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如何与当地环境相互作用并塑造它们。典型的肺炎克雷伯菌菌株是我们正常菌群的一部分，但当我们的免疫系统被削弱时，它会引起危及生命的感染。这些感染在加拿大和国外变得越来越难以治疗，因为肺炎克雷伯菌已经获得了对大多数，有时甚至是所有抗生素具有耐药性的基因。在当前Covid-19大流行的背景下，使用抗生素治疗患者并增加住院率将增加医院获得性感染和肺炎克雷伯菌等耐多药细菌传播的风险。当代耐多药肺炎克雷伯菌特定菌株的全球传播表明，除了耐药性外，它们还具有有助于其成功的特征。识别和描述这些特征对我们如何进行微生物控制和治疗耐多药肺炎克雷伯菌具有广泛的意义。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用分子生物学工具不能使用或由于抗菌素耐药性而受到限制。我的研究计划的长期目标是表征当代肺炎克雷伯菌菌株与当地环境相互作用和塑造的机制。II型分泌系统被许多革兰氏阴性菌用于将折叠蛋白以活性形式运输到环境中。这些转运蛋白允许细菌与周围环境相互作用，促进细菌发病和/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，尚未对肺炎克雷伯菌进行全面的研究，唯一已知的转运蛋白是一种用于营养获取的淀粉脱支酶，称为普鲁兰酶。我们假设肺炎克雷伯菌的II型分泌系统转运不止一种蛋白质，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。具体而言，我们将应用分子遗传学技术和功能分析来(1)生成与现有分子生物学工具兼容的当代肺炎克雷伯菌模型菌株。(2)明确肺炎克雷伯菌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。我们将生成一种模型菌株，这将简化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。总的来说，这项工作将为耐多药肺炎克雷伯菌如何与当地环境相互作用提供见解，这对其微生物控制具有广泛意义。

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.