离出了rfaH突变的直接和间接影响。我们发现LPS本身的截短是导致DeltarfaH菌株细胞内产量降低的原因。LPS突变体在复制能力上没有差异；相反，它们在细胞内环境中对抗菌肽的敏感性增加。另一方面，有证据表明，rfaH以及其他一些参与LPS生物合成的基因的缺失会导致对各种哺乳动物细胞的侵袭增强。在没有血清型特异性抗原的情况下，常见次要抗原的暴露可能是疫苗接种后所引发的免疫反应具有交叉反应性的原因。沙门氏菌rfaH突变体对抗抗原呈递细胞的侵袭性增加，再加上细胞内杀伤作用增强以及引发交叉保护性免疫反应的潜力，使得rfaH突变体成为理想的疫苗候选物。
