The RNA binding protein hnRNP A/B controls exosomal miR-27a-5p loading during Salmonella infection.

Salmonella infections cause significant economic losses in poultry and livestock production. Salmonella infection reprograms the proteomic and miRNA profiles of intestinal macrophage-derived exosomes, which subsequently modulates the inflammatory responses of surrounding recipient cells. However, the mechanisms underlying the selective packaging of specific miRNAs into exosomes during inflammation are still incompletely understood. In this study, we employed Tandem Mass Tag (TMT)-based quantitative proteomics to analyze exosomes derived from macrophages following Salmonella infection versus uninfected controls. Furthermore, RNA immunoprecipitation (RIP) assays were conducted to investigate potential interactions between a candidate exosomal protein and miR-27a-5p. The proteomic analysis revealed that Salmonella infection induced significant remodeling of the exosomal protein cargo secreted by RAW 264.7 macrophage-like cells. Quantitative comparison with uninfected controls showed 383 proteins were significantly upregulated and 666 were downregulated in exosomes at 12 h post-infection, demonstrating infection-dependent reprogramming of exosomal composition. ​Limited differential protein expression was observed at 3 h post-Salmonella infection group. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis indicated that the target genes are mainly associated with bacterial infectious diseases, translation, and molecular binding. Notably, the RNA binding protein heterogeneous nuclear ribonucleoprotein A/B (hnRNP A/B) was significantly upregulated in exosomes derived from Salmonella-infected macrophages. RIP assays confirmed specific binding of hnRNP A/B to miR-27a-5p. Furthermore, knockout (KO) of hnRNP A/B markedly decreased exosomal miR-27a-5p export while increasing its cellular retention. These findings establish hnRNP A/B as a critical regulator of miR-27a-5p sorting during Salmonella infection, suggesting its potential as a therapeutic target to interfere with bacterial pathogenesis and host immune evasion mechanisms in poultry and livestock medicine.