Table_3_Metabolic Perturbations Caused by the Over-Expression of mcr-1 in Escherichia coli.docx

Rapid dissemination of the plasmid-born polymyxin resistance gene mcr-1 poses a critical medical challenge. MCR-1 expression is tightly controlled and imposes a fitness cost on the bacteria. We used growth studies and metabolomics to examine growth and metabolic changes within E. coli TOP10 at 8 and 24 h in response to different levels of expression of mcr-1. Induction of mcr-1 greatly increased expression at 8 h and markedly reduced bacterial growth; membrane disruption and cell lysis were evident at this time. At 24 h, the expression of mcr-1 dramatically declined with restored growth and membrane integrity, indicating regulation of mcr-1 expression in bacteria to maintain membrane homeostasis. Intermediates of peptide and lipid biosynthesis were the most commonly affected metabolites when mcr-1 was overexpressed in E. coli. Cell wall biosynthesis was dramatically affected with the accumulation of lipids including fatty acids, glycerophospholipids and lysophosphatidylethanolamines, especially at 8 h. In contrast, levels of intermediate metabolites of peptides, amino sugars, carbohydrates and nucleotide metabolism and secondary metabolites significantly decreased. Moreover, the over-expression of mcr-1 resulted in a prolonged reduction in intermediates associated with pentose phosphate pathway and pantothenate and CoA biosynthesis. These findings indicate that over-expression of mcr-1 results in global metabolic perturbations that mainly involve disruption to the bacterial membrane, pentose phosphate pathway as well as pantothenate and CoA biosynthesis.质粒介导的多粘菌素耐药基因mcr-1的快速传播，已构成严峻的医学挑战。MCR-1的表达受到严格调控，且会给细菌带来适合度代价。本研究借助生长实验与代谢组学（metabolomics）技术，检测了大肠杆菌TOP10（E. coli TOP10）在8小时与24小时时间节点下，针对不同表达水平的mcr-1所产生的生长与代谢变化。在8小时时，mcr-1的诱导表达显著提升了其转录水平，并大幅抑制细菌生长；此时可观察到明显的细胞膜破坏与细胞裂解现象。至24小时，mcr-1的表达量显著下调，细菌生长与细胞膜完整性得以恢复，这表明细菌可通过调控mcr-1的表达以维持膜稳态（membrane homeostasis）。当大肠杆菌中mcr-1过表达时，肽与脂质生物合成的中间代谢物是受影响最为普遍的代谢物类别。细胞壁生物合成过程受到显著干扰，伴随脂肪酸、甘油磷脂、溶血磷脂酰乙醇胺等脂质的积累，该效应在8小时时尤为显著。与之相反，肽类、氨基糖、碳水化合物、核苷酸代谢的中间代谢物以及次级代谢产物的含量则显著降低。此外，mcr-1的过表达还导致磷酸戊糖途径（pentose phosphate pathway）与泛酸及辅酶A生物合成相关中间代谢物的含量持续降低。上述研究结果表明，mcr-1的过表达会引发全局性代谢扰动，其主要涉及细菌细胞膜破坏、磷酸戊糖途径以及泛酸与辅酶A生物合成过程。