Time-Resolved Proteomic and Phenotypic Analysis of the MRSA Response to (+)-Usnic Acid

Abstract

To characterize the antibacterial response of methicillin-resistant Staphylococcus aureus (MRSA) to (+)-usnic acid using time-resolved proteomics and phenotypic assays. MRSA cultures were exposed to (+)-usnic acid at the minimum inhibitory concentration (MIC; 8 mg/L) and sampled at 30, 60, and 120 min. Protein abundance profiles were analyzed by label-free LC–MS/MS, followed by fuzzy C-means clustering and GO and KEGG enrichment analyses. Membrane permeability and peptidoglycan biosynthetic activity were evaluated using propidium iodide uptake and HADA incorporation assays, respectively. (+)-Usnic acid inhibited MRSA growth in a concentration-dependent manner and induced extensive time-dependent changes in protein abundance. Among 1,037 identified proteins, those showing sustained decreases were enriched in functions related to ribosomal structure, translation, redox homeostasis, carbon metabolism, the pentose phosphate pathway, and peptidoglycan biosynthesis. (+)-Usnic acid exposure also caused a time-dependent increase in membrane permeability and a progressive reduction in HADA incorporation. (+)-Usnic acid exerts growth-inhibitory activity against MRSA and is associated with coordinated disruption of translation-related functions, redox homeostasis, membrane integrity, and peptidoglycan biosynthetic activity.