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

抗菌肽在机体对微生物的天然体液免疫中起重要作用。 多项体内和体外研究突出了人IIA组分泌的重要功能 磷脂酶A2（hGIIA）在保护免受革兰氏阳性细菌感染中的作用，包括组 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 通过催化膜甘油磷脂的水解。革兰氏阳性细胞 包膜，由多个肽聚糖层组成，修饰有各种糖共聚物， 是hGIIA的一个重大障碍，人们对hGIIA如何获得 破坏细菌的细胞膜，造成致命的伤害。我们最近发现了一种 与GBS密切相关的细菌中针对hGIIA的细菌耐药机制，组 链球菌（GAS）。我们的工作修订了目前的模型GAS细胞壁结构， 强调了这些结构决定因素对hGIIA耐药性的重要性。有趣的是， GBS被浓度比GAS低约500倍的hGIIA杀死 菌株本提案中描述的实验旨在了解 hGIIA对GBS效力的机制。为了实现我们的目标，我们将使用两个 接近。在第一种方法中，我们将对最近建造的高度 使用致死和亚致死浓度的hGIIA对饱和的GBS转座子突变体文库进行分析。 在所鉴定的基因中构建缺失突变体后，我们将进行抗微生物和 冷冻替代的机械分析和透射电子显微镜分析 细胞，以确认突变体的hGIIA易感性表型，并了解 抗性/易感性机制。在第二种方法中，我们将研究 GBS的主要肽聚糖连接的糖共聚物，即B族碳水化合物（GBC）， 通过在GBS中用GAS细胞壁糖共聚物交换GBC来测定hGIIA易感性。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.