Antibiotic tolerance: membraneless organelles and autolysin regulation

抗生素耐受：无膜细胞器和自溶素调节

• 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
• 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

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主要威胁， 疾病肺炎链球菌是肺炎、败血症和脑膜炎的主要原因 在世界范围内，是一种模式生物，用于了解植物诱导的自溶和失败， 抗生素耐受性疗法：细菌停止生长但未被杀死的表型。 主要的肺炎球菌自溶素，LytA，驱动自溶，但它的下调模式， 宽容是未知的。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监管机构的候选人， 解释导致耐受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，对于荚膜脱落，LytA被激活， 以响应于抗微生物肽而裂解细胞壁而不裂解，所述抗微生物肽典型但不限于LL- 37.相反，LytA在保护性反应中去除表面附着的胶囊以避免LL-37。我们 已经鉴定了3个调节LytA这些活性的基因座。这些LytA调节的突变 (Lym)位点概括了临床分离株的青霉素耐药表型： 生物活性LytA，但在青霉素处理后未能引发自溶。 我们的发现提供了工具，使重要的进展，以界定机制， 控制抗生素裂解反应（LytA是如何控制的第一个机制发现）。 从一般链球菌病原体的基因组分析中，显然lym基因座是 分布广泛，并具有与不同的青霉素耐受表型聚集的等位基因。新的见解 进入自溶素和青霉素反应需要推进细菌的领域， 生理学和传染病。 我们在目标1中建议采取生物化学、遗传学和显微镜相结合的方法 分析Lym蛋白和Lym基因座在LytA调控中的作用。在目标2中，我们将利用 我们的新发现，3 Lym蛋白是第一个显示出形成生物分子的细菌蛋白质， 冷凝并引发相分离。这种特性在真核生物中被广泛用于调节 复杂的时空多蛋白质过程，因此特别适合作为一个高度 新的机制，支持LytA调节3 Lyms。在目标3中，我们将检查lym等位基因 在耐药的肺炎球菌临床分离株中。这些分离物来自于死于 在脑膜炎的动物模型中重现的治疗失败导致的脑膜炎。我们将 将Lyms定义为由于耐受性导致抗生素的有效杀菌作用失败的原因。