Delineating genetic determinants of polymyxin resistance in Serratia marcescens

本研究旨在明确粘质沙雷氏菌对多粘菌素耐药的分子机制。 这种医院内病原菌对碳青霉烯类耐药的出现给治疗带来了两难境地 治疗方案是有的。长期以来，粘质葡萄球菌一直被认为对多粘菌素具有内在抗性， 治疗碳青霉烯类耐药肠杆菌的主要药物。然而，我们意外地注意到，一个 相当大比例的耐碳青霉烯类粘质葡萄球菌(CR-SM)对多粘菌素完全敏感。在……里面 此外，抗性菌株表现出两种不同的表型(低水平抗性(R1)和高水平抗性(R2))。 多粘菌素是以细菌外膜负电荷成分为靶标的阳离子多肽， 尤其是脂类A，主要是通过酶促L-Ara4N转移到脂类A的化学修饰。 多粘菌素抗性的贡献者，并由氨基阿拉伯糖转移酶Arnt催化。A类脂蛋白分析 代表3个表型群的分离株中，完全缺乏L-Ara4N修饰 敏感菌株和R1和R2表型之间不同的脂类A谱。通过全基因组 通过测序，我们已经开始建立多粘菌素易感性的推测遗传决定因素。这包括 一种独特的arnC样基因只存在于敏感菌株中，而与Arne相关的一种翻转酶仅在 高抗性的R2分离物。此外，比较了R1和R2分离株具有独特的ArnD和EPTA等位基因 对敏感的菌株。在这里，我们的目标是详细定义多粘菌素易感性的机制 提供关于多粘菌素作为CR-SM感染治疗选择的潜在适宜性的关键信息。 我们组建了一支技术精湛的团队，在微生物学、细菌基因组学方面具有互补的专业知识 和膜蛋白生物化学。在目标1中，我们将确定多粘菌素易感性在 全面收集临床多药耐药(MDR)和非MDR粘质链球菌分离株。穿过 全基因组测序我们将提炼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是否重复发生或代表稳定的子谱系。到时候我们会的 通过互补、基因编辑和基因编辑的组合验证PR上的候选遗传标记 删除实验。在目标2中，我们将评估等基因突变体的脂多糖结构和脂类A的修饰 使用放射性标记技术和质谱学在AIM 1中生成。我们将评估基因表达 类脂A修饰酶和不同表型间转录图谱的总体差异。最后，我们 将研究PR突变对细菌适应性和毒力的影响。我们的研究结果将填补 在内在PR的基因组和分子决定因素的知识方面的显著差距，提供了信息 破坏脂质A修饰的机制，为快速筛选提供分子标记 CR-SM的多粘菌素易感性表型；并提高我们对进化权衡的理解 导致固有PR表型丧失的原因。

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.