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Functional homologs of the Escherichia coli cytolysin A (clyA, hlyE, sheA) gene have recently been detected in Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A). In this study, analysis of a collection of Salmonella strains showed that all S. Typhi and S. Paratyphi A strains tested harbor an intact copy of the corresponding clyA variant, i.e. clyA(STy) and clyA(SPaA), respectively. On the other hand, clyA proved to be absent in the S. enterica serovar Paratyphi B and serovar Paratyphi C strains, in various non-typhoid S. enterica subsp. enterica serovars (Typhimurium, Enteritidis, Choleraesuis, Dublin, and Gallinarum), and in S. enterica subsp. arizonae and Salmonella bongori strains. When grown under normal laboratory conditions, the S. Typhi and S. Paratyphi A strains produced only basal amounts of ClyA protein and did not exhibit a clyA-dependent hemolytic phenotype. RT-PCR and immunoblot analyses as well as phenotypic data revealed, however, that the expression of clyA(STy) and clyA(SPaA) can be activated by the Salmonella transcription factor SlyA. In addition, osmotic protection assays and lipid bilayer experiments demonstrated that the hemolytic ClyA(STy) and ClyA(SPaA) proteins are effective pore-forming toxins which, similar to E. coli ClyA, generate large, stable, moderately cation-selective channels in target membranes. Taken together with our recent serological findings which have indicated that S. Typhi and S. Paratyphi A strains produce substantial amounts of ClyA during human infection, these data suggest that ClyA may play a role in S. Typhi and S. Paratyphi A pathogenesis.

Introduction of the Borrelia burgdorferi blyAB locus into Escherichia coli has recently been reported to cause a hemolytic phenotype that is dependent on the E. coli clyA (hlyE, sheA) gene (a cytolysin gene present in many E. coli strains, including E. coli K-12, which is repressed under standard in vitro growth conditions). The blyA gene product has been suggested to be a prophage-encoded holin, but the processes triggered in E. coli by the expression of blyA and/or blyB, which lead to the hemolytic phenotype, remained unclear. Here we show that expression of blyA in E. coli causes damage to the E. coli cell envelope and a clyA-dependent hemolytic phenotype, regardless whether blyB is present or absent. The expression of blyB in E. coli, on the other hand, did not have obvious phenotypic effects. Transcriptional studies demonstrated that the clyA gene is not induced in E. coli cells expressing blyA. Furthermore, protein analyses suggested that the impairment of the E. coli cell envelope by BlyA is responsible for the emergence of the hemolytic activity as it allows latent intracellular ClyA protein, derived from basal-level expression of the clyA gene, to leak into the medium and to lyse erythrocytes. These findings are compatible with the presumption that BlyA functions as a membrane-active holin.