The miR-744-5p/connexin 43 axis modulates Salmonella-containing vacuole association with the Golgi network

MicroRNAs are increasingly recognised as important regulators of host-pathogen interactions. Using a large-scale functional screening approach, we identified miR-744-5p as a strong inhibitor of Salmonella infection. Time-course analyses in epithelial cells revealed that miR-744-5p moderately restricts early infection but exerts a much stronger inhibitory effect on late intracellular replication. This activity is conserved across Salmonella strains of distinct sequence types, whereas inhibition of Shigella flexneri is limited to early infection stages. Integrated transcriptomic analyses identified connexin 43 as a functionally relevant miR-744-5p target. Connexin 43 expression is strongly upregulated during Salmonella infection, and functional studies demonstrated that connexin 43 depletion selectively impaired late-stage intracellular replication, while CRISPRa-mediated connexin 43 upregulation enhanced bacterial proliferation. Notably, this function was independent of the canonical gap junction intercellular communication. Mechanistically, connexin 43 is required to maintain Golgi network organisation, enabling proper positioning of the Salmonella-containing vacuole. Thus, infection-induced downregulation of miR-744-5p promotes connexin 43 expression to support the formation of a permissive vacuolar replicative niche, revealing an unanticipated role for connexin 43 in Golgi homeostasis.