Mechanisms of resistance against the human group IIA secreted phospholipase A2 in Group B Streptococcus

抗菌肽在体液免疫中发挥着重要作用。 多项体内和体外研究强调了人IIA组分泌的重要功能 磷脂酶A2(HGIIA)对革兰阳性细菌感染的保护作用 B链球菌(GBS)，新生儿败血症和脑膜炎的主要原因。HGIIA杀灭细菌 通过催化膜上甘油磷脂的水解。革兰氏阳性细胞 信封，由多个肽多糖层组成，装饰着各种糖共聚物， 对hGIIA来说是一个很大的障碍，人们对hGIIA如何获得 对细菌质膜产生致命性破坏。我们最近确定了一部小说 与GBS密切相关的细菌对hGIIA的耐药机制 一种链球菌(GAS)。我们的工作修正了现有的气室壁结构模型和 强调了这些结构性决定因素对抵抗hGIIA的重要性。有趣的是， 在浓度比气体低约500倍的情况下，gbs被hGIIA杀死 菌株。本提案中描述的实验旨在理解潜在的 HGIIA抗GBS的作用机制。为了实现我们的目标，我们将使用两个 接近了。在第一种方法中，我们将对最近建造的、高度 利用致死浓度和亚致死浓度的hGIIA饱和GBS转座子突变体库。 在构建了已鉴定基因的缺失突变体后，我们将进行抗菌和 冷冻取代物的力学分析和透射电子显微镜分析 以确定突变体的hGIIA易感表型并了解 抗性/敏感性的机制。在第二种方法中，我们将调查 GBS的主要肽聚糖连接的糖共聚物是B组碳水化合物(GBC)，在 用GBS中的气态细胞壁糖共聚物交换GBC对hGIIA的敏感性。成功 结果将指导未来针对GBS的新药开发工作。

Antimicrobial peptides play a major role in humoral innate immunity against microorganisms. Multiple in vivo and in vitro studies highlight the important function of human group IIA secreted phospholipase A2 (hGIIA) in protection against Gram-positive bacterial infection including Group B Streptococcus (GBS), a leading cause of neonatal sepsis and meningitis. hGIIA kills bacteria by catalyzing the hydrolysis of the membrane glycerophospholipids. The Gram-positive cell envelope, consisting of multiple peptidoglycan layers decorated with a variety of glycopolymers, represents a substantial barrier to hGIIA and it is poorly understood how hGIIA gains access to the bacterial plasma membrane to produce lethal damage. We have recently identified novel bacterial resistance mechanisms against hGIIA in the bacterium closely-related to GBS, Group A Streptococcus (GAS). Our work revised the current models of GAS cell wall architecture and highlighted the importance of these structural determinants for resistance to hGIIA. Interestingly, GBS is killed by hGIIA at concentrations that are approximately 500-fold lower, than the GAS strains. The experiments described in this proposal are designed to understand the underlying mechanisms for hGIIA potency against GBS. To accomplish our goal, we will use two approaches. In the first approach we will conduct screens of a recently constructed, highly saturated GBS transposon mutant library using lethal and sub-lethal concentrations of hGIIA. After construction of deletion mutants in the identified genes we will perform antimicrobial and mechanistic assays and transmission electron microscopy analysis of the freeze-substituted cells to confirm the hGIIA susceptibility phenotype of the mutants and understand the mechanisms of resistance/susceptibility. In the second approach, we will investigate the role of the major peptidoglycan-attached glycopolymer of GBS, the Group B Carbohydrate (GBC), in hGIIA susceptibility by swapping GBC with the GAS cell wall glycopolymer in GBS. Successful outcomes will guide future efforts for new drug development against GBS.