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 infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of pie genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium 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 different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of Al kansasii and ill, marinum homologous to the genes encoding phospholipase C from Al tuberculosis and ill. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, ill. intracellulare, ill. ulcerans, Al marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in Al. fortuitum. No hemolytic activity was detected in Al. smegmatis, Al gordonae, and ill. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.