Identification and characterization of the type II secretion system in carbapenemase-producing Klebsiella pneumoniae ST258

我的研究项目旨在了解细菌是如何与当地环境相互作用并塑造它们的。经典的肺炎克雷伯菌菌株构成了我们正常菌群的一部分，但当我们的免疫系统减弱时，会导致危及生命的感染。在加拿大和国外，这些感染正变得越来越难以治疗，因为肺炎克雷伯菌已经获得了使它们对大多数甚至偶尔对所有抗生素产生抗药性的基因。在持续的新冠肺炎大流行背景下，使用抗生素治疗患者，加上住院率上升，将增加医院获得性感染和肺炎克雷伯菌等多药耐药细菌传播的风险。当代特定的耐多药肺炎克雷伯菌菌株在全球的传播表明，除了耐药性之外，它们还有促成它们成功的特征。识别和表征这些特征对于我们如何处理耐多药肺炎克雷伯菌的微生物控制和治疗具有广泛的意义。研究这些当代肺炎克雷伯菌菌株的生物学在技术上具有挑战性，因为依赖抗生素的常见分子生物学工具不能使用，或者由于抗菌素耐药性而受到限制。我研究计划的长期目标是描述当代肺炎克雷伯菌菌株与当地环境相互作用和塑造当地环境的机制。许多革兰氏阴性细菌使用II型分泌系统将折叠蛋白以其活性形式输送到环境中。这些运输的蛋白质允许细菌与周围环境相互作用，促进细菌的致病和/或环境生存。编码II型分泌系统的基因在克雷伯氏菌中广泛存在。然而，还没有对肺炎克雷伯菌进行全面的研究，唯一已知的运输蛋白是一种被称为普鲁兰酶的淀粉去分支酶，用于获取营养。我们假设肺炎克雷伯菌的II型分泌系统运输不止一种蛋白质，这些蛋白质允许肺炎克雷伯菌塑造其局部环境。我的短期目标是开发所需的工具，以确定肺炎克雷伯菌如何利用其II型分泌系统与当地环境相互作用。具体地说，我们将应用分子遗传学技术和功能分析来(1)产生与现有分子生物学工具兼容的当代肺炎克雷伯菌模式菌株。(2)确定肺炎克雷伯菌II型分泌机制各组成部分对蛋白质分泌的个体贡献；(3)鉴定新的分泌蛋白质。我们将产生一个模型菌株，它将简化对耐多药肺炎克雷伯菌的研究，并对其II型分泌系统进行首次系统分析。总之，这项工作将深入了解耐多药肺炎克雷伯菌如何与其当地环境相互作用，这对其微生物控制具有广泛的影响。

My research program is aimed at understanding how bacteria interact with and shape their local environment. Classic Klebsiella pneumoniae strains form part of our normal flora, but can cause life-threatening infections when our immune system is weakened. These infections are becoming more difficult to treat in Canada and abroad because K. pneumoniae have acquired genes that make them resistant to most, and occasionally, all antibiotics. In the context of the ongoing Covid-19 pandemic, use of antibiotics to treat patients combined with increased hospitalisation rates will increase the risk of hospital-acquired infections and spread of multi-drug resistant (MDR) bacteria like K. pneumoniae. The global spread of specific contemporary MDR K. pneumoniae strains, suggests that in addition to drug resistance they have features that contribute to their success. Identifying and characterizing these features has broad implications for how we approach microbiological control and treatment of MDR K. pneumoniae. Studying the biology of these contemporary K. pneumoniae strains is technically challenging because commonly available molecular biology tools that rely on antibiotics cannot be used or are limited due to antimicrobial resistance. The long-term goal of my research program is to characterise mechanisms used by contemporary K. pneumoniae strains to interact with and shape their local environment. Type II secretion systems are used by many Gram-negative bacteria to transport folded proteins in their active form into the environment. These transported proteins allow bacteria to interact with their surrounding environment to promote bacterial pathogenesis and/or environmental survival. The genes encoding the type II secretion system are widespread among Klebsiella. Yet, no comprehensive studies have been performed in K. pneumoniae and the only known transported protein is a starch debranching enzyme used for nutrient acquisition called pullulanase. We hypothesize that the type II secretion system from K. pneumoniae transports more than one protein and that these proteins allow K. pneumoniae to shape its local environment. My short-term objectives are to develop the tools needed to define how K. pneumoniae uses its type II secretion system to interact with its local environment. Specifically, we will apply molecular genetics techniques and functional assays to (1) generate a contemporary model strain of K. pneumoniae compatible with existing molecular biology tools. (2) Define the individual contribution of each component of the K. pneumoniae type II secretion machinery to protein secretion and (3) identify novel secreted proteins. We will generate a model strain that will simplify the study of MDR K. pneumoniae and perform the first systemic analysis of its type II secretion system. Altogether this work will provide insight into how MDR K. pneumoniae interacts with its local environment, which has broad implications for its microbiological control.