Quantitative Proteomics Investigating the Molecular Responses of <i>Aeromonas hydrophila</i> to Zinc Stress

The aquaculture industry has recently reported the emergence of antibiotic and multimetal-resistant <i>Aeromonas hydrophila</i> strains. However, the intrinsic adaptation mechanisms of <i>A. hydrophila</i> under Zn²⁺ stress remain poorly understood. In this study, we employed a data-independent acquisition (DIA)-based quantitative proteomics approach to investigate global protein expression changes in <i>A. hydrophila</i> exposed to Zn²⁺ stress. A total of 338 proteins were upregulated and 388 were downregulated in response to Zn²⁺ exposure. Bioinformatic analyses revealed that some of the differentially expressed proteins (DEPs) were enriched in several Gene Ontology (GO) terms and KEGG pathways, such as metabolic processes (nucleotide metabolism, ribonucleotide biosynthesis, purine nucleotide metabolism), membrane-associated processes (ABC transporter complex), and signaling and energy pathways (quorum sensing, ABC transporters, and oxidative phosphorylation). Seven genes encoding protein secretion- and signaling-related proteins were selected for RT-qPCR analysis to examine whether their transcriptional responses corresponded to the proteomic trends, providing complementary insight rather than direct validation of the proteomic data. Furthermore, we assessed the tolerance of five gene-deletion mutants to Zn²⁺ stress. Among them, the Δ<i>AHA_2968</i> mutant, lacking a GGDEF-domain protein, displayed pronounced sensitivity to ZnSO₄, implicating c-di-GMP signaling in zinc stress resistance. These findings have shed light on the intrinsic adaptive mechanisms of <i>A. hydrophila</i>, indicating that they play an important role in resistance to Zn²⁺.