Application Of The Monoclonal Antibodies Against The Pyolysin Of Trueperella Pyogenes For Analysis Of The Structure Of Pyolysin

Trueperella pyogenes(T. pyogenes) is ubiquitous and is usually found on the skin, oropharynx, upper respiratory, urogenital, and gastrointestinal tracts of animals. T. pyogenes is also an important organism that is pathogenic to humans and animals, especially to economically important livestock, such as cattle, sheep and swine. T. pyogenes infections have been usually described involving pyogenic infections in livestock. Herds infected with T. pyogenes can incur serious economic losses.Pyolysin(PLO), the hemolytic exotoxin secreted by Trueperella pyogenes, and is the only hemolysin secreted by T. pyogenes, belongs to the homologous group of cholesterol-dependent cytolysins(CDCs). Our previous study showed that inoculation of recombinant pyolysin(PLO) could induce pyesis in skin and muscle around the injection site of mice. These suggested that PLO is the major virulence factor in the processes of pyogenic infections caused by T. pyogenes.In this study, two monoclonal antibodies(m Abs) against PLO were first prepared for the analysis of this toxin. AP-4F12 can markedly inhibit the hemolytic activity of PLO, whereas AP-1A3 showed no or undetectable hemolysis-inhibiting activity. The potential antigenic epitopes within PLO region, targeted by these two mAbs, were then analyzed. The epitope for AP-1A3 lies in the PLO ranging from 64 to 79 amino acids(91 to 106 amino acids including the signal peptide), the epitope for AP-1A3 is V P V T K D Q L K D G T Y T V F, whereas the epitope for AP-4F12 is ranging from 58 to 75 amino acids(85 to 102 amino acids including the signal peptide), the epitope for AP-4F12 is G E S I E N V P V T K D Q L K D G T. To determine the importance of the amino acids ranging from 58 to 63 on the hemolytic activity of PLO, six mutants of PLO were produced. Each of these mutants with a single amino acid substitution to alanine was constructed, and their hemolytic activities were tested. Results show that amino acids ranging from 58 to 62(85 to 89 amino acids including the signal peptide), the existence of glycine 58(G58), glutamic 59(E59), serine 60(S60), isoleucine 61(I61), glutamic 62(E62) is important for the hemolytic activity of PLO. Notably, isoleucine 61 is the most important amino acid for the hemolytic activity of PLO.In summary, this study was designed by investigating the role of the amino acid residues in the functional epitopes of monoclonal antibodies against PLO for which playing in the process of PLO as its biological effect, to provid some basis data to clarify the relationship between the molecular structure of PLO and its pore forming activity.