Shigella species are unique among enteric pathogens in the low infective dose required for disease. Whereas over 10-6 Vibrio cholera or 10-5 Salmonella species are required to produce disease, 200 or less shigellae are able to do so. Shigella species are able to survive a pH of less than 3.0 for several hours; the ability to survive low pH is an important determinant of infective dose. The objectives of this project are 1) to characterize the genetic and physiologic basis of acid resistance in Shigella species and 2) to utilize this information in the construction of better oral vaccine strains. We have isolated a clone from a library of S. flexneri DNA which confers acid resistance on an acid sensitive Escherichia coli. Deletion analysis and complementation studies identified the acid resistance determinant as a homologue of the E. coli stationary phase sigma factor, rpos. We have sequenced the gene and found over 90% DNA identity between the Shigella and E. coli open reading frame for rpos. However, there are significant differences between two in the upstream regulatory portion of the gene. Measurement of internal pH in the face of falling external pH show that wild type S. flexneri is able to maintain a significantly higher internal pH when exposed to an external pH below 4.0 than is a S. flexneri rpos:Tn10. This suggests that rpos may regulate the expression of proteins important for transport of ions across the cytoplasmic membrane. We are in the process of identifying rpos regulated genes required for survival at low pH. Twenty-nine acid sensitive TnPhoA mutants have been isolated and are being characterized. An analysis of acid survival in S. flexneri will provide a basis for designing better oral vaccine strains since delivery of antigens to the intestines will be enhanced by better survival of strains through the stomach.
