Elucidating the genotypic determinants and phenotypic effects of polymyxin resistance in Klebsiella pneumoniae utilizing CRISPR-Cas9

项目摘要/摘要：基本原理：耐碳青霉烯类肠杆菌科(CRE)，尤其是 肺炎克雷伯氏菌(Kp)仍然是一个重大的公共卫生威胁。由于缺乏治疗选择， 多粘菌素仍然是治疗的主要手段。多粘菌素耐药KP(PRKP)的兴起威胁到这些至关重要的 抗生素。而对细菌脂多糖(LPS)的修饰已证明其主要机制是 PR，三个双组分系统(TCS)中的广泛突变，Phop/Q，crrA/B，pmrA/B和mgrB，是 被认为可以诱导PR，但大多数还没有得到功能验证。此外，人们对它们是如何形成的知之甚少 突变会影响细菌的适合性和毒力，如果它们能增加多粘菌素的抑制率 浓度(MIC)不受内毒素修饰的影响。这个辅导式职业发展奖旨在 阐明这些基因中广泛选择的突变的下游表型效应。候选人：AS 我是一名传染病临床医生，在细菌学和分子生物学方面有很强的背景，我非常适合 开展以抗生素耐药性决定因素为重点的翻译研究。关于细菌的进一步培训 致病机制、抗菌素耐药性、细菌基因组学和生物统计学将是完成 提出的研究和我职业生涯的进步。在主要导师安妮-卡特琳·乌尔曼博士的帮助下，我 组建了一支多学科的专家团队，指导我的培训和研究进展。我的长期目标是 成为美国国立卫生研究院资助的独立研究人员，利用新的分子生物学技术来表征 细菌耐药性的决定因素和改善临床实践。环境：乌尔曼 哥伦比亚大学欧文医学中心的实验室有微生物学、分子生物学和测序 完成拟议研究的工具。该实验室包含大量CRE和PRKP临床资料 已完成全基因组测序的分离株。哥伦比亚大学长期以来一直支持 青年侦查员的职业发展。方法：我们的中心假设是 PR级联中的多个突变导致MIC上升，并通过激活改变细菌的毒力 独特的细胞路径。为了阐明各种突变的作用，我们将系统地插入这些 利用我们的CRISPR-Cas9系统(目标1)将其转化为两个CRKP临床分离株。我们将描述这些特性是如何 变化会影响MIC和内毒素。在目标2中，我们将评估PR是否可以通过 生长曲线、共孵育分析、梅隆格列氏菌杀灭试验和小鼠肺炎模型。在AIM 3我们将利用rna-seq通过定义细胞来鉴定PR突变的不同表型。 塔塔咨询服务的目标。通过这一点，我们的目标是识别参与PR和毒力的新途径，并验证 这些靶点通过CRISPR介导的修饰。除了阐明TCS的变化如何导致 PR和影响细菌适合性，这项工作有可能确定参与PR和毒力的新途径。 这将产生诊断、治疗和预防PRKP感染所需的关键信息。

PROJECT SUMMARY / ABSTRACT: Rationale: Carbapenem resistant Enterobacteriaceae (CRE), especially Klebsiella pneumoniae (KP) remain a significant public health threat. With a lack of treatment options, the polymyxins remain a mainstay of therapy. The rise of polymyxin resistant KP (PRKP) threatens these vital antibiotics. While modifications in the bacterial lipopolysaccharide (LPS) have proven the major mechanism of PR, a wide range of mutations in three two components systems (TCS), phoP/Q, crrA/B, pmrA/B, and mgrB, are thought to induce PR, but most have not been functionally validated. Additionally, little is known about how these mutations affect bacterial fitness and virulence, and if they can increase polymyxin minimum inhibitory concentration (MIC) independent of LPS modification. This mentored career development award aims to elucidate the downstream phenotypic effects of a broad selection of mutations in these genes. Candidate: As an infectious diseases clinician with a strong background in bacteriology and molecular biology, I am well suited to pursue translational research focusing on the determinants of antibiotic resistance. Further training in bacterial pathogenesis, anti-microbial resistance, bacterial genomics, and biostatistics will be crucial for the completion of the proposed research and advancement of my career. With primary mentor Dr. Anne-Catrin Uhlemann, I have assembled a multi-disciplinary team of experts to guide my training and research progress. My long-term goal is to become an independent NIH-funded researcher utilizing novel molecular biology techniques to characterize the determinants of bacterial antibiotic resistance and improve clinical practice. Environment: The Uhlemann laboratory at Columbia University Irving Medical Center has the microbiology, molecular biology and sequencing tools to complete the proposed research. The laboratory contains a large collection of CRE and PRKP clinical isolates that have undergone whole genome sequencing. Columbia has a long track record of supporting the career development of young investigators. Approach: Our central hypothesis is that the accumulation of multiple mutations in the PR cascade leads to rising MICs and changes in bacterial virulence through activation of unique cellular pathways. To elucidate the contribution of various mutations we will systematically insert these into two CRKP clinical isolates utilizing our CRISPR-Cas9 system (Aim 1). We will characterize how these changes affect MIC and LPS. In Aim 2 we will evaluate if PR can alter bacterial fitness and virulence through growth curves, co-incubation analyses, Galleria mellonella killing assays and a mouse pneumonia model. In Aim 3 we will utilize RNA-seq to characterize the differential phenotypes of the PR mutants by defining the cellular targets of the TCS. Through this we aim to identify novel pathways involved in PR and virulence and validate these targets through CRISPR mediated modification. In addition to elucidating how changes in the TCS induce PR and affect bacterial fitness, this work has the potential to identify novel pathways involved in PR and virulence. This would yield crucial information necessary for the diagnosis, treatment and prevention of PRKP infections.