Novel Class of Phospholipases-Molecular Pathogenesis

描述（由申请人提供）：磷脂酶C （plc）在生物学和医学中发挥的作用的多样性是非凡的。在过去的十年中，这类磷脂水解酶已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，包括肿瘤发生、细胞凋亡和炎症，已日益得到重视。同样，plc在微生物发病机制中也有许多重要的功能。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主义制剂。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的基因的细菌（基因拷贝数在括号中显示）包括：铜绿假单胞菌(2)，结核分枝杆菌(4)，土拉菌弗朗西斯菌(1)，假马利氏伯克氏菌和马利氏菌（各3）和百日咳博德氏菌(1)。我们和其他人提供了令人信服的证据，证明这类新型plc的成员在由这些病原体引起的传染病中发挥着重要而多样的作用。虽然这些plc具有相当大的氨基酸同源性，但每个成员具有不同的性质。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，可能在产生它们的生物体的发病机制中起特定的功能作用。本应用程序将主要集中在这种新型plc （PIcH）的范例上。除了其PLC活性外，PIcH是迄今为止鉴定的第一个具有鞘磷脂合成酶活性的原核或真核蛋白。PIcH的底物（如磷脂酰胆碱和鞘磷脂）或其产生的产物（如二酰基甘油、神经酰胺或鞘磷脂）可能具有深远的生物学效应，特别是在真核细胞的信号传导过程和宿主对这种感染因子的反应方面。我们也提供了证据表明PIcH对内皮细胞具有高度的细胞毒性，并且可能通过与整合素受体的相互作用进入内皮细胞。该研究项目将采用微生物学、遗传学、生物化学、结构和细胞生物学方法来研究PIcH和这类新型酶的其他成员如何影响产生它们的生物体的毒力。此外，从我们的努力中获得的信息很可能也将提供关于plc的生物化学和生物学的额外见解。

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.