Korbsrisate Sunee, Tomaras Andrew P, Damnin Suwat, Ckumdee Jutturong, Srinon Varintip, Lengwehasatit Idsada, Vasil Michael L, Suparak Supaporn
Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Department of Microbiology, University of Colorado at Denver and Health Sciences Center Aurora, CO 80045, USA.
Microbiology (Reading). 2007 Jun;153(Pt 6):1907-1915. doi: 10.1099/mic.0.2006/003004-0.
Burkholderia pseudomallei is a serious bacterial pathogen that can cause a lethal infection in humans known as melioidosis. In this study two of its phospholipase C (PLC) enzymes (Plc-1 and Plc-2) were characterized. Starting with a virulent strain, two single mutants were constructed, each with one plc gene inactivated, and one double mutant with both plc genes inactivated. The single plc mutants exhibited decreased extracellular PLC activity in comparison to the wild-type strain, thereby demonstrating that the two genes encoded functional extracellular PLCs. Growth comparisons between the wild-type and PLC mutants in egg-yolk-supplemented medium indicated that both PLCs contributed to egg-yolk phospholipid utilization. Both PLCs hydrolysed phosphatidylcholine and sphingomyelin but neither was haemolytic for human erythrocytes. Experimental infections of eukaryotic cells demonstrated that Plc-1 itself had no effect on plaque-forming efficiency but it had an additive effect on increasing the efficiency of Plc-2 to form plaques. Only Plc-2 had a significant role in host cell cytotoxicity. In contrast, neither Plc-1 nor Plc-2 appeared to play any role in multinucleated giant cell (MNGC) formation or induction of apoptotic death in the cells studied. These data suggested that PLCs contribute, at least in part, to B. pseudomallei virulence and support the view that Plc-1 and Plc-2 are not redundant virulence factors.
类鼻疽伯克霍尔德菌是一种严重的细菌病原体，可导致人类发生一种致命感染，即类鼻疽。在本研究中，对其两种磷脂酶C（PLC）酶（Plc-1和Plc-2）进行了特性分析。从一株强毒株开始，构建了两个单突变体，每个单突变体的一个plc基因失活，以及一个双突变体，其两个plc基因均失活。与野生型菌株相比，单plc突变体的细胞外PLC活性降低，从而证明这两个基因编码功能性细胞外PLC。在添加蛋黄的培养基中对野生型和PLC突变体进行生长比较表明，两种PLC均有助于利用蛋黄磷脂。两种PLC都能水解磷脂酰胆碱和鞘磷脂，但对人红细胞均无溶血作用。对真核细胞的实验性感染表明，Plc-1本身对噬斑形成效率没有影响，但对提高Plc-2形成噬斑的效率有累加作用。只有Plc-2在宿主细胞细胞毒性中起重要作用。相比之下，在研究的细胞中，Plc-1和Plc-2似乎都在多核巨细胞（MNGC）形成或凋亡死亡诱导中不起任何作用。这些数据表明，PLC至少部分地促成了类鼻疽伯克霍尔德菌的毒力，并支持Plc-1和Plc-2不是冗余毒力因子的观点。
