Mycobacterium tuberculosis modulates phosphorylation of host ATP6V1E1 to promote intracellular survival

Domain: neuroscience
Published: 2026-02-06
Editorial outcome: minor_revision

Abstract
Intracellular pathogens such as Mycobacterium tuberculosis (Mtb) can promote their survival within infected cells by preventing lysosomal acidification. Here, we report that Mtb secretes a protein (Rv1184, or acyltransferase Chp2) that inhibits lysosomal acidification by targeting the host vacuolar ATPase (V-ATPase). We show that phosphorylation of the V-ATPase E1 subunit (ATP6V1E1) at Tyr56/57 suppresses lysosomal acidification through inhibition of V-ATPase assembly. Further investigation reveals that tyrosine kinase BMX promotes phosphorylation of ATP6V1E1. Strikingly, Chp2 increases BMX-dependent phosphorylation of ATP6V1E1, apparently by directly binding ATP6V1E1 and facilitating its interaction with BMX. Furthermore, inhibition of BMX impairs Mtb growth within macrophages and in mice. Thus, our work reveals a mechanism for the regulation of lysosomal acidification and suggests lysosomal acidification modulation as a potential approach for host-directed therapy against Mtb.

Introduction
Mycobacterium tuberculosis (Mtb) has become a major killer among infectious diseases1,2. In 2024 global tuberculosis report, approximately 10.8 million people develop tuberculosis (TB) and 1.25 million deaths are caused by TB3. Mtb droplets are routinely phagocytosed by host alveolar macrophages to form phagosomes, and phagosomes undergo a series of maturation steps including fuse with lysosomes and thus develop into an increasingly acidified phagolysosomes, resulting in the effective degradation of Mtb, which is an essential process for the elimination of Mtb4.

As an extremely successful intracellular pathogen, Mtb can survive and persist within macrophages by suppressing phagosome maturation to avoid lysosomes5–7 hydrolases8. Mtb has evolved several strategies to suppress each stage of phagosome maturation including modulation of phagosome fusion and inhibition of phagosome acidification. Accumulating evidence indicate that mycobacterial cell wall components, such as lipoarabinomannan (LAM)9, phosphatidylinositol mannoside (PIM)10, and secretion proteins such as EsxG/EsxH11, PknG12, SapM12 and EsxA13, inhibit phagosome maturation through interfering with fusion processes, including phagosome-endosome fusion or phagosome-lysosome fusion. However, the mechanisms by which Mtb prevents lysosomal acidification to suppress phagosome maturation remain largely unclear.

Lysosomal acidification is fundamentally important for the degradation of macromolecules, and its dysfunction is associated with cancer, aging and neurodegenerative diseases14–17. Lysosomal acidification is regulated by the coordinated function of multiple ion channels and, most remarkably, vacuolar ATPase (V-ATPase), a macromolecular complex responsible for pumping protons (H+) into lysosomes using metabolic energy in the form of ATP. The V-ATPase reduces intraluminal pH to the acidic range that is required for the activation of dozens of hydrolases with acidic pH optima in lysosomes14,15. Currently, only two studies report that Mtb prevents lysosomal acidification through modulating V-ATPase18,19. Mtb infection induces CISH expression, which triggers ubiquitination and degradation of the A subunit of V-ATPase, thereby shutting down the proton pump18. Additionally, the Mtb secreted protein PtpA disrupts the incorporation of V-ATPase into the phagosomal membrane, preventing phagosome acidification19. However, whether Mtb employes additional immune evasion mechanisms to achieve survival through V-ATPase remains unclear.

In the present study, by screening of Mtb secretory proteins that inhibit the lysosomal acidification, we observe that Mtb acyltransferase (Chp2, Rv1184)20 inhibits lysosomal acidification through V-ATPase. Moreover, we uncover that E1 subunit of V-ATPase (ATP6V1E1) is an essential regulator for lysosomal acidification and phosphorylation of ATP6V1E1 mediated by BMX inhibits V-ATPase assembly to suppress lysosomal acidification, regulating the anti-mycobacterial immune response. However, Mtb Chp2 enhances the binding of ATP6V1E1 to BMX, increasing the BMX-mediated phosphorylation of ATP6V1E1 to promote Mtb survival.

Results
Chp2 inhibits lysosomal acidification through V-ATPase
Lysosomal acidification is essential for the clearance of Mtb21. To explore which Mtb protein inhibits host lysosomal acidification, 201 plasmids encoding Mtb-secreted proteins or lipoproteins were transfected into HEK293T cells, and their effects on lysosomal acidification were measured using LysoTracker staining, an acidotrophic fluorescent dye that accumulated in acidic organelles22. Chp2, an acyltransferase of Mtb, was the leading first protein of inhibiting lysosomal acidification (Fig. 1a-d). Chp2 was detected in both lysates and supernatants from cultures of H37Rv by using anti-Chp2 antibody, but not in the cultures of the H37RvΔChp2 (Supplementary Fig. 1a, b), conforming that Chp2 is a secreted protein. In Mtb-infected macrophages, H37RvΔChp2 markedly promoted the lysosomal acidification, and complementation of H37RvΔChp2 with Chp2 restored the its inhibition effect on lysosomal acidification (Fig. 1e, f, Supplementary Fig. 1c, d), validating that Chp2 inhibited lysosomal acidification. Furthermore, we assessed the co-localization of H37Rv, H37RvΔChp2, and the complemented strain with lysosomes using LAMP1 and evaluated lysosomal acidification using LysoTracker. All strains showed lysosomal co-localization without significant differences (Supplementary Fig. 1e, f). In parallel, H37RvΔChp2 notably enhanced lysosomal acidification compared to H37Rv, whereas complementation restored the acidification blockade (Supplementary Fig. 1g, h), demonstrating that Mtb inhibits lysosomal acidification via Chp2. V-ATPase is essential for lysosomal acidification, as it is the sole transporter that pumps protons into the organelle23. The use of bafilomycin A1 (Baf-A1)20, a specific inhibitor of V-ATPase, to inhibit lysosomal acidification and perform functional rescue experiments is a well-established and widely adopted strategy to validate whether a molecular mechanism acts through V-ATPase-mediated acidification. Given this, macrophages were pre-treated with Baf-A1 prior to infection with H37Rv and H37RvΔChp2 and then stained with LysoTracker to assess lysosomal acidification. As expected, Baf-A1 treatment significantly reduced lysosomal acidification in H37Rv-infected macrophages, confirming that the Mtb-induced acidification is mediated by V-ATPase. Furthermore, V-ATPase inhibition abolished the difference in acidification levels observed between H37Rv and H37RvΔChp2 (Fig. 1g, h, Supplementary Fig. 1i), demonstrating that Chp2 inhibited lysosomal acidification via V-ATPase.

Chp2 promotes Mtb survival through V-ATPase
Initially, we found no significant difference between the in vitro growth rate of wild-type H37Rv and H37RvΔChp2 (Supplementary Fig. 2a). We then investigated the functional role of Chp2 in the clearance of Mtb. Using a colony-forming unit (CFU) assay, we found similar intracellular bacterial loads for H37Rv and H37RvΔChp2 at 2 hours post-infection in peritoneal macrophages, suggesting Chp2 has no effects on the phagocytosis of Mtb by macrophages. However, at 24 to 96 hours post-infection, deletion of Chp2 dramatically reduced the intracellular bacteria CFU count compared to H37Rv controls (Fig. 2a, b). In addition, deletion of Chp2 also reduced Mtb survival in BMDMs (Supplementary Fig. 2b). As a weak base precursor, NH4Cl inhibits lysosomal acidification by diffusing into acidic compartments and neutralizing them24. Therefore, it can serve as a positive control for inducing a lysosomal impairment phenotype. The treatment of macrophages with Baf-A1 (Fig. 2c, d) or NH4Cl (Fig. 2e, f), eliminated the difference in intracellular survival between H37Rv and H37RvΔChp2. In vivo, the mice were infected with H37Rv, H37RvΔChp2 and H37Rv(ΔChp2+Chp2) for 4 weeks (Supplementary Fig. 2c). Chp2 deletion dramatically reduced histopathological changes in lung tissues (Fig. 2g, h). The multiplex immunohistochemistry demonstrated a significant reduction in both innate and adaptive immune cells, including monocytes/macrophages, neutrophils, T cells and B cells (Supplementary Fig. 2d, e). Consistently, Chp2 deletion comparably had a much lower bacterial loads in the lungs of mice (Fig. 2i, j). Complementation of H37RvΔChp2 with Chp2 restored the lung tissue pathological damage and bacterial burden (Fig. 2g-j), suggesting Chp2 is a virulence factor and exacerbated Mtb infection. The reduction in overall immune cell infiltration is a in vivo consequence of the enhanced bacterial clearance mediated by the Chp2 deletion. This superior early control leads to a lower overall bacterial burden and a subsequently attenuated inflammatory response in the lungs. Furthermore, mice were intraperitoneally injected with Baf-A1 prior to infection with H37Rv or H37RvΔChp2 for 3 days, followed by the weekly Baf-A1 injections for 4 weeks (Supplementary Fig. 2f). In H37Rv-infected mice, Baf-A1 treatment significantly increased the bacterial load in the lungs compared to the untreated group, demonstrating that the clearance of Mtb in vivo is dependent on V-ATPase. Furthermore, V-ATPase inhibition by Baf-A1 abolished the difference in lung bacterial burden between H37Rv and H37RvΔChp2 infection (Fig. 2k), indicating that Chp2 enhances bacterial survival in mice by targeting the V-ATPase. Together, Chp2 promoted the Mtb survival both within macrophages and in vivo through V-ATPase mediated lysosomal acidification.

Chp2 interacts with subunit E1 of human V-ATPase
The V-ATPases are large, multi-subunit protein complexes essential for cellular acidifications25. To explore how Chp2 regulates lysosomal acidification, we investigated its interaction with V-ATPase subunits by immunoprecipitation. We found E1 subunit of V-ATPase (ATP6V1E1) in the Chp2 precipitates (Fig. 3a–c, Supplementary Fig. 3a). Immunofluorescence assay confirmed the colocalization of Chp2 with ATP6V1E1 (Supplementary Fig. 3b), and the in vitro precipitation assay further demonstrated that recombinant Chp2 directly binds to ATP6V1E1 (Fig. 3d, e). Moreover, the surface plasmon resonance (SPR) assay showed that Chp2 had a strong affinity for ATP6V1E1 with an affinity constant of 21.3 nM (Fig. 3f). ATP6V1E1 is a subunit that forms a peripheral stalk with a stator function for stabilizing the V1 domains of V-ATPase complexes. Among 11 subunit isoforms of V1 domain, ATP6V1E1 had the highest intensity of lysotracker staining, suggesting its potential role in regulating acidification (Supplementary Fig. 3c, Fig. 3g, h). To better characterize the function role of ATP6V1E1 in Mtb infection in vivo, we therefore generated Atp6v1e1-deficient mice by CRISPR/Cas9-mediated genome editing (Supplementary Fig. 3d-f). The homozygous deletion of Atp6v1e1 results in embryonic lethality, which is consistent with the Mouse Genome Informatics (MGI) database. The Atp6v1e1 heterozygous mice appear normal and do not exhibit lysosomal storage disorder phenotypes in the lung (Supplementary Fig. 3g). Therefore, we used Atp6v1e1 heterozygous mice in our study. Upon Mtb infection, silencing of Atp6v1e1 with small interfering RNA (siRNA) decreased Mtb induced lysosomal acidification (Supplementary Fig. 3h–j). Deletion of Atp6v1e1 resulted in a decreased LysoTracker staining (Fig. 3i, j). In addition, using pHrodo™-dextran, a pH-sensitive probe that exhibits a strong increase in fluorescence specifically upon acidification, also shown an attenuated oregon-green dextran (OGD) staining (Supplementary Fig. 3k), and an increased lysosomal pH in macrophages infected with Mtb (Supplementary Fig. 3l). Intriguingly, the deletion of Atp6v1e1 markedly increased the intracellular survival of Mtb (Fig. 3k, l). Treatment with NH4Cl attenuated Atp6v1e1 deletion mediated enhancement of Mtb survival in macrophages (Fig. 3m). In vivo, the Atp6v1e1+/- mice infected with Mtb showed an increased bacterial burden in their lungs compared to the wild type mice (Fig. 3n, Supplementary Fig. 3m, n) and much severer pathological impairment (Fig. 3o, p). These results suggested ATP6V1E1 was an essential regulator for lysosomal acidification to defense against Mtb infection.

Given that Chp2 regulates lysosomal acidification via the V-ATPase, we hypothesized that it might specifically target the E1 subunit of V-ATPase. To test this, macrophages from WT and Atp6v1e1+/- mice were infected with H37Rv or H37RvΔChp2 and then stained with LysoTracker. The results were revealed that deletion of Atp6v1e1 attenuated H37RvΔChp2-induced enhancement of lysosomal acidification in the infected macrophages (Fig. 3q, r, Supplementary Fig. 3o). Furthermore, deletion of Atp6v1e1 eliminated H37RvΔChp2-mediated inhibition of Mtb intracellular survival in macrophages (Fig. 3s). After treating cells with NH4Cl, no difference in Mtb intracellular survival was observed in WT or Atp6v1e1+/- macrophages infected with H37Rv or H37RvΔChp2 (Fig. 3t), suggesting Chp2 inhibits lysosomal acidification to promote intracellular survival of Mtb through ATP6V1E1. Moreover, we infected Atp6v1e1+/- mice with H37Rv or H37RvΔChp2 to further validate in vivo relevance of Chp2 and ATP6V1E1. Knockout of Atp6v1e1 markedly increased bacterial burden of the Mtb H37Rv-infected mice, and abolished the increased bacterial burden by Chp2 in lung tissues of the Mtb-infected mice (Fig. 3u, Supplementary Fig. 3p). To assess the direct pathogenic potential of the Chp2 protein, we administered purified recombinant Chp2 intravenously or intranasally to naive C57BL/6 mice over a three-week period. Histopathological analysis of H&E-stained lung sections revealed that, compared to control groups treated with PBS, Chp2 alone did not induce significant pulmonary damage (Supplementary Fig. 3q, r). Given the lack of direct pathology, the significant impact of Chp2 on bacterial survival and pathogenesis likely depends on ATP6V1E1. Together, our results demonstrate that Chp2 promotes intracellular survival of Mtb in macrophages and in vivo by targeting ATP6V1E1 to inhibit lysosomal acidification.

Phosphorylation of ATP6V1E1 inhibits lysosomal acidification by suppressing V-ATPase assembly
It has been reported that the phosphorylation of V-ATPase subunits affects lysosomal acidification. To examine whether post-translational modifications of ATP6V1E1 regulate lysosomal acidification, we analyzed the phosphorylation modification of ATP6V1E1 by phosphorylation site prediction and found four potential phosphorylation sites (Fig. 4a). We mutated these sites to alanine and overexpressed them in HEK293T cells. After Lysotracker staining, we found that a dual-site mutation at residues 56 and 57 to alanine promoted lysosomal acidification, while a dual-site mutation to glutamic acid, mimicking the phosphorylated state, inhibited lysosomal acidification. Mutations at other individual sites had no effect on lysosomal acidification (Fig. 4b, c). Sequence alignment analysis revealed that tyrosine 56 and tyrosine 57 of ATP6V1E1 were highly conserved cross species (Fig. 4d). Using phosphor-tyrosine antibody 4G10, we confirmed dual-site mutation at tyrosine 56 and 57 to alanine markedly decreased phosphorylation of ATP6V1E1 in the recombinant proteins purified both from TKB1 (Fig. 4e) or HEK293T cells (Fig. 4f). We then generated the specific antibody against the tyrosine 56 and 57 and further confirmed ATP6V1E1 was phosphorylated at Tyr56/57 in HEK293T cells that overexpressed ATP6V1E1 and mutants (Fig. 4g). These results suggested that Tyr56/57-dependent phosphorylation of ATP6V1E1 suppressed lysosomal acidification.

V-ATPase assembly through reversible dissociation of its V1 and V0 domains was the most important forms of V-ATPase regulation26,27. To test whether phosphorylation of ATP6V1E1 regulates V-ATPase assembly, western blotting was performed on membrane and cytosolic fractions isolated from HEK293T cells that overexpressed ATP6V1E1 and mutants. Since the peripheral V1 domain was only present in membranes in fully assembled V-ATPase complexes, the abundance of V1 subunits in the membrane fraction indicated the level of V-ATPase assembly14,28. The results were shown Tyr56/57A mutant of ATP6V1E1 led to a significant increase in V-ATPase assembly, while Tyr56/57E mutant attenuated V-ATPase assembly (Fig. 4h, i), indicating the phosphorylation of ATP6V1E1 suppresses lysosomal acidification might through inhibiting V-ATPase assembly.

Through structural analysis, we further examined the influence of phosphorylation at Tyr56/57 on the structure and functionality of ATP6V1E1 and V-ATPase. The findings revealed that ATP6V1E1 and ATP6V1G1 form two straight, parallel α-helical structures, which subsequently organized into a robust, zipper-like arrangement. Notably, Tyr56/57 resided in the central region of the helices (Fig. 4j). AlphaFold3 simulations indicated that phosphorylation of Tyr57 facilitated an electrostatic interaction with Arg48 in ATP6V1G1, contributing to enhanced structural stability and reinforcing the overall zipper-like conformation (Fig. 4k, l). Together, the phosphorylation of the E1 subunit may induce a conformational change in the V-ATPase peripheral stalk, regulating its reversible assembly.

BMX regulates ATP6V1E1 phosphorylation to inhibit lysosomal acidification and promote Mtb survival
To investigate the regulatory mechanism of ATP6V1E1 phosphorylation, we screened a short hairpin RNA (shRNA) library containing 38 non-receptor tyrosine kinases to identify the kinase responsible for phosphorylating ATP6V1E1. Transfection of HEK293T cells with shRNA targeting bone marrow tyrosine kinase on chromosome X (BMX), a TEC family kinase associated with numerous pathological pathways in cancer cells29,30, substantially reduced the phosphorylation of ATP6V1E1 at Tyr56/57 (Fig. 5a). To confirm BMX as a kinase of ATP6V1E1, we performed an in vitro phosphorylation assay. The recombinant protein of ATP6V1E1 and ATP6V1E1Y56/57A purified from HEK293T cells, were incubated with purified GFP-BMX protein in optimized kinase buffer. The results demonstrated that ATP6V1E1 was phosphorylated by BMX at Tyr56/57 site (Fig. 5b). Upon Mtb infection, silencing of Bmx (Supplementary Fig. 4a, Fig. 5c) or inhibition of BMX using the specific kinase inhibitor BMX-IN-1 (Fig. 5d), markedly reduced the Tyr56/57 phosphorylation of ATP6V1E1 in macrophages. Consistently, deletion of Bmx resulted in decreased phosphorylation of ATP6V1E1 at Tyr56/57 in macrophages infected with Mtb (Fig. 5e), suggesting that Mtb regulates phosphorylation of ATP6V1E1 via BMX. To determine if Bmx-mediated phosphorylation of ATP6V1E1 impacts the assembly of the V-ATPase complex at the lysosomal membrane, we analyzed the lysosomal fractions isolated from BMX-IN-1 treated vs. untreated macrophages, as well as from Bmx+/- vs. wild-type macrophages. The presence of key V1 subunit ATP6V1E1 and V0 subunit ATP6V0d1 in the lysosomal membrane was assessed by immunoblotting. The results showed that Bmx inhibition either by pharmacologically or genetically alter the abundance of V-ATPase subunits at the lysosomal membrane (Fig. 5f). This suggests that Bmx inhibits V-ATPase assembly likely via changing the phosphorylation status of ATP6V1E1 subunit. Silencing of Bmx markedly increased the lysosome acidification in macrophages infected with Mtb as revealed by LysoTracker staining (Fig. 5g, h, Supplementary Fig. 4b) and dramatically reduced the intracellular survival of Mtb as determined by the CFU assay (Fig. 5i). Moreover, in Atp6v1e1+/- macrophages, treatment with BMX inhibitor abrogated the BMX-mediated increase in intracellular bacteria load (Fig. 5j). We also detect Mtb intracellular survival in WT and Bmx+/- macrophages infected with H37Rv and H37RvΔChp2. Deletion of either Bmx robustly reduced Mtb intracellular survival only in H37Rv-infected murine peritoneal macrophages and eliminated the difference in intracellular survival between H37Rv and H37RvΔChp2 (Fig. 5k, l). Additionally, after 4 weeks Mtb infection, Bmx+/- mice exhibited a dramatically reduction in histopathological changes (Fig. 5m, n) as well as the bacterial burden in their lungs compared to the WT animals (Fig. 5o, p). Taking together, these results suggested that BMX might promote phosphorylation of ATP6V1E1 to inhibit lysosome acidification, thus promoting intracellular survival of Mtb.

Chp2 promotes phosphorylation of ATP6V1E1 by enhancing its binding with BMX
To explore whether and how Chp2 regulates the phosphorylation of ATP6V1E1, we assessed the phosphorylation level of ATP6V1E1 in macrophages infected with H37Rv and H37RvΔChp2. Deletion of Chp2 led to a decrease in ATP6V1E1 phosphorylation (Fig. 6a). The inhibition of Bmx by siRNA reduced the difference in ATP6V1E1 phosphorylation between H37Rv and H37RvΔChp2-infected macrophages (Fig. 6b). Moreover, we detected the ATP6V1E1 phosphorylation in peritoneal macrophages from WT and Bmx+/- mice infected with H37Rv and H37RvΔChp2. The results showed deletion of Chp2 led to a decrease in ATP6V1E1 phosphorylation and Bmx deletion abolished the inhibition of ATP6V1E1 phosphorylation that is induced by deletion of Chp2 (Fig. 6c). This genetic evidence powerfully confirms that Bmx is the primary kinase responsible for phosphorylating ATP6V1E1 in response to Mtb infection. The in vitro phosphorylation assay showed that Chp2 enhanced the BMX mediated phosphorylation of ATP6V1E1 (Fig. 6d). We then examined the role of Chp2 in the assembly of V-ATPase. The results showed that the deletion of Chp2 markedly increased the levels of V1 domain of V-ATPase in membrane fractions (Fig. 6e, f), indicating Rv1184 inhibits the assembly of the V-ATPase at lysosomal membranes. Moreover, the inhibition of Bmx by siRNA reduced the difference in intracellular bacterial loads between H37Rv and H37RvΔChp2-infected macrophages (Fig. 6g, h). Treatment with NH4Cl reversed the effect of Chp2 deletion on Mtb survival in macrophages transfected with control siRNA, and while in Bmx knockdown macrophages, NH4Cl treatment promoted the growth of both H37RvΔChp2 and H37Rv (Fig. 6g, h), suggesting that Chp2 promotes intracellular survival of Mtb by increasing BMX-mediated phosphorylation of ATP6V1E1 to inhibit lysosomal acidification.

We next investigated the mechanism underlying the promotion of ATP6V1E1 phosphorylation by Chp2. Surface plasmon resonance (SPR) measurements demonstrated that BMX binds ATP6V1E1 with a dissociation constant (Kd) of 190 nM, indicating a high-affinity interaction. (Fig. 6i). Given that Chp2 interacts with ATP6V1E1 and promotes its phosphorylation, we next investigated whether Chp2 regulates the binding of the ATP6V1E1 and BMX. Overexpression of Chp2 in HEK293T cells increased the interaction between ATP6V1E1 and BMX (Fig. 6j). Moreover, we observed that the recruitment of endogenous BMX to ATP6V1E1 was reduced in macrophages infected with H37Rv compared to uninfected, and that deletion of Chp2, significantly reduced endogenous interaction of BMX with ATP6V1E1 (Fig. 6k). The immunofluorescence experiments confirmed that Chp2 is present in the host cytoplasm and partially co-localizes with LysoTracker, providing spatial evidence for its translocation into the host cell (supplementary Fig. 5a). Consistently, we also observed that deletion of Chp2 significantly reduced the endogenous phosphorylation level of ATP6V1E1 in macrophages (Fig. 6k), suggesting that Chp2 enhances the binding of ATP6V1E1 and BMX to promote the phosphorylation of ATP6V1E1. Using AlphaFold3 structural simulations, we revealed a potential interaction model in which Chp2, BMX, ATP6V1E1, and ATP6V1G1 form a stable complex. In this model, Chp2 acts as a large scaffold protein, promoting the spatial proximity between the catalytic domain of BMX and Tyr56/57 of ATP6V1E1, thereby facilitating their interaction and the phosphorylation process (Fig. 6l, supplementary data 1). Taken together, these data suggest that Chp2 acts as a scaffold, facilitating the formation of the Chp2-ATP6V1E1-BMX complex, which enhances the binding of ATP6V1E1 to BMX and increases the phosphorylation levels of ATP6V1E1. This phosphorylation inhibits V-ATPase assembly and suppresses lysosomal acidification, ultimately promoting the intracellular survival of Mtb.

Inhibition of BMX promotes Mycobacterium clearance
Lysosomal acidification is essential for the Mtb clearance, making lysosome acidification modulators promising targets for host-directed therapy (HDT) against tuberculosis. BMX-IN-1, the first successfully synthesized selective BMX inhibitor31, was evaluated for its therapeutic effect on the pathogenesis of Mtb. The mice were infected with the Mtb for 14 days, followed by treatment with BMX-IN-1 every two days for an additional 14 days (Fig. 7a). Treatment with BMX-IN-1 led to a significant reduction in bacterial burden and much less pathological damage in the wild-type mice (Fig. 7b-e). However, the therapeutic benefits of BMX-IN-1 were abolished in Atp6v1e1+/- mice (Fig. 7b-e), indicating that BMX-IN-1 has a therapeutic role for Mtb infection through targeting ATP6V1E1. Moreover, to investigate whether the therapeutic efficacy of BMX inhibitor involves targeting Chp2, we infected mice with H37Rv or H37RvΔChp2. Two weeks post-infection, we administered the inhibitor and assessed pulmonary pathology and bacterial load two weeks after treatment. The data showed that the administration of the BMX inhibitor significantly reduced the bacterial burden and pathological damage in mice infected with H37Rv. However, this therapeutic effect was abolished in mice infected with the H37RvΔChp2 strain (Fig. 7f-i), suggesting that BMX inhibitor play a role via Chp2, indicating that the efficacy of the BMX inhibitor is dependent on the presence of the bacterial effector Rv1184c. It supports a model wherein the inhibitor acts by targeting the host pathway that is subverted by Rv1184c, thus neutralizing the bacterium’s virulence strategy. This suggests that host-directed therapy (HDT) with BMX inhibitors could be particularly effective against Mtb strains that rely on this specific mechanism. Collectively, targeting lysosomal acidification by BMX-IN-1 holds potential as a strategy for host-directed therapy in tuberculosis.

Methods
Mice
C57BL/6 mice were purchased from the Shanghai Laboratory Animal Center (Shanghai, China) and maintained under specific pathogen free (SPF) conditions at the Laboratory Animal Center of Tongji University. Female mice aged 6-8 weeks were used for all experiments. Atp6v1e1+/- mice and Bmx+/- mice were generated using the CRISPR/Cas9 method49. Briefly, in vitro-translated Cas9 mRNA and guide RNAs (gRNAs) were co-microinjected into fertilized C57BL/6 zygotes. The sequences of gRNA were listed in Supplementary Table 1. Frameshift mutations in the founders were screened by T7E1 assay and confirmed by DNA sequencing. Three F1 founders were obtained for each line, and a single founder from each was selected for backcrossing to C57BL/6 mice to establish the strain. Atp6v1e1+/- mice and Bmx+/- mice were identified by PCR using genomic DNA isolated from tail tissue. The sequences of PCR primes were listed in Supplementary Table 2.

Plasmids, antibodies and reagents
Plasmids used in this study were described in Supplementary Table 3. Antibodies against phosphorylated V1E1Y56/57, total anti-V1E1 and anti-Chp2 were produced by ABclonal Technology (Wuhan, China). Antibodies against BMX 24733S) and GAPDH (2118S) were obtained from Cell Signaling Technology (Beverly, MA) and used at 1:1000 for Western blotting. The anti-phosphotyrosine monoclonal antibody (4G10) (05-321) was obtained from Merck. Anti-hemagglutinin (HA, H6908) and mouse monoclonal anti-Flag (F3165) from Sigma-Aldrich. Rabbit monoclonal anti-His (AE086), rabbit monoclonal anti-Myc (AE070), goat anti-rabbit IgG secondary antibody (AS014) and goat anti-mouse IgG secondary antibody (AS003) were purchased from ABclonal. Anti-V0d1 antibody (18274-1-AP), anti-tubulin antibody (80762-1-RR), anti-Na-K antibody (13884-1-AP) were purchased from Proteintech. TSA 7-color kit (abs50015-100T) was purchased from Absinbio. Antibodies against CD3 (78588) and CD20 (70168T) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against MPO (ab208670) and CD11b (ab133357) were purchased from Abcam. Anti-SigA (663205) were purchased from BioLegend. Antibody against LAMP1 (S-952-18) was purchased from STARTER. Fluorescent secondary antibodies were purchased from Thermo Fisher and used according to the manufacturers’ instructions. FITC-dextran (A642107) was purchased from Sangon Biotech. RPMI 1640 medium (C11875500CP), DMEM (SH30249.01), fetal bovine serum (FBS) (26170043), penicillin and streptomycin (15140163) were purchased from Invitrogen (Shanghai, China). LysoTrackerTM Red DND-99 (L7528) and pHrodo™ Green Dextran (P35368) were obtained from InvitrogenTM. The Magic Red™ Cathepsin B Assay Kit (ICT-937) was purchased from Enzo Life Sciences. The shRNA library of non-receptor tyrosine kinases was originally from the MISSION® LentiPlex® Human Pooled shRNA Library TRC2 from Sigma Aldrich. The siRNA targeting mouse Bmx (sc-38942) and control scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology, and a siRNA pool specifically targeting mouse Atp6v1e1 was obtained from RiboBio (Guangzhou, China). Bafilomycin A1 (Baf-A1, HY-100558) was purchased from MedChemExpress. BMX-IN-1 (1431525-23-3) was purchased from ApexBio.

Bacterial culture and infection
Mycobacterium tuberculosis H37Rv strain was cultured to mid-log phase in Middlebrook 7H9 broth (Becton Dickinson) supplemented with 0.05% Tween-80 and 10% oleic acid-albumin-dextrose-catalase (OADC, 211886, Becton Dickinson, Sparks, MD). H37RvΔChp2 was constructed by Shanghai Gene-optimal Science & Technology Co., Ltd. The shuttle vector pMV261 (provided by K. Mi, Institute of Microbiology, Beijing, China) was used to complement the strain H37RvΔChp2 with wild-type Chp2. Expression of Chp2 in mycobacteria was examined by immunoblot analysis. For H37RvΔChp2, 50 μg/mL hygromycin B was added to culture. For H37Rv(ΔChp2+Chp2), 50 μg/mL hygromycin B and kanamycin were added to culture.
For macrophages infection, mice peritoneal macrophages were seeded in 12-well plates, and cultured for 24 h at 37°C in a 5% CO2 incubator. Mtb strains were added to cells at a multiplicity of infection (MOI) of 5.

FITC labeling of Mtb
Mid-log phase Mtb cultures were harvested by centrifugation, washed twice, and resuspended in pre-chilled 0.1 M carbonate-bicarbonate buffer (pH 9.0). FITC isomer I was added to a final concentration of 0.1 mg/mL, and the suspension was incubated at 37°C for 1 hour in the dark with gentle agitation. After labeling, bacteria were washed thoroughly 4-5 times with pre-chilled phosphate-buffered saline (PBS, pH 7.4) until the supernatant was fluorescence-free to remove unbound dye. Finally, the labeled bacteria were resuspended in appropriate infection medium and quantified using McFarland standards coupled with colony-forming unit (CFU) plating. All labeling and infection steps were performed under light-protected conditions. In the image analysis phase, fields containing visible large clumps (>5 µm in diameter or containing clearly fused bacterial signals) were systematically excluded from quantification.

Cells culture
HEK293T cells (ATCC CRL-3216) were obtained from the American type culture collection (ATCC). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) supplemented with 10% (v/v) heated-inactivated fetal bovine serum (FBS, Gibco) and 100 U/ml penicillin-streptomycin. Routine testing was conducted to ensure the cells were free of mycoplasma contamination.

Mice macrophages isolation
Peritoneal macrophages were isolated as previously described. Briefly, mice were injected intraperitoneally (IP) with 2.0 ml of 4% Brewer’s thioglycollate medium (Sigma‒Aldrich). Three days later, primary macrophages were harvested by peritoneal lavage with 10 mL cold PBS from euthanized animals. Subsequently, the cells were plated in 12-well plates at 10^6 cells per-well in RPMI 1640 supplemented with 10% FBS and penicillin-streptomycin, and then incubated at 37°C in 5% CO2 for 2 h. The cultures were then washed three times with PBS to remove nonadherent cells, and the remaining adherent monolayer cells were used as primary peritoneal macrophages. The bone marrow-derived monocytes (BMDMs) were generated by isolating mouse bone marrow cells and culturing them for 7 days in medium supplemented with 10% (v/v) FBS, 40 ng/ml M-CSF (Peprotech), and 20 ng/ml IL-4 (Peprotech).

Cell fractionation
To measure the V-ATPase assembly, the cells fractionation was performed to obtain cytosolic and membrane fractions. HEK293T cells were transfected with plasmids encoding HA-ATP6V1E1, HA-ATP6V1E1Y56/57A and HA-ATP6V1E1Y56/57E for 48 h, or primary peritoneal macrophages from wild-type and Bmx+/- mice pretreated with BMX-IN or not were prepared. Monolayers were rinsed with ice-cold PBS and scraped into 500 μL ice-cold fractionation buffer (250 mM sucrose, 10 mM HEPES, pH 7.2, 1 mM EDTA, 1 mM PMSF, 5 μg/mL leupeptin, 2 μg/mL aprotinin, 1 μg/mL pepstatin, 2 mM β-glycerophosphate, 1 mM NaF). Cell suspensions were centrifuged at 300 × g for 5 min at 4°C, and the pellets were resuspended in 500 μL fresh ice-cold fractionation buffer. Cells were lysed using a cell homogenizer, and crude lysates were cleared of unbroken cells and nuclei by centrifugation at 1000 × g for 10 min at 4°C. The resulting supernatants were centrifuged at 100,000 × g for 30 min at 4°C in the ultracentrifuge to pellet membrane fractions. The supernatants (cytosolic fractions) were concentrated, and membrane pellets were solubilized in fractionation buffer supplemented with 1% SDS.

Immunoblotting and immunoprecipitation
For immunoblotting analysis, the cells were lysed in RIPA buffer (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% Na-deoxycholate, 1 mM EDTA, 0.05% SDS) supplemented with freshly added protease inhibitors. Protein concentration was determined by the Bradford assay (Bio-Rad). Equal amounts of protein were separated by SDS–PAGE and transferred to nitrocellulose membranes (Millipore). Membranes were blocked with 5% bovine serum albumin and incubated with primary antibodies at 4°C overnight, followed by HRP-conjugated secondary antibodies Signals were detected with ECL (GE Healthcare) or SuperSignal West Dura (Thermo Scientific) and visualized on autoradiographic film.
For immunoprecipitation (IP), the cells were lysed in Western blot and IP cell lysate buffer supplemented with protease inhibitor cocktail (MedChemExpress). Following lysis, the samples were subjected to centrifugation, and the resulting supernatants were incubated overnight at 4°C with either anti-FLAG M2 Magnetic Beads or HA Beads (Sigma-Aldrich). The samples were washed three times with PBST (KH2PO4 2 mM, Na2HPO4 8 mM, NaCl 136 mM, KCL 2.6 mM, 1% Triton X-100) and subjected to Western blot analysis.

Immunofluorescence staining and confocal microscopy analysis
HEK293T cells were grown in glass bottom cell culture dish (NEST-SCIENTIFIC 801002). Adherent primary peritoneal macrophages were infected with Mtb at MOI of 5 for 4 h, fixed in 4% formaldehyde overnight, and then washed twice with PBS for 5 min. Cells were blocked with 1% BSA for 30 min at room temperature and then incubated with a primary antibody (1:200) overnight at 4°C. Subsequently, samples were incubated with a secondary antibody (1:400) for 2 h at 37°C. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Images were acquired on a Leica SP5 confocal microscope using a 63× objective.
For all microscopy-based LysoTracker experiments, fluorescence intensity was quantified in a minimum of 100 individual cells from multiple randomly selected fields of view. This analysis was repeated across three independent biological replicates. The sample size (n) stated in the figure legends refer to the number of biological replicates (each containing data from >100 cells).

High-throughput fluorescence screening and analysis
HEK293T cells were seeded onto 96-well microplates and allowed to attach overnight. The next day, cells were transfected with plasmids encoding Mtb-secreted proteins or lipoproteins for 48 h. A miniaturized fluorescence-based Lyso-tracker was performed in 96-well microplates with a final volume of 50 μL per well to assess lysosomal acidification. After the staining, fluorescence (Ex/Em = 577/590 nm) was measured on a Thermo Scientific™ ArrayScan™ multimode microplate reader using top-read optics; instrument sensitivity was fixed at a pre-optimized gain to ensure consistency across the screen. Raw fluorescence units (RFU) were normalized per plate to percent inhibition relative to the median uninhibited enzyme control wells (maximum signal) and median no-enzyme control wells (background), which were included in designated columns on each plate. Normalized data were processed and analyzed for hit identification.

Multiplex Immunohistochemistry (mIHC)
Multiplexed immunohistochemistry (mIHC) was performed by staining 4-um-thick formalin-fixed, paraffin-embedded whole tissue sections with standard, primary antibodies sequentially and paired with Five Color Multiplex Fluorescent lmmunostaining Kit (abs50013, Absinbio, Shanghai). Then by staining with DAPI. The deparaffinized slides from lung tissues of mice were incubated with anti-CD3 antibody (CST78588), for 30 minutes and then treated with anti-rabbit/mouse horseradish peroxidase-conjugated (HRP) secondary antibody (abs50015-02, Absinbio, Shanghai) for 10 min. Then labelling was developed for a strictly observed 10 minutes, using TSA 570 per manufacturer’s direction. Slides were washed in TBST buffer and then transferred to preheated citrate solution (90°C) before being heat-treated using a microwave set at 20% of maximum power for 15 min. Slides were cooled in the same solution to room temperature. Between all steps, the slides were washed with Tris buffer. The same process was repeated for the following antibodies/fluorescent dyes, in order: anti-MPO (ab208670)/ TSA 620, anti-CD11b (ab133357)/ TSA 700, anti-CD20 (70168T)/ TSA 520. Each slide was then treated with 2 drops of DAPI, washed in distilled water, and manually cover slipped. Slides were air dried, and take pictures with Pannoramic MIDI II (3DHISTECH). Images were analyzed using Indica Halo software.

Lysosomal content assay
To assess lysosomal acidification, primary peritoneal macrophages were infected with Mtb at MOI of 5 for 4 h and then stained with two fluorescent dextrans: Oregon Green™ 488-conjugated Dextran (10,000 MW, Anionic, quenched in acidic environments), and Tetramethylrhodamine-conjugated Dextran (10,000 MW, Anionic, Lysine Fixable, pH-independent) (Thermo Fisher). After the addition of these two dextrans, cells were incubated in a glass-bottom dish (NEST) at 37°C for 6 h, the medium was replaced, and incubation was continued overnight. Labeled cells were then washed into HBSS, and imaged by fluorescence microscope. Green and red fluorescence intensities were quantified using a BZ-X800 (KEYENCE).
As an alternative approach, LysoTracker Red DND-99 was used to evaluate lysosomal acidification. Primary peritoneal macrophages plated in glass-bottom culture dishes (NEST) were infected with Mtb at MOI of 5 for 4 h, and then treated with 25 μM monensin for 20 minutes, incubated with 1 μM LysoTracker for 5 min, washed twice with PBS and nuclei were stained with DAPI. Images were captured by confocal microscopy (Leica TCS SP8).
To generate a lysosomal pH calibration curve, primary peritoneal macrophages were equilibrated in calibration buffer of varying pH (5 mM NaCl, 115 mM KCl, 1.2 mM MgSO4, 25 mM MES; pH 3.5–7.5). Samples were excited with 357–373 nm and emission were collected in the blue (W1; 417–483 nm) and yellow (W2; 490–530 nm) channels. Ratiometric (W1/W2) images were computed using the Image Calculator function in MetaMorph (Molecular Devices).

RNA interference and transfection
Mice peritoneal macrophages were transfected with small interfering RNAs using the transfection reagent (AM4511, Thermo Fisher) according to the manufacturer’s instructions. The individual siRNAs targeting mouse Bmx, Atp6v1e1 and a scrambled siRNA were each diluted in RNase-free water to final volume of 330 μL.

Intracellular CFU assay
Adherent primary murine peritoneal macrophages were infected with the H37Rv strain or other H37Rv strains at a multiplicity of infection (MOI) of 5 for 2 hours at 37°C. Following infection, the cells were washed twice with phosphate-buffered saline (PBS). To determine the number of viable intracellular bacteria, the infected cells were lysed at 2 h or 24 h post-infection using 0.1% (v/v) Triton X-100 (Thermo Fisher, Cat#85111) in PBS. The lysates were then serially diluted, plated on agar, and the bacterial burden was quantified by counting the resulting colony-forming units (CFU). The BMDMs were infected with the H37Rv strain or H37RvΔChp2 for 2 h, 24 h, and 48 h to detect CFU.

Generation of phospho-ATP6V1E1Y56/57 antibodies
A rabbit polyclonal antibody recognizing ATP6V1E1 phosphorylated at Y56/57 (designated p-ATP6V1E1Y56/57) was generated in collaboration with ABclonal Biotech. Two rabbits were immunized with a 1:1 mixture of the phosphopeptide c(KLH)52KIMEY(p-Y)Y(p-Y)EK59 and c(KLH)54MEY(p-Y) Y(p-Y) EKKE61, (KLH conjugation via an N-terminal cysteine; p-Y denotes phosphotyrosine). The corresponding non-phosphorylated peptide c(KLH)-KIMEYYEKKE was used for affinity purification and antibody validation.

In vitro kinase assay
Recombinant BMX (1 μg) was incubated with recombinant ATP6V1E1 (1 μg) in 3 μL of 10× phosphorylation buffer (200 mM Tris-Cl, pH 7.5, 500 mM KCl, 100 mM MgCl2), 1 μL 2 pM of ATP, and complete protease inhibitor mixture (Roche). The reaction was brought to a final volume of 30 μL with distilled water and incubated for 45 min at 30°C. Then, the reaction mixture was subjected to SDS-PAGE.

Intranasal infection (i.n.) of mice with Mtb
Mice were infected intranasally with 4×10^5 viable CFUs of H37Rv, H37RvΔChp2 or H37Rv (ΔChp2 + Chp2). The inoculum (40 μL per mouse) was applied to the nares, alternating between the left and right nostrils, in a Biosafety Level-3 (BSL-3) laboratory. Mice were sacrificed 28 days post-infection, and bacterial burden in the lungs was determined by plating serial tenfold dilutions of individual lung homogenates on Middlebrook 7H10 agar supplemented with 10% OADC. Mtb colonies were incubated at 37°C and counted after 21 days. For BMX-IN-1 intervention, after 2 weeks of infection, mouse was given a dose of 20 mg/kg BMX-IN-1 working solution through oral administration for 2 weeks. Four weeks after infection, the mice were sacrificed, and the bacterial loads in the lungs were determined by CFU counts. To assess the effect of Baf-A1, mice were intraperitoneally injected with Baf-A1 prior to infection for 3 days, followed by the weekly Baf-A1 (10 mg/kg, 200 uL/mice) injections for 4 weeks. Finally, the mice were sacrificed, and CFU counts in the lungs were measured to determine the bacterial load.

Chp2 protein administration
To assess the direct pathogenic potential of the Chp2 protein, C57BL/6 mice were injected with recombinant Chp2 protein via either the intravenous (100 μg/mL, 200 μL per mouse) or intranasal (100 μg/mL, 30 μL per mouse) route. After three weeks, histopathological damage in the lungs from both treatment groups was assessed by H&E staining.

Histological analysis and acid-fast staining
Lung tissues from infected mice were fixed in 4% phosphate-buffered formalin for 24 h, then embedded in paraffin. Serial sections were cut from the tissue blocks, and five non-continuous sections were selected for Hematoxylin and eosin (H&E) staining to assess inflammatory cell infiltration. The stained slides were examined by light microscopy, and histopathological evaluation was performed by pathologists blinded to group identity. The pathology scores (0-10) were based on the ratio of inflammatory cell infiltration area to the total lung section area on the maximal cross-section of a lung lobe, with heavily stained areas indicating infiltration and damage, and lighter areas representing normal alveolar structure. Acid-fast staining (Ziehl–Neelsen) was performed to detect mycobacteria.

Surface plasmon resonance (SPR)
Interaction between ATP6V1E1 and BMX or Chp2 was analyzed using OpenSPRTM (Nicoya Lifesciences, Waterloo, Canada). A carboxyl-functionalized sensor chip (COOH chip) was installed, and the instrument was primed with PBST (pH 7.4) at a maximum flow rate of 150 μL/min until the baseline signal stabilized. The sample loop was then rinsed and emptied. The flow rate was reduced to 30 μL/min prior to chip activation with a fresh 1:1 EDC/NHS solution. Ligand (200 μL) diluted in activation buffer was injected for 4 min. After ligand immobilization and buffer rinses, the surface was blocked with 200 μL blocking solution, rinsed and dried with air. Baseline stability was confirmed for 5 min. The flow rate was returned to 150 μL/min and a regeneration solution was used as required to remove residual analyte. Analytes were prepared at the concentrations indicated in the results and injected at 30 μL/min. Association and dissociation phases were 120 s and 300 s, respectively. Sensorgrams were fitted using TraceDrawer (Ridgeview Instruments AB, Sweden) with a one-to-one binding model.

AlphaFold3 structural simulations
The human V-ATPase structure (PDB: 7U4T), previously published, was utilized to analyze the structure of V1E1. Structural simulations were conducted using the AlphaFold3 online server (https://golgi.sandbox.google.com/), and the results were visualized with PyMOL software. The model is available in Model Archive at https://www.modelarchive.org/doi/10.5452/ma-3dlmq.

Statistical analysis
Data are presented as mean ± SEM. Differences between two groups were assessed by unpaired two-tailed t tests. For comparisons involving more than two groups, Two-sided Mann-Whitney U test was performed with correction for multiple comparisons where appropriate. All statistical analyses were conducted in GraphPad Prism 8.0. A P value < 0.05 was considered statistically significant.

Ethics consideration
All animal experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Experiment Administration Committee of Tongji University School of Medicine (Approval No. TJAA06520101).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this article, the authors explore how Mycobacterium tuberculosis (Mtb) manipulates macrophages to inhibit lysosomal acidification and promote its own replication. They show that Mtb secretes the effector Rv1184c, which targets the V-ATPase acidification machinery, specifically the V1 subunit. A wide range of techniques was used, and substantial experimental data were generated to support their hypothesis. The study is well-constructed and very interesting. Mechanisms by which Mtb blocks lysosomal acidification remain poorly understood, making this work valuable. Furthermore, the in vivo inhibition of BMX appears to be a promising host-directed therapy (HDT) strategy.\nBelow are some suggestions to improve the quality of the manuscript:\n• In the first section of the results, the authors conclude that the use of bafilomycin to inhibit V-ATPase demonstrates that Rv1184c inhibits lysosomal acidification via this pathway. This conclusion seems a bit premature and could benefit from an additional experimental approach to confirm the hypothesis, even if later experiments do support it further.\n• Line 129: Infection in macrophages was monitored for 24 hours. This time point may be too early—what happens at later time points, such as 3 or 4 days post-infection?\n• Line 131: Please explain the role of the NH₄Cl control.\n• The results show increased acidification during infection with the Rv1184c-deficient mutant strain, and that this strain is attenuated in macrophages. Is it possible to provide imaging data showing co-localization between bacteria and Lysotracker under these conditions? For instance, is there increased co-localization of the Rv1184c deletion mutant with Lysotracker compared to wild-type Mtb?\n• Line 135: Could you comment on the reduced immune cell infiltration? Which immune cell types are affected? Could this be assessed by flow cytometry?\n• Line 145: Again, I am not fully convinced by the bafilomycin experiment. Inhibiting V-ATPase will necessarily favor infection and may mask the effect of the mutation. Could you consider an alternative readout?\n• Is it possible to visualize or measure lysosomal acidification in alveolar macrophages from infected mice?\n• Line 180: The in vivo results are interesting. Have you considered infecting Atp6v1E1 knockout mice with the Rv1184c mutant strain? It would be interesting to discuss the expected results.\n• BMX inhibition shows very promising data. Could this therapy be combined with standard antibiotic treatment to test for potential synergy?\n• What would be the effect of BMX inhibition in mice infected with the Rv1184c mutant strain?\n• Similarly, what would be the effect of PLGA-aNP treatment in Atp6v1e1 knockout", "reviewer_2": "This manuscript by Chen et al, titled “Mycobacterium tuberculosis manipulates phosphorylation of ATP6V1E1 to promote its intracellular survival”, shows an important phosphorylation-dependent regulation of lysosomal acidification during Mtb infection, with the potential to regulate Mtb survival inside the infected macrophages. The authors show that Mtb secreted protein Rv1184c facilitates interaction between tyrosine kinase BMX and the vATPase subunit ATP6v1e1, leading to increased phosphorylation of ATP6v1e1 and corresponding decline in the lysosomal acidification, resulting in less efficient bacterial killing. In view of that, inhibiting BMX kinase in the animal models shows a decrease in lung CFU burden, which is dependent on the protein ATP6v1e1.\nThe regulation of lysosomal activity by Mtb during infection has been under immense focus for investigation due to its direct role in killing intracellular bacterial infections. The current manuscript provides interesting insights into this key battle between the host and the pathogen. The authors generate useful models to address the specific questions asked in this study.\nHowever, the study suffers from some serious limitations. There are some general observations and specific concerns pointed out below.\nGeneral observations: There is a lack of details on the experiments and results. For studies on Mtb infection, cell/cell-lines used, the time points post-infection, duration of treatment, etc., are vital details. These details are missing even in the methods section. Some of the results reflect a less robust experimental strategy, leading to deviations from the expected line. Different experiments are done on different cell lines/types. The rationale for those is not obvious. Cells used for the experiments are not explicitly mentioned.\nMajor limitations:\n1) The ability of Mtb to block phagosome maturation and lysosomal acidification is well-documented. However, throughout this study, a significantly higher number of bacilli is seen in acidified lysosomes, which, of course, increases upon loss of Rv1184c. The observed phenotype is difficult to interpret due to the absence of any staining for the Mtb, which would allow assessing bacterial presence in acidified lysosomes.\n2) Conclusions for a few results and interpretations made thereof are not aligned with the experimental observations.\n3) The phenotype of ATP6v1e1 knockout mice is not well explained. With a loss of lysosomal function, in these animals, even without infection, they should show lysosomal storage disorder phenotype.\nSpecific points:\n• How ATP6v1e1 overexpression alone is able to cause significant lysosomal acidification in 293T cells? The vacuolar ATPase is a multi-subunit protein, and the catalytic site, formed by the V1 domain, consists of all subunits. This needs more clarity. What happens to ATP6ve1 expression upon Mtb infection, and whether that differs from the expression of other subunits?\n• For lysosomal acidification during Mtb infection, there is no staining for Mtb (Fig. 1g, 3q, fig. S3g, etc.). The virulent Mtb is known to block lysosomal acidification through PMA. Since MOI used is 5, it is important to show that the cells analysed/being shown are also Mtb- infected.\n• Why should H37Rv CFU decline in WT cells from 0 hour to 24 hours post-infection? This is a critical observation, Mtb CFU is generally stable or grows during 24 hours, however a decline in CFU indicates loss of virulence. Have the authors checked PDIM levels of the strains used? The observed dip in Mtb CFU in the WT cells needs to be analysed more robustly.\n• Tissue sections from uninfected ATP6v1e1 should also be shown. With the loss of lysosomal function, severe pathology may develop due to lysosomal storage disorder like condition.\n• Line 178: Lysotracker staining shown in WT H37Rv infection is too high. H37Rv blocks lysosomal acidification. Secondly, fig. 3q-r: the lysotracker staining upon H37Rv infection visible in the images at 0 hours and 4 hours in the WT and ATP6v1e1-/- cells (3q) is not reflected in the corresponding quantification plot (3r). This is very concerning.\n• Fig 6a: Rv1184c deletion: the impact on ATP6v1e1 phosphorylation is not very distinct from the WT infection.\n• The Rv1484c knockout and complementation data is clean. However, authors need to show that sufficient Rv1484c gets secreted during infection in macrophages to be able to interact with and alter ATP6v1e1 phosphorylation. So far all such evidences are shown using overexpression experiments but whether expression and secretion of native protein is sufficient to alter ATP6v1e1 phosphorylation and lysosome acidification?\n• siBMX treatment: How many hours after BMX knockdown infection was given? What does 0 hour mean here? What is the knockdown efficiency of BMX siRNA? It is strange to see a complete loss of ATP6v1e1 phosphorylation.\n• For all phosphorylation immunoblots, GAPDH is not a correct loading control. The control should be the ATP6v1e1 protein itself.\n• The PLGA-aNP experiment is not relevant to the current study unless it is shown in combination with the BMX inhibitor.\nMinor points:\nTypo in line 233: “numerous pathological pathways”?\nAuthors should use Grammarly or similar software for written English.", "reviewer_3": "This manuscript by Chen J et al. uncovers a mechanism by which Mycobacterium tuberculosis H37Rv (Mtb) manipulates phagolysosomal maturation to avoid killing by macrophages. The authors show that Mtb protein Rv1184 promotes BMX interaction with the v-H+-ATPase subunit ATP6V1E1 thereby enhancing phosphorylation of ATP6V1E1 at tyrosine residues. This inhibits the v-ATPase assembly at the membrane, dampening phagolysomal acidification and allowing Mtb survival. This manuscript therefore highlights an additional mechanism by which Mtb manipulate phagosome maturation.\nIn its large part, I found this MS very interesting and convincing. It provides many details in the mechanism described with very neat experiments. My enthusiasm was somehow dampened by some flows in the logic that brought the authors to realized some experiments. For example, what brought the authors to assess phosphorylation fo ATP6V1E1 in the first place? Furthermore, there is a duplication of images in Fig 7b and 7g: 1) Mock controls are the same images but it is not mentioned in the legend ; 2) BMX-IN-1 #3 is the same image as PLGA-aNP #2.\nI must say that my expertise in histological HandE staining in the context of Mtb infection is limited. Thus I could not really evaluate this part of the work.\nSome suggestions/comments that may improve the study:\n1) Fig 1b, do the authors found CISH and PtpA in their screen?\n2) What is the effect of Rv1184c by itself upon injection in mice?\n3) In Fig 3B, I understand that the authors over-expressed FLAG-Rv1184c together with HA-tagged subunits of the v-ATPase. However, it might be good to provide more details in the text. It is hard to apprciate the relevance of the blot shown since MW are missing (Fig 3B and C). Whole lysate indicates a problem of expression of some HA-tagged v-ATPase subunits. How does this affect the conclusions made by the authors? Temporality of the interaction? Why the other subunits do not appear in the immuno precipitate?\n3) The reason for using Bmx +/- mice instead of Bmx-/- mice is not clear? Is the KO lethal? Is there a reason why these heterozygote mice were not uses in addtion of BMX-IN1 in key experiments of Mtb burden.\n4) Fig 6a and 6b are not convincing, and lack proper quantification. Since Bmx +/- are available, why the authors did not use them to assess ATP6V1E1 phosphorylation in response to Mtb?\n5) In Fig 7, proper controls on PLGA-aNP on lysosomal pH stability are missing. How PLGA-aNP can end up in the same phagolysosomes as Mtb?\n6) Although fluorescence intensity is widly use for lyso-Tracker-mediated evaluation of lysosomal acidification, I found this microscopy-mediated approach a little bit weak for this type of analysis (how many cells were used to determine fluorescence activity?)\nMinor points:\n1) Rephrase sentences in line 261-262, Line 276-277, and Lines 142-145. These sentences are particularly confusing.\n2)English writing could be improved for clarity.\n3) Is Rv1184c constitutively secreted by mtb or is it secreted at the time of infection?\nAdd the MW and proper quantifications of all the blots.\n4) Methods are poorly described."}, "author_response": "Point to point response\nReviewers' comments:\nReviewer #1: (Remarks to the Author):\nIn this article, the authors explore how Mycobacterium tuberculosis (Mtb)\nmanipulates macrophages to inhibit lysosomal acidification and promote its\nown replication. They show that Mtb secretes the effector Rv1184c, which\ntargets the V-ATPase acidification machinery, specifically the V1 subunit. A\nwide range of techniques was used, and substantial experimental data were\ngenerated to support their hypothesis. The study is well-constructed and very\ninteresting. Mechanisms by which Mtb blocks lysosomal acidification remain\npoorly understood, making this work valuable. Furthermore, the in vivo\ninhibition of BMX appears to be a promising host-directed therapy (HDT)\nstrategy.\nResponse: We are grateful to you for your favorable comments on our manuscript.\nBelow are some suggestions to improve the quality of the manuscript:\nIn the first section of the results, the authors conclude that the use of\nbafilomycin to inhibit V-ATPase demonstrates that Rv1184c inhibits\nlysosomal acidification via this pathway. This conclusion seems a bit\npremature and could benefit from an additional experimental approach to\nconfirm the hypothesis, even if later experiments do support it further.\nResponse: We sincerely thank you for raising this important point. We fully agree\nthat lysosomal acidification is a highly regulated process involving multiple\ncoordinated mechanisms. Among these, V-ATPase is essential for lysosomal\nacidification, as it is the sole transporter that pumps protons into the organelle.\nGiven this, we first investigated the role of the V-ATPase in the mechanism of\nlysosomal acidification regulated by Rv1184c.\nThe use of Bafilomycin A1 (Baf-A1), a specific inhibitor of V-ATPase, to inhibit\nlysosomal acidification and perform functional rescue experiments is a well-\nestablished and widely adopted strategy to validate whether a molecular\nmechanism acts through V-ATPase-mediated acidification[1]. Our results\ndemonstrate that treatment with Baf-A1 rescues the lysosomal acidification defect\nphenotype caused by the H37RvΔRv1184c mutant strain (Fig. 1g, please see\nbelow). This supports the conclusion that Rv1184c inhibits lysosomal acidification\nspecifically via the V-ATPase pathway.\nNote：Chp2 (Rv1184c)\nIn addition to the inhibitor experiments, we have also provided genetic evidence\nand specified subunit involved. We generated Atp6v1e1 knockout mice and\nevaluated lysosomal acidification in macrophages infected with either H37Rv or\nH37RvΔRv1184c. The results clearly indicate that the regulatory effect of Rv1184c\non lysosomal acidification is dependent on the E1 subunit of the V-ATPase (Fig.\n3q, r, please see below). The detail description of this analysis was provided in the\nrevised results section (Line 200-205).\nNote：Chp2 (Rv1184c)\nLine 129: Infection in macrophages was monitored for 24 hours. This time\npoint may be too early—what happens at later time points, such as 3 or 4 days\npost-infection?\nResponse: Thank you for this valuable comment regarding the infection time\ncourse. We agree that assessing later time points is crucial to ensure the observed\nphenotype is sustained and not a transient effect. In response to your suggestion,\nwe extended the infection time of the intracellular survival assays to 4 days. The\nresults consistently demonstrated that the increased clearance phenotype (reduced\nCFU) compared to the H37Rv was maintained at 96 hours for H37RvΔRv1184c\n(Fig. 2a, b), confirming that the inhibitory effect of Rv1184c defect on the\nintracellular survival of Mtb observed at 24 hours is robust and representative.\nThe new data (please see below) from the extended time course have been replaced\nthe original data and figure legend have been updated in the revised manuscript.\nNote：Chp2 (Rv1184c)\nLine 131: Please explain the role of the NH₄Cl control.\nResponse: Thank you for raising this point. We apologize for the lack of clarity in\nour manuscript.\nThe NH Cl control was included to chemically inhibit lysosomal acidification and\n4\nthereby block lysosomal function. As a weak base, NH Cl diffuses into acidic\n4\ncompartments (mainly lysosomes) and raises the intravascular pH, which disrupts\nthe activity of acid-dependent hydrolases and impairs the degradative capacity of\nthe lysosome[2-4].\nThe purpose of including this control was to serve as a positive functional\nbenchmark for a lysosomal impairment phenotype. Our hypothesis was that if\nRv1184c promotes bacterial survival primarily by compromising lysosomal\nfunction, then in macrophages with intact lysosomal function (untreated with\nNH Cl), the Rv1184c mutant, which preserves lysosomal function, should be\n4\ncleared more efficiently than the wild-type strain. Artificially impairing lysosomes\nwith NH Cl should phenocopy the effect of the Rv1184c protein and rescue the\n4\nsurvival defect of the mutant. In other words, in NH Cl-treated macrophages where\n4\nlysosomes are inhibited, the difference in survival between the wild-type bacteria\nand the Rv1184c mutant should be abolished.\nFurthermore, this initial pharmacological evidence with NH Cl laid the\n4\ngroundwork for our subsequent, more specific investigations using both\npharmacological (Baf-A1) and genetic (Atp6v1e1+/-) inhibition of the V-ATPase,\nwhich collectively and robustly demonstrate that Rv1184c functions through this\npathway.\nWe have added an explanation of the NH Cl control's function in the result section\n4\nof the revised manuscript (Line 139-141).\nThe results show increased acidification during infection with the Rv1184c-\ndeficient mutant strain, and that this strain is attenuated in macrophages. Is\nit possible to provide imaging data showing co-localization between bacteria\nand Lysotracker under these conditions? For instance, is there increased co-\nlocalization of the Rv1184c deletion mutant with Lysotracker compared to\nwild-type Mtb?\nResponse: Thank you for this excellent suggestion. We agree that providing direct\nimaging evidence for the increased lysosomal acidification and colocalization\nwould greatly strengthen our conclusions.\nAs you recommended, we performed confocal microscopy experiments on\nmacrophages infected with either wild-type H37Rv or the Rv1184c deletion mutant\nstrains. Both bacterial strains were pre-labeled with FITC prior to infection. At 4\nhours post-infection, the macrophages were stained with LysoTracker-Red to\nvisualize acidic compartments. Consistent with our LysoTracker staining intensity\ndata, we observed a significant increase in the colocalization of the Rv1184c\nmutant with LysoTracker-positive compartments compared to the wild-type\nbacteria (Supplementary Fig. 1c, d).\nWe have incorporated these new findings (please see below) in the revised\nmanuscript, which provide direct visual and quantitative evidence that the Rv1184c\ndeletion mutant is targeted to acidic lysosomes more frequently than the wild-type\nstrain, which strongly supports our model that the Rv1184c protein functions to\ninhibit acidification.\nNote：Chp2 (Rv1184c)\nLine 135: Could you comment on the reduced immune cell infiltration? Which\nimmune cell types are affected? Could this be assessed by flow cytometry?\nResponse: We thank you for this question regarding the specific immune cell types\naffected.\nThe observed reduction in overall immune cell infiltration is a key in vivo\nconsequence of the enhanced bacterial clearance mediated by the Rv1184c deletion\nmutant. By promoting lysosomal acidification and degradation within\nmacrophages during the critical early stage of infection, the mutant strain is\ncontained more effectively. This superior early control leads to a lower overall\nbacterial burden and a subsequently attenuated inflammatory response in the lungs,\nwhich manifests as reduced recruitment of immune cells.\nTo identify the specific cell populations affected, we performed multiplex\nimmunohistochemistry (mIHC) on the same lung sections from Fig. 2g. The results\n(Supplementary Fig. 2c, d) quantitatively demonstrate a significant reduction in\nboth innate and adaptive immune cells, including CD11B+\nmonocytes/macrophages, MPO+ neutrophils, CD3+ T cells and CD20+ B cells,\nconsistent with the resolution of a more effectively controlled infection.\nThank you for this insightful suggestion regarding the use of flow cytometry to\nassess immune cell infiltration. We agree that flow cytometry is an excellent\nmethod for immune cell profiling. In our study, we ultimately chose multiplex\nimmunohistochemistry instead of flow cytometry primarily because it allowed us\nto use the same formalin-fixed, paraffin-embedded lung tissue sections from the\nsame batch of animal experiments as those analyzed in Fig. 2g. This approach\nensured sample source consistency and strengthened the comparability of results\nacross different parts of the study.\nWe have incorporated these new findings (please see below) and detail description\nof this analysis in the revised result section (Line 146-148).\nNote：Chp2 (Rv1184c)\nLine 145: Again, I am not fully convinced by the bafilomycin experiment.\nInhibiting V-ATPase will necessarily favor infection and may mask the effect\nof the mutation. Could you consider an alternative readout?\nResponse: We sincerely appreciate the your constructive feedback. Bafilomycin\nA1 (Baf-A1) is a V-ATPase inhibitor. As such, employing Baf-A1 to inhibit\nlysosomal acidification for functional rescue experiments represents the most\ncommon strategy for validating whether a molecule exerts its role by regulating V-\nATPase-mediated lysosomal acidification[5]. Based on our hypothesis that\nRv1184c promotes Mtb intracellular survival by suppressing lysosomal\nacidification, we reasoned that Baf-A1 treatment should rescue the survival defect\nof the H37RvΔRv1184c mutant, restoring its bacterial burden (CFU) to a level\ncomparable to that of the wild-type H37Rv infection (Fig. 2d).\nNote：Chp2 (Rv1184c)\nHowever, we also recognize that Baf-A1 treatment causes a global V-ATPase\ninhibition, thereby potentially obscuring the specific role of Rv1184c. Therefore,\nafter identifying ATP6V1E1 as the specific V-ATPase subunit targeted by Rv1184c,\nwe employed a more targeted genetic approach to substantiate our findings. We\ngenerated Atp6v1e1+/- mice and evaluated both lysosomal acidification and\nintracellular bacterial survival in macrophages from these mice. The results\ndemonstrated that the regulatory effect of Rv1184c on lysosomal acidification and\nintracellular bacterial clearance is specifically dependent on ATP6V1E1(Fig. 3q-\ns), thereby confirming that Rv1184c exerts its function through this subunit of the\nV-ATPase complex.\nAdditionally, we recognize that lysosomal function is closely linked to multiple\ncellular processes-such as lipid metabolism, energy balance, and\napoptosis/necrosis-all of which can directly or indirectly influence Mtb survival\nwithin host cells. Although we don’t explicitly investigate whether these pathways\nare differentially affected, we indeed cannot exclude the possibility that Rv1184c-\nmediated suppression of lysosomal acidification may indirectly modulate these\nprocesses, thereby contributing to enhanced bacterial survival in vivo.\nThe detail description of this analysis was provided in the revised result section\n(Line 200-207).\nNote：Chp2 (Rv1184c)\nIs it possible to visualize or measure lysosomal acidification in alveolar\nmacrophages from infected mice?\nResponse: Thank you for this excellent and challenging question. We agree that\ndirectly assessing lysosomal acidification in alveolar macrophages from infected\nmice would provide invaluable in vivo validation of our model. Unfortunately,\nusing LysoTracker or pHrodo to measure lysosomal acidification require live-cell\nimaging. Our Biosafety Level 3 (BSL-3) laboratory is unable to perform this test.\nWe sincerely appreciate your understanding and have acknowledged this limitation\nin the discussion section of the manuscript.\nLine 180: The in vivo results are interesting. Have you considered infecting\nAtp6v1E1 knockout mice with the Rv1184c mutant strain? It would be\ninteresting to discuss the expected results.\nResponse: Thank you for this excellent suggestion. We agree that using\nAtp6v1e1+/- mice infected with the Rv1184c mutant strain would be a fascinating\nand direct experiment to further solidify our proposed mechanism.\nAs per your suggestion, we infected Atp6v1e1+/- mice with H37Rv or\nH37RvΔRv1184c to further validate in vivo relevance of Rv1184c and ATP6V1E1.\nThe results showed knockout of Atp6v1e1 markedly increased bacterial burden in\nlung tissues of the Mtb H37Rv-infected mice, and abolished the increased bacterial\nburden by Rv1184c in lung tissues of the Mtb-infected mice (Fig. 3u,\nSupplementary Fig. 3p), suggesting Rv1184c promotes intracellular survival of\nMtb in vivo by targeting ATP6V1E1. The new data (please see below) and analysis\n(Line210-214) were included in the revised result section.\nNote：Chp2 (Rv1184c)\nBMX inhibition shows very promising data. Could this therapy be combined\nwith standard antibiotic treatment to test for potential synergy?\nResponse: We thank you for this excellent suggestion regarding potential\ncombination therapy of BMX inhibitor. To investigate this, we treated Mtb-infected\nmacrophages with the BMX inhibitor alone or in combination with rifampicin, the\nmost commonly used first-line anti-tuberculosis drug. The results demonstrated\nthis combination failed to enhance bactericidal efficacy against intracellular Mtb\n(please see below), suggesting there is no synergistic interaction between the BMX\ninhibitor and rifampicin. While we acknowledge the limited scope of drugs tested\nin combination with the BMX inhibitor, this does not rule out potential synergy\nwith other anti-tuberculosis drugs, which we will explore in future work.\nONPPONPP\nD M B S M X-I\nX\nR -I N F + R F D M B S M X-I\nX\nR -I N F + R F\nM M\nB B\nWhat would be the effect of BMX inhibition in mice infected with the Rv1184c\nmutant strain?\nResponse: We thank you for this excellent suggestion. To address this, we infected\nmice with either H37Rv or H37RvΔRv1184c. After infection two weeks, we\ninitiated BMX inhibitor treatment and assessed the outcomes two weeks later. The\nresults showed that the BMX inhibitor significantly reduced the bacterial burden\nand pathological damage in mice infected with wild-type H37Rv. Crucially, this\ntherapeutic effect was abolished in mice infected with the H37RvΔRv1184c mutant\n)401(UFC\n8\n6\n4\n2\n0\n4\n=\nh\n0 .0 0 0 5\nP = 0 .1\n4 8\nP =\n2 7\nh\n0 .0 0 0 5\nP = 0 .1 5 2 3\n(Fig. 7f-i).\nThis finding indicates that the efficacy of the BMX inhibitor is dependent on the\npresence of the bacterial effector Rv1184c. It supports a model wherein the\ninhibitor acts by targeting the host pathway that is subverted by Rv1184c, thus\nneutralizing the bacterium's virulence strategy. This suggests that host-directed\ntherapy (HDT) with BMX inhibitors could be particularly effective against Mtb\nstrains that rely on this specific mechanism.\nWe have added these new data (please see below) and the corresponding analysis\n(Lines 370-384) to the revised manuscript.\nNote：Chp2 (Rv1184c)\nSimilarly, what would be the effect of PLGA-aNP treatment in Atp6v1E1\nknockout)\nResponse: Thank you for this insightful suggestion. Investigating the effect of\nPLGA-aNP treatment in an Atp6v1e1 knockout model would indeed be a\nfascinating experiment to further dissect the mechanism of action, and we\nappreciate you raising this idea.\nDuring the revision process, and in an effort to sharpen the focus of the manuscript\non the novel Rv1184c-BMX-ATP6V1E1 signaling axis we have identified, we\nmade the decision to remove the PLGA-aNP treatment data. While these data were\ninteresting, we agreed that its mechanism was not directly upstream or downstream\nof the Rv1184c-BMX-ATP6V1E1 pathway that is the central focus of this study,\nand its inclusion diluted the main narrative, which is also pointed out by reviewer\n2#.\nAlthough this specific experiment is not included in the current manuscript, we\nhave carefully noted your suggestion regarding the Atp6v1e1 knockout. It\nrepresents an excellent direction for a future study to understand how modulating\nlysosomal pH intersects with specific host pathways for antimicrobial activity. We\nsincerely appreciate your guidance on this point and plan to pursue it in our\nsubsequent work.\nReviewer #2: (Remarks to the Author):\nThis manuscript by Chen et al, titled “Mycobacterium tuberculosis\nmanipulates phosphorylation of ATP6V1E1 to promote its\nintracellular survival”, shows an important phosphorylation-dependent regulation of\nlysosomal acidification during Mtb infection, with the potential to regulate\nMtb survival inside the infected macrophages. The authors show that Mtb\nsecreted protein Rv1184c facilitates interaction between tyrosine kinase BMX\nand the vATPase subunit ATP6v1e1, leading to increased phosphorylation of\nATP6v1e1 and corresponding decline in the lysosomal acidification, resulting\nin less efficient bacterial killing. In view of that, inhibiting BMX kinase in the\nanimal models shows a decrease in lung CFU burden, which is dependent on\nthe protein ATP6v1e1.\nThe regulation of lysosomal activity by Mtb during infection has been under\nimmense focus for investigation due to its direct role in killing intracellular\nbacterial infections. The current manuscript provides interesting insights into\nthis key battle between the host and the pathogen. The authors generate useful\nmodels to address the specific questions asked in this study.\nResponse: We appreciate your supportive assessment of our work.\nHowever, the study suffers from some serious limitations. There are some\ngeneral observations and specific concerns pointed out below.\nGeneral observations: There is a lack of details on the experiments and results.\nFor studies on Mtb infection, cell/cell-lines used, the time points post-infection,\nduration of treatment, etc., are vital details. These details are missing even in\nthe methods section. Some of the results reflect a less robust experimental\nstrategy, leading to deviations from the expected line.\nDifferent experiments are done on different cell lines/types. The rationale for\nthose is not obvious. Cells used for the experiments are not explicitly\nmentioned.\nResponse: We thank you for this critical assessment and the opportunity to\nstrengthen our manuscript. We sincerely apologize for the lack of essential details\nand the perceived lack of robustness in our experimental strategy.\nWe have thoroughly revised the manuscript to address these general concerns\ncomprehensively. The changes are detailed below.\nWe have now extensively expanded the Methods section to include all missing\ncritical details, including the complete cell line information, the precise time points,\nthe treatment durations, and the step-by-step protocols. Furthermore, we have\nrevised that all figure legends now explicitly state the specific cell type and key\nexperimental conditions (e.g., MOI, time points).\nWe have revised the main text to clearly articulate the scientific rationale behind\neach cell model selection, demonstrating how their complementary use provides a\nmore robust and physiologically relevant validation of our findings.\nAll the modifications mentioned above have been incorporated into the manuscript\nand are highlighted in red for easy identification. We believe they significantly\nimprove the manuscript's rigor and clarity.\nMajor limitations:\nThe ability of Mtb to block phagosome maturation and lysosomal acidification\nis well-documented. However, throughout this study, a significantly higher\nnumber of bacilli is seen in acidified lysosomes, which, of course, increases\nupon loss of Rv1184c. The observed phenotype is difficult to interpret due to\nthe absence of any staining for the Mtb, which would allow assessing bacterial\npresence in acidified lysosomes.\nResponse: We thank you for this critical insight. We agree that bacterial staining\nis essential to definitively link the observed increase in lysosomal acidification to\nthe location of the bacteria themselves.\nAs per your suggestion, we performed confocal microscopy with FITC-labeled\nbacteria and LysoTracker-Red in macrophages. We observed a significantly higher\ndegree of colocalization between the FITC-labeled H37RvΔRv1184 and the\nLysoTracker-positive compartments compared to the wild-type H37Rv\n(Supplementary Fig. 1c). In addition, we have re-examined the Mtb staining for\nthe other lysosomal acidification (Supplementary Fig. 1d, Supplementary Fig.\n3j, o and Supplementary Fig. 4b)\nThese new data directly confirm that the increased lysosomal acidification we\nreported previously is indeed associated with the bacilli themselves, and that loss\nof Rv1184c leads to a significantly higher proportion of bacteria being trapped in\nacidified lysosomes. This provides strong visual and quantitative support for our\nmodel that the Rv1184c protein is essential for Mtb to inhibit phagolysosomal\nmaturation.\nNote：Chp2 (Rv1184c)\nConclusions for a few results and interpretations made thereof are not aligned\nwith the experimental observations.\nResponse: We thank you for this general comment regarding the alignment of our\nconclusions with the experimental data. We agree that maintaining this precision is\ncritical for the scientific rigor of the manuscript.\nWe therefore have conducted a thorough, line-by-line review of the entire text to\nensure all interpretations are strictly constrained by the data presented. This\ninvolved refining quantitative analyses, ensuring all controls are appropriately\nreferenced, and calibrating the language in the text to ensure all interpretations are\nfully and clearly supported by the observations. We believe these revisions have\nsignificantly improved the accuracy and rigor of the manuscript. We are happy to\nprovide a point-by-point response if you could kindly specify any particular\ninstances where they perceived a misalignment, so that we may address them with\nutmost precision.\nThe phenotype of ATP6v1e1 knockout mice is not well explained. With a loss\nof lysosomal function, in these animals, even without infection, they should\nshow lysosomal storage disorder phenotype.\nResponse: We sincerely thank you for emphasizing this critical point, which was\nraised both as a major concern in this section and again in the specific comments.\nAs rightly highlighted, the homozygous deletion of Atp6v1e1 results in embryonic\nlethality, a finding consistent with the MGI database. Cyagen, which is a global\nprovider of genetically modified rodent models, also reported Atp6v1e1 complete\nknockout mice is embryonic lethality (official link:\nhttps://www.cyagen.com/mouseatlas/S-KO-01171). This phenotype underscoring\nthe gene's essential role in development and lysosomal function, much like what\nhas been observed with other core lysosomal genes such as LAMP2 and CSTD.\nAlthough complete knockout of Atp6v1e1 results in embryonic lethality, Atp6v1e1\nheterozygous mice appear normal and do not exhibit lysosomal storage disorder\nphenotypes (Supplementary Fig. 3g). Detection of V1E1 protein expression in\nperitoneal macrophages of uninfected mice showed that the protein level in\nheterozygous mice was significantly reduced, but approximately 50% of the wild-\ntype level remained (Supplementary Fig. 3e, f). We propose that the reduction in\nprotein dosage from a single allele is sufficiently compensated under basal\nconditions, likely through adaptive mechanisms that maintain minimal functional\nV-ATPase activity.\nWe are grateful for the opportunity to clarify this important matter and have revised\nthe manuscript to include the new data (please see below) and a detailed discussion\nof these points (Line 179-182).\nSpecific points:\nQuestion 1: How ATP6v1e1 overexpression alone is able to cause significant\nlysosomal acidification in 293T cells? The vacuolar ATPase is a multi-subunit\nprotein, and the catalytic site, formed by the V1 domain, consists of all\nsubunits. This needs more clarity.\nResponse: We sincerely thank you for this insightful question. You rightly points\nout V-ATPase is indeed a multi-subunit complex. Our data showed that among the\nindividual V1 subunits overexpressed, only E1 subunit significantly enhanced\nlysosomal acidification. We propose the explanations for this specific effect of\nV1E1 as below:\nThe functional V-ATPase complex requires the assembly of subunits in a precise\nstoichiometry. Research by Mark A. Compton et al. using a yeast model\ndemonstrated that Vma9p (subunit e) acts as a rate-limiting factor in V-ATPase\nassembly. Disruption of the yeast VMA9 gene prevents V₁ and V₀ subunits from\nassembling at the vacuolar membrane, while its overexpression enhances complex\nformation and boosts proton pump activity [6]. Vma9p is a small hydrophobic\nprotein that is conserved from fungi to animals. Therefore, overexpression of the\nE1 subunit may indirectly enhance lysosomal acidification capacity by promoting\nthe efficiency of V-ATPase assembly. Our findings indicate that phosphorylation\nof V1E1 can significantly influence the efficiency of V-ATPase assembly. We\nhypothesize that V1E1 overexpression, unlike other subunits, may bypass the\nnormal stoichiometric constraints by acting as a rate-limiting assembly factor or a\nnucleating agent, thereby promoting the assembly of more functional complexes\nand enhancing proton pumping. We have added the analysis in the Discussion\nsection of the revised manuscript (Line 404-409).\nQuestion 2: What happens to ATP6ve1 expression upon Mtb infection, and\nwhether that differs from the expression of other subunits?\nResponse: Thank you for raising this important question. As per your suggestion,\nwe infected peritoneal macrophage with Mtb and detect the subunits expression.\nWe found that the mRNA expression level of the ATP6V1E1 subunit did not change\nsignificantly at the time points post-Mtb infection that we examined. More\nimportantly, the mRNA expression of other V-ATPase subunits (including other V1\ndomain subunits such as ATP6V1A, ATP6V1B2, ATP6V1C1, ATP6V1D, etc.) also\nshowed no statistically significant differences before and after infection (please see\nbelow).\nTogether, these data indicate that, at the mRNA level and under our experimental\nconditions, Mtb infection does not significantly regulate the expression of\nATP6V1E1 or other V-ATPase subunits. Mtb is unlikely to affect V-ATPase\nfunction via transcriptional modulation of these subunits.\nQuestion 3: For lysosomal acidification during Mtb infection, there is no\nstaining for Mtb (Fig. 1g, 3q, fig. S3g, etc.). The virulent Mtb is known to block\nlysosomal acidification through PMA. Since MOI used is 5, it is important to\nshow that the cells analysed/being shown are also Mtb- infected.\nResponse: Thank you for this critical insight, which is also concerned by reviewer\n1#. We agree that Mtb staining is important to confirm the analyzed cells are\ninfected by Mtb and the increased lysosomal acidification is indeed associated with\nthe bacilli themselves. As per your suggestion, we performed additional\nexperiments to assess the co-localization of Mtb with lysosomes. This was achieved\nby pre-labeling Mtb with FITC and staining the cells with LysoTracker Red. Mtb\nstaining for the other lysosomal acidification data was also re-examine. The new\ndata demonstrate the presence of Mtb within the analyzed cell population\n(Supplementary Fig. 1c, d, Supplementary Fig. 3 j, o, Supplementary Fig. 4b),\nthus confirming that the measured lysosomal acidification occurs in infected cells.\nThis supplemental data strengthens our interpretation by directly demonstrating the\nco-localization of Mtb with acidified compartments in the analyzed cell population.\nWe apologize for this omission in the original submission and are grateful for the\nopportunity to clarify this crucial point.\nNote：Chp2 (Rv1184c)\nQuestion 4: Why should H37Rv CFU decline in WT cells from 0 hour to 24\nhours post-infection? This is a critical observation, Mtb CFU is generally\nstable or grows during 24 hours, however a decline in CFU indicates loss of\nvirulence. Have the authors checked PDIM levels of the strains used? The\nobserved dip in Mtb CFU in the WT cells needs to be analysed more robustly.\nResponse: Thank you for raising this important point. We appreciate the\nopportunity to clarify this question. Indeed, Jennifer A. Philips et al [7] and Dai Y\net al [8] reported an increase in Mtb CFU in H37Rv-infected bone marrow derived\nmarcrophages(BMDMs) from WT mice for 48h or more. Ge P et al.[9] found Mtb\nCFU in H37Rv-infected U937 cells had increased within 0 to 24 hours. However,\nour data exhibited a decrease in Mtb CFU in H37Rv-infected primary peritoneal\nmacrophages from mice at 24 hours. Consistent with our findings, several other\nresearch using peritoneal macrophages have also reported a declining trend in\nH37Rv CFU post-infection[10] [11]. Thus, the difference of our data with some\npublished literature could be due to infection of different type of macrophages.\nFurthermore, studies by Mosser, D. M., & Edwards, J. P.[12] have also confirmed\nthat the bactericidal capacity of macrophages is associated with their specific type.\nThis early CFU drop in peritoneal macrophages reflects the host's innate immune\nresponse, including phagolysosomal maturation and acidification, which\nsuccessfully clears a portion of the intracellular Mtb. Subsequent bacterial\nreplication, often observed after 24-48 hours, signifies the ability of surviving\nbacilli to overcome these host defenses. Therefore, the initial CFU decrease we\nobserved is not indicative of a loss of virulence in our H37Rv strain but rather\nconfirms the expected and validated response of competent macrophages to Mtb\nchallenge.\nFurthermore, we repeated the intracellular survival assay in BMDMs. The results\nshowed an increasing trend in H37Rv CFU between 24 and 48 hours post-infection,\nwhile the CFU of H37RvΔRv1184c remained relatively stable. Deletion of\nRv1184c dramatically reduced the intracellular bacterial load compared to the\nH37Rv control (please see below), suggesting that Rv1184c promotes the\nintracellular survival of Mtb in BMDMs, consistent with the observations in\nprimary peritoneal macrophages.\nP<0.0001\n) 10\n1\n\n(\nU\nF\nC 5\n0\n2h 24h 48h\nBMDM\nNote：Chp2 (Rv1184c)\nQuestion 5: Tissue sections from uninfected ATP6v1e1 should also be shown.\nWith the loss of lysosomal function, severe pathology may develop due to\nlysosomal storage disorder like condition.\nResponse: We thank you for this important suggestion. We agree that assessing\nbasal pathology is crucial.\nAs we mentioned in the previous response, the homozygous Atp6v1e1 knockout is\nembryonically lethal (MGI: 6148313), a definitive severe phenotype. Although we\ndid not further investigate the exact cause of death, we speculate that it may be\nattributed to severe impairment of lysosomal function caused by the absence of\nATP6V1E1. In our study, we used heterozygous Atp6v1e1 deletion mice. As per\nyour suggestion, we examined lung sections from uninfected Atp6v1e1+/- mice and\nfound no signs of spontaneous pathology or storage disease, indicating that the\npartial gene dosage reduction is functionally compensated under baseline\nconditions (Supplementary Fig. 3g).\nThe phenotypic consequence of this haploinsufficiency is, however, unmasked\nunder stress. We propose that the heightened lysosomal demand during Mtb\ninfection exceeds this compensatory capacity, leading to the specific defects in\nacidification and bacterial clearance that we report. This clarifies that the\nAtp6v1e1+/- model reveals a context-dependent vulnerability, not a constitutive\ndisorder. We have added this data and discussion to the manuscript (Lines 182-\n186).\nLine 178: Lysotracker staining shown in WT H37Rv infection is too high.\nH37Rv blocks lysosomal acidification. Secondly, fig. 3q-r: the lysotracker\nstaining upon H37Rv infection visible in the images at 0 hours and 4 hours in\nthe WT and ATP6v1e1-/- cells (3q) is not reflected in the corresponding\nquantification plot (3r). This is very concerning.\nResponse: We thank you for these critical observations, which allow us to clarify\nkey aspects of our experimental system and data presentation.\nRegarding the level of LysoTracker signal in H37Rv-infected cells, you are correct\nthat H37Rv employs strategies to inhibit lysosomal acidification. However, the\nfinal lysosomal pH represents a dynamic equilibrium between the host's efforts to\nacidify the compartment and the bacterium's efforts to neutralize it. In our\nexperimental conditions, the equilibrium pH remains within the acidic range\ndetectable by LysoTracker. This is consistent with findings from other groups.\nFor instance, Gaur RL,et.al [13] and Sachdeva K et al.,[14] have also reported\npositive LysoTracker staining in H37Rv-infected cells with similar fluorescence\nintensity.\nOur model posits that the wild-type strain, secreting Rv1184c and other effectors,\nshifts this balance toward a more neutral pH. In contrast, the ΔRv1184c mutant is\ndefective in this inhibition, resulting in a failure to counteract host acidification and\nthus a higher observed LysoTracker signal. The signal in WT infection is therefore\nnot artifactually high but reflects the outcome of this active host-pathogen struggle.\nRegarding the apparent discrepancy between the representative images in Fig. 3q\nand the quantification in Fig. 3r, we sincerely apologize for the confusion caused\nby the suboptimal presentation of the data in the original Fig. 3r. To address this\nissue, we have revised the presentation of this panel. The revised figure (please see\nbelow) clearly shows that in WT macrophages, H37RvΔ1184 infection\nsignificantly promotes lysosomal acidification compared with H37Rv; however,\nthis enhancing effect was attenuated in Atp6v1e1+/- macrophages. Although the\nconclusion in the revised Fig. 3r remains consistent with the previous version, the\ndata presentation has been improved and is now clearer.\nWe have incorporated the revised figure into the updated manuscript. We sincerely\nthank you for this valuable suggestion, which has significantly improved the clarity\nand quality of our data presentation.\nNote：Chp2 (Rv1184c)\nFig 6a: Rv1184c deletion: the impact on ATP6v1e1 phosphorylation is not very\ndistinct from the WT infection.\nResponse: Thank you for your careful observation, which is highly valuable.\nDuring our assessment of ATP6v1e1 phosphorylation levels, we also\nsimultaneously measured the level of ATP6V1E1 protein, although those results\nwere not shown. To address this concern directly and unequivocally, we have\nconducted exact densitometric analysis of the phospho-ATP6V1E1 signal with\nnormalization to total ATP6V1E1.\nThe quantified data demonstrated a decrease in ATP6V1E1 phosphorylation in\nmacrophages infected with the ΔRv1184c mutant strain compared to those infected\nwith H37Rv (Fig. 6a). These results indicate that Rv1184c protein is indeed\nrequired for the full induction of ATP6V1E1 phosphorylation during Mtb infection.\nThe quantitative data now provides clear support for our conclusion.\nTo avoid the limitations of visual assessment in evaluating ATP6V1E1\nphosphorylation, we have provided the total ATP6V1E1 protein levels and\nsupplemented all of the densitometric quantification data for the phospho-\nATP6V1E1.\nWe have updated the text in the Results section to reflect this quantification (please\nsee below). We are grateful to you for prompting us to strengthen this critical piece\nof evidence.\nNote：Chp2 (Rv1184c)\nThe Rv1484c knockout and complementation data is clean. However, authors\nneed to show that sufficient Rv1484c gets secreted during infection in\nmacrophages to be able to interact with and alter ATP6v1e1 phosphorylation.\nSo far all such evidences are shown using overexpression experiments but\nwhether expression and secretion of native protein is sufficient to alter\nATP6v1e1 phosphorylation and lysosome acidification?\nResponse: Thank you for this critical question regarding the secretion and\nfunctionality of natively expressed Rv1184c. We agree that demonstrating this\nunder physiological infection conditions is essential.\nIn our original submission, we included a Co-IP experiment (Fig. 6j) from\nmacrophages infected with wild-type Mtb H37Rv, which showed that endogenous\nATP6V1E1 co-precipitates with Rv1184c. This provided initial evidence for a\nnative interaction.\nTo directly address your point and further strengthen this conclusion, we have now\nperformed additional immunofluorescence experiments. As shown in the new data,\nstaining with a specific anti-Rv1184c antibody visually confirms that the protein is\nsecreted into the host cytoplasm during infection and partially co-localizes with\nLysoTracker, providing spatial confirmation of its translocation (Supplementary\nFig. 5a).\nTogether, the Co-IP (Fig. 6j) and new IF data (Supplementary Fig. 5a) form a\ncomplementary and robust body of evidence, demonstrating that natively\nexpressed and secreted Rv1184c is present at functionally relevant levels to interact\nwith its host target. We have added this new data and analysis to the revised\nmanuscript (Line 344-346) and are grateful for your suggestion, which has\nsignificantly strengthened this part of our study.\nNote：Chp2 (Rv1184c)\nsiBMX treatment: How many hours after BMX knockdown infection was\ngiven? What does 0 hour mean here? What is the knockdown efficiency of\nBMX siRNA? It is strange to see a complete loss of ATP6v1e1 phosphorylation.\nResponse: We thank you for these important questions, which allow us to clarify\nthe experimental details and provide additional validation data. In our study, cells\nwere transfected with siRNA targeting Bmx (or non-targeting control siRNA) for\n48 hours to achieve optimal gene silencing prior to Mtb infection. The time point\nin the immunoblot analysis refers to the timing of Mtb infection in cells transfected\nwith Bmx siRNA for 48 hours. The\"0 hour\" time point refers to cells transfected\nwith Bmx siRNA for 48 hours but not infected with Mtb.\nWe apologize for not including the Bmx siRNA knockdown efficiency data from\nthe original submission. In our study, we used a commercially available Bmx\nsiRNA (product code sc-38942, as noted in the Method section) from Santa Cruz\nBiotechnology, Inc. It is a pool of three target-specific 19-25 nt siRNAs designed\nfor gene knockdown and is a well-established and widely used reagent (Thomas,\nJ.D., et al.[15]; August, A., et al.[16]). Upon acquisition of the Bmx siRNA, we\nsubsequently assessed its knockdown efficiency at the mRNA levels. The results\ndemonstrated that the knockdown efficiency exceeds 50% (supplementary Fig.\n4a) (please see below).\nWe thank you for this keen observation for p-ATP6V1E1 blotting. We agree that\nthe original blot for p-ATP6V1E1 in the siRNA-Bmx group appeared unexpectedly\nweak, likely due to suboptimal lane ordering and exposure conditions. We have\nsince repeated the experiment with careful adjustments. The new, clear data\nconsistently show a significant decrease in p-ATP6V1E1 upon Bmx knockdown,\nrobustly supporting our initial conclusion (Fig. 6b) (please see below).\nThe consistency of this result-across both pharmacological inhibition (BMX-IN-1)\nand genetic knockdown (siRNA)-provides compelling and complementary\nevidence that BMX is the essential kinase for Mtb-induced ATP6V1E1\nphosphorylation.\nNote：Chp2 (Rv1184c)\nFor all phosphorylation immunoblots, GAPDH is not a correct loading control.\nThe control should be the ATP6v1e1 protein itself.\nResponse: We thank you for emphasizing the importance of showing total\nATP6V1E1 levels. We fully agree that showing the ATP6V1E1 protein itself is\ncrucial for confirming the specific changes in its phosphorylation. We would like\nto clarify that the ATP6V1E1 protein levels were in fact measured in parallel in all\nrelevant experiments and served as an internal control to confirm that the\nalterations in phosphorylation were not attributable to variations in total protein\nabundance. In the initial manuscript, to highlight the main findings, we primarily\npresented the phospho- ATP6V1E1 results and don’t show the total ATP6V1E1\nprotein data.\nFollowing your suggestion, we have added total ATP6V1E1 and performed\ndensitometric quantification of the phospho-ATP6V1E1 signal with normalization\nto total ATP6V1E1 for each sample lane. The results confirm that total protein\nlevels remain constant across conditions, solidifying our conclusion that the\nobserved band intensity differences are due to specific phosphorylation changes.\nThis revision has fully addressed the point and strengthened our manuscript.\nThe PLGA-aNP experiment is not relevant to the current study unless it is\nshown in combination with the BMX inhibitor.\nResponse: We thank you for this feedback. We agree with your assessment\nregarding the weak relevance of the PLGA-aNP (acid-sensitive nanoparticle) data\nwith the current manuscript, which is firmly focused on elucidating the role of the\nRv1184c-BMX-ATP6V1E1 axis in modulating lysosomal acidification.\nThe PLGA-aNP experiment was initially included as a broader conceptual proof\nthat enhancing lysosomal acidification could be a potential therapeutic strategy.\nHowever, during the revision process, we realized its mechanism is distinct from\nthe specific Rv1184c-BMX-ATP6V1E1 pathway that is the focus of this work, and\nits inclusion diluted the main narrative. We decided to remove it to maintain focus\nand prevent distraction from the core narrative.\nInvestigating the effect of PLGA-aNP combined with the BMX inhibitor would\nindeed be a fascinating experiment to further dissect the mechanism of PLGA-aNP\naction, and we appreciate you raising this idea. While those combination\nexperiments would be more appropriate as a separate and extensive study. It\nrepresents a promising strategy to explore the potential synergistic effects of\ncombining multiple HDT drugs that have proximate targets. Although this specific\nexperiment is not included in the current manuscript, we sincerely appreciate your\nguidance and plan to pursue it in our subsequent work.\nMinor points:\nTypo in line 233: “numerous pathological pathways”?\nResponse: We sincerely thank you for their meticulous reading and for identifying\nthis typographical error. The word “ nμerous ” has been corrected to “numerous”\nin the revised manuscript.\nWe appreciate your attention to detail, which has helped improve the accuracy of\nour manuscript.\nAuthors should use Grammarly or similar software for written English.\nResponse: We sincerely thank you for this helpful suggestion. We apologize for\nany language-related issues in the initial submission.\nIn response, we have thoroughly revised the entire manuscript with a focus on\nimproving the clarity, flow, and precision of the English language. We have enlisted\nthe help of several colleagues proficient in academic English and utilized\nprofessional editing tools to refine the text.\nWe believe these revisions have significantly enhanced the readability of the\nmanuscript, and we appreciate your suggestion, which has helped us improve the\noverall quality of our work.\nReviewer #3 (Remarks to the Author):\nThis manuscript by Chen J et al. uncovers a mechanism by which\nMycobacterium tuberculosis H37Rv (Mtb) manipulates phagolysosomal\nmaturation to avoid killing by macrophages. The authors show that Mtb\nprotein Rv1184 promotes BMX interaction with the v-H+-ATPase subunit\nATP6V1E1 thereby enhancing phosphorylation of ATP6V1E1 at tyrosine\nresidues. This inhibits the v-ATPase assembly at the membrane, dampening\nphagolysomal acidification and allowing Mtb survival. This manuscript\ntherefore highlights an additional mechanism by which Mtb manipulate\nphagosome maturation.\nResponse: We appreciate your pertinent comments.\nIn its large part, I found this MS very interesting and convincing. It provides\nmany details in the mechanism described with very neat experiments. My\nenthusiasm was somehow dampened by some flows in the logic that brought\nthe authors to realized some experiments. For example, what brought the\nauthors to assess phosphorylation fo ATP6V1E1 in the first place?\nResponse: We are sincerely grateful to you for your overall positive assessment of\nour work and for their meticulous reading of the manuscript. We also deeply\nappreciate them pointing out the specific issues, which we have addressed below.\nThe decision to assess the phosphorylation of ATP6V1E1 was driven by the central\nand well-established role of phosphorylation as a primary mechanism for the rapid\nand reversible regulation of protein function in virtually all cellular processes,\nincluding signal transduction. While the V-ATPase is essential for general\nacidification, emerging evidence suggests that specific subunits can be subject to\npost-translational modifications, allowing for nuanced regulation. As we\nmentioned in the discussion section of the text, it has been reported that the\nphosphorylation of V-ATPase subunits affects lysosomal acidification. For\nexample, the A subunit of V-ATPase is phosphorylated by protein kinase A (PKA),\nAMP-activated protein kinase (AMPK) or Aurora kinase A (AURKA), while the C\nsubunit of V-ATPase is phosphorylated by WNK (with no lysine (K)) protein\nkinases[17-19]. This led us to assess phosphorylation of ATP6V1E1 in the first\nplace. We add the analysis in the revised result section (Line 227-228).\nFurthermore, there is a duplication of images in Fig 7b and 7g: 1) Mock\ncontrols are the same images but it is not mentioned in the legend ; 2) BMX-\nIN-1 #3 is the same image as PLGA-aNP #2.\nResponse: Thank you for your meticulous review of our manuscript. We are\ndeeply grateful for the time and effort you have dedicated to it.\nWe sincerely apologize for this serious oversight in our figure preparation. You are\nabsolutely correct. The duplication of the images was a serious mistake that\noccurred during the assembly of the figure panels. This does not reflect a lack of\nindependent biological replicates for each condition but was an error in image\nselection and labeling. To correct this, we have thoroughly re-examined all our\noriginal raw image data and identified the correct, independent images for each\nspecific experimental condition (Mock, BMX-IN-1, and PLGA-aNP). We have\ngenerated a completely revised Figure 7 with the correct images in panels 7b and\n7g. However, as one of the reviewers considered the PLGA-aNP results to be not\nclosely related to the main narrative of the article, this section has been removed\nfrom the revised manuscript. To facilitate your review and verify the correctness\nof our revision, we provide the revised image data in the response letter (please see\nbelow).\nWe sincerely apologize for this oversight, which was unintentional. The corrected\nfigure is now included in the revised manuscript (Fig.7b). We are grateful that this\nwas caught during review, allowing us to correct the record and present an accurate\nrepresentation of our data.\nThank you again for your time and for providing feedback that has significantly\nimproved the clarity and accuracy of our manuscript.\nI must say that my expertise in histological HandE staining in the context of\nMtb infection is limited. Thus I could not really evaluate this part of the work.\nResponse: We thank you for this comment and sincerely apologize for the lack of\nsystematic interpretation of the H&E staining results in our original manuscript,\nwhich limited the clarity of our pathological analysis.\nTo ensure our pathological analysis is rigorous and clear, we have now provided a\ndetailed description of our scoring method in the Methods section (Line742-745).\nBriefly, pathology scores (0-10) were based on the ratio of inflammatory cell\ninfiltration area to the total lung section area on the maximal cross-section of a lung\nlobe, with heavily stained areas indicating infiltration and damage, and lighter areas\nrepresenting normal alveolar structure. Furthermore, to quantitatively characterize\nthe inflammatory infiltration, we performed multiplex immunohistochemistry for\nkey immune cell markers: CD3 (T cells), CD20 (B cells), MPO (neutrophils), and\nCD11b (monocytes/macrophages) (supplementary Fig. 2c, d). The consolidated\ndata robustly demonstrate that the deletion of Rv1184c limits the overall\ninflammatory infiltration in the host lungs.\nNote：Chp2 (Rv1184c)\nSome suggestions/comments that may improve the study:\nFig 1b, do the authors found CISH and PtpA in their screen?\nResponse: We thank you for raising the point about CISH and PtpA, two known\ninhibitors of phagosome acidification. Our initial screening plasmid library, while\nlarge-scale and validated in previous studies[10, 11], did not have 100% coverage\nof the Mtb secretome and unfortunately did not include CISH and PtpA.\nImportantly, our screening system was robust enough to identify other well-\nestablished regulators of lysosomal acidification, such as LpqH (Rv3763). This\nconfirms the effectiveness of our approach, which successfully led us to identify\nRv1184c as a novel player in this process. We have noted the library's coverage in\nthe revised manuscript.\nWhat is the effect of Rv1184c by itself upon injection in mice?\nResponse: We thank you for this question. To test the systemic effect of Rv1184c\nalone, we intravenously or intranasally injected purified protein into naive mice.\nHistopathological analysis revealed no significant lung damage compared to\ncontrols (Supplementary Fig. 3q, r) (please see blew). This indicates that\nRv1184c is not a general toxin but a precise effector whose major function, as our\nstudy shows, depends on its specific interaction with host ATP6V1E1 during\ninfection. Again, we thank you for suggesting this experiment, which has provided\nvaluable insight into the specific nature of this host-pathogen interaction.\nIn Fig 3B, I understand that the authors over-expressed FLAG-Rv1184c\ntogether with HA-tagged subunits of the v-ATPase. However, it might be good\nto provide more details in the text. It is hard to apprciate the relevance of the\nblot shown since MW are missing (Fig 3B and C). Whole lysate indicates a\nproblem of expression of some HA-tagged v-ATPase subunits. How does this\naffect the conclusions made by the authors? Temporality of the interaction?\nWhy the other subunits do not appear in the immuno precipitate?\nResponse: We thank you for these insightful comments, which have allowed us to\nsignificantly strengthen this part of our study.\nFor the molecular weights and specificity, we apologize for the initial omission of\nmolecular weight markers. In the revised manuscript, molecular weight markers\nhave been added to all western blot results. Regarding the variable expression of\nthe HA-tagged subunits of V1 domain, we have repeated the co-IP experiments\nwith an expanded panel including all of the V1 subunits. The new data confirm that\nRv1184c specifically co-immunoprecipitates with ATP6V1E1, but not with other\nV-ATPase subunits-including those that were robustly expressed. This underscores\nthe specificity of the Rv1184c-ATP6V1E1 interaction, rather than a general\nassociation with the V-ATPase complex (Fig. 3b) (please see blew).\nFor the temporality of the interaction, you raises a valuable point about the\ndynamics of this interaction. We are pleased to clarify that this was already\naddressed in our original submission. The direct protein-protein interaction assay\n(Fig. 3e) and the endogenous co-IP during infection (Fig. 6k) provide compelling\nevidence that the Rv1184c-ATP6V1E1 interaction is specific and occurs in a\nphysiological context, rather than being a transient or overexpression-induced\nartifact. We have incorporated these clarifications and new data into the revised\nmanuscript.\nNote：Chp2 (Rv1184c)\nThe reason for using Bmx +/- mice instead of Bmx-/- mice is not clear? Is the\nKO lethal? Is there a reason why these heterozygote mice were not uses in\naddtion of BMX-IN1 in key experiments of Mtb burden.\nResponse: We appreciate your comments. The generation of Bmx homozygous\nknockout (Bmx-/-) mice for our experiments was not feasible due to challenges in\nobtaining a sufficient number of age-matched knockout and wild-type littermate\ncontrols from the breeding colony, largely attributable to low fertility. To enable a\nrobust and practical investigation of Bmx function, we therefore utilized Bmx-/-\nmice. Importantly, our results demonstrate that heterozygous loss of Bmx in\nmacrophages is sufficient to produce a significant phenotype in enhanced lysosome\nacidification during H37Rv infection. This phenotype not only confirms the\nfunctional impact of reduced Bmx dosage but also aligns with and strengthens our\nconclusions from pharmacological or siRNA inhibition.\nAs per your suggestion, we further assessed the Mtb intracellular survival using\nprimary peritoneal macrophages derived from Bmx+/- mice. We found that\nheterozygous loss of BMX significantly enhanced the macrophages’ capacity to\nclear Mtb. This result provides further evidence that BMX acts as a negative\nregulator of V-ATPase-mediated lysosomal acidification (Fig. 5k, l). We have\nincluded this new data (please see below) in the revised manuscript.\nNote：Chp2 (Rv1184c)\nFig 6a and 6b are not convincing, and lack proper quantification. Since Bmx\n+/- are available, why the authors did not use them to assess ATP6V1E1\nphosphorylation in response to Mtb?\nResponse: We thank you for raising the need for proper quantification. We would\nlike to clarify that during our assessment of ATP6V1E1 phosphorylation levels, we\nalso simultaneously measured the level of ATP6V1E1 protein, although those\nresults were not shown. As per your suggestion, we have performed densitometric\nquantification of the phospho-ATP6V1E1 signal with normalization to total\nATP6V1E1 for Fig 6a and 6b. This new quantitative analysis demonstrates that\nRv1184c promotes the phosphorylation of ATP6V1E1 via BMX, which is\nconsistent with our previous conclusions. We have updated the figures (Fig. 6a, b)\n(please see below).\nNote：Chp2 (Rv1184c)\nAs per your suggestion, we assessed ATP6V1E1 phosphorylation in peritoneal\nmacrophages from WT and Bmx+/- mice. The new data (Fig. 6c) confirmed that the\nimpaired ATP6V1E1 phosphorylation caused by Rv1184c deletion is abolished in\nBmx+/- macrophages. Critically, this genetic phenotype replicates the effect we\nobserved with Bmx siRNA knockdown, powerfully confirming through an\nindependent method that BMX is the primary kinase responsible for\nphosphorylating ATP6V1E1 in response to Mtb infection. This new data (please\nsee below) was included in the revised manuscript.\nNote：Chp2 (Rv1184c)\nFinally, does BMX deciciency alter v-ATPase assembly at the lysosomal\nmembrane?\nResponse: To determine if BMX-mediated phosphorylation of ATP6V1E1 impacts\nthe assembly of the V-ATPase complex at the lysosomal membrane, we performed\nadditional experiments as suggested by you. We analyzed lysosomal fractions from\nboth BMX-IN-1 treated macrophages and Bmx+/– macrophages, comparing them to\ntheir respective controls. Immunoblotting for key V1 (represented by ATP6V1E1)\nand V0 (represented by ATP6V0d1) subunits revealed that Bmx deficiency, whether\nachieved pharmacologically or genetically, reduces the abundance of these\nsubunits at the lysosomal membrane (Fig. 5f). This result indicates that BMX is\nrequired for efficient V-ATPase assembly or stability at the lysosome, likely\nthrough its role in phosphorylating the ATP6V1E1 subunit. The new data (please\nsee below) and the detail description (Line286-293) were included in the revised\nmanuscript.\nIn Fig 7, proper controls on PLGA-aNP on lysosomal pH stability are missing.\nHow PLGA-aNP can end up in the same phagolysosomes as Mtb?\nResponse: We thank you for this insightful comment and we agree that these are\ncritical points to validate our drug delivery strategy. In our original results on\nPLGA-aNP, the lack of proper controls indeed prevents us from demonstrating\ntheir stability in response to lysosomal pH. To directly address this question, we\nshould employ bafilomycin A1 to inhibit lysosomal acidification and confirm the\npH-dependency of PLGA-aNP drug release and efficacy. Additionally, we should\nconduct confocal microscopy studies using green fluorescently labeled PLGA-aNP\nand using LysoTracker Red to stain acidic compartments to detect co-localization\nbetween PLGA-aNP and acidic lysosomes, verifying their delivery to the intended\ncompartment. These experiments will be a central part of our follow-up study.\nRegarding your question on how PLGA-aNP can co-localize with Mtb, our\nunderstanding is as follows. The fate of most internalized particles, including our\nPLGA-aNP, is to undergo progressive maturation from early endosomes to late\nendosomes and finally to acidic phagolysosomes. This is a default and dominant\npathway for phagocytosed material and is also a cell's innate clearance machinery.\nEven if a single Mtb bacterium successfully arrests its phagosome, the\nnanoparticles, being trafficked en masse via the cell's innate clearance machinery,\nhave a high probability of being delivered to the same compartment. Of course, to\ndirectly address this point, we should perform additional experiment to provide\ndirect visual evidence of their co-localization using labelled Mtb, PLGA-aNP and\nlysosomal markers.\nThe results of the PLGA-aNP were also concerned by the other two reviewers. One\nof the reviewers considered the weak relevance of the PLGA-aNP data with the\ncurrent manuscript, which is firmly focused on elucidating the role of the\nRv1184c-BMX-ATP6V1E1 axis in modulating lysosomal acidification. We decided\nto remove this section from the revised manuscript to maintain focus and prevent\ndistraction from the core narrative.\nInvestigating the lysosomal pH stability of our PLGA-aNP and their co-localization\nwith Mtb would indeed be a critical experiment to further validate our drug delivery\nstrategy. Although this specific experiment is not included in the current\nmanuscript, we sincerely appreciate your guidance and plan to pursue it in our\nsubsequent work.\nAlthough fluorescence intensity is widely use for lyso-Tracker-mediated\nevaluation of lysosomal acidification, I found this microscopy-mediated\napproach a little bit weak for this type of analysis (how many cells were used\nto determine fluorescence activity?)\nResponse: We thank you for raising this important point regarding the potential\nlimitations of using fluorescence intensity-based microscopy for evaluating\nlysosomal acidification. We fully agree your opinion. In our original manuscript,\nto robustly corroborate our findings obtained with LysoTracker, we performed an\nadditional, independent assay using pHrodo™-dextran. pHrodo™-dextran is a pH-\nsensitive probe that exhibits a strong increase in fluorescence specifically upon\nacidification within the lysosome, and its signal is less dependent on lysosomal\nloading compared to LysoTracker. This method is widely regarded as a more direct\nand quantitative measure of phagolysosomal acidification. Importantly, the results\nfrom the pHrodo™-dextran assay were entirely consistent with the trends observed\nin our LysoTracker experiments (Supplementary Fig. 3k-l) (please see blew). In\nthe original manuscript, we only briefly mentioned these confirmatory pHrodo™-\ndextran results. In response to your insightful comment, we have now substantially\nexpanded the description of the pHrodo™-dextran methodology in the results\nsection (Line189-192) of the revised manuscript.\nFor all microscopy-based LysoTracker experiments, fluorescence intensity was\nquantified in a minimum of 40 individual cells from multiple randomly selected\nfields of view. This analysis was repeated across three independent biological\nreplicates. The sample size (n) stated in the figure legends refers to the number of\nbiological replicates (each containing data from >40 cells). We added the\ndescription in the revised method section (Line629-633).\nMinor points:\nRephrase sentences in line 261-262, Line 276-277, and Lines 142-145. These\nsentences are particularly confusing.\nResponse: Thank you for this helpful suggestion. We agree that the indicated\nsentences were confusing and have revised them for clarity and precision. The\nchanges are detailed below.\nRevision for Lines 261-262:\nOriginal:\"Knockdown of Bmx abolished Rv1184c deletion induced inhibition of\nATP6V1E1 phosphorylation.\"\nRevised: \" the inhibition of Bmx by siRNA reduced the difference in ATP6V1E1\nphosphorylation between H37Rv and H37RvΔRv1184c-infected macrophages.”\nRevision for Lines 276-277:\nOriginal:\"By SPR assay, we found BMX has a strong affinity for ATP6V1E1 with\na strong affinity (Kd=190 nM).\"\nRevised: \"Surface plasmon resonance (SPR) measurements demonstrated that\nBMX binds ATP6V1E1 with a dissociation constant (Kd) of 190 nM, indicating a\nhigh-affinity interaction.”\nRevision for Lines 142-145:\nOriginal:\"Compared to the untreated group, Baf-A1 administration significantly\nattenuated the increase in Mtb survival in the lung induced by Rv1184c (Fig. 2k).\"\nRevised: \" In H37Rv-infected mice, Baf-A1 treatment significantly increased the\nbacterial load in the lungs compared to the untreated group, demonstrating that the\nclearance of Mtb in vivo is dependent on V-ATPase. Furthermore, V-ATPase\ninhibition by Baf-A1 abolished the difference in lung bacterial burden between\nH37Rv and H37RvΔRv1184c infection (Fig.2k), indicating that Rv1184c enhances\nbacterial survival in mice by targeting the V-ATPase.\nThank you again for your careful review, which has greatly improved the clarity of\nour manuscript in blue highlight.\nEnglish writing could be improved for clarity.\nResponse: We thank you for this important feedback. We sincerely apologize for\nany lack of clarity in our original submission.\nIn response, we have thoroughly revised the entire manuscript with a focus on\nimproving the clarity, flow, and precision of the English language. We have enlisted\nthe help of several colleagues proficient in academic English and utilized\nprofessional editing tools to refine the text.\nWe believe these revisions have significantly enhanced the readability of the\nmanuscript, and we appreciate your suggestion, which has helped us improve the\noverall quality of our work.\nIs Rv1184c constitutively secreted by mtb or is it secreted at the time of\ninfection?\nResponse: We thank you for the comment. In our study, Rv1184c was detected in\nboth lysates and supernatants from cultures of H37Rv (Supplementary Fig. 1a),\nconforming that Rv1184c constitutively secreted by Mtb. To directly demonstrate\nits secretion during the infectious process, we performed a co-immunoprecipitation\nassay using macrophages infected with wild-type Mtb. We successfully\nimmunoprecipitated endogenous host ATP6V1E1 and detected co-precipitating\nRv1184c (Fig. 6j). To further provide spatial confirmation of its translocation, we\nconducted new immunofluorescence experiments. The results clearly showed\nsecreted Rv1184c in the host cytoplasm, with partial co-localization with\nLysoTracker (Supplementary Fig. 5a), suggesting Rv1184c could be secreted by\nMtb into macrophages during infection. The new data have added to the revised\nmanuscript.\nNote：Chp2 (Rv1184c)\nAdd the MW and proper quantifications of all the blots.\nResponse: We thank you for highlighting the need for complete blot data. We have\nnow revised all immunoblot figures to address these omissions. Specifically,\nmolecular weight markers have been added to each blot, and we have included\ndensitometric quantifications above the corresponding bands.\nMethods are poorly described.\nResponse: We appreciate your insightful suggestion, which is also concerned by\nreviewer 2#. We have comprehensively expanded the methods section to ensure\nfull reproducibility, now including complete cell line specifications, exact\nexperimental timepoints, detailed treatment protocols, and step-by-step procedures.\nThe revised sections have been highlighted in red."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have taken my suggestions into account, and the manuscript is now suitable for publication.", "reviewer_2": "I appreciate the extensive revision carried out by the authors. The authors have satisfactorily addressed most of my original comments.\nI have the following comments on the experiment using FITC-labelled H37Rv, which the authors should address:\n• Using FITC-labelled or any other labelled H37Rv has limitations. The best way is to use a GFP or mCherry-expressing Mtb strain. The protocol for FITC labelling is missing.\n• In a few cells, large clumps of Mtb are visible. This could be due to the shedding of FITC or leftover clumps during single-cell suspension preparation. The presence of large clumps usually interferes with the quantification. How did the authors address this while analysing the data?\n• Secondly, whether only confirmed infected cells from every field were used for quantifying the lysotracker staining?\n• Finally, was there any effort to calculate the co-localisation of H37Rv, the Chp2-deleted strain and the complemented strain with lysotracker and any other lysosomal marker? This is important to demonstrate that WT Mtb, even if it ends up in the lysosome, blocks lysosomal acidification through Chp2.\nThe CFU experiment in BMDM is very good, but the authors have only presented it in the response letter. I wonder why this has not been included in either the main figures or in the supplementary information.", "reviewer_3": "The authors have adequately responded to the critiques of the reviews and have performed additional studies to support their conclusions. The key findings are convincingly established.\nI have no additional concerns."}, "author_response": "Point to point response\nReviewers' comments:\nReviewer #1: (Remarks to the Author):\nThe authors have taken my suggestions into account, and the manuscript is\nnow suitable for publication.\nReviewer #1 (Remarks on code availability):\nThe authors have taken my suggestions into account, and the manuscript is\nnow suitable for publication.\nResponse: Thank you for the positive feedback and for your valuable suggestions\nthroughout the review process.\nReviewer #2 (Remarks to the Author):\nI appreciate the extensive revision carried out by the authors. The authors\nhave satisfactorily addressed most of my original comments.\nResponse: Thank you for the positive feedback. We gratefully appreciate for your\nvaluable suggestions.\nI have the following comments on the experiment using FITC-labelled H37Rv,\nwhich the authors should address:\n• Using FITC-labelled or any other labelled H37Rv has limitations. The best\nway is to use a GFP or mCherry-expressing Mtb strain. The protocol for FITC\nlabelling is missing.\nResponse: Thank you very much for raising this insightful point. We completely\nagree with your perspective that using Mtb strains stably expressing GFP or\nmCherry is the \"gold standard\" for intracellular localization and long-term tracking\nstudies, as it effectively avoids issues such as dye detachment or quenching.\nIn our study, we successfully constructed a GFP-expressing H37Rv strain.\nHowever, as the H37RvΔChp2 strain was constructed by Shanghai Gene-optimal\nScience & Technology Co., we unfortunately did not obtain a GFP-expressing\nChp2 knockout strain. To ensure consistency in the labeling method across all\nstrains, we opted for FITC labeling. Fortunately, we have mastered this technique\nand have utilized it in previous studies [1]. In the staining phase, we strictly\ncontrolled the dye concentration and staining duration to ensure optimal labeling.\nIn the washing phase, thorough PBS washes for four or five times were performed\nto maximally remove non-phagocytized bacteria and free dye attached to the cell\nsurface. In the image analysis phase, any diffuse, non-particulate fluorescent\nbackground was excluded from quantitative analysis. Through rigorous quality\ncontrol of the FITC labeling method, we ensured the reliability of our research\nconclusions.\nWe apologize for omitting the specific steps for FITC labeling in the initial\nmanuscript. We have now supplemented the protocol for FITC labeling in the\nMethods section (Line 549-560) and as detailed below.\nFITC labeling of Mtb\nMid-log phase Mtb cultures were harvested by centrifugation, washed twice, and\nresuspended in pre-chilled 0.1 M carbonate-bicarbonate buffer (pH 9.0). FITC\nisomer I was added to a final concentration of 0.1 mg/mL, and the suspension was\nincubated at 37°C for 1 hour in the dark with gentle agitation. After labeling,\nbacteria were washed thoroughly 4-5 times with pre-chilled phosphate-buffered\nsaline (PBS, pH 7.4) until the supernatant was fluorescence-free to remove\nunbound dye. Finally, the labeled bacteria were resuspended in appropriate\ninfection medium and quantified using McFarland standards coupled with colony-\nforming unit (CFU) plating. All labeling and infection steps were performed under\nlight-protected conditions. In the image analysis phase, fields containing visible\nlarge clumps (>5 µm in diameter or containing clearly fused bacterial signals) were\nsystematically excluded from quantification.\n• In a few cells, large clumps of Mtb are visible. This could be due to the\nshedding of FITC or leftover clumps during single-cell suspension preparation.\nThe presence of large clumps usually interferes with the quantification. How\ndid the authors address this while analysing the data?\nResponse: We sincerely thank the reviewer for raising this important technical\npoint regarding the presence of large Mtb clumps and their potential impact on\nquantification accuracy. We fully agree that large bacterial clumps can severely\ninterfere with quantitative analysis, as they may lead to misinterpretation of\nfluorescence signals. In our study, fields containing visible large clumps (>5 µm in\ndiameter or containing clearly fused bacterial signals) were systematically\nexcluded from quantification. Only fields with well-dispersed, single-cell or small-\n or small-\ncluster bacterial signals were analyzed. We have added this exclusion criterion to\nthe Methods section of the revised manuscript (Line 558-560).\n• Secondly, whether only confirmed infected cells from every field were used\nfor quantifying the lysotracker staining?\nThank you for raising this important concern. We agree that, in the absence of\nfluorescently labeled bacteria, it is technically difficult to unambiguously identify\nevery infected macrophage based solely on LysoTracker staining and bright-field\nmicroscopy, particularly at MOI of 5 where the infection rate cannot be precisely\ninferred at the single-cell level.\nFor this reason, in the revised version of the manuscript, we performed additional\nexperiments using FITC-labeled Mtb to explicitly define infected cells and to\nquantify lysosomal acidification specifically within these cells. This approach\nallowed us to directly address the reviewer’s concern and to rigorously validate our\nconclusions under conditions where infection status could be unambiguously\ndetermined.\nImportantly, the infected-cell-restricted analysis yielded results fully consistent\nwith our original observations, demonstrating that deletion of Chp2 enhances\nlysosomal acidification in infected macrophages. These data are now included in\nSupplementary Fig1d (please see below) and form an integral part of the revised\nmanuscript.\n• Finally, was there any effort to calculate the co-localisation of H37Rv,\nthe Chp2-deleted strain and the complemented strain with lysotracker and any\nother lysosomal marker? This is important to demonstrate that WT Mtb, even\nif it ends up in the lysosome, blocks lysosomal acidification through Chp2.\nResponse: Thank you for raising this insightful point. We fully agree that directly\nquantifying the co-localization of bacteria with lysosomes and with LysoTracker\nprovides key evidence to support the hypothesis that Chp2 functions within\nlysosomes to block acidification.\nAccordingly, we examined the co-localization of H37Rv, H37RvΔChp2, and the\ncomplemented strain with lysosomes using the canonical late-\nendosomal/lysosomal marker LAMP1, as well as their effects on lysosomal\nacidification using LysoTracker. The results showed that all strains showed\nlysosomal co-localization without significant differences (Supplementary Fig. 1e,\nf). In parallel, H37RvΔChp2 notably enhanced lysosomal acidification compared\nto H37Rv, whereas complementation restored the acidification blockade\n(Supplementary Fig. 1g, h), in line with our previous conclusion. Together, all\nthree strains reach a LAMP1-positive compartment to a similar significant extent,\nbut only the H37Rv and complemented strains, possessing functional Chp2, can\nsubsequently prevent lysosomal acidification, demonstrating that Mtb inhibits\nlysosomal acidification via Chp2.\nWe believe this integrated analysis robustly demonstrates wild-type Mtb trafficks\nto a lysosomal compartment, and its Chp2 is specifically responsible for blocking\nthe acidification of that compartment. We have incorporated these new findings\n(please see below) and detail description of this analysis in the revised result\nsection (Line 107-114). We thank the reviewer for the perceptive suggestions,\nwhich have significantly enhanced the rigor and depth of our work.\nThe CFU experiment in BMDM is very good, but the authors have only\npresented it in the response letter. I wonder why this has not been included in\neither the main figures or in the supplementary information.\nResponse: Thank you for your positive feedback on the CFU data in BMDMs and\nfor this excellent suggestion. We agree that these results are crucial for supporting\nour findings on bacterial survival. Accordingly, we have now included this dataset\nas a new panel in Supplementary Fig.2b of the revised manuscript (Line135-136).\nThe corresponding figure legend and Methods (Line 576-578, Line 692-694) have\nbeen updated accordingly.\nReviewer #3 (Remarks to the Author):\nThe authors have adequately responded to the critiques of the reviews and\nhave performed additional studies to support their conclusions. The key\nfindings are convincingly established.\nI have no additional concerns.\nResponse: We are grateful for your constructive feedback.\nReference\ntherapeutic target for Mycobacterium avium complex disease. Cell Rep Med. 6:101923\n(2025)."}]

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