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

B 族链球菌对人 IIA 族分泌磷脂酶 A2 的耐药机制

基本信息

批准号：
9979339
负责人：
Natalia Korotkova
金额：
$ 23.11万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-15 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9979339
关键词：
Acute Affect Anti-Bacterial Agents Antimicrobial Cationic Peptides Architecture Bacteria Bacterial Infections Biochemical Biological Assay Biology Body Fluids Candidate Disease Gene Carbohydrates Cations Cell Wall Cell membrane Cells Charge Chronic Disease Complex Data Elderly Enzymes Freeze Substitution Future Gene Deletion Genes Genetic Glycerol Glycerophospholipids Glycolipids Goals Gram-Positive Bacteria Histones Human Hydrolysis In Vitro Infection Inflammation Knowledge Libraries Link Membrane Modeling Modification Morphology Muramidase Natural Immunity Neonatal meningitis Newborn Infant Outcome Peptidoglycan Permeability Phase Phenotype Phosphatidylglycerols Phospholipase A2 Phospholipids Plasma Cells Play Polymers Polysaccharides Predisposition Proteins Publishing Research Resistance Rhamnose Role Serum Side Skin Streptococcus Streptococcus Group B Streptococcus mutans Streptococcus pyogenes Stress Structure Systemic infection Technology Testing Transgenic Mice Transmission Electron Microscopy Vertebral column Work antimicrobial antimicrobial peptide bacterial resistance base capsule cell envelope cell type design drug development experimental study extracellular human pathogen in vivo inorganic phosphate microorganism mouse model mutant neonatal sepsis novel novel therapeutics pathogen phosphodiester prevent resistance mechanism screening

项目摘要

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.

抗菌肽在对微生物的体液先天免疫中起着重要作用。多个体内和体外研究突出了人类IIA分泌的重要功能磷脂酶A2（HGIIA），以防止革兰氏阳性细菌感染（包括组） B链球菌（GBS）是新生儿败血症和脑膜炎的主要原因。 HGIIA杀死细菌通过催化膜甘油磷脂的水解。革兰氏阳性细胞信封，由多种肽聚糖层组成，装饰有各种糖聚合物，代表了HGIIA的实质性障碍，并且鲜为人知的HGIIA如何获得进入细菌质膜可产生致命的损伤。我们最近确定了小说细菌对HGIIA的细菌耐药性机制与GB密切相关的细菌，组链球菌（气体）。我们的工作修改了当前的气体墙体系结构的模型和强调了这些结构决定因素对HGIIA的抗性的重要性。有趣的是， GBS被HGIIA杀死，浓度比气体低约500倍菌株。本提案中描述的实验旨在了解基础针对GB的HGIIA效力的机制。为了实现我们的目标，我们将使用两个方法。在第一种方法中，我们将进行最近构造的，高度的屏幕饱和的GBS转座子突变库，使用HGIIA的致死和亚致死浓度。在确定基因中构建缺失突变体后，我们将执行抗菌和机械测定和透射电子显微镜分析，冻结的冻结细胞确认突变体的HGIIA敏感性表型，并了解抵抗/敏感性的机制。在第二种方法中，我们将研究 GB的主要肽聚糖糖糖聚合物B组B碳水化合物（GBC）通过将GBC与GBS中的燃气壁糖聚合物交换，HGIIA敏感性。成功的成果将指导未来针对GB的新药开发的努力。