Environmental Regulation of Salmonella typhi Invasion-Defective Mutants

Salmonella typhi is the etiologic agent of human typhoid. During infection, S. typhi adheres to and invades epithelial and M cells that line the distal ileum. To survive in the human host, S. typhi must overcome numerous complex extracellular and intracellular environments. Since relatively little is known about S. typhi pathogenesis, studies were initiated to identify S. typhi genes involved in the early steps of interaction with the host and to evaluate the environmental regulation of these genes. In the present study, TnphoA mutagenesis was used to study these early steps. We isolated 16 Salmonella typhi TnphoA mutants that were defective for both adherence and invasion of the human small intestinal epithelial cell line Int407. Twelve of sixteen mutations were identified in genes homologous to the S. typhimurium invG and prgH genes, which are known to be involved in the type III secretion pathway of virulence proteins. Two additional insertions were identified in genes sharing homology with the cpxA and damX genes from Escherichia coli K-12, and two uncharacterized invasion-deficient mutants were nonmotile. Gene expression of TnphoA fusions was examined in response to environmental stimuli. We found that the cpxA, invG, and prgH genes were induced when grown under conditions of high osmolarity (0.3 M NaCl). Expression of invG and prgH genes was optimal at pH 6.5 and strongly reduced at low pH (5.0). Transcription of both invG and prgH TnphoA gene fusions was initiated during the late logarithmic growth phase and was induced under anaerobic conditions. Finally, we show that both invG and prgH genes appear to be regulated by DNA supercoiling, a mechanism influenced by environmental factors. These results are the first to demonstrate that in S. typhi, (i) the prgH and cpxA genes are osmoregulated, (ii) the invG gene is induced under low oxygen conditions, (iii) the invG gene is pH regulated and growth phase dependent, and (iv) the prgH gene appears to be regulated by DNA supercoiling. Since our experimental conditions were designed to mimic the in vivo environmental milieu, our results suggest that specific environmental conditions act as signals to induce the expression of S. typhi invasion genes.

伤寒沙门菌（Salmonella typhi）是人类伤寒的致病原。感染过程中，伤寒沙门菌可黏附并侵袭回肠末端黏膜上皮细胞与M细胞。为在人体宿主体内存活，伤寒沙门菌必须克服多种复杂的细胞外与细胞内环境。鉴于目前对伤寒沙门菌致病机制的了解仍较为有限，本研究旨在筛选参与宿主早期相互作用的伤寒沙门菌基因，并评估这些基因的环境调控模式。本研究采用TnphoA诱变技术对上述早期过程展开研究，我们筛选得到16株同时存在黏附与侵袭人类小肠上皮细胞系Int407缺陷的伤寒沙门菌TnphoA突变株。其中12株的突变位点定位于与鼠伤寒沙门菌（S. typhimurium）invG及prgH基因同源的序列，已知这两个基因参与毒力蛋白的III型分泌通路。另外2株插入突变分别位于与大肠杆菌K-12（Escherichia coli K-12）cpxA及damX基因同源的区域，剩余2株未被鉴定的侵袭缺陷突变株呈现无动力表型。我们检测了TnphoA基因融合在不同环境刺激下的表达情况，发现cpxA、invG及prgH基因在高渗环境（0.3 M NaCl）中表达被诱导。invG与prgH基因的表达在pH 6.5时达到最佳水平，在低pH（5.0）条件下则显著下调。invG与prgH的TnphoA基因融合转录均起始于对数生长后期，且在厌氧条件下表达被诱导。最后，我们证实invG与prgH基因的表达似乎受DNA超螺旋调控，而该机制受环境因素影响。本研究结果首次在伤寒沙门菌中证实：(i) prgH与cpxA基因受渗透压调控；(ii) invG基因在低氧条件下表达上调；(iii) invG基因的表达受pH调控且呈生长阶段依赖性；(iv) prgH基因似乎受DNA超螺旋调控。由于本实验条件旨在模拟体内环境微环境，研究结果提示特定环境条件可作为信号分子，诱导伤寒沙门菌侵袭相关基因的表达。