Isoprenoid synthesis and allocation in Staphylococcus aureus

项目摘要 类异戊二烯是一种多样的、普遍存在的分子家族，具有无限的工业和临床潜力。作为 类异戊二烯是地球上产生的最大的次级代谢产物家族，也是一种基本的 生命的基石与此相一致，来自所有三个生命领域的生物体合成类异戊二烯， 支持重要的代谢和生理过程。在细菌中，类异戊二烯是生产 色素、呼吸辅助因子和细胞被膜的基本成分。阻碍生产 这些分子对细菌细胞具有毁灭性的后果，支持类异戊二烯前体 合成酶是治疗病原微生物的治疗靶点。虽然已经 确定了许多依赖于类异戊二烯的过程是毒力所必需的， 尚未直接评估类异戊二烯产生与细菌发病机理的关系。此外，机制 细菌合成和有效分配类异戊二烯前体的途径代表了我们在 知识在细菌中，类异戊二烯是通过一系列缩合和延伸反应产生的， 作为底物的通用前体以产生不同链长的分子。这些反应是由以下物质催化的： 称为异戊烯基二磷酸脱氢酶（PDS）的酶。越来越多的证据支持一种模式 由此短链PDS和长链PDS都合成用于生产聚异戊二烯的类异戊二烯前体， 最终产品。然而，长链PDS通常是必不可少的，使得难以测试该模型。在 在金黄色葡萄球菌中，短链和长链PDS分别由ispA和hepT编码。 值得注意的是，S。金黄色葡萄球菌ispA突变体不产生类异戊二烯依赖性色素葡萄黄素，然而， 其它类异戊二烯的生产似乎保持完整。我们的初步数据表明，S。金黄色 hepT突变体是可行的，使我们能够确定这种长链PDS如何有助于类异戊二烯的生产。 此外，我们分离出葡萄球菌素产生ispA抑制突变体，并将突变定位于 HepT。这些数据首次揭示了hepT在S.金黄色葡萄球菌类异戊二烯生成 和分配。我们假设IspA和HepT协同作用，有效地合成和分配 类异戊二烯前体在感染期间维持最大适应性。利用我们的初步数据和测试 根据我们的假设，我们将监测ispA和hepT中大量类异戊二烯衍生分子的产生 使用已建立的遗传学、生物化学和质谱法测定突变体。一只轮廓分明的老鼠 感染模型将量化ispA和hepT突变体的毒力。这些方法将共同建立 IspA和HepT对S.金黄色葡萄球菌在感染期间产生类异戊二烯。这项工作的完成将 定义IspA和HepT在重要人类病原体中的作用，并验证这些蛋白质作为新型治疗药物 目标的

PROJECT SUMMARY Isoprenoids are a diverse, ubiquitous family of molecules with limitless industrial and clinical potential. As the largest family of secondary metabolites produced on Earth, isoprenoids also represent a fundamental building block for life. Consistent with this, organisms from all three domains of life synthesize isoprenoids that support critical metabolic and physiological processes. In bacteria, isoprenoids are necessary for the production of pigments, respiratory cofactors, and essential components of the cell envelope. Obstructing production of these molecules has devastating consequences for bacterial cells, supporting the idea that isoprenoid precursor synthesis enzymes are therapeutic targets for the treatment of pathogenic microorganisms. Although it has been established that numerous processes that rely on isoprenoids are required for virulence, the importance of isoprenoid production to bacterial pathogenesis has not been directly assessed. Additionally, the mechanism(s) by which bacteria synthesize and efficiently allocate isoprenoid precursors represent significant gaps in our knowledge. In bacteria isoprenoids are produced via a series of condensation and elongation reactions that use universal precursors as substrates to produce molecules of varying chain length. The reactions are catalyzed by enzymes referred to as prenyl diphosphate synthases (PDS). A growing body of evidence supports a model whereby short chain PDS and long chain PDS both synthesize the isoprenoid precursors used to produce the final products. However, long chain PDS are typically essential, making it difficult to test this model. In Staphylococcus aureus, the short chain and long chain PDS are encoded by ispA and hepT, respectively. Notably, S. aureus ispA mutants do not produce the isoprenoid-dependent pigment staphyloxanthin, however, production of the other isoprenoids appears to remain intact. Our preliminary data demonstrates that S. aureus hepT mutants are viable, enabling us to determine how this long chain PDS contributes to isoprenoid production. Additionally, we isolated staphyloxanthin-producing ispA suppressor mutants and mapped the mutations to hepT. These data reveal for the first time that hepT plays an important role in S. aureus isoprenoid production and allocation. We hypothesize that IspA and HepT function cooperatively to efficiently synthesize and allocate isoprenoid precursors to maintain maximal fitness during infection. To capitalize on our preliminary data and test our hypothesis, we will monitor the production of numerous isoprenoid-derived molecules in ispA and hepT mutants using established genetic, biochemical, and mass spectrometry approaches. A well-defined murine model of infection will quantify virulence of ispA and hepT mutants. Together these approaches will establish the contributions of IspA and HepT to S. aureus isoprenoid production during infection. Completion of this work will define the roles of IspA and HepT in an important human pathogen and validate the proteins as novel therapeutic targets.