Biochemical and molecular analysis of phospholipase C and phospholipase D activity in mycobacteria

Abstract

Resurgence of mycobacterial infections in the United States has led to an intense effort to identify potential virulence determinants in the genus Mycobacterium, particularly ones that would be associated with the more virulent species (e.g., Mycobacterium tuberculosis). Thin-layer chromatography (TLC) using radiolabeled phosphatidylcholine and sphingomyelin as substrates indicated that cell extracts of M. tuberculosis contain both phospholipase C (PLC) and phospholipase D (PLD) activities. In contrast, only PLD activity was detected in cell extracts of M. smegmatis. Neither activity was detected in cell-free culture supernatants from these organisms. We and others recently identified two open reading frames in M. tuberculosis with the potential to encode proteins which are highly homologous to the nonhemolytic (PlcN) and hemolytic (PlcH) phospholipase C enzymes of Pseudomonas aeruginosa. In contrast to the plc genes in P. aeruginosa, which are considerably distal to each other (min 34 and 64 on the chromosome), the mycobacterial genes, designated mpcA and mpcB, are tandemly arranged in the same relative orientation and separated by only 191 bp. Both the mpcA and the mpcB genes were individually cloned in M. smegmatis, and PLC activity was expressed from each gene in this organism. Hybridization experiments using the mpcA and the mpcB genes as probes under conditions of moderate stringency identified sequences homologous to these genes in M. bovis, M. bovis BCG, and M. marinum but not in several other Mycobacterium species, including M. smegmatis, M. avium, and M. intracellulare. TLC analysis using radiolabeled substrates indicated that M. bovis and M. marinum cell extracts contain PLC and PLD activities, but only PLD activity was detected in M. bovis BCG cell extracts. Sphingomyelinase activity was also detected in whole-cell extracts of M. tuberculosis, M. marinum, M. bovis, and M. bovis BCG, but this activity was not detected in extracts of M. smegmatis. Sphingomyelinase activity was detected in cell extracts from M. smegmatis harboring either recombinant mpcA or mpcB. These data indicate that PLC and sphingomyelinase activities are associated with the most virulent mycobacterial species, while PLD activity was detected in both virulent and saprophytic strains.

The respiratory burst-inhibiting acid phosphatase AcpA is not essential for the intramacrophage growth or virulence of Francisella novicida

“…The mutation in 2L is predicted to truncate 486 bp from the 3P end of acpA, removing the C-terminal 162 amino acids of AcpA. This represents 31.5% of the protein and includes three of the ¢ve most highly conserved regions between AcpA and the PLCs of P. aeruginosa (Plc-N and Plc-H) [7] as well as two more recently identi¢ed PLCs of Mycobacterium tuberculosis (MpcA and MpcB) [12,13].…”

“…The di¡erence in substrate speci¢city has been proposed to be conferred by the C-terminal one-third of the proteins, which was found to have reduced homology compared to the N-terminal two-thirds [17]. MpcA and MpcB can both direct the hydrolysis of phosphatidylcholine and sphingomyelin when expressed in Mycobacterium smegmatis, a mycobacterial strain devoid of PLC activity [13]. It is interesting to note that AcpA, MpcA, and MpcB are substantially smaller (by about 200 amino acids) than Plc-H and Plc-N, although by comparison of their amino acid sequences with the Pseudomonas PLCs and AcpA, the Mycobacterium PLCs are more similar to Plc-H and Plc-N (36^38% identical vs. 18% identical to AcpA).…”

“…The 50% lethal dose (LD 50 ) of a plcS mutant of P. aeruginosa (plcS encodes Plc-H) is increased 200-fold over the wild-type in a mouse model of infection [18]. Analysis of PLC activity in various mycobacterial species only detected PLC activity in extracts of the most virulent species [13]. Our data do not suggest any role for AcpA in the virulence of F. novicida; however, the role of the new acid phosphatase found in this work needs to be investigated.…”

Bacterial Phospholipases

“…Second, the products of phospholipid hydrolysis can be converted to glycine betaine which accumulates within the bacterial cell and is thought to protect the bacterial cell from conditions of high osmolarity found in the lung (Landfald and Strom 1986;Shortridge et al 1992). The recent identification of tandemly organized genes in Mycobacterium tuberculosis (Johansen et al 1996), which would encode homologues of the Ps. aeruginosa phos-pholipases C will open new avenues of research with this pathogen.…”

“…aeruginosa (Ostroff et al 1990), only PlcH is able to hydrolyse both substrates and this enzyme is thought to play an important role in the pathogenesis of disease. In mycobacterial species, the most virulent strains © 1998 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement 84, 127S-137S produce both phospholipase C and sphingomyelinase activities (Johansen et al 1996).…”