Genome-Wide Identification of Genes Conferring Energy Related Resistance to a Synthetic Antimicrobial Peptide (Bac8c)

A fundamental issue in the design and development of antimicrobials is the lack of understanding of complex modes of action and how this complexity affects potential pathways for resistance evolution. Bac8c (RIWVIWRR-NH2) is an 8 amino acid antimicrobial peptide (AMP) that has been shown to have enhanced activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. We have previously demonstrated that Bac8c appears to interfere with multiple targets, at least in part through the disruption of cytoplasmic membrane related functions, and that resistance to this peptide does not easily develop using standard laboratory methods. Here, we applied a genomics approach, SCalar Analysis of Library Enrichement (SCALEs), to map the effect of gene overexpression onto Bac8c resistance in parallel for all genes and gene combinations (up to ∼ 10 adjacent genes) in the E. coli genome (a total of ∼ 500,000 individual clones were mapped). Our efforts identified an elaborate network of genes for which overexpression leads to low-level resistance to Bac8c (including biofilm formation, multi-drug transporters, etc). This data was analyzed to provide insights into the complex relationships between mechanisms of action and potential routes by which resistance to this synthetic AMP can develop.

抗菌药物研发设计中的核心挑战之一，在于对其复杂作用模式，以及该复杂性如何影响耐药性演化的潜在路径缺乏充分认知。Bac8c（RIWVIWRR-NH2）是一种由8个氨基酸组成的抗菌肽（antimicrobial peptide, AMP），已被证实对多种致病性革兰氏阳性菌、革兰氏阴性菌及酵母菌均具有增强的抗菌活性。本团队此前已证实，Bac8c似乎可通过多靶点发挥作用，其部分机制为破坏细胞质膜相关功能，且采用标准实验室方法难以诱导该肽类产生耐药性。本研究采用基因组学方法——文库富集标量分析（SCalar Analysis of Library Enrichement, SCALEs），对大肠杆菌（E. coli）基因组中所有基因及基因组合（最多包含约10个相邻基因）的过表达对Bac8c耐药性的影响进行并行图谱绘制，共计完成约50万个独立克隆的图谱构建。本研究成功鉴定出一个复杂的基因网络，其中基因过表达可赋予菌株对Bac8c的低水平耐药性，涵盖生物膜形成、多药转运蛋白等相关通路。本研究对该数据集进行分析，旨在深入解析该合成抗菌肽的作用机制与耐药性潜在演化路径之间的复杂关联。