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. pyogenes swine 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 coli by means of ZAP Express vector. Analysis by SDS-polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E. coli is 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. pyogenes swine isolate differed only slightly (97.6% identity) from the sequence of plo gene from A. pyogenes strain 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. pyogenes seems to be a novel member of the thiol-activated cytolysin family.
Ikegami
M
M
Department of Bioscience, Faculty of Biotechnology, Fukui Prefectural University, Japan.
Hashimoto
N
N
Kaidoh
T
T
Sekizaki
T
T
Takeuchi
S
S
D000191:Actinomycetaceae / Q000235:genetics*; D000193:Actinomycetales Infections / Q000382:microbiology / Q000662:veterinary; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006461:Hemolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; C108448:Pyolysin, Arcanobacterium pyogenes