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首页> 外文期刊>Infection and immunity >Biochemical and molecular analysis of phospholipase C and phospholipase D activity in mycobacteria.
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Biochemical and molecular analysis of phospholipase C and phospholipase D activity in mycobacteria.

机译：分枝杆菌中磷脂酶C和磷脂酶D活性的生化和分子分析。

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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.
机译：在美国，分枝杆菌感染的再次流行导致人们为确定分枝杆菌属中潜在的毒力决定因素，特别是与更具毒性的物种（例如，结核分枝杆菌）相关的决定因素付出了巨大的努力。使用放射性标记的磷脂酰胆碱和鞘磷脂作为底物的薄层色谱法（TLC）表明结核分枝杆菌的细胞提取物同时具有磷脂酶C（PLC）和磷脂酶D（PLD）活性。相反，在耻垢分枝杆菌的细胞提取物中仅检测到PLD活性。在这些生物的无细胞培养上清液中均未检测到任何活性。我们和其他人最近在结核分枝杆菌中发现了两个开放阅读框，它们可能编码与铜绿假单胞菌的非溶血性（PlcN）和溶血性（PlcH）磷脂酶C酶高度同源的蛋白质。与铜绿假单胞菌中的plc基因彼此相距很远（染色体上的第34和64位）相反，分枝杆菌基因（命名为mpcA和mpcB）以相同的相对方向串联排列，并且仅相距191 bp。将mpcA和mpcB基因分别克隆到耻垢分枝杆菌中，并从该生物中的每个基因表达PLC活性。在中等严格性条件下使用mpcA和mpcB基因作为探针的杂交实验确定了与牛分枝杆菌，牛分枝杆菌BCG和海事分枝杆菌中这些基因同源的序列，但未在其他几种分枝杆菌物种中（包括耻垢分枝杆菌，M鸟，和胞内分枝杆菌。使用放射性标记底物的TLC分析表明，牛分枝杆菌和海藻分枝杆菌细胞提取物含有PLC和PLD活性，但在牛分枝杆菌BCG细胞提取物中仅检测到PLD活性。在结核分枝杆菌，海分枝杆菌，牛分枝杆菌和牛分枝杆菌BCG的全细胞提取物中也检测到鞘磷脂酶活性，但是在耻垢分枝杆菌的提取物中未检测到该活性。在具有重组mpcA或mpcB的耻垢分枝杆菌的细胞提取物中检测到鞘磷脂酶活性。这些数据表明PLC和鞘磷脂酶活性与最强的分枝杆菌种类有关，而在强毒和腐生菌株中均检测到PLD活性。

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