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

Abstract

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 plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. 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 M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. 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, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.

FIG. 1

Autoradiography of thin-layer chromatography results showing phospholipase C and phospholipase D activity of whole-cell extracts on the radiolabeled phosphatidylcholine l-α-1-palmitoyl-2-linoleoyl-(linoleyl-1-¹⁴C). The solvent system included petroleum ether, ethyl ether, and acetic acid (50:50:1). Lanes: L1, H₂O (negative control); L2, phospholipase C control (phospholipase C from C. perfringens [Sigma, Bornem, Belgium]); L3, phospholipase D control (phospholipase D type I from cabbage [Sigma]); L4 to L9, M. avium isolates 98-920, 98-922, 98-924, 98-925, 98-926, and 98-927, respectively.

FIG. 2

Southern hybridization using the plc probe. Genomic DNA from each mycobacterial strain was digested with PvuII and probed with the plc probe. There is evidence of hybridization with M. kansasii 96-1073 (lane 3) M. marinum 7732 (lane 6), M. bovis 96-319 (lane 7), M. tuberculosis 8251 (lane 8), and M. ulcerans strains 5147 and 94-1326 (lanes 9 and 10). No hybridization is seen with M. smegmatis 4995 (lane 2), M. avium 1104 (lane 4), and M. intracellulare 4199 (lane 5). λ HindIII DNA marker is in lane 1. Sizes are given in kilobase pairs.

FIG. 3

Hemolytic activity in horse and sheep blood agar medium. The hemolytic activity of M. tuberculosis 8251 (Q2, sheep blood), M. ulcerans 5147 (Q3, horse blood), and M. avium 98-922 (Q4, horse blood), with clear zones of hemolysis surrounding the growth of the bacteria, is shown. Although extensive growth is observed in M. smegmatis MC2/155 (Q1, horse blood), there are no clear zones and, hence, there is no hemolytic activity.