Delineating genetic determinants of polymyxin resistance in Serratia marcescens

描述粘质沙雷氏菌多粘菌素抗性的遗传决定因素

• 批准号: 10317863
• 负责人: Anne-Catrin Uhlemann
• 金额: $ 25.54万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-05 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10317863
• 关键词: Adjuvant; Alleles; Antibiotics; Arabinose; Automobile Driving; Biochemical; Biological Assay; Cations; Charge; Chemicals; Clinical; Collection; Complement; Data; Enzymes; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Determinism; Genetic Markers; Genome; Genomics; Geography; Glucans; Gram-Negative Bacteria; Hospitals; Imipenem; Impairment; Infection; Knowledge; Lactamase; Lactams; Lead; Lipid A; Mass Spectrum Analysis; Membrane; Membrane Proteins; Methods; Microbiology; Minimum Inhibitory Concentration measurement; Modeling; Modification; Molecular; Monitor; Multi-Drug Resistance; Mutation; New York City; Operon; Organism; Peptides; Pharmaceutical Preparations; Phenotype; Phylogenetic Analysis; Polymyxin Resistance; Polymyxins; Predisposition; Prevalence; Protein Biochemistry; Proteins; Radiolabeled; Rapid screening; Recording of previous events; Regimen; Regulator Genes; Regulatory Pathway; Resistance; Resort; Resources; Role; Serratia marcescens; Structure; Techniques; Technology; Testing; Transferase; Urban Hospitals; Virulence; bacterial fitness; carbapenem resistance; carbapenem-resistant Enterobacteriaceae; design; effective therapy; experimental study; genome sequencing; improved; inhibitor/antagonist; inorganic phosphate; molecular diagnostics; molecular marker; mutant; novel; pathogen; periplasm; reconstruction; resistance mechanism; response; screening; structural biology; whole genome

This proposal aims to define the molecular mechanisms of resistance to polymyxins in Serratia marcescens. The emergence of carbapenem resistance in this nosocomial pathogen poses a treatment dilemma as few treatment options exist. S. marcescens has long been considered intrinsically resistant to polymxyins, a mainstay for treatment of carbapenem-resistant Enterobacteriales. However, we unexpectedly noted that a substantial proportion of carbapenem-resistant S. marcescens (CR-SM) are fully susceptible to polymyxin. In addition, resistant isolates displayed two distinct phenotypes (low-level (R1) and high-level resistance (R2). Polymyxins are cationic peptides targeting negatively charged components of the bacterial outer membrane, notably Lipid A. Chemical modification of Lipid A through enzymatic transfer of L-Ara4N to Lipid A is the major contributor to polymyxin resistance and is catalyzed by the aminoarabinose transferase ArnT. Lipid A analysis of isolates representing the 3 phenotypic groups demonstrated a complete lack of L-Ara4N modification in susceptible isolates and distinct lipid A profiles between R1 and R2 phenotypes. Through whole genome sequencing we have begun to establish putative genetic determinants of polymyxin susceptibility. This includes a unique arnC-like gene only present in susceptible isolates and a flippase related to arnE only encoded in highly-resistant R2 isolates. In addition, R1 and R2 isolates harbored unique arnD and eptA alleles compared to susceptible isolates. Here, we aim to define the mechanisms underlying polymyxin susceptibility in detail to provide critical information on the potential suitability of polymyxins as a treatment option for CR-SM infections. We have assembled a highly skilled team with complementary expertise in microbiology, bacterial genomics and membrane protein biochemistry. In Aim 1, we will determine the prevalence of polymyxin susceptibility in a comprehensive collection of clinical multi-drug resistant (MDR) and non-MDR S. marcescens isolates. Through whole genome sequencing we will refine putative genetic markers of PR phenotypes and through phylogenetic reconstruction determine if the loss or PR occurred repeatedly or represents a stable sublineage. We will then validate candidate genetic markers on PR through a combination of complementation, gene editing and deletion experiments. In Aim 2, we will assess LPS structure and lipid A modifications of isogenic mutants generated in Aim 1 using radiolabeling techniques and mass spectrometry. We will evaluate gene expression of lipid A modifying enzymes and overall differences in transcriptional profiles across phenotypes. Lastly, we will examine the impact of PR mutants on bacterial fitness and virulence. Findings from our study will fill significant gaps in knowledge of the genomic and molecular determinants of intrinsic PR, provide information on the mechanisms of disruption of lipid A modifications, deliver molecular markers for rapid screening of polymyxin susceptibility phenotypes in CR-SM; and improve our understanding of the evolutionary trade-offs underlying the loss of the intrinsic PR phenotype.

该提案旨在定义锯齿状铜质中对多碳素抗性的分子机制。 在这种医院病原体中碳青霉苯甲部耐药性的出现构成了治疗困境很少 存在治疗选择。长期以来，铜链球菌一直被认为对多晶型蛋白具有本质上的抗性 用于治疗碳苯甲酸肠杆菌肠杆菌的支柱。但是，我们出乎意料地指出 大量抗碳青霉烯链球菌（CR-SM）完全容易受到多染色素的影响。在 另外，抗性分离株显示了两种不同的表型（低水平（R1）和高级电阻（R2）。 多碳素是靶向细菌外膜负电荷成分的阳离子肽， 值得注意的是脂质A.通过将L-ARA4N酶转移至脂质A的化学修饰是主要的 促甲状腺素耐药性的贡献者，并由氨基酰胺糖转移酶ARNT催化。脂质分析 代表3个表型组的分离株显示出完全缺乏L-ARA4N的修饰 R1和R2表型之间的易感分离株和不同的脂质A谱。通过整个基因组 测序我们已经开始建立多粘蛋白易感性的假定遗传决定因素。这包括 仅存在于易感分离株和与Arne相关的Flippase中的独特类似ARNC样基因仅编码 高度抗性的R2分离株。此外，R1和R2分离出具有独特的ARND和EPTA等位基因 易感分离株。在这里，我们旨在详细介绍多聚酸性易感性的机制 提供有关多碳素作为CR-SM感染的治疗选择的潜在适合性的关键信息。 我们已经组建了一个高技能的团队，具有微生物学，细菌基因组学方面的互补专业知识 和膜蛋白生物化学。在AIM 1中，我们将确定在A 综合收集临床多药抗药性（MDR）和非MDR S. marcescens分离株。通过 整个基因组测序我们将完善PR表型的假定遗传标记，并通过系统发育 重建确定损失或PR是反复发生还是代表稳定的sublineage。然后我们会 通过互补，基因编辑和 删除实验。在AIM 2中，我们将评估LPS结构和脂质的质子突变体的修饰 使用放射性标记技术和质谱法在AIM 1中生成。我们将评估基因表达 脂质的修饰酶和跨表型转录曲线的总体差异。最后，我们 将检查PR突变体对细菌适应性和毒力的影响。我们研究的发现将填补 内在PR的基因组和分子决定因素的知识差距很大，提供信息 关于脂质A修饰的破坏机制，提供分子标记以快速筛选 CR-SM中的多霉质敏感性表型；并提高我们对进化权衡的理解 固有PR表型的损失的基础。