Background:
Antimicrobial resistance (AMR) poses a critical global health challenge. While proteomics has advanced our understanding of bacterial responses to antibiotics, the role of changing protein structure and its relationship to antibiotic binding and protein-protein interactions remains largely unexplored using mass spectrometry–based methods. Consequently, a comprehensive understanding of conformational changes across the proteome and their relationship to antibiotic treatment has yet to be achieved. Conventional bottom-up proteomics workflows primarily quantify changes in protein abundance and therefore often overlook conformational changes that can influence antibiotic efficacy. In recent years, several methodologies have been developed to investigate these structural changes on a proteome-wide scale, including Thermal Proteome Profiling (TPP)[1] and Limited Proteolysis–Mass Spectrometry (LiP-MS)[2].
Objective:
This study applied LiP-MS to investigate proteome-wide structural changes in Acinetobacter baumannii following meropenem treatment, aiming to uncover conformational mechanisms contributing to AMR.
Methodology:
During LiP-MS, proteins are extracted and maintained under near-native conditions, subjected to limited, non-specific proteolysis that preferentially cleaves solvent-exposed or flexible regions, and then undergo complete tryptic digestion for mass spectrometry-based analysis. In this study, variations in the detection of tryptic and half-tryptic peptides between treated and untreated samples were then analysed using FragPipe and FlippR[3] to infer which proteins were undergoing conformational changes in response to meropenem treatment. In parallel, a standard shotgun proteomics workflow was employed to quantify overall changes in protein abundance.
Results:
Pathway enrichment via STRING-DB of the conventional bottom-up proteomics data revealed upregulation of CTP and AMP biosynthetic processes, suggesting a metabolic shift that enabled A. baumannii to withstand antibiotic stress. Notably, no significant changes in abundance were detected for key meropenem resistance proteins, such as β-lactamases. LiP-MS further identified conformational changes in key metabolic proteins, including HemF and FabI. FabI (Enoyl-[acyl-carrier-protein] reductase [NADH]) is a promising antibiotic target that exhibits multiple conformations and binding partners, affecting its function [4]. HemF (oxygen-dependent coproporphyrinogen-III oxidase), involved in heme biosynthesis, has been proposed to catalyse through two distinct routes, each involving alternative binding partners, potentially explaining the observed structural changes[5].
Conclusion:
These findings demonstrate that LiP-MS provides insights into the structural dynamics of bacterial proteomes during antibiotic challenge. Conformational changes in FabI and HemF may influence metabolic processes in response to antibiotic treatment, thereby contributing to antibiotic resistance. This work represents an initial investigation into how structural changes across the proteome influence resistance; further metabolic analysis will be performed to elucidate these resistance mechanisms in greater detail.