Insights into the Klebsiella pneumoniae adaptive response mechanisms to colistin exposure using a label-free quantitative proteomics approach

The rise of MDR Klebsiella pneumoniae and its resistance to the last-resort antibiotic colistin poses a significant threat to global healthcare. While genomic studies have identified several resistance mutations, the transient proteomic shifts that occur during the initial exposure of sensitive strains to lethal antibiotic doses remain poorly characterised. In this study, we employed a label-free quantitative proteomics approach to investigate the protein expression profile of K. pneumoniae strain ATCC 13883 treated with colistin at its MIC. Membrane proteins were extracted at critical growth stages, and differentially abundant proteins (DAPs) were analysed using Gene Ontology and KEGG pathway enrichment analysis. Our proteomic analysis identified 718 DAPs (339 upregulated and 379 downregulated). The cellular response was characterised primarily by outer membrane remodelling and a significant upregulation of the capsule-associated kinase Wzc and the ArnBCADTEF operon, which facilitates lipid A modification with L-Ara4N moiety. Paradoxically, while RND-family efflux pumps (AcrAB) were significantly induced, the global activator RamA and major porins (OmpA, OmpX, LamB) were downregulated, possibly to minimise antibiotic entry. KEGG pathway enrichment analysis further revealed a synchronised metabolic shift, characterised by an intensified TCA cycle flux to fuel high-energy resistance processes despite a general slowdown in carbohydrate metabolism. Our findings demonstrate that K. pneumoniae responds to colistin stress through a rapid, multifaceted proteomic reorganisation involving charge neutralisation, structural reinforcement of the cell envelope, and metabolic re-routing. These results provide a molecular blueprint of the early adaptive response, identifying several proteins as potential therapeutic targets.