Rv1983 promotes mycobacterial dissemination by triggering ferroptosis through GPX4 ubiquitination

BACKGROUND: Ferroptosis, a unique form of regulated cell death induced by iron-dependent lipid peroxidation, has been implicated in the pathogenesis of Mycobacterium tuberculosis (Mtb). However, the role of Mtb proteins, particularly those encoded by the genomic regions of deletion (RDs), involved in mediating macrophage ferroptosis has not been thoroughly investigated. This study aimed to screen for Mtb RD region-encoded proteins that induce macrophage ferroptosis and elucidate the underlying molecular mechanisms. METHODS: We identified the Rv1983 protein with cytotoxic activity against murine bone marrow-derived macrophages (BMDMs) through screening of prokaryotically expressed Mtb RDs proteins, and subsequently determined the specific cell death modality induced by Rv1983 in BMDMs through flow cytometry, western blotting, and cytotoxicity assays following Rv1983 stimulation. To investigate the role of Rv1983 in Mtb infection, we constructed an Rv1983-knockout Mtb H37Ra strain (H37RaΔRv1983) and compared its intracellular and extracellular bacterial loads with wild-type H37Ra strain in infected BMDMs. Using coimmunoprecipitation, immunofluorescence, and flow cytometry, we identified and validated the target protein of Rv1983 in macrophages and its functional role in Rv1983-mediated macrophage death. For in vivo validation, wild-type H37Ra and H37RaΔRv1983 strains were used to infect both wild-type and macrophage-specific Rv1983-binding protein knockout mice, and the role of Rv1983 during Mtb infection was assessed through bacterial colony counting, flow cytometry analysis, and histochemical staining. RESULTS: This study identifies Rv1983 (PE_PGRS35), an RD2-encoded secreted protein of Mtb that acts as a ferroptosis effector to promote mycobacterial dissemination. Mechanistically, Rv1983 binds to E3 ubiquitin ligase tripartite motif 25 (TRIM25) in macrophages through its PE domain, especially on the Y62 site. The Rv1983-TRIM25 complex interacts with glutathione peroxidase 4 (GPX4), and subsequently promotes K48-linked ubiquitination degradation of GPX4 at the K75 site, ultimately inducing ferroptosis to promote mycobacterial dissemination. An Rv1983-deficient Mtb strain (H37RaΔRv1983) displayed significant suppression of Mtb dissemination both in vitro and in vivo. CONCLUSIONS: These findings provide a new insight into the molecular mechanism of Mtb-induced ferroptosis, and suggest that targeting ferroptosis mediated by the Rv1983-TRIM25-GPX4 signaling axis is a potential strategy for therapeutic control of tuberculosis.