The inactivation of enzymes belonging to the central carbon metabolism, a novel mechanism of developing antibiotic resistance

Fosfomycin is a bactericidal antibiotic, analogous to phosphoenolpyruvate (PEP) that exerts its activity by inhibiting the activity of MurA. This enzyme catalyzes the first step of peptidoglycan biosynthesis, the transfer of enolpyruvate from PEP to uridine- diphosphate-N-acetylglucosamine. Fosfomycin is increasingly used in the last years, mainly for treating infections caused by Gram-negative multidrug resistant bacteria as Stenotrophomonas maltophilia, an opportunistic pathogen characterized by its low susceptibility to antibiotics of common use. The mechanisms of mutational resistance to fosfomycin in Stenotrophomonas maltophilia were studied in the current work. None of the mechanisms so far described for other organisms, which include the production of fosfomycin inactivating enzymes, target modification, induction of alternative peptidoglycan biosynthesis pathway and the impaired entrance of the antibiotic, are involved in the acquisition of such resistance by this bacterial species. Rather the unique cause of resistance in the studied mutants is the mutational inactivation of different enzymes belonging to the Embden-Meyerhof-Parnas central metabolism pathway. The amount of intracellular fosfomycin accumulation did not change in any of these mutants showing that neither the inactivation nor the transport of the antibiotic were involved. Transcriptomic analysis also showed that the mutants did not present changes in the expression level of putative alternative peptidoglycan biosynthesis pathway genes neither any related enzyme. Finally, the mutants did not present an increased PEP concentration that might compete with fosfomycin for its binding to MurA. Based on these results, we describe a completely novel mechanism of antibiotic resistance based on the remodeling of S. maltophilia metabolism. Overall design: Stenotrophomonas maltophilia D457 and fosfomycin resistant mutants (FOS1, FOS4, FOS7 and FOS8) mRNA profiles measured by paired-end RNA-seq

磷霉素（Fosfomycin）是一种杀菌类抗生素，其结构与磷酸烯醇式丙酮酸（phosphoenolpyruvate, PEP）类似，通过抑制MurA酶（MurA）的活性发挥抗菌作用。该酶催化肽聚糖生物合成的第一步反应，即将烯醇式丙酮酸从PEP转移至尿苷二磷酸-N-乙酰葡糖胺（uridine-diphosphate-N-acetylglucosamine）。近年来磷霉素的临床应用愈发广泛，主要用于治疗由革兰氏阴性多重耐药菌——如嗜麦芽窄食单胞菌（Stenotrophomonas maltophilia）——引起的感染。嗜麦芽窄食单胞菌是一种机会致病菌，对常用抗生素的敏感性普遍较低。本研究聚焦于嗜麦芽窄食单胞菌对磷霉素的突变耐药机制。目前已在其他微生物中报道的磷霉素耐药机制包括：产生磷霉素灭活酶、靶标修饰、诱导替代肽聚糖生物合成通路以及抗生素摄入受损，但本研究发现嗜麦芽窄食单胞菌并未通过上述任一途径获得耐药性。本研究的耐药突变株的耐药唯一诱因，是属于Embden-Meyerhof-Parnas（EMP）中心代谢通路的不同酶类发生了突变失活。所有突变株的胞内磷霉素积累量均未发生改变，表明该菌的耐药性与抗生素的灭活或转运过程均无关。转录组分析结果显示，突变株的推定替代肽聚糖生物合成通路基因及其相关酶的表达水平均未出现变化。此外，突变株的PEP浓度并未升高，而PEP浓度升高可通过与磷霉素竞争结合MurA酶从而介导耐药。基于上述结果，本研究揭示了一种全新的抗生素耐药机制，其源于嗜麦芽窄食单胞菌的代谢重塑。实验设计：以嗜麦芽窄食单胞菌D457及其磷霉素耐药突变株（FOS1、FOS4、FOS7、FOS8）为研究对象，采用双端RNA测序（paired-end RNA-seq）检测其mRNA表达谱。