Deubiquitinase USP15 restricts LC3-dependent targeting of <i>Mycobacterium tuberculosis</i>

Macroautophagy/autophagy enables macrophages to degrade intracellular <i>Mycobacterium tuberculosis</i> (Mtb), and this defense depends on E3 ubiquitin ligases such as PRKN/PARKIN/PARK2 and SMURF1, which tag Mtb-associated structures for lysosomal clearance. Deubiquitinases (DUBs) counter ubiquitin ligases by removing ubiquitin from molecular targets. We hypothesized that DUBs might offset ubiquitin ligase activity and negatively regulate host immunity to Mtb. Here, we identify USP15 (ubiquitin specific peptidase 15) as a negative regulator of MAP1LC3/LC3-dependent targeting pathways (consistent with xenophagy or CASM/LAP-related ATG8ylation) that mediate macrophage immunity to Mtb. Using a targeted knockdown screen in mouse macrophages, we found that <i>Usp15</i> loss increased K63-linked ubiquitination and LC3 recruitment to Mtb-associated structures, leading to reduced bacterial replication. These effects required USP15's catalytic activity and were reversed by knockdown of PRKN or inhibition of autophagy initiation. In primary human macrophages, <i>USP15</i> knockdown similarly enhanced LC3 targeting and restricted Mtb growth. Importantly, pharmacological inhibition of USP15 with a selective small molecule decreased Mtb burden in human macrophages. Our findings identify USP15 as a suppressor of macrophage immunity and suggest that targeting deubiquitinases may represent a promising host-directed therapeutic strategy against tuberculosis.<b>Abbreviations</b>: CFU: colony-forming unit; DUBs: deubiquitinases; K48-Ub: K48-linked ubiquitin; K63-Ub: K63-linked ubiquitin; Mtb-pLux: luminescent Mtb strain Mtb; <i>Mycobacterium tuberculosis</i>; MOI: multiplicity of infection; NTC: non-targeting control; TB: tuberculosis.