Staphylocidal Mechanism of Platelet Microbicidal Protein

性状（由申请方提供）：金黄色葡萄球菌是一种毒性 病原体，其与广谱临床感染相关。其 能够定殖宿主组织，并在宿主内持续存在和增殖 组织需要生物体绕过先天宿主防御机制。我们 已经发现哺乳动物的血小板储存并分泌一个家族， 抗微生物肽在血管内损伤和微生物 定殖，其用于生长抑制和杀死S.金黄色。在 上一个赠款期间，我们已经划定，主要抗菌 由血小板分泌的肽（凝血酶诱导的血小板 tPMP-1）与S.初始金黄色葡萄球菌体外试验 附着在细胞质膜上，之后， 在对这种肽固有敏感的菌株中引发。与此相反， 那些被工程改造为在体外对tPMP-1具有抗性的菌株（例如， 通过转座子诱变）通过改变它们的基本生物学来做到这一点。 tPMP-1的细胞质膜靶点。对tPMP-1的体外敏感性是 这反映在血管内植入动物模型中这些菌株的清除率提高 感染;相反，体外对tPMP-1的耐药性与感染相关。 在相同的动物模型中增加生存优势。的总体目的 这些建议是：i）定义tPMP-1执行其 杀微生物作用，特别关注细胞内靶向， 应激反应系统的激活;和ii）描述机制， 遗传途径和膜生物化学适应，生物体是 能够成功应对暴露于tPMP-1的生存。为这些 目的，我们将利用一系列特征良好的等基因菌株 对S.金黄色葡萄球菌（包括定点质粒突变体，以及突变体 与质粒报告基因融合），这将使我们能够定义 tPMP-1的杀菌作用机制以及体内平衡 生物体在暴露于tPMP-1后生存所使用的适应性途径。此外，委员会认为， 我们将采用蛋白质组学的方法来揭示新的基因和代谢 由tPMP-1触发的途径，作为其杀微生物级联的一部分，或作为 是生物体适应性策略的一部分这些研究将提供一个坚实的 为将来设计独特的血小板肽同源物奠定了基础， 更好地瞄准S。金黄色葡萄球菌菌株杀死，以及规避 生物体为了生存而使用的先天自我平衡机制。

DESCRIPTION (provided by the applicant): Staphylococcus aureus is a virulent pathogen which is associated with a broad-spectrum of clinical infections. Its ability to colonize host tissues, and to persist and proliferate within host tissues requires the organism to circumvent innate host defense mechanisms. We have discovered that mammalian platelets store and secrete a family of antimicrobial peptides at potential sites of endovascular damage and microbial colonization that serve to both growth inhibit and kill S. aureus. In the previous grant period, we have delineated that the principal antimicrobial peptide which is secreted from platelets (thrombin-induced platelet microbicidal protein-1 [tPMP-1]), interacts with S. aureus in vitro by initial attachment to the cytoplasmic membrane, after which a microbicidal cascade is triggered in strains intrinsically susceptible to this peptide. In contrast, those strains which were engineered to be resistant to tPMP-1 in vitro (e.g., by transposon mutagenesis) do so by changing the basic biology of their cytoplasmic membrane target for tPMP-1. In vitro susceptibility to tPMP-1 is mirrored by enhanced clearance of such strains in animal models of endovascular infection; in contrast, in vitro resistance to tPMP-1 is correlated with an augmented survival advantage in the same animal models. The overall purposes of this proposal are: i) to define the mechanisms by which tPMP-1 executes its microbicidal effects, particularly focusing on intracellular targeting and activation of stress response systems; and ii) to delineate the mechanisms, genetic pathways and membrane biochemical adaptations by which the organism is able to successfully respond to exposures to tPMP-1 for survival. For these purposes, we will utilize a series of well-characterized and isogenic strain pairs of S. aureus (including site-directed plasmid mutants, as well as mutants with plasmid reporter fusions) that will enable us to define both the mechanisms of microbicidal action of tPMP-1, as well as the homeostatic adaptive pathways used by the organism to survive tPMP-1 exposures. Moreover, we will employ proteomics approaches to divulge novel genes and metabolic pathways triggered by tPMP-1 as part of either its microbicidal cascade, or as part of the organism's adaptive strategies. These studies will provide a solid foundation for the future design of unique platelet peptide congeners which are better able to target S. aureus strains for killing, as well as to circumvent innate homeostatic mechanisms used by the organism for survival.