Transcriptional regulation of pyolysin production in the animal pathogen, Arcanobacterium pyogenes.

Arcanobacterium pyogenes is an opportunistic pathogen of a number of important livestock species, and usually infects from an endogenous, commensal source. Thus, as with other normal flora opportunistic pathogens, the regulation of A. pyogenes virulence factors is likely important during both commensal and pathogenic interactions with the host. The aim of this study was to investigate the regulation of a key A. pyogenes virulence factor, the cholesterol dependent cytolysin, pyolysin (PLO), under in vitro conditions, as a first step to understanding its regulation during the disease process. Analysis of PLO production in broth culture indicated that expression of PLO was induced during early stationary phase, and that this correlated with an increase in plo-specific mRNA. Analysis of a plo-cat transcriptional fusion indicated that transcription of plo was also induced during early stationary phase. Primer extension analysis and 5' RACE suggested that two putative promoter sequences, P1 and P2 were active. Analysis of site-directed mutants of these promoters in the plo-cat fusion indicated that P2 was the major stationary phase promoter. Deletions of the plo promoter region from the plo-cat fusion implicated three direct repeat (DR) sequences as important for plo transcription. Mutagenesis of both DR1 and DR2 resulted in reduction in plo transcription, while the presence of only DR3 in deletions of the plo promoter region repressed transcription from P2. Gel shift experiments indicated that a soluble factor from A. pyogenes binds to the plo promoter region and that the DRs may act as binding sites for a transcriptional regulator.

The gene encoding pyolysin, the pore-forming toxin of Arcanobacterium pyogenes, resides within a genomic islet flanked by essential genes.

The plo gene, encoding the Arcanobacterium pyogenes cholesterol-dependent cytolysin, pyolysin (PLO), was localized to a 2.7-kb genomic islet of reduced %G+C content and alternate codon usage frequency. This islet, conserved among isolates from diverse hosts and geographical locations, separated the housekeeping genes smc and ftsY, which are found adjacent in many prokaryotes. The ftsY and ffh genes, located downstream of the plo islet, encode components of the signal recognition particle. Mutational analysis suggested that these genes were essential for viability in A. pyogenes. The A. pyogenes ffh gene was unable to complement a conditional ffh mutant of Escherichia coli and its overexpression was toxic in E. coli. Mutagenesis of the islet-encoded orf121 did not affect plo expression, indicating that it may not be involved directly in the regulation of plo expression. Regardless, the presence of the plo gene as part of a genomic islet inserted between genes essential for normal growth may provide selective pressure for the retention of this important virulence factor.