Genetic and Biochemical Properties of a Hemolysin (Pyolysin) Produced by a Swine Isolate of Arcanobacterium (Actinomyces) pyogenes

Arcanobacterium (Actinomyces) pyogenes, a causative agent of various pyogenic diseases in domestic animals, produces a hemolysin which is thought to be an important virulence factor. This hemolysin was purified from the culture supernatant of A. pyogenesswine isolate. The purified hemolysin showed a single band with a molecular mass of 56 kDa on SDS‐polyacrylamide gel electrophoresis, and its isoelectric point was 9.2. The activity of this hemolysin was not enhanced by the addition of L‐cysteine or sodium thioglycolate, but it was inhibited by cholesterol. The gene encoding the hemolysin was cloned, sequenced and expressed in Escherichia coliby means of ZAP Express vector. Analysis by SDS‐polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E. coliis the same as that of the hemolysin purified from A. pyogenes. Nucleotide sequence analysis revealed an open reading frame of 1,605 bp encoding a 534 amino acid protein of 57,989 Da. The nucleotide sequence of the hemolysin gene from A. pyogenesswine isolate differed only slightly (97.6% identity) from the sequence of plogene from A. pyogenesstrain BBR1 reported by Billington et al (J. Bacteriol. 179: 6100–6106, 1997). The cysteine residue existed in the undecapeptide region of the hemolysin, which is highly conserved in thiol‐activated cytolysins (cholesterol‐binding cytolysins), and is replaced with alanine. Therefore, the hemolysin of A. pyogenesseems to be a novel member of the thiol‐activated cytolysin family.