Analysis of the SlyA‐controlled expression, subcellular localization and pore‐forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K‐12

Abstract

SummaryEscherichia coli K-12 harbours a chromosomal gene, clyA (sheA, hlyE ), that encodes a haemolytic 34 kDa protein. Recombinant E. coli overexpressing the cloned clyA gene accumulated this haemolysin in the periplasm and released only very small amounts of it into the external medium. The secretion of ClyA was confined to the log phase and paralleled by the partial release of several other periplasmic proteins. Sequencing of ClyA revealed the translational start point of the clyA gene and demonstrated that the clyA gene product is not N-terminally processed during transport. The transcription of clyA from its native promoter region was positively controlled by SlyA, a regulatory protein found in E. coli, Salmonella typhimurium and other Enterobacteriaceae. SlyA-controlled transcription started predominantly 72 bp upstream from clyA, as shown by primer extension. The corresponding putative promoter contains an unusual ¹10 sequence (TATGAAT) that is separated from a conventional ¹35 sequence by a GC-rich spacer. Site-directed deletion of the G in the ¹10 sequence abrogated the SlyA requirement for strong ClyA production, whereas a reduction in the GþC content of the spacer diminished the capability of SlyA to activate the clyA expression. Osmotic protection assays and lipid bilayer experiments suggested that ClyA forms stable, moderately cation-selective transmembrane pores that have a diameter of about 2.5-3 nm.

A Non-classical Assembly Pathway of Escherichia coli Pore-forming Toxin Cytolysin A

“…The activation of ClyA may be regulated by the change of its redox status during the secretion of ClyA into OMVs (10,21,43). ClyA in the periplasm contains a C87-C285 disulfide bond (6,21,46), whereas ClyA within OMV is reduced (21). The disulfide bond may prevent assembly of functional pore complexes, as oxidized ClyA showed a decreased hemolytic activity compared with reduced ClyA, which is consistent with previous studies (10,21,43).…”

“…3a). Similar to the results of the electrophysiological studies of E. coli ClyA and its homologs from Salmonella typhi (6,39,40), the ClyA channels formed by monomer show a broad distribution from 5-15 nS with a major peak at around 11 nS (Fig. 3b), suggesting that ClyA might create pores of variable size.…”

Interaction of the Salmonella typhimuriumTranscription and Virulence Factor SlyA with Target DNA and Identification of Members of the SlyA Regulon

“…Overproduction of S. typhimurium SlyA, Actinobacillus pleuropneumoniae FNR (HlyX), or E. coli MprA proteins activate expression of hlyE , thereby conferring a hemolytic phenotype on E. coli (8 -11). Site-directed mutagenesis led to the suggestion that a GC-rich sequence located between an unusual heptameric Ϫ10 element (TATGAAT) and a conventional Ϫ35 element in the hlyE promoter might be the site of SlyA interaction and thereby mediate activation of hlyE transcription (12). Here, we report that a slyA mutant strain is impaired in associating with macrophages and in starvation survival.…”

Mecanismos de virulencia de Escherichia coli enteropatógena

“…Según estudios, inhibe la síntesis de ADN en células de cáncer de colon, que depende de los niveles de calcio intracelulares (Figura 3) 79,80 . La citolisina A (ClyA: cytolysin A) expresada del gen clyA también ha sido descrita como citotoxina de ECET cuyas funciones son la formación de poros, inducción de apoptosis en los macrófagos y actividad hemolítica [81][82][83] .…”