Abstract

Brucella spp. achieve intracellular parasitism by hijacking host autophagy pathways. Sheep Leydig cells (SLCs) are important intracellular parasitic target cells for Brucella melitensis (B. melitensis). To investigate the molecular regulatory network induced by B. melitensis in SLCs, particularly whether the autophagic pathway response differs from that in other cell types, transcriptome sequencing analysis was performed on SLCs treated with B. melitensis BA0711. According to the criteria of |log2FC| > 0.582 and p < 0.05, a total of 6,036 differentially expressed genes (DEGs) and 100 differentially expressed miRNAs (DE miRNAs) were identified, both of which were significantly enriched in the autophagy–animal pathway. RT-qPCR confirmed that the expression trends of validated DEGs were consistent with mRNA sequencing results. In the autophagy–animal pathway, ULK1, ATG13, ATG14, PIK3C3, ATG5, ATG101, and ATG16L2 were predicted as the top seven hub DEGs. Overexpression and knockdown experiments of miR-1, together with dual-luciferase reporter assays, demonstrated that miR-1 inhibits the mRNA expression of its downstream target ATG14 by binding to the 3’UTR of ATG14. This study revealed the potential regulatory role of the miR-1–ATG14 axis in this process. These findings provide a novel, cell-specific perspective on the early host–pathogen interactions elicited by B. melitensis within a tissue microenvironment relevant to brucellosis, offering a foundation for future mechanistic studies in more complex systems.