ENVIRONMENTAL-REGULATION OF SALMONELLA-TYPHI INVASION-DEFECTIVE MUTANTS

Salmonella typhi is the etiologic agent of human typhoid. During infec tion, S. typhi adheres to and invades epithelial and M cells that line the distal ileum. To survive in the human host, S. typhi must overcom e 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 in teraction with the host and to evaluate the environmental regulation o f these genes. In the present study, TnphoA mutagenesis was used to st udy 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 w ere identified in genes homologous to the S. typhimurium invG and prgH genes, which are known to be involved in the type III secretion pathw ay 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 non motile. Gene expression of TnphoA fusions was examined in response to environmental stimuli, We found that the cpxA, invG, and prgH genes we re 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 g ene fusions was initiated during the late logarithmic growth phase and was induced under anaerobic conditions. Finally, we show that both in vG and prgH genes appear to be regulated by DNA supercoiling, a mechan ism influenced by environmental factors. These results are the first t o demonstrate that in S. typhi, (i) the prgH and cpxA genes are osmore gulated, (ii) the invG gene is induced under low oxygen conditions, (i ii) 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 environment al milieu, our results suggest that specific environmental conditions act as signals to induce the expression of S. typhi invasion genes.