Roles of novel cationic lipids in bacterial pathogenesis

摘要 脂质是细菌细胞膜的主要成分，在细菌的生存、宿主-病原体关系中起着重要作用 相互作用和发病机制。细菌用来与宿主相互作用并应对 抗菌应激是通过改变膜脂组成来调节表面电荷。然而，在这方面， 许多重要的细菌病原体的膜脂仍然没有特征，我们对如何 细菌细胞表面对致病的贡献仍然非常有限。我们的目标是解决这些重要的 我们在革兰氏阳性病原体研究方面的知识空白。我们最近发现了一种新的，非常积极的 一系列革兰氏阳性病原体（包括新生儿脑膜炎病原体）中的带电糖脂 无乳链球菌（B组链球菌; GBS）。该项目的首要假设是， 阳离子糖脂影响革兰氏阳性菌细胞表面的生化特性， 发病机制使用GBS作为模式生物，在目的1中，我们提出研究阳离子 GBS对糖脂依赖性血脑屏障的破坏。我们将研究参与屏障的关键机制， 破坏，包括紧密连接的丧失、直接细菌入侵和胞吞作用以及炎症损伤。 我们还将描述GBS膜囊泡在脑膜炎发病过程中的作用。在目标2中， 我们将研究阳离子糖脂和脂磷壁酸（LTA）生物合成之间的调节相互作用。 我们的遗传学和脂质组学分析表明，这些途径具有相反的关系，因为它们利用 相同的基板。这是一种调节细胞表面结构-功能的新机制， 阳性病原体，并具有超越GBS的相关性，因为我们还发现了新的阳离子糖脂， 多重致病性肠球菌我们将进一步研究这一拟议机制，并量化这一共同- 调控影响GBS LTA的结构和水平，使用化学，生物化学和遗传学的组合 接近。在目标3中，我们将研究阳离子糖脂合成的分子机理。酶 合成新型阳离子糖脂的是多肽抗性因子（MprF），一种广泛分布的 细菌中的毒力因子。MprF酶选择脂质底物的分子机制尚不清楚。 利用分子进化、全局统计模型和机器学习方法， MprF家族，我们提出了一个严格的解剖MprF脂质底物特异性在GBS，这将有助于研究 MprF在GBS和其他致病菌中的结构-功能，并指导其他致病菌的发现。 产生阳离子糖脂的物种。总的来说，这个项目将阐明生物合成，分布和 新型阳离子脂质在GBS和其他革兰氏阳性菌中的生理功能，并揭示其 在细菌致病机制中的作用。

Abstract Lipids are major components of bacterial membranes and play vital roles in bacterial survival, host-pathogen interactions and pathogenesis. An important mechanism used by bacteria to interact with hosts and cope with antimicrobial stresses is the modulation of surface charge via alteration of membrane lipid composition. However, membrane lipids remain uncharacterized for many significant bacterial pathogens, and our understanding of how the bacterial cell surface contributes to pathogenesis is still very limited. We aim to address these important knowledge gaps in our research on Gram-positive pathogens. We recently discovered novel, highly positively charged glycolipids in a range of Gram-positive pathogens, including the neonatal meningitis pathogen Streptococcus agalactiae (Group B Streptococcus; GBS). The overarching hypothesis of the project is that cationic glycolipids impact Gram-positive bacterial cell surface biochemical properties and play crucial roles in pathogenesis. Using GBS as a model organism, in Aim 1 we propose to investigate mechanisms for cationic glycolipid-dependent blood-brain barrier disruption by GBS. We will examine key mechanisms involved in barrier disruption, including loss of tight junctions, direct bacterial invasion and transcytosis, and inflammatory damage. We will also characterize the role of GBS membrane vesicles during the pathogenesis of meningitis. In Aim 2, we will investigate regulatory interactions between cationic glycolipids and lipoteichoic acid (LTA) biosynthesis. Our genetic and lipidomic analysis show that these pathways have an inverse relationship because they utilize the same substrates. This is a novel mechanism for regulating the cell surface structure-function of a Gram- positive pathogen, and has relevance beyond GBS, as we have also identified novel cationic glycolipids in multiple pathogenic enterococci. We will investigate this proposed mechanism further and quantify how this co- regulation impacts the structure and level of GBS LTA using a combination of chemical, biochemical and genetic approaches. In Aim 3, we will investigate the molecular mechanism for cationic glycolipid synthesis. The enzyme that synthesizes novel cationic glycolipids is the Multiple Peptide Resistance Factor (MprF), a broadly distributed virulence factor in bacteria. The molecular mechanism for lipid substrate selection by MprF enzymes is unknown. Using molecular evolution, global statistical models, and machine learning approaches that analyze the entire MprF family, we propose a rigorous dissection of MprF lipid substrate specificity in GBS, which will inform studies of MprF structure-function in GBS and other pathogenic bacteria, and guide the discovery of other pathogenic species producing cationic glycolipids. Overall, this project will elucidate the biosynthesis, distribution and physiological functions of the novel cationic lipids in GBS and other Gram-positive bacteria and uncover their roles in bacterial pathogenesis.