In the present report we characterized the outer membrane proteome, genomic, and lipid A remodelling changes following the evolution of a colistin-susceptible K. pneumoniae ATCC 13883 strain into an extremely colistin-resistant strain. Lipid A profiling revealed the outer membrane of the colistin-susceptible strain is decorated primarily by hexa- and hepta-acylated lipid A species and a minor tetra-acylated species. In the lipid A profile of the extremely colistin-resistant strain, in addition to the aforementioned lipid A species, the obligatory 4-amino-4-deoxy-l-arabinose modification of the hexa-acylated lipid A was detected. Comparative genomic analysis revealed that the mgrB gene of the colistin-resistant strain is inactivated by a single nucleotide insertion which produces a frame-shift, resulting in premature termination. We also detected two synonymous mutations in the two-component system genes phoP and phoQ. Comparative profiling of the outer membrane proteome of each strain revealed that outer membrane proteins from bacterial stress response, glutamine degradation, pyruvate, aspartate, and asparagine metabolic pathways were over-represented in the extremely colistin-resistant K. pneumoniae ATCC 13883 strain. In comparison, in the sensitive strain, outer membrane proteins from carbohydrate metabolism, H⁺-ATPase, cell division, and peptidoglycan biosynthesis were over-represented. Notably, there were no discernible differences between the OmpK35 and OmpK36 major outer membrane porins between the polymyxin-susceptible and -resistant strains suggesting porin deficiency is not involved in the colistin resistance in the ATCC 13883 strain. These findings shed new light on the outer membrane remodelling events accompanying the development of extremely high levels of colistin resistance in K. pneumoniae.

中文翻译：

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Colistin易感配对和极耐Colistin肺炎克雷伯菌配对的外膜蛋白质组的比较研究

在本报告中，我们表征了大肠埃希菌敏感的肺炎克雷伯氏菌ATCC 13883菌株进化为极耐大肠菌素的菌株后，外膜蛋白质组，基因组和脂质A重塑变化。脂质A分析揭示了粘菌素敏感菌株的外膜由主要装饰六-和庚-acylated脂质A物种和次要四-acylated物种。在对大肠粘菌素抗性极强的菌株的脂质A谱中，除了上述脂质A种类之外，还检测到六酰化脂质A的强制性的4-氨基-4-脱氧-1-阿拉伯糖修饰。比较基因组分析表明大肠菌素抗性菌株的mgrB基因通过单核苷酸插入而失活，该插入产生移码，导致过早终止。我们还检测到两组分系统基因phoP和phoQ中的两个同义突变。每个菌株的外膜蛋白质组的比较分析表明，来自细菌应激反应，谷氨酰胺降解，丙酮酸，天冬氨酸和天冬酰胺代谢途径的外膜蛋白在耐大肠菌素性肺炎克雷伯氏菌ATCC 13883菌株中被过度表达。相比之下，在敏感菌株中，碳水化合物代谢产生的外膜蛋白H ⁺-ATPase，细胞分裂和肽聚糖的生物合成被过度代表。值得注意的是，多粘菌素敏感性和抗性菌株之间的OmpK35和OmpK36主要外膜孔蛋白之间没有明显差异，这表明孔蛋白缺乏与ATCC 13883菌株的大肠菌素抗性没有关系。这些发现为肺炎克雷伯氏菌产生极高水平的大肠菌素抵抗的外膜重塑事件提供了新的启示。