UFMylation suppresses Type I IFN signaling during M. tuberculosis infection of human macrophages

Type I Interferons (IFN-I) promote host defense against a wide range of viral infections, but inhibit control of several bacterial pathogens, including Mycobacterium tuberculosis (Mtb). Given the significance of IFN-I signaling in determining Mtb infection outcomes, we sought to uncover new molecular mechanisms that regulate IFN-I during mycobacterial infection, specifically focusing on how IFN-I signaling is regulated at the earliest stages of mycobacterial infection as modeled by macrophage infection. To comprehensively identify these host genes, we performed a reporter-based, genome- wide CRISPR-interference screen in human macrophages infected with Mycobacterium marinum, a close relative of Mtb. Our screen detected 2035 significantly enriched genes (p<0.05), which included many known regulators of IFN-I, but also many unexpected genes as potentially novel regulators of IFN-I signaling. One of these unexpected genes was UFL1, an E3-like ligase that catalyzes conjugation of the ubiquitin-like protein, UFM1, in a process termed UFMylation. UFL1-deficiency during Mtb macrophage infection resulted in increased expression of IFN-{beta}, interferon-stimulated genes, and other pro-inflammatory genes, such as TNF and IL-6, at both the transcript and protein level. Depletion of other UFMylation components phenocopied UFL1-deficiency, suggesting that UFMylation activity is required for IFN-I repression. Full transcriptional profiling revealed a broad increase in the inflammatory response of UFL1-deficient cells during Mtb infection, including both protective inflammatory cytokines and potentially detrimental interferon-stimulated genes. Our results suggest a role for UFMylation in suppressing IFN-I signaling and inflammatory responses during the earliest stages of Mtb infection. IMPORTANCEMycobacterium tuberculosis was estimated to have caused over 1 million deaths in 2025 - the most deaths globally by a single bacterial pathogen. Past studies in mice and humans have shown that some host immune responses, such as IFN-I signaling, can enhance susceptibility to M. tuberculosis. We report the results of our genome-wide CRISPR-interference screen to determine regulators of IFN-I signaling during the earliest stages of mycobacterial infection. This screen identified new regulators of IFN-I signaling, including the UFMylation pathway, which appears to play an unexpected role in suppressing both IFN-I signaling and a broader inflammatory response. These findings may be relevant for the development of therapeutics and prophylactics for tuberculosis that impact IFN-I signaling - a known determinant of tuberculosis susceptibility.