Characterization of a Pore-Forming Cytotoxin Expressed by Salmonella enterica Serovars Typhi and Paratyphi A

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Abstract
Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene that has been characterized so far only in Escherichia coli . Using DNA sequence analysis and PCR, we established that clyA is conserved in the human-specific typhoid Salmonella enterica serovars Typhi and Paratyphi A and that the entire clyA gene locus is absent in many other S. enterica serovars, including Typhimurium. The gene products, designated ClyA STy and ClyA SPa , show ≥90% amino acid identity to E. coli cytolysin A, ClyA EC , and they are immunogenically related. The Salmonella proteins showed a pore-forming activity and are hence functional homologues to ClyA EC . The chromosomal clyA STy gene locus was expressed at detectable levels in the serovar Typhi strains S2369/96 and S1112/97. Furthermore, in the serovar Typhi vaccine strain Ty21a, expression of clyA STy reached phenotypic levels, as detected on blood agar plates. The hemolytic phenotype was abolished by the introduction of an in-frame deletion in the clyA STy chromosomal locus of Ty21a. Transcomplementation of the mutant with a cloned clyA STy gene restored the hemolytic phenotype. To our knowledge, Ty21a is the first reported phenotypically hemolytic Salmonella strain in which the genetic determinant has been identified.