Deubiquitinase USP15 restricts autophagy and macrophage immunity to Mycobacterium tuberculosis

10 Autophagy enables macrophages to degrade intracellular Mycobacterium tuberculosis 11 (Mtb), and this defense depends on E3 ubiquitin ligases such as PARKIN and SMURF1, 12 which tag Mtb-associated structures for lysosomal clearance. Deubiquitinases (DUBs) 13 counter ubiquitin ligases by removing ubiquitin from molecular targets. We hypothesized 14 that DUBs might offset ubiquitin ligase activity and negatively regulate host immunity to 15 Mtb. Here, we identify ubiquitin-specific protease 15 (USP15) as a negative regulator of 16 autophagy-mediated macrophage immunity to Mtb. Using a targeted knockdown screen 17 in mouse macrophages, we found that Usp15 loss increased K63-linked ubiquitination 18 and LC3 recruitment to Mtb-associated structures, leading to reduced bacterial 19 replication. These effects required USP15’s catalytic activity and were reversed by 20 knockdown of PARKIN ( Park2) or inhibition of autophagy initiation. In primary human 21 macrophages, USP15 knockdown similarly enhanced LC3 targeting and restricted Mtb 22 growth. Importantly, pharmacologic inhibition of USP15 with a selective small molecule 23 decreased Mtb burden in human macrophages. Our findings identify USP15 as a 24 suppressor of macrophage immunity and suggest that targeting deubiquitinases may 25 represent a promising host-directed therapeutic strategy against tuberculosis. 26 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint

27 Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is 28 responsible for an estima ted 10 million new cases and 1.3 million deaths annually 29 worldwide (1). The airborne route of transmission, combined with its ability to persist 30 within infected individuals, contribute to its global success (2, 3). Mtb primarily infects 31 macrophages or inflammatory monocytes following inhalation (4, 5) and can enter a 32 long-lived persistent state that later reactivates to cause disease and transmission (3, 33 6). While antibiotics are effective against active TB, they are less effective against latent 34 bacteria, and the emergence of multidrug-resistant Mtb strains further complicates 35 treatment (1, 7). These challenges underscore the need for host-directed therapies that 36 enhance immune control of Mtb replication (8-14). However, a detailed understanding of 37 the molecular mechanisms used by host innate immune cells to restrict bacterial 38 replication is incomplete. 39 Xenophagy, a form of selective autophagy that targets large intracellular 40 structures, is a critical component of macrophage cell-autonomous immunity against 41 Mtb (15-18). Pathogen- and damage-associated molecular patterns (PAMPs and 42 DAMPs) stimulate xenophagy by recruiting E3 ubiquitin ligases such as PARKIN 43 (encoded by the Park2 gene) and SMURF1 to cytosolic Mtb or damaged phagosomal 44 membranes (19-26). These ligases attach ubiquitin to Mtb-associated structures, 45 particularly K63- and K48-linked polyubiquitin chains, which are recognized by adaptor 46 proteins like p62, NDP52, NBR1 and Optineurin (OPTN). In turn, adaptors recruit LC3 47 and other autophagy machinery, leading to the formation of autophagosomes and 48 subsequent bacterial degradation through lysosomal fusion (27-29). While some 49 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint controversy remains regarding the role of autophagy in Mtb control (30, 31), genetic 50 ablation of Park2, Smurf1, or autophagy pathway components in murine (25, 26, 32-35) 51 or human (26, 36) macrophages results in increased Mtb growth and reduced mouse 52 survival (25, 26, 32-35). 53 Although the role of ubiquitin ligases in promoting xenophagy is well established, 54 the contribution of opposing deubiquitinases (DUBs) remains poorly understood. DUBs 55 remove ubiquitin chains from proteins and are categorized into five major families based 56 on structural and functional characteristics (37). These enzymes are known to regulate 57 key immune and stress response pathways, but only a few have been implicated in 58 macrophage immunity during infection. For example, DUB30 negatively regulates 59 mitophagy by delaying mitochondrial recruitment of PARKIN (38, 39), and ubiquitin-60 specific protease (USP) 18 modulates type I interferon signaling and host defense 61 during Mtb and Salmonella infections (40). Finally, a recent study identified USP8 as a 62 negative regulator of cell-autonomous immunity to Mtb by facilitating the repair of Mtb-63 induced membrane damage that would normally trigger Mtb degradation (41). However, 64 the broader roles of DUBs in regulating host responses to Mtb remain largely 65 unexplored. 66 To address this gap, we performed a knockdown screen targeting murine DUBs 67 in BV2 macrophages (42) and identified multiple candidates that alter Mtb replication. 68 USP15 emerged as a potent negative regulator of host immunity to Mtb. Genetic 69 deletion or knockdown of USP15 in mouse and human macrophages enhanced K63-70 linked ubiquitination and LC3 recruitment to Mtb-associated structures, resulting in 71 impaired bacterial replication. These effects required USP15’s catalytic activity and 72 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint were reversed by knockdown of Park2 or pharmacologic inhibition of autophagy 73 initiation. In primary human macrophages, pharmacologic inhibition of USP15 similarly 74 reduced Mtb burden. These findings identify USP15 as a key regulator of autophagy 75 and macrophage defense against Mtb and suggest that targeting deubiquitinases may 76 represent a promising strategy for host-directed therapy against tuberculosis. 77 78

79 Depletion or deletion of Usp15 inhibits Mtb replication 80 To identify deubiquitinases (DUBs) that regulate Mycobacterium tuberculosis 81 (Mtb) replication in macrophages, we generated a knockdown library in the genetically 82 tractable murine BV2 macrophage cell line. We selected BV2 microglial macrophages 83 due to their robust genetic tractability, their previously validated use in autophagy 84 studies, and their established utility in modeling mycobacterial pathogenesis (43-50). 85 We used a luminescent Mtb strain (Mtb-pLux) to measure intracellular bacterial growth 86 across cell lines, each stably expressing one of three distinct shRNAs per target gene 87 (Figure 1A). Among the DUBs screened, knockdown of 33 genes increased Mtb 88 replication, whereas knockdown of 6 genes decreased bacterial growth by at least 1.25-89 fold compared to a non-targeting control (NTC) (Figure 1A, 1B). Notably, Usp18 90 knockdown increased Mtb growth, consistent with prior reports and validating our 91 screening strategy (40). 92 We focused on USP15 based on its significant suppression of Mtb replication 93 and its known interactions with PARKIN and roles in autophagy regulation (51-53). We 94 first validated Usp15 knockdown (KD) in BV2 macrophages using two independent 95 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint shRNAs, which effectively reduced USP15 protein levels by immunoblotting 96 (Supplemental Figure 1A). We next quantified Mtb replication in BV2 Usp15 KD cell 97 lines using a traditional colony-forming unit (CFU) assay. Compared to control BV2 98 cells, BV2 Usp15 KD macrophages exhibited static Mtb growth and significantly 99 reduced CFU over a 3-day infection (Figure 1C). 100 To confirm this phenotype, we generated BV2 Usp15 knockout (KO) cells using 101 CRISPR/Cas9 and validated USP15 loss by sequencing and immunoblotting 102 (Supplemental Figure 1B). In agreement with the knockdown data, BV2 Usp15 KO 103 macrophages infected with Mtb Erdman (MOI 1) showed reduced CFU at two and three 104 days post-infection compared to wild-type BV2 macrophages (Figure 1D). Bacterial 105 burden remained static in BV2 Usp15 KO cells over time, while it increased steadily in 106 wild-type controls. These results demonstrate that Usp15 suppresses cell-autonomous 107 immunity to Mtb in BV2 macrophages. 108 Usp15 deletion in BV2 cells increases K63 ubiquitin co-localization with Mtb 109 Because USP15 has been reported to remove K63-linked ubiquitin (K63-Ub) and 110 counteract PARKIN activity (51), we next tested whether loss of Usp15 affected K63 111 ubiquitination of Mtb-associated structures. We use the term "Mtb-associated 112 structures" because it remains unclear whether ubiquitin directly attaches to bacteria or 113 the membranes surrounding the bacteria. We infected wild-type and BV2 Usp15 KO 114 cells with mCherry-expressing Mtb and performed immunofluorescence microscopy 115 using an anti-K63 ubiquitin antibody. Compared to wild-type cells, BV2 Usp15 KO cells 116 exhibited a significant increase in the colocalization of K63-Ub with Mtb (Figure 1E, 1F). 117 As a control, we examined K48-linked ubiquitin (K48-Ub), which has also been reported 118 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint to localize to Mtb-associated structures (25). In contrast to K63-Ub, K48-Ub 119 colocalization with mCherry Mtb was unchanged between wild-type and BV2 Usp15 KO 120 cells (Supplemental Figure 1C, 1D). These results suggest that USP15 specifically limits 121 the accumulation of K63-linked ubiquitin at Mtb-associated structures, consistent with its 122 role in countering PARKIN-mediated ubiquitination. 123 Usp15 deletion in BV2 cells increases LC3-I to LC3-II conversion and recruitment 124 of LC3 to Mtb-associated structures 125 Because K63-linked ubiquitination has been implicated in promoting recruitment 126 of autophagy machinery (25), we next tested whether loss of Usp15 altered LC3 127 dynamics during Mtb infection. One hallmark of autophagy activation is the conversion 128 of cytosolic LC3-I to the lipidated LC3-II form, which becomes membrane-associated 129 during autophagosome formation (54, 55). BV2 Usp15 KO cells infected with Mtb 130 exhibited increased LC3-II relative to LC3-I, as measured by immunoblotting (Figure 2A, 131 2B). We next examined whether this increase in LC3-II was accompanied by enhanced 132 recruitment of LC3 to Mtb-associated structures. Immunofluorescence microscopy 133 revealed greater colocalization of endogenous LC3 with mCherry Mtb in BV2 Usp15 KO 134 cells compared to wild-type controls (Figure 2C, 2D). 135 To determine whether this phenotype was dependent on canonical autophagy 136 initiation, we treated wild-type and BV2 Usp15 KO macrophages with PIK-III, an 137 inhibitor of the phosphoinositide 3-kinase complex component VPS34 essential for 138 autophagy initiation (Figure 2E) (56). As expected, inhibition of autophagy increased 139 Mtb replication in wild-type BV2 cells (Figure 2F). Importantly, PIK-III treatment also 140 reversed the reduced Mtb burden in BV2 Usp15 KO cells (Figure 2F), indicating that 141 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint autophagy is required for the enhanced bacterial control observed in the absence of 142 Usp15 in macrophages. 143 Usp15 deletion in mouse bone marrow-derived macrophages leads to decreased 144 bacterial replication and increased autophagy 145 To determine whether Usp15 deletion affects macrophage immunity in primary 146 cells, we examined bone marrow-derived macrophages (BMDMs) from wild-type and 147 Usp15 knockout mice (57, 58). Following infection with Mtb, BMDMs from Usp15 KO 148 mice exhibited significantly reduced bacterial burden compared to wild-type controls at 149 day 3 post-infection (Figure 3A). We next tested whether Usp15 deletion in BMDMs 150 enhanced autophagy activation. Immunofluorescence microscopy showed that 151 endogenous LC3 was more frequently colocalized with mCherry Mtb in Usp15 KO 152 BMDMs (Figure 3B, 3C), consistent with enhanced recruitment of autophagy machinery. 153 Together, these results confirm that the autophagy-dependent restriction of Mtb 154 observed in BV2 cells also occurs in primary mouse macrophages lacking Usp15. 155 The deubiquitinase activity of USP15 is necessary for its role in regulating Mtb 156 replication in BV2 macrophages 157 USP15 contains a conserved catalytic triad (Cys269, His862, Asp879) typical of 158 the USP family of deubiquitinases (59). To determine whether USP15 suppresses 159 macrophage immunity to Mtb through its enzymatic function, we generated BV2 Usp15 160 KO macrophages stably complemented with either wild-type (WT) USP15 or a 161 catalytically inactive mutant, C269A, which targets the catalytic cysteine within the USP 162 domain triad (59). Specifically, we complemented BV2 Usp15 KO cells with either an 163 empty vector, a vector containing a cDNA encoding full-length USP15 ( Usp15WT), or a 164 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint vector containing a cDNA encoding the catalytically inactive USP15 C269A allele, each 165 also containing an N-terminal 3X-FLAG tag (Figure 4A, 4B). Complementation with WT 166 USP15 fully restored intracellular Mtb replication to levels observed in parental BV2 167 cells, whereas expression of the USP15C269A mutant did not (Figure 4C). 168 We next examined whether USP15 enzymatic activity was required to suppress 169 K63-ubiquitination and LC3 recruitment to Mtb-associated structures. 170 Immunofluorescence microscopy showed that only WT USP15 reversed the increased 171 K63-ubiquitination and LC3 colocalization with mCherry Mtb observed in BV2 Usp15 KO 172 cells, while the USP15 C269A mutant failed to do so (Figure 4D-4G). These results 173 indicate that USP15 requires its catalytic activity to restrict autophagy and promote 174 intracellular Mtb growth in macrophages. 175 USP15 counters the activity of PARKIN on K63 ubiquitination and Mtb survival 176 Previous studies suggest that USP15 interacts with and counteracts PARKIN, an 177 E3 ubiquitin ligase that promotes K63-linked polyubiquitination of Mtb and enhances 178 autophagy-mediated clearance (Figure 5A) (25, 26, 51). We therefore tested whether 179 the effects of Usp15 deletion on Mtb ubiquitination and intracellular growth were 180 dependent on PARKIN. First, we performed shRNA-mediated knockdown of Park2 in 181 BV2 Usp15 KO cells and confirmed loss of PARKIN expression by immunoblotting 182 (Figure 5B). As expected, Park2 knockdown in wild type BV2 cells resulted in increased 183 bacterial burden over time compared to wild type BV2 cells expressing the non-targeting 184 control (Figure 5C). In addition, compared to non-targeting controls, Park2 knockdown 185 restored Mtb replication in BV2 Usp15 KO cells nearly to levels observed in wild-type 186 BV2 cells at 3 days after infection (Figure 5C). 187 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint Next, we examined the impact of Park2 knockdown on K63-linked ubiquitination 188 and LC3 recruitment to Mtb associated structures. Immunofluorescence microscopy 189 showed that in BV2 Usp15 KO cells expressing a Park2 shRNA, the frequency of K63-190 ubiquitin-positive and LC3-positive Mtb-associated structures was significantly reduced 191 compared to BV2 Usp15 KO cells encoding a non-targeting control (Figure 5D-5G). 192 These findings demonstrate that USP15 restricts macrophage immunity to Mtb, at least 193 in part, by opposing PARKIN-mediated K63 ubiquitination and downstream engagement 194 of autophagy machinery components. 195 USP15 depletion in human monocyte-derived macrophages leads to decreased 196 Mtb replication 197 To further validate the role of USP15 in cell-autonomous immunity to Mtb, we 198 tested the impact of genetic depletion of USP15 on Mtb replication in primary human 199 monocyte-derived macrophages (hMDM). We isolated peripheral blood mononuclear 200 cells (PBMCs) from buffy coat preparations obtained from healthy blood donors, 201 knocked down USP15 through lentiviral transduction of USP15-specific shRNA, and 202 differentiated the cells into macrophages over seven days. We achieved approximately 203 40–50% knockdown of USP15 as determined by immunoblot (Figure 6A), which is 204 consistent with our prior results targeting SMURF1 (26). 205 Despite partial knockdown, hMDMs from multiple donors transduced with 206 USP15-specific shRNA exhibited reduced Mtb CFU compared to those transduced with 207 a non-targeting control shRNA (Figure 6B, 6C). To determine whether this phenotype 208 correlated with autophagy induction, we assessed LC3 colocalization with Mtb-209 associated structures by immunofluorescence. USP15 knockdown resulted in increased 210 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint LC3 recruitment relative to control cells (Figure 6D, 6E; Supplemental Figure 2). We 211 also attempted to assess K63-linked ubiquitination of Mtb-associated structures in 212 hMDMs using multiple antibodies but were unable to detect K63-Ub reliably by 213 immunofluorescence, preventing us from evaluating whether USP15 knockdown altered 214 ubiquitination dynamics in this setting. 215 Together, these data show that USP15 suppresses autophagy-mediated control 216 of Mtb in primary human macrophages, further supporting its role as a negative 217 regulator of macrophage cell-autonomous immunity in both murine and human systems. 218 Pharmacologic inhibition of USP15 leads to decreased bacterial replication 219 Based on the findings that USP15 suppresses antibacterial autophagy in both 220 mouse and human macrophages, we next tested whether pharmacologic inhibition of 221 USP15 could recapitulate these effects. We used a recently described small molecule 222 inhibitor, USP15-IN-1 (60), to assess whether pharmacologic inhibition of USP15 223 restricts Mtb replication in macrophages. First, we confirmed that USP15-IN-1 had no 224 direct effect on axenic Mtb growth (Supplemental Figure 3A). Next, to test specificity of 225 USP15-IN-1, we treated wild-type and BV2 Usp15 KO cells with increasing 226 concentrations of USP15-IN-1 and infected them with Mtb-pLux. In wild-type BV2 cells, 227 USP15-IN-1 reduced Mtb replication by approximately two-fold at the highest 228 concentration tested (60 µM) (Figure 7A). This reduction was not observed in BV2 229 Usp15 KO cells, indicating the effect was USP15-dependent. Cell viability was 230 unaffected by USP15-IN-1 at all tested concentrations (Supplemental Figure 3B, 3C). 231 We next tested the efficacy of USP15-IN-1 in hMDMs derived from four healthy 232 donors. USP15-IN-1 treatment resulted in a dose-dependent decrease in Mtb replication 233 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint in three of four donors (Figure 7B-F). Notably, Mtb replication was reduced by nearly 234 50% with as little as 3.75 µM USP15-IN-1 in responsive donors (Figure 7C-E), without 235 evidence of cytotoxicity as determined by LDH release assay (Supplemental Figure 3D, 236 3E). 237 To assess whether these effects were associated with autophagy induction, we 238 evaluated LC3 colocalization with Mtb. USP15-IN-1 increased LC3 recruitment in 239 hMDMs from two of the three donors (Figure 7G, 7H; Supplemental Figure 3F-I). No 240 increase in LC3 recruitment was observed in hMDMs from donor 9, which also failed to 241 respond to USP15-IN-1 with reduced bacterial replication (Figure 7F; Supplemental 242 Figure 3F, 3G). 243 Together, these data demonstrate that pharmacologic inhibition of USP15 with 244 USP15-IN-1 enhances autophagy-mediated restriction of Mtb in human macrophages, 245 supporting its potential as a host-directed therapeutic strategy for tuberculosis. 246 247 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint

248 Host-directed therapies that harness or enhance innate immune pathways offer a 249 promising avenue to improve outcomes in tuberculosis (TB), particularly in the face of 250 rising drug resistance (61-63). In this study, we identify the deubiquitinase USP15 as a 251 conserved and targetable suppressor of autophagy-mediated immunity to Mtb in both 252 murine and human macrophages. Our findings reveal that USP15 removes PARKIN-253 dependent K63-linked ubiquitin from Mtb-associated structures, reducing LC3 254 recruitment and autophagic clearance. Genetic deletion or pharmacologic inhibition of 255 USP15 increases K63 ubiquitination, enhances LC3 colocalization, and restricts Mtb 256 replication in multiple macrophage systems. While USP15 has been linked to mitophagy 257 and mitochondrial quality control through interactions with PARKIN (38, 51), our study 258 newly defines its role in pathogen-directed autophagy, extending current understanding 259 of USP15’s role in host-pathogen interactions. 260 Beyond its role in xenophagy, USP15 may regulate additional immune signaling 261 pathways relevant to Mtb infection (64, 65). For instance, USP15, with the aid of COP9 262 signalosome (CSN), is a negative regulator of NF- κ B via deubiquitination of I κ Bα (66, 263 67). Furthermore, loss of USP15 has been suggested to impact lipid droplet 264 accumulation while decreasing HCV virus propagation (52). Lipid droplets are 265 considered an additional nutrient source that can influence the balance of Mtb between 266 dormancy and active growth (68, 69). In addition, USP15 stabilizes TRIM25 to enhance 267 production of IFN-β (70, 71), which itself is considered a key regulator of Mtb replication 268 (72). Thus, USP15 may act as a direct negative regulator of xenophagy and influence 269 additional signaling pathways during Mtb infection. 270 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint By comparing our screen to that of Chandra et al. (41), we validate USP15 as a 271 key negative regulator of macrophage immunity and highlight the broader relevance of 272 deubiquitinases in host-pathogen interactions. Notably, Chandra et al. showed that 273 knockdown of Usp15 in immortalized BMDMs results in increased ubiquitination of Mtb 274 and reduced bacterial burden (41). Additionally, like Chandra et al., we observed that 275 depletion of Cyld, a DUB that counters the activity of the linear ubiquitin assembly 276 complex (LUBAC) (73), resulted in decreased Mtb replication compared to a 277 nontargeting control or scramble. However, in contrast to the Chandra et al. study that 278 demonstrated a reduced CFU upon Usp8 depletion, we observed a modest but 279 reproducible increase in bacterial replication when Usp8 was knocked down in BV2 280 cells. The functional divergence regarding Usp8 between our studies underscores the 281 importance of cellular context (BV2 cells versus BMDM), screen design (luminescent 282 growth assay versus ubiquitination/CFU), and bacterial strain (Mtb Erdman versus 283 H37Rv) when interpreting DUB function. Nevertheless, both studies converge on the 284 therapeutic potential of modulating the host ubiquitin system to combat intracellular 285 pathogens. 286 Importantly, USP15-IN-1, a small molecule inhibitor, mimics the effects of USP15 287 deletion in both murine and human macrophages, with enhanced bacterial clearance 288 observed at non-cytotoxic concentrations. The specificity of this effect, absent in 289 USP15-deficient cells, highlights its therapeutic potential. Remarkably, hMDMs were 290 sensitive to much lower concentrations of USP15-IN-1 than murine BV2 cells, despite 291 95% sequence identity between human and mouse USP15. This observation suggests 292 that modest USP15 inhibition may be sufficient to enhance antimicrobial activity in 293 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint human macrophages, and that USP15-IN-1 or related compounds may warrant further 294 investigation as host-directed therapeutic agents in combination with antibiotics. 295 While we were unable to test USP15 function in vivo due to the perinatal lethality 296 of global Usp15 knockout mice, our results in primary cells strongly suggest physiologic 297 relevance. Future studies using conditional or inducible knockout models of USP15 (74) 298 or administration of USP15 inhibitors with established pharmacokinetics and 299 pharmacodynamics will be essential to determine whether these findings extend to host 300 defense in the context of whole-animal infection. 301 In conclusion, our data define USP15 as a key negative regulator of bacterial 302 xenophagy in both mouse and human macrophages and establish proof-of-concept that 303 pharmacologic inhibition of USP15 enhances control of Mtb in human macrophages. 304 These findings have implications for understanding the roles of deubiquitinases in 305 innate immunity against intracellular pathogens and support further development of 306 USP15-targeted strategies as a component of host-directed therapy for TB and 307 potentially other infections. 308

309 Bacterial Strains 310 We used the Mtb Erdman strain for all Mtb experiments. The Mtb mCherry-expressing 311 strain was previously described (22). We received the pLux plasmid 312 (pMV306hsp+LuxG13, BEI plasmid #26161) as a gift from Brian Robertson and 313 Siouxsie Wiles and electroporated it into Mtb Erdman as described (75). We cultured all 314 strains in 7H9 medium supplemented with 0.5% glycerol, 0.05% Tween 80, and 10% 315 Middlebrook OADC (BD Biosciences), as previously described (22). For the USP15-IN-316 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint 1 growth curve, we added 0 µM, 3.75 µM, 15 µM, or 60 µM of inhibitor to 7H9. We 317 diluted Mtb to an initial OD600 of 0.15 and then exposed to USP15-IN-1 for four days. 318 Cells and Cell Culture 319 Mouse cells: We cultured the BV2 murine microglial cell line in DMEM (Gibco, 11965-320 092) supplemented with 10% fetal bovine serum (FBS; Gibco, 16000-044) and 1% 321 HEPES (cytiva, SH30237.01). For the initial screen, because the efficacy of each 322 shRNA was unpredictable, we generated three unique stable BV2 cell lines per gene, 323 each expressing a distinct shRNA sequence (Supplemental Table 1). The same BV2 324 cell line transduced with a non-targeting shRNA served as the control. Cells were 325 infected with Mtb-pLux at an MOI of 3 to ensure strong luminescence signal. BV2 326 Usp15 KO cells were obtained from GEiC Washington University using the gRNA 327 sequence 'TCTTATAAGCAGTATATGACNGG'. BMDMs were extracted from mouse 328 femurs and tibias and differentiated in DMEM supplemented with 30% L929 cell-329 conditioned media, 20% FBS, and 1% HEPES (22). After seven days, differentiated 330 BMDMs were harvested for experiments. 331 Human cells: Buffy coats from anonymous donors were obtained from a local blood 332 bank. PBMCs were isolated using SepMate-50 tubes (Stemcell Technology, 85450) 333 following the manufacturer’s protocol. CD14-positive cells were selected using CD14 334 Microbeads (Miltenyi Biotec, 130-050-201). Adherent monocytes were differentiated in 335 RPMI supplemented with 1% HEPES, 1% sodium pyruvate, 10% heat-inactivated 336 human serum, 10% FBS, and 50 ng/mL GM-CSF (Peprotech, 300-03) for four days. On 337 day five, the media was changed to RPMI with 10% FBS. 338 Mice 339 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint Usp15-/- mice on a C57BL/6J background were obtained from Dr. Yue Xiong with 340 permission from Taconic (TF2834). All experiments used Usp15 -/- mice and littermate 341 controls from heterozygous crosses. Mice were housed under specific pathogen-free 342 conditions. All studies were approved by the IACUC of UT Southwestern, an AAALAC-343 accredited institution. 344 Lentiviral Transduction for Knockdown in BV2 cells 345 shRNAs targeting mouse DUBs were obtained from Sigma MISSION (Supplemental 346 Table 1); a non-targeting sequence served as the control. Lentiviruses were generated 347 by transfecting HEK293T cells with lentiviral vectors and packaging plasmids (psPAX2 348 and pMD2.G). After three days, viral supernatant was collected and filtered through 0.45 349 µm filters. BV2 cells were exposed to lentiviral supernatant diluted in DMEM and 350 incubated at 37°C with 5% CO ₂ . After infection, the media was replaced with fresh 351 DMEM containing 4 µg/mL puromycin (Fisher Scientific, BP2956-100). Selection was 352 maintained for at least seven days, with media changes every other day. 353 Lentiviral Transduction for Knockdown in hMDM 354 CD14+ cells were differentiated as described above. On day three of differentiation, 355 lentivirus containing USP15-targeting shRNA or non-targeting control was added. Cells 356 were centrifuged for 1 hour at 800 × g. The next day, the media was changed to RPMI 357 with 2 µg/mL puromycin, 1% HEPES, 1% sodium pyruvate, and 10% FBS. Selection 358 was continued for three days. On the day of infection, media was replaced with RPMI 359 supplemented with 1% HEPES, 1% sodium pyruvate, and 10% FBS. 360 Macrophage Infections 361 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint BV2, BMDMs, and hMDMs were infected with Mtb as previously described (22). Briefly, 362 Mtb cultures were grown to OD600 0.4–0.6, washed three times, and sonicated for three 363 rounds of 7 seconds on and 7 seconds off (total 21 seconds). OD 600 was remeasured 364 and bacteria diluted to an MOI of 1 for all CFU and immunofluorescence experiments. 365 For Mtb-pLux experiments, an MOI of 3 was used to ensure sufficient luminescence. 366 BV2 cells were seeded at 10 /i4 cells per well in white 96-well plates (ThermoFisher 367 Scientific, 165306). Mtb was centrifuged onto cells for 10 minutes at 1500 rpm and 368 incubated for 30 minutes at 37°C with 5% CO ₂ . Plates were read at days 0, 1, 2, 3, and 369 4. For CFU, BMDMs and hMDMs were seeded at 10 /i4 cells/well in 48-well plates 370 (Corning, 3548). For immunofluorescence, 2 × 10 /i4 cells were seeded on sterile 371 coverslips in 24-well plates. PIK-III (MCE, HY-12794) was added to inhibit autophagy 372 immediately post-infection. USP15-IN-1 (MCE, HY-148046) was added at 0 µM (DMSO 373 control), 3.75 µM, 15 µM, or 60 µM. Cytotoxicity was measured using the CyQUANT 374 LDH assay (Fisher Scientific, C20300). 375 Immunofluorescence 376 Sixteen hours post-infection with mCherry Mt b, cells were was hed twice with PBS and 377 fixed with 4% paraformaldehyde in PBS for 4 hours (26). After fixation, we washed and 378 permeabilized the cells. For LC3, cells were permeabilized with 100% methanol for 5 379 minutes and blocked with 3% BSA for 30 minutes at room temperature. For K63 and 380 K48 staining, cells were permeabilized with 0.5% saponin for 30 minutes and blocked in 381 3% BSA with 0.5% saponin at room temperature. Primary antibodies were diluted and 382 incubated at room temperature for 1 hour: anti-LC3 (Sigma, L7543-200UL) at 1:250; 383 anti-K63 (EMD Millipore, 05-1306) and anti-K48 (EMD Millipore, 05-1307) at 1:1000. 384 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint Secondary antibody (anti-mouse AlexaFluor 488, Invitrogen, A11008) was diluted 385 1:2000 and incubated for 1 hour at room temperature. Coverslips were mounted with 5 386 µL ProLong Diamond with DAPI and dried overnight. Images were acquired using a 387 Nikon W1 spinning disk confocal microscope and analyzed using Imaris 10 (Bitplane); 388 image panels were assembled using ImageJ. 389 Immunoblots 390 BMDMs, hMDMs, and BV2 cells were seeded at 10 /i4 cells/well in 6-well plates and 391 either infected at MOI 5 or left uninfected. After 24 hours, cells were washed three times 392 with PBS and lysed in RIPA buffer with protease inhibitors (Roche, 11836170001) for 5 393 minutes. Lysates were homogenized by pipetting and scraping, then filtered twice 394 through 0.22 µm filters. Protein concentration was determined using a BCA assay 395 (ThermoFisher, 23225). Lysates (20 µg) were separated on 8–20% SDS-PAGE gels 396 (Bio-Rad, 4561096) and transferred to PVDF membranes (Bio-Rad, 1620174) using 397 semi-dry transfer (Bio-Rad, 1704150). Membranes were blocked in 3% BSA in TBST for 398 1 hour at room temperature. Primary antibodies were used at the following dilutions in 399 3% BSA: anti-USP15 (Novus Biologicals, H00009958-M01) 1:500, anti-PARKIN (Cell 400 Signaling, 4211) 1:2000, anti-B-ACTIN (Santa Cruz, sc-47778) 1:10000, and anti-LC3B 401 (Novus Biologicals, NB100-2220) 1:250. After three washes, membranes were 402 incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, 115-403 035-003 or 111-035-003), washed again, and developed using Clarity ECL Substrate 404 (Bio-Rad, 1705060). Blots were imaged using a Bio-Rad ChemiDoc MP system. 405 Statistical Analysis 406 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint Statistical analyses were performed using GraphPad Prism 9 (version 9.5.0). For CFU 407 and Mtb-pLux experiments involving multiple comparisons, ordinary two-way ANOVA 408 followed by Tukey’s multiple comparisons test was used. For comparisons between two 409 groups, Student’s t-test was used. For co-localization experiments involving more than 410 two groups, ordinary one-way ANOVA was used. All experiments were independently 411 repeated at least three times. 412 Authorship contribution statement 413 KCR: Conceptualization, Formal analysis, Investigation, Writing – original draft, Writing 414 – review and editing, PCC: Investigation, Formal Analysis, Writing – review and editing, 415 BRD: Investigation, Formal Analysis, Writing – review and editing, PKP: Investigation, 416 Formal Analysis, Writing – review and editing, MUS: Conceptualization, Formal 417 analysis, Funding acquisition, Project administration, Supervision, Writing – original 418 draft, Writing – review and editing. 419 Declaration of competing interests 420 All authors declare that they have no competing interests. 421 Acknowledgments 422 The authors would like to thank all members of the Shiloh lab for their support and 423 constructive feedback on the manuscript. We also thank members of the Animal 424 Resources Center at UT Southwestern for providing support with all animal welfare and 425 husbandry. We thank Dr. Yue Xiong, formerly of the University of North Carolina, for the 426 Usp15-/- mice. We would like to thank Dr. Kate Luby-Phelps and Dr. Marcel Mettlen of 427 the Quantitative Light Microscopy Core Facility at UT Southwestern for their assistance 428 with fluorescence microscopy. The spinning disk confocal microscope was funded 429 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint through the NIH 1S10OD028630 grant to Dr. Kate Luby-Phelps. This work was funded 430 by NIH T32HL098040 (K.C.R.) and 5U19AI142784 (M.U.S.) grants. Michael Shiloh 431 would also like to acknowledge support from the Disease Oriented Clinical Scholar 432 program at UT Southwestern. 433 Figure Legends 434 Figure 1. Loss of Usp15 inhibits Mtb replication and increases K63 ubiquitination 435 co-localization. A) Schematic for shRNA screen in the BV2 cell line infected with 436 luminescent Mtb. B) Heat map shows the average of relative luminescence units when 437 normalized to day 0 and then to the nontargeting control (NTC). C) Colony forming unit 438 (CFU) values of WT Erdman Mtb infected into non-targeting control shRNA (NTC) and 439 Usp15 KD shRNA BV2 cells were normalized for each day to the count on Day 0. D) 440 CFUs of WT or CRISPR Usp15 KO BV2 infected with WT Mtb. E) Images show Mtb 441 (gray or red) and K63 (gray or green) with DAPI (blue) in WT or Usp15 KO BV2 cells. 442 Scale bar is 5 µM. F) Quantification of K63 immunofluorescence co-localizing with 443 mCherry Mtb infected WT or Usp15 KO BV2 cells at 16h post-infection. For statistical 444 analysis of CFU, we used two-way ANOVA and Tukey’s multiple comparison test. For 445 statistical analysis of co-localization, we used Student’s t-test. * p<0.05, ** p<0.01, *** 446 p<0.001, **** p<0.0001. Data shown are representative of at least 3 independent 447 experimental replicates. 448 Figure 2. Usp15 deletion in BV2 cells increases LC3-II conversion and 449 colocalization of LC3 with Mtb-associated structures. A) Western blot of LC3-I and 450 LC3-II and ACTIN in WT and Usp15 KO BV2 cells infected with Mtb. B) Quantification of 451 LC3-II normalized to ACTIN. C) Representative immunofluorescence images of 452 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint mCherry Mtb (grey or red), LC3 (grey or green) in WT (upper panel) or Usp15 KO 453 (lower panel) BV2 cells. Scale bar is 5 µM. D) Quantification of LC3 co-localization with 454 mCherry Mtb in BV2 cells. E) Schematic of PIK-III inhibition of upstream autophagy 455 initiation via the PI3KC3 complex. F) CFU of WT Mtb in WT or Usp15 KO cells with or 456 without 5 µM of PIK-III normalized for each day to the count on Day 0. For statistical 457 analysis, we used two-way ANOVA and Tukey’s multiple comparison test.* p<0.05, ** 458 p<0.01, *** p<0.001, **** p<0.000.1. Data shown are representative of at least 3 459 independent experimental replicates. 460 Figure 3. Loss of Usp15 in BMDM results in reduced CFU and increased co-461 localization of LC3 with Mtb-associated structures. A) CFU of WT Mtb in WT or 462 Usp15-/- BMDMs normalized to the count at Day 0. B) Representative images of 463 mCherry (grey or red) and LC3 immunofluorescence (grey or green) in WT or Usp15-/- 464 BMDMs. Scale bar is 5 µM. C) Quantification of LC3 co-localization with mCherry Mtb in 465 BMDMs 16 at 16h post-infection. For statistical analysis of CFU, we used two-way 466 ANOVA and Tukey’s multiple comparison test. For statistical analysis of co-localization, 467 we used Student's t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Data shown 468 are representative of at least 3 independent experimental replicates. 469 Figure 4. The catalytic activity of USP15 is necessary for regulating Mtb 470 replication in BV2 cells. A) Schematic of Usp15 with the location of the catalytic dead 471 mutation noted in red. Domain abbreviations are as follows: DUSP (domain present in 472 USP) and UBL (ubiquitin-like domain). The catalytic domains (CD) are noted in blue. B) 473 Complementation of BV2 Usp15 KO cells with 3XFlag-Usp15WT (Usp15 KO::WT) or 474 3XFlag-Usp15C269A ( Usp15 KO::C269A). Western blot demonstrating 3XFlag-475 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint Usp15WT or 3XFlag - Usp15C269A in BV2 cell lines. C) CFU of Mtb infection in BV2 WT 476 with empty vector (WT Empty), BV2 Usp15 KO with empty vector ( Usp15 KO Empty), 477 BV2 Usp15 KO complemented with 3XFlag- Usp15WT (Usp15 KO::WT) or BV2 Usp15 478 KO complemented with 3XFlag- Usp15C269A ( Usp15 KO:: C269A). CFU values were 479 normalized to Day 0. D) Representative image of mCherry (grey or red) and K63 480 immunofluorescence (grey or green) with DAPI (blue) in WT Empty, BV2 Usp15 KO 481 Empty, BV2 Usp15 KO::WT, or BV2 Usp15 KO::C269A. Scale bar is 5 µM. E) 482 Quantification of K63 co-localization with mCherry Mtb in WT Empty, BV2 Usp15 KO 483 Empty, BV2 Usp15 KO::WT, or BV2 Usp15 KO::C269A. F) Representative image of 484 mCherry (grey or red) and LC3 immunofluorescence (grey or green) with DAPI (blue) in 485 WT Empty, BV2 Usp15 KO Empty, BV2 Usp15 KO::WT, or BV2 Usp15 KO::C269A. 486 Scale bar is 5 µM. G) Quantification of LC3 co-localization with mCherry Mtb in WT 487 Empty, BV2 Usp15 KO Empty, BV2 Usp15 KO::WT, or BV2 Usp15 KO::C269A. For 488 statistical analysis of CFU, we used two-way ANOVA and Tukey’s multiple comparison 489 test. For statistical analysis of colocalization, we used one-way ANOVA and Tukey’s 490 multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Data shown 491 are representative of at least 3 independent experimental replicates. 492 Figure 5. USP15 counters the activity of PARKIN. A) Schematic of PARKIN’s activity 493 in the context of ubiquitination of Mtb. B) Western blot confirms knockdown (KD) of 494 PARKIN in WT or Usp15 KO compared to non-targeting controls (NTC). The 495 percentage of KD is shown below each KD. C) CFU of WT Mtb infected in WT with 496 NTC, Usp15 KO with NTC, WT with Parkin KD, and Usp15 KO with Parkin KD. CFU 497 values were normalized to the count at Day 0. D) Representative image of mCherry 498 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint (grey or red) and K63 immunofluorescence (grey or green) with DAPI (blue) in WT with 499 NTC, Usp15 KO with NTC, WT with Parkin KD, and Usp15 KO with Parkin KD. Scale 500 bar is 5 µM E) Quantification of K63 co-localization with mCherry Mtb in WT with NTC, 501 Usp15 KO with NTC, WT with Parkin KD, and Usp15 KO with Parkin KD. F) 502 Representative image of mCherry (grey or red) and LC3 immunofluorescence (grey or 503 green) with DAPI (blue) in WT with NTC, Usp15 KO with NTC, WT with Parkin KD, and 504 Usp15 KO with Parkin KD. Scale bar is 5 µM. G) Quantification of LC3 co-localization 505 with mCherry Mtb in WT with NTC, Usp15 KO with NTC, WT with Parkin KD, and 506 Usp15 KO with Parkin KD. For statistical analysis of CFU, we used two-way ANOVA 507 and Tukey’s multiple comparison test. For statistical analysis of colocalization, we used 508 one-way ANOVA and Tukey’s multiple comparison test. * p<0.05, ** p<0.01, *** 509 p<0.001, **** p<0.0001. Data shown are representative of at least 3 independent 510 experimental replicates. 511 Figure 6. USP15 depletion in hMDM leads to decreased Mtb burden and increased 512 LC3 co-localization. A) Representative western blot of knockdown of USP15 in human 513 monocyte-derived macrophages (hMDMs) from Donor 1 and Donor 2. B) 514 Representative time course from day 0 to day 3 of Mtb CFU in hMDMs from Donor 2. C) 515 Combined normalized CFU to day 0 from day 3 of Donor 1 (D1), Donor 2 (D2), Donor 3 516 (D3), and Donor 4 (D4). D) Representative image of mCherry Mtb (grey or red) and LC3 517 immunofluorescence (grey or green) in hMDMs from Donor 5 (D5) with NTC or USP15 518 KD. Scale bar is 5 µM. E) Quantification of LC3 co-localization in hMDMs from Donor 5 519 with NTC or USP15 KD. For statistical analysis of CFU, we used two-way ANOVA and 520 Tukey’s multiple comparison test. For statistical analysis of colocalization, we used 521 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint Student’s t-test.* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Data shown are 522 representative of at least 3 independent experimental replicates. 523 Figure 7. Inhibition of USP15 by USP15-IN-1 leads to decreased Mtb burden in 524 BV2 cells and hMDM. A) A 4-point dose response of USP15-IN-1 in WT or Usp15 KO 525 BV2 cells infected with Mtb-pLux. B) Combined Relative Luminescence Units (RLU) of 526 Day 3 from Donor 6, Donor 7, Donor 8, and Donor 9. C-F) The 4-point dose response of 527 USP15- IN-1 in C) Donor 6, D) Donor 7, E) Donor 8, F) Donor 9. G) Representative 528 immunofluorescence image of mCherry Mtb (grey or red) and LC3 (grey or green) in 529 hMDMs from Donor 7 with 0 µM (DMSO control) or 60 µM of 15 IN 1 at 18 hours post-530 infection. Scale bar is 5 µM. H) Quantification of LC3 co-localization in hMDMs from 531 donor 7. For statistical analysis for Mtb-pLux experiments, we used two-way ANOVA 532 and Tukey’s multiple comparison test. For colocalization analysis, we used Student’s t-533 test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Data shown are representative of 534 at least 3 independent experimental replicates. 535 Supplemental Figure 1. Validation of USP15 loss and its effect on K48 ubiquitination. 536 A) Western blot of USP15 in BV2 NTC cells and in two of the three BV2 Usp15 537 knockdown (KD) cells. B) Western blot of USP15 in WT or Usp15 K O B V 2 c e l l s . C ) 538 Representative immunofluorescence images of K48-Ub with mCherry Mtb in WT BV2 539 cells. Scale bar is 5 µM. D) Quantification of K48 co-localization with mCherry-540 expressing Mtb in WT or Usp15 KO BV2 cells. Data shown are representative of at least 541 3 independent experimental replicates. 542 Supplemental Figure 2. USP15 depletion in hMDMs leads to increased LC3 co-543 localization with Mtb-associated structures in two donors. A,C) Representative 544 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint immunofluorescence images of mCherry Mtb (grey or red) and LC3 (grey or green) in 545 hMDMs from A) Donor 10 or C) Donor 11 with NTC or USP15 KD. Scale bar is 5 µM. 546 B,D) Quantification of LC3 co-localization in hMDMs from B) Donor 10 or D) Donor 11 547 with NTC or USP15 KD. For statistical analysis, we used Student’s t-test.** p<0.01. 548 Supplemental Figure 3. Impact of USP15-IN-1 on BV2 and hMDM viability and LC3 549 colocalization with Mtb-associated structures. A) Growth curve of Mtb Erdman in 7H9 550 with different concentrations of USP15-IN-1. B) 10X image of BV2 WT or BV2 Usp15 551 KO exposed to DMSO or 60 µM USP15-IN-1 at day 3 after Mtb infection. C,D) LDH 552 assay showing the 4-point dose response of C) BV2 WT and BV2 Usp15 KO cells or D) 553 hMDMs treated with USP15-IN-1 at day 3 after Mtb infection. E) 10X images from each 554 donor in the RLU experiments with 0 µM (DMSO) or 60 µM USP15-IN-1. F, H) 555 Representative immunofluorescence images of mCherry Mtb (grey or red) and LC3 556 (grey or green) in hMDMs from F) Donor 8 or H) Donor 12 treated with 0 µM (DMSO 557 control) or 60 µM USP15-IN-1 at 18 hours post-infection. Scale bar is 5 µM. G, I) 558 Quantification of LC3 co-localization in hMDMs from G) donor 8 or I) donor 12. For 559 statistical analysis, we used Student’s t-test. *** p<0.001. For non-human donor 560 experiments, data shown are representative of 3 independent experimental replicates. 561 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint

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It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 7, 2025. ; https://doi.org/10.1101/2025.08.06.668987doi: bioRxiv preprint