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

项目总结/摘要：依据：碳青霉烯耐药肠杆菌科（CRE），尤其是 肺炎克雷伯氏菌（Klebsiella pneumoniae，KP）是一种严重的公共卫生威胁。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）的增加威胁着这些重要的 抗生素虽然细菌脂多糖（LPS）中的修饰已经证明了 PR是在三个双组分系统（TCS）phoP/Q、crrA/B、pmrA/B和mgr B中的广泛突变， 被认为可以诱导PR，但大多数尚未得到功能验证。此外，我们对这些人如何 突变会影响细菌的适应性和毒力，如果它们能增加多粘菌素的最小抑制 浓度（MIC）独立于LPS修饰。这一指导性职业发展奖旨在 阐明这些基因中广泛选择的突变的下游表型效应。候选人： 作为一名在细菌学和分子生物学方面有很强背景的传染病临床医生，我非常适合 进行转化研究，重点是抗生素耐药性的决定因素。进一步培训细菌 致病机理、抗微生物耐药性、细菌基因组学和生物统计学对于完成 我的研究和职业发展计划与主要导师Anne-Catrin Uhlemann博士，我有 组建了一个多学科专家团队，指导我的培训和研究进展。我的长期目标是 成为一名独立的NIH资助的研究人员，利用新的分子生物学技术来表征 细菌抗生素耐药性的决定因素，并改善临床实践。地点：The Uhlemann 哥伦比亚大学欧文医学中心的一个实验室拥有微生物学、分子生物学和测序 工具来完成所提出的研究。该实验室包含大量的CRE和PRKP临床 已经进行了全基因组测序的分离株。哥伦比亚有着长期的支持记录， 青年调查员的职业发展。方法：我们的中心假设是， PR级联中的多个突变导致MIC升高，并通过激活改变细菌毒力 独特的细胞通路。为了阐明各种突变的贡献，我们将系统地插入这些 利用我们的CRISPR-Cas9系统（Aim 1）将CRKP转化为两种CRKP临床分离株。我们将描述这些 变化影响MIC和LPS。在目标2中，我们将评估PR是否可以通过以下方式改变细菌适应性和毒力： 生长曲线、共孵育分析、Galleria mellonella杀伤测定和小鼠肺炎模型。在Aim中 3我们将利用RNA-seq通过定义细胞内的表型来表征PR突变体的差异表型。 TCS的目标。通过这一点，我们的目标是确定新的途径参与PR和毒力，并验证 这些目标通过CRISPR介导的修饰。除了阐明TCS的变化如何诱导 PR和影响细菌适应性，这项工作有可能确定新的途径参与PR和毒力。 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.