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内毒素的外膜和内质膜来克服防腐挑战。由于膜活性杀菌剂在临床上的广泛应用，导致细菌对这些杀菌剂失去敏感性的适应变得越来越常见。使这个问题雪上加霜的是，这些由此产生的适应可能导致对人类免疫系统的关键成分产生交叉耐药性，包括膜活性防御素抗菌肽，甚至是最后的抗生素。这个项目集中在铜绿假单胞菌中的PSSA基因，一个被注释为磷脂酰丝氨酸合成酶的基因。我们最近发现，PSSA的两个突变使这种多药耐药菌的几个临床分离株对辛替尼定产生耐药性，并对洗必泰、其他防腐剂和至少一种抗菌肽产生交叉耐药性。现在必须了解这些突变在对更广泛的防腐剂(交叉)抗药性中的作用，以便为诊断和监测方案提供信息，例如在临床或农业环境中。具体地说，该项目试图了解PSSA中两个已知突变的影响，这些突变产生的条件，以及这些突变导致不同临床来源的铜绿假单胞菌不同耐药表型的机制。如果时间允许，该项目的元件将在鲍曼不动杆菌中复制，据预测，鲍曼不动杆菌的PSSA蛋白具有与铜绿假单胞菌相似的单域跨膜结构，这与大多数其他革兰氏阴性病原体不同。该项目依赖于专业知识、技能和技术的结合，只有通过KCL和英国公共卫生(PHE)之间的广泛合作才能获得。该项目将结合经典的分子微生物学技术、下一代测序、核磁共振代谢组学和膜生物物理方法。第1年：在Phe-在各种铜绿假单胞菌谱系中创建PSSA的定点突变(包括/不具有可疑的smvR增强突变，发现smvR是参与阳离子化合物耐药的外排泵)，评估(交叉)耐药表型，并确定PSSA突变本身是否增强了进一步适应防腐剂的突变。使用报告融合和/或标记的PSSA来了解与点突变相关的PSSA活性改变的分子基础。技术：细菌培养和药敏测试，重组工程，实验进化，qPCR(PSSA和smvR表达)，全基因组测序和MALDI-Biotyper。2-3年：在KCL-确定每个PSSA突变对每个铜绿假单胞菌分离株的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(hBD2和hBD3)的耐药性/敏感性，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。技术：HR-MAS核磁共振代谢组学、高效液相-质谱仪脂质组学(离子陷阱MS和蒸发光散射检测)、RNAseq、生物物理实验(主要是荧光光谱以及不同防腐剂与细菌膜相互作用的成像和计算模型，适当时还得到液体和固体核磁共振以及圆二色谱的支持)，哺乳动物细胞培养和共聚焦显微镜。第4年：在PHE和KCL-取决于项目结果1)使用结果为鲍曼不动杆菌对辛替尼定适应的初步研究提供信息，或2)将结果转换为基于qPCR和/或MALDI-TOF的诊断分析，可用于从常规应用辛替尼定和相关防腐剂的临床或农业环境中筛查PSSA突变导致铜绿假单胞菌耐药的标志物。

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.