Department of Infectious Diseases, Institute for Biomedicine, Sahlgrenska Academy of the University of Gothenburg, Gothenburg, Sweden.
Centre for Antibiotic Resistance Research, University of Gothenburg, Gothenburg, Sweden.
Microbiol Spectr. 2023 Feb 14;11(1):e0386722. doi: 10.1128/spectrum.03867-22. Epub 2023 Jan 9.
Resistance to β-lactams is known to be multifactorial, although the underlying mechanisms are not well established. The aim of our study was to develop a system for assessing the phenotypic and proteomic responses of bacteria to antibiotic stress as a result of the loss of selected antimicrobial resistance genes. We applied homologous recombination to knock out plasmid-borne β-lactamase genes (, , and ) in Escherichia coli CCUG 73778, generating knockout clone variants lacking the respective deleted β-lactamases. Quantitative proteomic analyses were performed on the knockout variants and the wild-type strain, using bottom-up liquid chromatography tandem mass spectrometry (LC-MS/MS), after exposure to different concentrations of cefadroxil. Loss of the gene had the greatest impact on the resulting protein expression dynamics, while losses of and affected fewer proteins' expression levels. Proteins involved in antibiotic resistance, cell membrane integrity, stress, and gene expression and unknown function proteins exhibited differential expression. The present study provides a framework for studying protein expression in response to antibiotic exposure and identifying the genomic, proteomic, and phenotypic impacts of resistance gene loss. The critical situation regarding antibiotic resistance requires a more in-depth effort for understanding underlying mechanisms involved in antibiotic resistance, beyond just detecting resistance genes. The methodology presented in this work provides a framework for knocking out selected resistance factors, to study the adjustments of the bacterium in response to a particular antibiotic stress, elucidating the genetic response and proteins that are mobilized. The protocol uses MS-based determination of the proteins that are expressed in response to an antibiotic, enabling the selection of strong candidates representing putative resistance factors or mechanisms and providing a basis for future studies to understand their implications in antibiotic resistance. This allows us to better understand how the cell responds to the presence of the antibiotic when a specific gene is lost and, consequently, identify alternative targets for possible future treatment development.
β-内酰胺类抗生素耐药性是多因素的，尽管其潜在机制尚未完全确定。我们的研究目的是开发一种系统，以评估由于选定的抗菌耐药基因丢失而导致细菌对抗生素应激的表型和蛋白质组反应。我们应用同源重组敲除大肠杆菌 CCUG 73778 中质粒携带的β-内酰胺酶基因（、和），生成缺失相应缺失β-内酰胺酶的敲除克隆变体。在不同浓度头孢羟氨苄暴露后，对敲除变体和野生型菌株进行基于 Bottom-up 的液相色谱串联质谱（LC-MS/MS）定量蛋白质组分析。基因的缺失对蛋白质表达动力学的影响最大，而基因的缺失和的缺失则影响较少蛋白质的表达水平。涉及抗生素耐药性、细胞膜完整性、应激和基因表达以及未知功能蛋白的蛋白质表现出差异表达。本研究为研究抗生素暴露后蛋白质表达以及确定耐药基因丢失的基因组、蛋白质组和表型影响提供了框架。抗生素耐药的危急情况需要更深入地努力了解抗生素耐药性涉及的潜在机制，而不仅仅是检测耐药基因。本工作中提出的方法为敲除选定的耐药因子提供了框架，以研究细菌对特定抗生素应激的调整，阐明遗传反应和动员的蛋白质。该方案使用基于 MS 的方法确定对抗生素有反应的蛋白质，从而选择代表潜在耐药因子或机制的强候选物，并为未来研究提供基础，以了解它们在抗生素耐药性中的意义。这使我们能够更好地了解当特定基因丢失时细胞如何对抗生素的存在做出反应，从而确定可能用于未来治疗开发的替代靶标。
