Quantitative proteomic analysis of cadmium chloride tolerance mechanisms in Aeromonas hydrophila

Aeromonas hydrophila, an antibiotic-and multi-metal-resistant pathogen, threatens aquaculture, yet its adaptation to cadmium chloride (CdCl₂) remains poorly understood. A.hydrophilla was grown with CdCl₂ exposure, and data-independent acquisition (DIA)-based proteomics was employed to identify the 253 upregulated and 163 downregulated proteins. GO and KEGG enrichment analyses revealed upregulated proteins involved in sulfate metabolism, siderophore- and iron transport, and other transition metal transport processes, while downregulated proteins were linked to amino acid metabolism, the tricarboxylic acid (TCA) cycle, and fatty acid metabolism. Gene Set Enrichment Analysis (GSEA) indicated positive enrichment of ribosome, RNA degradation, sulfur metabolism, and riboflavin metabolism, and negative enrichment of valine, leucine, and isoleucine degradation, two-component system, and bacterial chemotaxis. CdCl₂ tolerance assays of four gene-deletion mutants confirmed proteomics predictions: ΔAHA_3605 (uncharacterized protein) and ΔAHA_1796 (GGDEF-domain deletion) were significantly more sensitive to CdCl₂, suggesting a role for cyclic di-GMP (c-di-GMP) signaling in cadmium resistance. Collectively, these results provide novel insights into the adaptive strategies of A. hydrophila, revealing key molecular determinants of CdCl₂ stress tolerance. SIGNIFICANCE: Cadmium contamination increasingly co-selects for antibiotic-resistant pathogens in aquaculture, yet the molecular basis of cadmium tolerance in Aeromonas hydrophila-one of the most problematic Gram-negative fish pathogens-has been unknown. Here, we provide the first comprehensive, DIA-based quantitative proteome map of A. hydrophila under CdCl₂ stress, uncovering a tightly orchestrated stress response that simultaneously remodels metal acquisition, down-regulates energy-intensive metabolism (TCA cycle, branched chain amino acid and fatty acid degradation), and activates sulfur-riboflavin-ribosome pathways to repair oxidative damage. Functional validation using gene-deletion mutants demonstrated that loss of AHA_3605 (uncharacterized) or AHA_1796 (GGDEF domain protein) significantly increased CdCl₂ sensitivity, implicating cyclic di-GMP signaling in cadmium resistance. These findings advance our mechanistic understanding of heavy-metal adaptation in aquatic pathogens and offer proteome informed targets to disrupt metal-driven co-selection of multidrug-resistant A. hydrophila in aquaculture ecosystems.