Novel Class of Phospholipases-Molecular Pathogenesis

描述(申请人提供)：磷脂酶C(PLC)在生物学和医学中扮演的角色的多样性是非同寻常的。在过去的十年中，这类磷脂水解酶被证明比最初认为的要复杂得多，它们对真核生物中广泛的基本细胞过程的影响已经被越来越多地认识到，包括肿瘤发生、细胞凋亡和炎症。同样，PLC在微生物发病机制中也有许多不同的重要功能。我们鉴定并鉴定了一类新的同源PLC的第一个成员--铜绿假单胞菌的溶血性磷脂酶C(PICH)。这类PLC的成员是由一系列机会主义和坦率的病原体产生的，包括潜在的生物恐怖分子。其中一些生物的基因组编码多达4个这类PLC的同源物。携带编码这些PLC的基因(基因拷贝数见括号)的细菌包括：铜绿假单胞菌(2)、结核分枝杆菌(4)、图拉氏弗朗西斯杆菌(1)、假腮腺炎伯克霍尔德氏菌和马利氏杆菌(各3)和百日咳杆菌(1)。我们和其他人提供了令人信服的证据，证明这类新的PLC成员在由这些病原体引起的传染病中发挥着重要和不同的作用。虽然这些PLC有相当大的氨基酸同源性，但每个成员都有不同的性质。它们的底物特异性有一些重要的差异，许多成员具有独特的结构特征，这些特征可能在产生它们的生物体的发病机制中发挥特定的功能作用。本应用将主要关注这类新型PLC(PICH)的范例。除了具有PLC活性外，PICH还是第一个被鉴定出具有鞘磷脂合成酶活性的原核或真核蛋白质。PICH的底物(如磷脂酰胆碱和鞘磷脂)或其产生的产物(如二酰甘油、神经酰胺或鞘磷脂)可能具有深刻的生物学效应，特别是在真核细胞的信号传递过程和宿主对这种感染性物质的反应方面。我们还提供了PICH对内皮细胞具有高度细胞毒性的证据，可能是通过与整合素受体的相互作用进入这些细胞的。这项研究项目将使用微生物学、遗传学、生化、结构和细胞生物学方法来研究Pich和这一新型酶的其他成员如何影响产生它们的有机体的毒力。此外，我们从我们的努力中获得的信息很可能还将提供关于PLC的一般生物化学和生物学的更多见解。

DESCRIPTION (provided by applicant): The diversity of roles that phospholipases C (PLCs) play in biology and medicine is extraordinary. In the past decade this class of phospholipid hydrolyzing enzymes has been shown to be considerably more complex than initially perceived and their impact on a wide range of basic cellular processes in eukaryotes, including oncogenesis, apoptosis, and inflammation has been increasingly appreciated. Likewise, there are many sundry and important functions for PLCs in microbial pathogenesis. We identified and characterized the first member of a novel class of homologous PLCs, the hemolytic phospholipase C (PIcH) of Pseudomonas aeruginosa. Members of this class of PLCs are produced by an array of opportunistic and frank pathogens, including potential bioterrorist agents. The genomes of some of these organisms encode as many as 4 homologs of this class of PLCs. Bacteria carrying genes encoding these PLCs (gene copy number shown in parentheses) include: P. aeruginosa (2), Mycobacterium tuberculosis (4), Francisella tularensis (1), Burkholderia pseudomallei and mallei (3 each) and Bordetella pertussis (1). We, as well as others, provided cogent evidence that members of this novel class of PLCs play significant and diverse roles in the infectious diseases caused by those agents. Although these PLCs share considerable amino acid homology, each member has distinct properties. There are some important differences in their substrate specificities, and many members have unique structural features that probably play a specific functional role in the pathogenesis of the organisms that produce them. This application will mainly focus on the paradigm of this novel class of PLCs (PIcH). In addition to its PLC activity, PIcH is the first prokaryotic or eukaryotic protein yet identified that has Sphingomyelin Synthase activity. The substrates (e.g. phosphatidylcholine & sphingomyelin) of PIcH or the products (e.g. diacylglycerol, ceramide or sphingomyelin) that it generates could have profound biological effects, particularly with respect to signaling processes in eukaryotic cells and the host responses to this infectious agent. We have also provided evidence that PIcH is highly cytotoxic for endothelial cells and probably enters these cells through interaction with integrin receptors. This research project will employ microbiological, genetic, biochemical, structural and cell biology methods to examine how PIcH and other members of this novel class of enzymes affect the virulence of the organisms that produce them. Furthermore, it is likely that information we derive from our efforts will also provide additional insights about the biochemistry and biology of PLCs in general.