TBC1D14 positively regulates autophagy induced by Brucella melitensis vaccine strain BA0711 in Sheep Leydig Cells

Brucella is an intracellular Gram-negative bacterium that primarily infects the host reproductive and immune systems, inducing autophagy and facilitating pathogen replication. TBC (Tre2-Bub2-Cdc16) domain-containing proteins are important in membrane trafficking, cell polarity, and signal transduction as regulators of Rab small GTPases. Previously, we demonstrated that B. melitensis M5-90 modulates the expression of miR-146b-5p, which targets TBC1D14, in RAW264.7 cells. In this study, CRISPR-Cas9 was used to generate TBC1D14-knockout (KO) Sheep Leydig cells (SLCs), and B. melitensis BA0711 treatment experiment was conducted at a multiplicity of infection (MOI) of 100. After confirming that SLCs retain autophagic activity, Western blot, autophagy flux assays, transmission electron microscopy (TEM), and RT-qPCR were performed to identify the function of TBC1D14. We found that autolysosome fluorescence was significantly enhanced in NC-SLCs compared with TBC1D14-KO-SLCs following BA0711 treatment at 8 and 12 hpi. Notably, autophagy flux in NC-SLCs remained consistently higher than that in KO-SLCs from 8 hpi onwards. Consistently, Western blot revealed a decreased LC3-II/LC3-I ratio, and TEM confirmed a reduced number of autolysosomes in KO-SLCs at 12 hpi. These results indicate that TBC1D14 positively regulates autophagy in SLCs by BA0711 stimulation. Although transcriptomic and proteomic analyses indicated activation of autophagy- and phagocytosis-related pathways during BA0711 exposure, this response was significantly attenuated in TBC1D14-KO cells. Furthermore, TBC1D14 knockout led to downregulation of phagosome-related proteins and altered chemotaxis pathways, whereas re-expression of TBC1D14 in KO cells upregulated RAB28. In conclusion, this study demonstrates that TBC1D14 positively regulates autophagy and provides insights into the response of SLCs treated by B. melitensis BA0711. Furthermore, it suggests that RAB28 may serve as a downstream effector of TBC1D14.