Studies on the expression and secretion of pyolysin, the cholesterol-dependent cytolysin of Arcanobacterium pyogenes

Abstract

Arcanobacterium pyogenes is a gram-positive, fastidious, facultative anaerobe that can persist in a number of animal species both as a commensal and as a pathogen. The economic impact from A. pyogenes -related bovine mastitis and liver abscess infections makes the characterization of possible targets for vaccine development important. One such target, pyolysin (PLO), is a member of the cholesterol-dependent cytolysin (CDC) family and a major virulence factor and host protective antigen in A. pyogenes pathogenesis. Here, sequence analysis of the chromosomal region surrounding plo, which encodes PLO, indicated that the plo gene and an upstream open reading frame, orf121, constitute a genomic islet conserved across geographically diverse isolates. Two genes downstream of plo, ftsY and ffh, encode homologues of the protein components of the signal recognition particle (SRP) pathway which may be involved in plo expression and secretion. However, the role of ftsY and ffh in plo expression could not be defined as they were found essential in A. pyogenes, and ffh was unable to complement a heterologous system. Analysis of plo mRNA and PLO levels across a growth curve indicated that plo is transcriptionally or post-transcriptionally regulated. Sequence analysis of the plo promoter region identified three 11-mer repeats, R1, R2 and R3, and two putative sigma70-like promoter sequences, P1 and P2. Primer extension experiments suggested active transcription from P1 and P2 in vitro. Analysis of transcriptional fusions of successive truncations of the plo promoter region and site-directed mutations in the promoter motifs to a chloramphenicol acetyl transferase (cat) gene, indicated a role for each motif in the regulation of plo expression in vitro, with R2 required for activation and P2 being the regulated promoter. While a null mutation in orf121 did not have a significant effect on plo expression in vitro, gel shift analysis confirmed the ability of some factor within A. pyogenes soluble cellular extracts to bind specifically to the plo promoter region. The identification of cis- and trans-acting factors involved in plo expression provides a basis for characterizing PLO expression in vivo, and may lead to a better understanding of the global regulation of pathogenesis in A. pyogenes.