Antibiotic tolerance: membraneless organelles and autolysin regulation

抗生素耐受：无膜细胞器和自溶素调节

基本信息

批准号：
10333641
负责人：
Elaine I Tuomanen
金额：
$ 45.5万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-05 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10333641
关键词：
Affect Algorithms Alleles Amino Acid Sequence Animal Model Antibiotic Resistance Antibiotics Autolysin Autolysis Bacteria Bacterial Physiology Bacterial Proteins Binding Biochemical Biochemical Genetics Biological Biological Assay Brain Cell Nucleolus Cell Wall Cessation of life Cleaved cell Clinical Communicable Diseases Complex Cytolysis Data Dissection Doctor of Philosophy Down-Regulation Engineering Enzymes Equilibrium Eukaryota Excision Failure Family Genetic Genome Goals Grant Growth Host Defense Immune Immunocompromised Host Individual Infection Laboratories Lead Lytic Meningitis Methods Microscopic Modeling Molecular Multiprotein Complexes Mus Mutation Nyssa Organelles Pathway interactions Patients Penicillins Peptide Antibiotics Phase Phenotype Physiological Pneumonia Process Production Property Proteins Regulation Relapse Role Sepsis South Africa Streptococcus Streptococcus pneumoniae Study models Sum Surface System Testing Time Tissues Treatment Failure Variant antibiotic tolerance antimicrobial peptide bactericide base biological adaptation to stress capsule clinical phenotype combat genome analysis in vivo insight multidrug tolerance mutant novel pathogen pressure response skills spatiotemporal suicidal tool

项目摘要

Antibiotic resistance and tolerance are a major increasing threat to treating infectious diseases. Streptococcus pneumoniae, the leading cause of pneumonia, sepsis, and meningitis worldwide, is a model organism for understanding antibiotic-induced autolysis and failure of therapy due to antibiotic tolerance: a phenotype when bacteria stop growing but are not killed. The main pneumococcal autolysin, LytA, drives autolysis but its mode of downregulation during tolerance is unknown. We have discovered a new LytA activity: capsule shedding and through its analysis have, for the first time, discovered candidates for regulators of LytA that also could explain the modulation of penicillin responses that lead to tolerance and treatment failure. Using the pneumococcus as a model, our lab has revealed that, for capsule shedding, LytA is activated to cleave cell wall without lysis in response to antimicrobial peptides, typified but not limited to LL- 37. Rather, LytA removes surface attached capsule in a protective response to avoid LL-37. We have identified 3 loci that regulate these activities of LytA. Mutation of these LytA modulating (Lym) loci recapitulates the penicillin tolerance phenotype of clinical isolates: production of bioactive LytA, but a failure to trigger autolysis after penicillin treatment. Our discovery provides tools to make important inroads into defining the mechanisms governing antibiotic lytic responses (the first mechanistic discovery of how LytA is controlled). From analysis of genomes of streptococcal pathogens in general, it is apparent that lym loci are widespread and have alleles that cluster with distinct penicillin tolerant phenotypes. New insights into autolysin and penicillin responses are needed to advance both the fields of bacterial physiology and infectious diseases. We propose in Aim 1 to take a combined biochemical, genetic, and microscopic approach to analyze the roles of the Lym proteins and lym loci on LytA regulation. In aim 2, we will exploit our new discovery that 3 Lym proteins are the first bacterial proteins shown to form biomolecular condensates and initiate phase separation. This property is widely used in eukaryotes to regulate complex spatiotemporal multi-protein processes and is thus especially well-suited as a highly novel mechanism to underpin LytA regulation by 3 Lyms. In Aim 3, we will examine lym alleles in tolerant clinical isolates of pneumococcus. These isolates are derived from patients dying of meningitis due to treatment failure that is recapitulated in the animal model of meningitis. We will define Lyms as a cause of failure of potent bactericidal action of antibiotics due to tolerance.

抗生素耐药性和耐受性是治疗感染性疾病的一个日益严重的威胁疾病。肺炎链球菌，肺炎、败血症和脑膜炎的主要原因在世界范围内，是一种模型生物体，用于了解抗生素诱导的自溶和失效由于抗生素耐受性而进行的治疗：细菌停止生长但未被杀死的一种表型。主要的肺炎球菌自溶素 LytA 驱动自溶，但其在耐受性未知。我们发现了一种新的 LytA 活性：胶囊脱落并通过其分析首次发现了 LytA 监管机构的候选者，这些候选者也可以解释导致耐受性和治疗失败的青霉素反应的调节。使用以肺炎球菌为模型，我们的实验室发现，在胶囊脱落时，LytA 被激活响应抗菌肽，典型但不限于 LL-，裂解细胞壁而不裂解 37. 相反，LytA 以保护性反应去除表面附着的胶囊以避免 LL-37。我们已经确定了 3 个调节 LytA 活性的基因座。这些 LytA 调节突变 (Lym) 位点概括了临床分离株的青霉素耐受表型：产生 LytA 具有生物活性，但青霉素治疗后未能引发自溶。我们的发现提供了在定义机制方面取得重要进展的工具控制抗生素裂解反应（第一个关于如何控制 LytA 的机制发现）。从对链球菌病原体基因组的分析来看，很明显，lym 位点是广泛存在，并且具有与独特的青霉素耐受表型聚集的等位基因。新见解为了推进细菌领域的发展，需要对自溶素和青霉素反应进行研究生理学和传染病。我们在目标 1 中建议采取生化、遗传和微观相结合的方法分析 Lym 蛋白和 lym 位点对 LytA 调节的作用。在目标 2 中，我们将利用我们的新发现，3 个 Lym 蛋白是第一个被证明可形成生物分子的细菌蛋白冷凝并引发相分离。该特性广泛应用于真核生物中以调节复杂的时空多蛋白过程，因此特别适合作为高度通过 3 个 Lyms 支持 LytA 调节的新机制。在目标 3 中，我们将检查 lym 等位基因肺炎球菌的耐受性临床分离株。这些分离株来自死于以下疾病的患者由于治疗失败而导致的脑膜炎在脑膜炎动物模型中得到重现。我们将将 Lyms 定义为由于耐受性而导致抗生素有效杀菌作用失败的原因。