How do mutations in pssA lead to antiseptic/antibiotic resistance in Pseudomonas aeruginosa?

越来越清楚的是，革兰氏阴性菌可以通过重塑富含脂多糖的外膜和内膜来克服防腐剂的挑战。由于膜活性杀生物剂广泛用于临床，导致对这些防腐剂失去敏感性的细菌适应变得普遍。使这一问题更加复杂的是，这些产生的适应性可能导致对人类免疫系统的关键组成部分的交叉耐药性，包括膜活性防御素抗菌肽，甚至是最后的手段antibiotics.This project focused on pssA，一个被注释为编码磷脂酰丝氨酸合成酶的基因，在铜绿假单胞菌中。我们最近已经确定，在pssA的两个突变赋予耐药性在几个临床分离的这种多药耐药生物体奥替尼啶和交叉耐药性洗必泰，其他防腐剂和至少一种抗菌肽。现在必须了解这些突变在对更广泛的防腐剂的（交叉）抗性中的作用，以告知诊断和监测计划，例如在临床或农业环境中。具体而言，该项目旨在了解pssA中两种已知突变的影响，这些突变出现所需的条件以及这些突变导致不同临床来源的铜绿假单胞菌分离株中不同耐药表型的机制。如果时间允许，该项目的元素将在鲍曼不动杆菌中复制，预计其pssA蛋白具有与铜绿假单胞菌类似的单域跨膜结构，这与大多数其他革兰氏阴性病原体不同。该项目依赖于只有通过KCL和英国公共卫生（PHE）之间的广泛合作才能获得的专业知识，技能和技术的组合。该项目将结合经典的分子微生物学技术，下一代测序，NMR代谢组学和膜生物物理方法。第一年：在PHE -在多种铜绿假单胞菌谱系中创建pssA的定点突变体（在smvR中存在/不存在疑似增强突变，smvR是一种外排泵，被发现与阳离子化合物耐药性有关），评估（交叉）抗性表型，并确定pssA突变本身是否是进一步适应防腐剂的增强突变。使用报告融合和/或标记的PssA来理解与点突变相关的PssA活性改变的分子基础。技巧：细菌培养和药敏试验，重组工程，实验进化，qPCR（pssA和smvR表达），全基因组测序和MALDI-Biotyper。第2-3年：在KCL -确定每种pssA突变对每种铜绿假单胞菌分离株背景中细菌代谢和膜组成的影响，了解对一系列防腐剂和膜活性宿主防御肽的抗性/敏感性（hBD 2和hBD 3），并使用Transwell肺上皮细胞共培养系统研究pssA突变对感染模型的可能影响。技巧：HR-MAS NMR代谢组学、HPLC-MS脂质组学（离子阱MS和蒸发光散射检测）、RNAseq、生物物理学实验（主要是荧光光谱法以及不同防腐剂与细菌膜相互作用的成像和计算建模，适当时由液体和固体NMR和圆二色光谱法支持）、哺乳动物细胞培养和共聚焦显微镜。第4年：在PHE和KCL -取决于项目成果1）使用结果通知A的初步研究。在一些实施方案中，所述方法包括以下步骤：1）将鲍曼不动杆菌适应性转化为奥替尼啶，或2）将结果转化为基于qPCR和/或MALDI-TOF的诊断测定，其可用于从常规应用奥替尼啶和相关防腐剂的临床或农业环境中筛选铜绿假单胞菌中pssA突变诱导的抗性的标志物。

It is increasingly clear that Gram-negative bacteria can overcome antiseptic challenges by remodelling both the lipopolysaccharide rich outer membrane and the inner, plasma membrane. Since membrane active biocides are widely used in the clinic, bacterial adaptions that lead to loss of sensitivity to these antiseptics are becoming common. Compounding this issue, these resulting adaptations can lead to cross-resistance to key components of the human immune system including membrane-active defensin antimicrobial peptides as well as even last-resort antibiotics.This project focuses on pssA, a gene annotated as coding for phosphatidylserine synthase, in Pseudomonas aeruginosa. We have recently identified that two mutations in pssA confer resistance in several clinical isolates of this multidrug resistant organism to octenidine and cross-resistance to chlorhexidine, other antiseptics and at least one antimicrobial peptide. It is now essential to understand the role of these mutations in (cross)resistance to a wider range of antiseptics to inform diagnostic and surveillance programmes e.g. in clinical or agricultural settings. Specifically, this project seeks to understand the effects of two known mutations in pssA, the conditions required for these mutations to arise and the mechanisms by which these mutations lead to varying resistance phenotypes in P. aeruginosa isolates of varying clinical origin. If time allows, elements of the project will be replicated in Acinetobacter baumanii, the pssA protein of which is predicted to have a similar single domain trans-membrane structure to that of P. aeruginosa, which is unlike that of most other Gram-negative pathogens. The project relies on a combination of expertise, skills and techniques available only through extensive collaboration between KCL and Public Health England (PHE). The project will incorporate classical molecular microbiology techniques, next-generation sequencing, NMR metabolomics and membrane biophysical approaches. Year 1: At PHE - Create site-directed mutants of pssA in a variety of P. aeruginosa lineages (both with/without suspected potentiating mutations in smvR, an efflux pump found to be involved in cationic compound resistance.), evaluate (cross)resistance phenotypes and determine whether mutations in pssA are themselves potentiating mutations for further adaptation to antiseptics. Use reporter fusions and/or tagged PssA to understand the molecular basis for alteration in PssA activity associated with point mutations. Techniques: bacterial culture and susceptibility testing, recombineering, experimental evolution, qPCR (of pssA and smvR expression), whole genome sequencing and MALDI-Biotyper.Years 2-3: At KCL - Determine the effect of each pssA mutation on bacterial metabolism and membrane composition in each P. aeruginosa isolate background, understand resistance/susceptibility to a range of antiseptics and membrane active host defence peptides (hBD2 and hBD3) and investigate the possible effect of pssA mutations on infection models using a Transwell lung epithelial cell co-culture system. Techniques: HR-MAS NMR metabolomics, HPLC-MS lipidomics (ion-trap MS and evaporative light scattering detection), RNAseq, biophysical experiments (primarily fluorescence spectroscopy and imaging and computational modelling of interaction of different antiseptics with bacterial membranes supported where appropriate by liquid and solid-state NMR and circular dichroism spectroscopy), mammalian cell culture and confocal microscopy. Year 4: At PHE and KCL - Depending on project outcomes 1) use results to inform preliminary study of A. baumanii adaptation to octenidine or 2) translate results to qPCR and/or MALDI-TOF based diagnostic assays that can be deployed to screen for markers of pssA mutation induced resistance in P. aeruginosa from clinical or agricultural environments where octenidine and related antiseptics are applied routinely.