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Escherichia coli, a Gram-negative bacterium, which can be classified into three groups: the commensal, intestinal pathogenic (IPEC) and extra-intestinal pathogenic (ExPEC) E. coli. The cytolysin A (ClyA) protein, a 34-kDa pore-forming toxin, encoded by a gene found in both non-pathogenic and pathogenic E. coli and in Salmonella enterica serovars Typhi and Paratyphi. It mediates a cytotoxic effect on various mammalian cells. ClyA is released by E. coli via outer membrane vesicles (OMVs) after reaching the periplasm via an unknown mechanism through the inner membrane. The gene is silenced by mutations in some of the most studied ExPEC strains suggesting that the locus would be subject to patho-adaptive alterations.

To study if the mutations of the clyA gene in E. coli strains was particular to certain strains, the sequences of the clyA gene locus of a set of ExPEC isolates and of the E. coli collection of reference strains (ECOR) were compared. The ExPEC strains – uropathogenic and neonatal meningitis E. coli (UPEC and NMEC) strains contained various ΔclyA alleles. Next, a functional clyA gene locus was restored and tagged with luxAB in the chromosome of the UPEC strain 536. Luciferase activity of the bacteria carrying the restored gene showed that the clyA gene expression is highly increased at the late logarithmic growth phase when compared to the non-pathogenic E. coli K-12 strain. A higher transcriptional level of the clyA+ gene was observed when the SfaX regulatory protein was heterologously overproduced. It was concluded that the clyA+ gene is expressed at elevated levels in the UPEC strain and this is at least in part due to the SfaX/PapX transcriptional regulators.

Studies of clyA::phoA fusions obtained by transposon TnphoA insertion mutagenesis showed that the first 12 amino acid residues of ClyA was sufficient for translocation of the protein chimera into the periplasm and to the OMVs. The role of the two cysteine residues in ClyA for protein translocation was tested by introducing substitution mutations. The results indicated that the C-terminal Cys (ClyAC 285S) is important for localization and/or stability of the protein in the periplasm. Structural analysis of ClyAwt purified from the periplasm revealed that the protein forms dimeric complexes. Upon treatment with the reducing agent DTT the ClyA protein readily assembled into typical pore complexes as revealed by electron miscroscopic analysis. In conclusion, the ClyA protein is present in the periplasm in a conformation that prevents it from forming pores in the bacterial membranes.

Vibrio cholerae cytolysin (VCC) is a pore-forming toxin which induces lysis of mammalian cells by forming transmembrane channels. Although the biophysical activities of VCC were well studied, there was no detailed analysis of VCC secretion. Our study demonstrated that a fraction of the VCC was secreted in association with OMVs. OMV-associated VCC from the wild type V. cholerae strain V:5/04 is biologically active as shown by toxic effects on mammalian cells, interestingly, OMV-associated VCC was more active than purified VCC. Both environmental and clinical V. cholerae isolates transport VCC via OMVs. In addition, when the vcc gene is heterologously expressed in E. coli, OMV-associated secretion of VCC was also observed. We suggest that OMV-mediated release of VCC is a feature shared with ClyA.