The development of novel antimicrobials provides additional treatment options for infectious diseases, including antimicrobial resistant infections. There are many hurdles to antimicrobial development and identifying an antimicrobial's mechanism of action is a crucial step in progressing candidate molecules through the drug discovery pipeline. We used the genome wide screening method TraDIS-Xpress to identify genes in two model Gram-negative bacteria that affected sensitivity to three analogues of a novel antimicrobial compound (OPT-2U1). TraDIS-Xpress identified that all three analogues targeted the lipid IVA biosynthetic pathway in E. coli and Salmonella Typhimurium. Specifically, we determined that the antimicrobial target was likely to be LpxD, and validated this by finding a 5 log2-fold increase in the MIC of the OPT-2U1 analogues in E. coli when lpxD was overexpressed. Synergies were identified between OPT-2U1 analogues combined with rifampicin or colistin, to varying strengths, in both E. coli and S. Typhimurium. LPS composition was a likely reason for differences between E. coli and S.Typhimurium, as perturbation of LPS synthesis affected synergy between antibiotics and OPT-2U1 analogues. Finally, genes involved in ATP synthesis and membrane signalling functions were also found to affect the synergy between colistin and OPT-2U1 analogues. TraDIS-Xpress has proven a powerful tool to rapidly assay all genes (and notably, essential genes) within a bacterium for roles in dictating antimicrobial sensitivity. This study has confirmed the predicted target pathway of OPT-2U1 and identified synergies which could be investigated for development of novel antimicrobial formulations.

中文翻译：

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新型脂质IVA生物合成抑制剂的作用机制和协同作用

新型抗菌药物的开发为传染病（包括抗菌药物耐药性感染）提供了额外的治疗选择。抗菌药物的开发存在许多障碍，确定抗菌药物的作用机制是在药物发现管道中推进候选分子的关键一步。我们使用全基因组筛选方法 TraDIS-Xpress 来鉴定两种模型革兰氏阴性细菌中影响新型抗菌化合物 (OPT-2U1) 的三种类似物敏感性的基因。TraDIS-Xpress 发现所有三种类似物均靶向大肠杆菌和鼠伤寒沙门氏菌中的脂质 IVA 生物合成途径。具体来说，我们确定抗菌靶标可能是 LpxD，并通过发现大肠杆菌中 OPT-2U1 类似物的 MIC 增加了 5 log2 倍来验证这一点。lpxD 过表达时的大肠杆菌。在大肠杆菌和鼠伤寒沙门氏菌中，OPT-2U1 类似物与利福平或粘菌素联合使用时，发现了不同强度的协同作用。LPS 组成可能是大肠杆菌和鼠伤寒沙门氏菌之间差异的一个原因，因为 LPS 合成的干扰会影响抗生素和 OPT-2U1 类似物之间的协同作用。最后，还发现参与 ATP 合成和膜信号传导功能的基因会影响粘菌素和 OPT-2U1 类似物之间的协同作用。TraDIS-Xpress 已被证明是一种强大的工具，可以快速分析细菌内的所有基因（尤其是必需基因），以确定其在决定抗菌敏感性方面的作用。这项研究证实了 OPT-2U1 的预测靶标途径，并确定了可用于开发新型抗菌制剂的协同作用。