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对感染性治疗的主要威胁疾病。肺炎链球菌，肺炎，败血症和脑膜炎的主要原因在全球范围由于抗生素耐受性而引起的治疗：细菌停止生长但未杀死时的表型。 Lyta的主要肺炎球菌自动肌蛋白驱动自溶液，但其下调模式公差是未知的。我们发现了一种新的Lyta活动：胶囊脱落及其通过它的分析首次发现了Lyta监管机构的候选人解释青霉素反应的调节，从而导致耐受性和治疗失败。使用肺炎球菌作为模型，我们的实验室表明，对于胶囊脱落，Lyta被激活裂解细胞壁，而无需裂解，以响应抗菌肽，但不限于ll- 37。相反，Lyta在保护性响应中去除表面附着的胶囊以避免LL-37。我们已经确定了调节Lyta活动的3个基因座。这些Lyta调节的突变（LYM）基因座概括了临床分离株的青霉素耐受性表型：产生生物活性的Lyta，但在青霉素治疗后未能触发自溶。我们的发现提供了使重要侵害定义机制的工具管理抗生素裂解反应（对Lyta的控制方式的第一个机理发现）。从一般而言的链球菌病原体的基因组分析中，很明显淋巴结基因座是普遍存在的等位基因簇聚集，具有不同的青霉素耐受性表型。新见解需要进入自脂蛋白和青霉素反应以推进细菌的两个领域生理和传染病。我们在AIM 1中提议采用合并的生化，遗传和微观方法分析淋巴结蛋白和淋巴结基因座在Lyta调节中的作用。在AIM 2中，我们将利用我们的新发现，即3叶蛋白是表现出生物分子的第一个细菌蛋白冷凝并开始相分离。该特性广泛用于真核生物来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调节的新机制。在AIM 3中，我们将检查LYM等位基因在肺炎球菌的耐受临床分离株中。这些分离株来自死于脑膜炎动物模型中概括的治疗衰竭引起的脑膜炎。我们将将LYM定义为耐受性抗生素有效杀菌作用失败的原因。