A genome-wide CRISPR screen defines host determinants of early <i>Brucella</i> infection in human macrophage-like cells

ABSTRACT Brucella spp. are widespread intracellular animal pathogens that cause brucellosis, a significant zoonosis. Despite the global impact of brucellosis on animal and human health, the host genes that support Brucella infection remain incompletely defined. To address this knowledge gap, we developed a flow cytometry-based infection assay with fluorescent Brucella and performed a genome-wide CRISPR-Cas9 loss-of-function screen in human macrophage-like cells. Disruption of &gt;150 host genes significantly reduced intracellular B. abortus signal at 3 hours post-infection. In addition to recovering known host factors, the screen revealed previously unappreciated genes linked to endosomal trafficking, cytoskeletal remodeling, and lipid homeostasis. The screen was robust, as validation within these functional categories confirmed that the small GTPase RAB14, the Src-family kinase regulator CSK, and the phospholipid flippase subunit TMEM30A support the B. abortus and B. ovis infection process at a post-entry step. Gene set enrichment analysis further identified positive regulators of mTORC1 signaling as host factors. This result was validated by genetic disruption of LAMTOR2 and AKT1, and pharmacologic inhibition of AKT1. Together, these data indicate that the AKT-Ragulator-mTORC1 axis contributes to establishing a permissive intracellular niche. Finally, to assess whether these host requirements extend beyond Brucella , we examined infection by the unrelated intracellular pathogen Mycobacterium abscessus . CSK, AKT1, and LAMTOR2 were required for efficient M. abscessus infection, whereas RAB14 was dispensable. Together, these results define host genes that impact Brucella infection and distinguish shared versus pathogen-specific host dependencies exploited by intracellular bacteria.