Utilization of a CRISPRi-based ex vivo challenge model to reveal temporally dependent gene essentiality in intracellular Mycobacterium tuberculosis.

UNLABELLED: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. Using a library comprising ~20,000 sgRNAs covering >96% of Mtb open reading frames, we identified genes required for growth within the changing immune microenvironment. Mutant depletion patterns varied across immune environments sampled at 2, 4, and 6 weeks post-infection, which revealed a weighted dependency on cell wall biosynthesis genes early and the reliance on cholesterol catabolism and iron acquisition across all time points. Functional validation of three genes-embB, fadE29, and mbtI-confirmed their temporal significance in vivo. This screen provides increased resolution of the differential metabolic vulnerabilities in Mtb in the evolving immune environments during infection, stressing the temporal nature of conditional essentiality in vivo.

IMPORTANCE: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. This novel approach enabled us to identify how different bacterial metabolic pathways were of greater importance to the bacterium at different time points post-infection. The results provide insights into how the evolving immune response to infection shapes the metabolic and replicative status of the bacterium. This information has significance in the design of therapeutic strategies toward cure.