Detection of phospholipase C in nontuberculous mycobacteria and its possible role in hemolytic activity

Phospholipase C plays a key role in the pathogenesis of several bacterial i nfections, for example, those caused by Clostridium perfringens and Listeri a monocytogenes. Previous studies have reported multiple copies of pie gene s homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hem olytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacte rium tuberculosis, Al. marinum, ill. bovis, and Al ulcerans, In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine di fferent species. Detection of phospholipase C enzymatic activity was carrie d out using thin-layer chromatography to detect diglycerides in the hydroly sates of radiolabeled phosphatidylcholine. DNA sequences of Al kansasii and ill, marinum homologous to the genes encoding phospholipase C from Al tube rculosis and ill. ulcerans were identified by DNA-DNA hybridization and seq uencing. Finally, we developed a direct and simple assay to detect mycobact erial hemolytic activity. This assay is based on a modified blood agar medi um that allows the growth and expression of hemolysis of slow-growing mycob acteria. Hemolytic activity was detected in M. avium, ill. intracellulare, ill. ulcerans, Al marinum, M. tuberculosis, and M. kansasii mycobacteria wi th phospholipase C activity, but not in Al. fortuitum. No hemolytic activit y was detected in Al. smegmatis, Al gordonae, and ill. vaccae. Whether or n ot phospholipase C enzyme plays a role in the pathogenesis of nontuberculou s mycobacterial diseases needs further investigation.