The ClyA protein is a pore-forming cytotoxin expressed by Escherichia coli and some other enterobacteria. It confers cytotoxic activity toward mammalian cells, but it has remained unknown how ClyA is surface exposed and exported from bacterial cells. Outer-membrane vesicles (OMVs) released from the bacteria were shown to contain ClyA protein. ClyA formed oligomeric pore assemblies in the OMVs, and the cytotoxic activity toward mammalian cells was considerably higher than that of ClyA protein purified from the bacterial periplasm. The redox status of ClyA correlated with its ability to form the oligomeric pore assemblies. In bacterial cells with a defective periplasmic disulphide oxidoreductase system, the ClyA protein was phenotypically expressed in a constitutive manner. The results define a vesicle-mediated transport mechanism in bacteria, and our findings show that the localization of proteins to OMVs directly may contribute to the activation and delivery of pathogenic effector proteins.
Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene of Escherichia coli K-12. Genetic analysis suggested that clyA is silenced by the nucleoid protein H-NS. Purified H-NS protein showed preferential binding to clyA sequences in the promoter region, as evidenced by DNase I footprinting and gel mobility shift assays. Transcriptional derepression and activation of a chromosomal clyA::luxAB operon fusion were seen under conditions of H-NS deficiency and SlyA overproduction, respectively. In H-NS-deficient bacteria neither the absence nor the overproduction of SlyA affected the derepressed ClyA expression any further. Therefore, we suggest that overproduction of SlyA in hns ؉ E. coli derepresses clyA transcription by counteracting H-NS. The cyclic AMP receptor protein (CRP) was required for ClyA expression, and it interacted with a predicted, albeit suboptimal, CRP binding site in the clyA upstream region. Site-specific alterations of the CRP binding site to match the consensus resulted in substantially higher levels of ClyA expression, while alterations that were predicted to reduce CRP binding reduced ClyA expression. During anaerobic growth the fumarate and nitrate reduction regulator (FNR) was important for ClyA expression, and the clyA gene could be activated by overexpression of FNR. A major clyA transcript having its 5 end (؉1) located 72 bp upstream of the translational start codon and 61 bp downstream of the CRP-FNR binding site was detected in the absence of H-NS. The clyA promoter was characterized as a class I promoter that could be transcriptionally activated by CRP and/or FNR. According to DNA bending analyses, the clyA promoter region has high intrinsic curvature. We suggest that it represents a regulatory region which is particularly susceptible to H-NS silencing, and its features are discussed in relation to regulation of other silenced operons.
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.
We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.
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