Identification and analysis of bacterial virulence genes in vivo

Signature-tagged mutagenesis is a mutation-based screening method for the i dentification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical functio n, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are avai lable to elucidate the function of genes of the second and third classes. W e describe the use of some of these approaches to study the function of thc Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium. This virulence determinant is required for intracellular survi val. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella-containing vacuole. Use of a temperature-sensitive non-replicating plasmid and competitive index tests w ith other genes show that in vivo phenotypes do not always correspond to th ose predicted from in vitro studies.