Rab14 restricts pathogens by promoting V-ATPase lysosomal delivery to drive lysosomal acidification

Abstract

Host restriction factors mediate intrinsic immunity against infections, thus serving as promising targets for host-directed therapy (HDT) against drug-resistant pathogens. While restriction factors counteracting viruses have been extensively studied, those targeting bacteria, particularly those with broad-spectrum activity, remain largely unexplored. Here, through screening for host factors promoting lysosomal acidification, a crucial process clearing pathogens, we identify the host small GTPase Rab14 as a restriction factor with broad-spectrum activity against multiple bacteria and viruses. Mechanistically, upon pathogen infections, GTP-bound Rab14 increases and binds to the calcium/calmodulin-dependent protein kinase type 2 delta (CAMK2D), suppressing CAMK2D-mediated phosphorylation of V0a1, the critical subunit determining V-ATPase localization, thus promoting V0a1 binding to the COPⅡ complex to facilitate V-ATPase trafficking from the endoplasmic reticulum to lysosomes, resulting in lysosomal acidification and pathogen clearance. Taken together, our data demonstrate an unrecognized intrinsic immune mechanism mediated by Rab14-CAMK2D-V-ATPase axis, which might be a promising target for infectious diseases.

Broad-spectrum host restriction factors against bacteria and viruses remain largely unclear. Here, the authors identify the small GTPase Rab14 as a restriction factor that promotes lysosomal acidification by delivering the V-ATPase to lysosomes.

Introduction

Infectious diseases continue to pose major global health challenges, exacerbated by the rise of drug-resistant pathogens that are projected to kill approximately 39 million people by 2050^1,2. This problem arises because that traditional antibiotics or antivirals directly target pathogens, exerting lethal selective pressure that drives resistance mutations. Against this backdrop, host-directed therapy (HDT) has emerged as a transformative strategy that leverages host biological mechanisms to enhance pathogen clearance while minimizing resistance development³. Intrinsic immunity functions as the cell-autonomous first-line defense mechanism against pathogen infections, which is mediated by the constitutively expressed host restriction factors^4,5. In the past decade, studies have reported multiple host restriction factors (such as TRIM5, APOBEC3G, IFITMs) against virus infections through directly inhibiting viral replication or reprogramming host anti-viral immune responses^6–8. However, restriction factors that counteract bacterial infections, as well as those with broad-spectrum activity against both viral and bacterial pathogens, remain poorly defined. In particular, their specific and dynamic regulatory mechanisms during infection remain elusive. This gap impedes the development of HDT strategies targeting bacterial and polymicrobial infections.

Lysosomes are membrane-bound organelles that serve as the primary degradative hubs in cells, playing a pivotal role in the cell-intrinsic defense against a wide range of pathogens⁹. Hydrolytic enzymes, such as cathepsins, are the main executors of lysosomal degradation functions, and their optimal activity is critically dependent on the maintenance of an acidic luminal pH within lysosomes, which is established and maintained by the vacuolar ATPase (V-ATPase), a proton pump that actively transports protons into the lysosomal lumen^10,11. The activity of V-ATPase is tightly controlled by its assembly of cytosolic V1 and membrane-bound V0 domains, as well as its trafficking between organelles. Previous studies have characterized the assembly mechanisms of V-ATPase. For example, the cryoelectron microscopy structure analysis of human V-ATPase showed that ATP6AP1 interacts with multiple subunits of V-ATPase, positioning it as a central hub for facilitating V-ATPase assembly¹². The amino acid sensor mechanistic target of rapamycin complex 1 (mTORC1) was also identified to dynamically modulate the assembly/disassembly of V-ATPase in response to nutrient variations¹³. However, the mechanisms underlying V-ATPase trafficking to the specific organelle such as lysosomes remain largely unclear. Elucidating the specific host factors governing V-ATPase trafficking and deciphering the underlying mechanisms would significantly enhance our understanding on V-ATPase-mediated lysosomal acidification and its regulatory dynamics. Such insights could provide potential intervention targets and strategies against infectious diseases through improving host V-ATPase-dependent intrinsic immunity.

The Rab family of small GTPases serves as a highly conserved molecular switch governing vesicle budding, trafficking, and fusion in eukaryotic membrane trafficking systems, and thus has emerged as a pivotal regulatory hub that orchestrates multiple stages of host anti-infection immunity^14,15. However, the role of Rab proteins in regulating V-ATPase trafficking and lysosomal acidification, as well as the underlying mechanisms remain largely unexplored. Here, we demonstrate that Rab14 is essential for delivering V-ATPase to lysosomes for promoting acidification, thus functioning as a broad-spectrum antimicrobial factor against bacterial and viral pathogens. Mechanistic studies demonstrated that Rab14 competitively binds to calcium/calmodulin-dependent protein kinase type 2 delta (CAMK2D) with V0a1, the critical subunit protein determining V-ATPase localization¹⁶, followed by inhibiting CAMK2D-catalyzed V0a1 phosphorylation, ultimately eliciting V0a1 interaction with the COPⅡ complex to mediate the trafficking of V-ATPase from the endoplasmic reticulum (ER) to the Golgi apparatus and lysosomes. These data reveal that Rab14 is a broad-spectrum host restriction factor promoting V-ATPase trafficking and lysosomal acidification, suggesting that the Rab14-CAMK2D-V-ATPase axis serves as a promising target for treating infectious diseases.

Results

Rab14 is required for lysosomal acidification and pathogen clearance

To identify the critical Rab protein modulating lysosomal acidification during infections, we screened Rab proteins highly expressed in macrophages (> 50 nTPM, Supplementary Data 1), given their essential role as the primary immune defense against intracellular pathogens¹⁷. Then, the intracellular bacterium Mycobacterium smegmatis dually labeled with the pH-sensitive pHrodo fluorescent dye and the pH-insensitive Alexa Fluor 488 was used to infect macrophages with Rab gene knockdown for lysosomal pH analysis (Supplementary Fig. 1a, b and Supplementary Fig. 2). Interestingly, our system recapitulated the role of Rab32, which was reported to promote lysosomal acidification in osteoclasts¹⁸, confirming the system’s validity. Moreover, we also observed a significant increase in lysosomal pH upon Rab14 knockdown (Fig. 1a), indicating that Rab14 is a previously unrecognized critical factor in promoting lysosomal acidification. Additionally, Rab2, a protein that has been reported to have overlapping functions with Rab14 in promoting autophagosome-lysosome fusion in Madin-Darby canine kidney (MDCK) epithelial cells¹⁹, did not exhibit an effect on lysosomal pH in our experimental system. Thus, we focused on Rab14 to further explore its regulatory roles in lysosomal acidification. To further investigate whether the promoting effect of Rab14 on lysosomal acidification is unique to M. smegmatis infection, we constructed Rab14 knockout (Rab14^-/-) macrophages (Supplementary Fig. 3a), and then infected wild-type (Rab14^+/+) and Rab14^-/- macrophages with another intracellular bacterium Listeria monocytogenes, as well as two types of viruses including DNA virus HSV-1 and RNA virus SeV. Through examining the fluorescence intensity of LAMP1 and LysoTracker, which are two biomarkers indicating total lysosomes and acidified lysosomes, rspectively^20,21, we found that while the LysoTracker fluorescence intensity was much higher in infected Rab14^+/+ macrophages than that in infected Rab14^-/- macrophages, the LAMP1 intensity remained comparable between the two group (Fig. 1b–d), suggesting that Rab14 depletion specifically compromises lysosomal acidification without affecting lysosomal number. Moreover, we performed live-cell imaging assays using the LysoSensor Yellow/Blue probe, which mainly produces yellow fluorescence in acidic lysosomes (low pH), but blue fluorescence in less acidic lysosomes (high pH)²². The results demonstrated that compared with Rab14^-/- macrophages, Rab14^+/+ macrophages exhibited stronger yellow fluorescence but weaker blue fluorescence (Supplementary Fig. 3b), indicating a lower lysosomal pH in Rab14^+/+ macrophages than that in Rab14^-/- macrophages. Taken together, Rab14 is required for lysosomal acidification during a broad range of pathogen infections.

Fig. 1. Rab14 promotes lysosomal acidification and pathogen clearance.

a Lysosomal pH analysis of RAW264.7 cells interfering with Rab genes. RAW264.7 cells were transfected with control siRNA (siNC) or siRNA targeting Rab genes for 24 hours and were then infected with M. smegmatis dually labeled with the pH-sensitive pHrodo fluorescent dye and the pH-insensitive Alexa Fluor 488 at a MOI of 10 for 4 hours. The lysosomal pH of the infected macrophages was measured with fluorescence-activated cell sorting (FACS). b Representative images showing the LAMP1 and LysoTracker staining in Rab14^+/+ and Rab14^-/- cells uninfected or infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV. Cells were uninfected or infected with M. smegmatis (MOI = 10), L. monocytogenes (MOI = 5), HSV-1 (MOI = 1), or SeV (MOI = 1) for 6 hours and were then incubated with 75 nM LysoTracker (red) for an additional 30 minutes. Thereafter, cells were fixed and stained with the antibody against LAMP1 (green). Scale bars: 5 µm. Quantification of the fluorescent intensity of LAMP1 (c) and LysoTracker (d) in Rab14^+/+ and Rab14^-/- cells treated as in (b). Immunoblotting analysis of CTSD in Rab14^+/+ and Rab14^-/- RAW264.7 cells infected with M. smegmatis (e), L. monocytogenes (f), HSV-1 (g), or SeV (h). Cells were infected as in (b) and were then lysed for immunoblotting analysis with antibodies against CTSD and cleaved CTSD light chain (LC). TUBA1A was used as the loading control. The ratio of mature CTSD heavy chain (HC) or light chain to TUBA1A was calculated for quantification. The intracellular survival of pathogens in Rab14^+/+ and Rab14^-/- RAW264.7 cells infected with M. smegmatis (i), L. monocytogenes (j), HSV-1 (k), or SeV (l). Data are mean ± s.e.m. with each symbol representing a biological replicate [n = 3 in (a, c, d); n = 4 in (i–l)]. Statistical significance was determined using one-way ANOVA with Dunnett’s post-hoc test (a), and two-way ANOVA with Tukey’s post-hoc test (c, d, i–l). ns, nonsignificant, P > 0.05. Source data are provided as a Source Data file.

Lysosomal acidification is essential for the maturation and activation of lysosomal hydrolytic enzymes, especially the broadly specific aspartyl protease cathepsin D (CTSD)²³. Consistent with the defective lysosomal acidification in Rab14-deficient macrophages, we observed decreased mature CTSD levels in Rab14^-/- macrophages infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV than that in infected Rab14^+/+ macrophages (Fig. 1e–h). In vitro CTSD activity assays also confirmed a marked reduction in proteolytic activity in Rab14^-/- macrophages compared to Rab14⁺/⁺ macrophages, both under uninfected conditions and following infection with bacterial or viral pathogens (Supplementary Fig. 3c). Furthermore, as lysosomal acidification is critical for pathogen clearance, we detected the intracellular survival of pathogens in Rab14^+/+ and Rab14^-/- macrophages. The results showed that Rab14^-/- macrophages exhibited increased intracellular pathogen survival than Rab14^+/+ macrophages (Fig. 1i–l), indicating that Rab14 contributes to pathogen clearance. Moreover, it should be mentioned that lysosomal acidification is involved in multiple cellular processes including autophagy²⁴, and we expectedly found an increased protein level of the 16-kD lipidated autophagosome-bound form of LC3B (LC3-II) in Rab14^-/- macrophages infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV than that in infected Rab14^+/+ macrophages, while the observed difference was abrogated by the autophagy flux inhibitor bafilomycin A1 (Baf)²⁵, suggesting a disrupted autophagy flux in Rab14^-/- macrophages (Supplementary Fig. 4a–d). Consistently, pathogen intracellular survival analysis demonstrated that Rab14 promotes the elimination of pathogens in a manner dependent on autophagy (Supplementary Fig. 4e–h). Collectively, Rab14 serves as an essential host restriction factor that orchestrates lysosomal acidification to promote pathogen clearance.

Rab14 elicits the lysosomal targeting of V-ATPase to promote lysosomal acidification

Next, we sought to investigate the mechanism by which Rab14 promotes lysosomal acidification. Given that V-ATPase is the master regulator of lysosomal acidification¹⁰, we wondered whether Rab14 affects the activation of V-ATPase to promote the acidification of lysosomes. V-ATPase is a multisubunit protein complex consisting of the cytosolic V1 domain and the lysosomal membrane-embedded V0 domain. The V1 domain is composed of eight different subunits (A, B, C, D, E, F, G, H) and is in charge of ATP hydrolysis, whereas the V0 domain contains six different subunits (a, d, e, c, c’, c”) and is responsible for the translocation of protons from the cytoplasm to the organelle lumen¹⁶. The reversible assembly of V1/V0 on the lysosomal membrane is essential for regulating lysosomal pH. Prior to this, the V0 sector needs to be transported to the lysosomal membrane, which is primarily regulated by the subunit a of V0 (V0a)¹⁶. As Rab14 is a small GTPase contributing to membrane trafficking^26–28, we speculated that Rab14 might be involved in the trafficking of V0a to lysosomes. There are four isoforms of V0a (a1-a4). Among them, V0a1, V0a2, and V0a3, but not V0a4, can be expressed in macrophages (https://www.proteinatlas.org/). Therefore, we detected the subcellular localization of V0a1, V0a2, and V0a3 in Rab14^+/+ and Rab14^-/- cells, respectively. The double-immunofluorescence labeling analysis revealed strong colocalization of V0a1 with LAMP1-positive lysosomes in Rab14^+/+ cells, whereas in Rab14^-/- cells, V0a1 primarily colocalized with the ER-integral protein calnexin (Fig. 2a–e). Moreover, V0a1 showed minimal colocalization with GM130 (the biomarker for Golgi apparatus), Rab5 (the biomarker for early endosomes), and Rab7 (the biomarker for late endosomes) in Rab14^+/+ cells, and these colocalizations further reduced in Rab14^-/- cells (Fig. 2a–e). Furthermore, we isolated ER and lysosomal fractions from Rab14^⁺/⁺ and Rab14^⁻/⁻ cells to examine the distribution of V0a1, and consistently found that V0a1 was predominantly enriched on lysosomes in Rab14^⁺/⁺ cells, whereas it was largely retained in the ER in Rab14^⁻/⁻ cells (Supplementary Fig. 5a). Unlike the different pattern of V0a1 subcellular localization in Rab14^+/+ and Rab14^-/- cells, there was no significant difference in the subcellular localization of V0a2 and V0a3 between Rab14^+/+ and Rab14^-/- cells (Supplementary Fig. 5b–k). Taken together, Rab14 promotes the lysosomal targeting of V0a1.

Fig. 2. Rab14 promotes the lysosomal trafficking of V0a1.

Representative images showing the colocalization of V0a1 with calnexin (a), GM130 (b), Rab5 (c), Rab7 (d), or LAMP1 (e) in Rab14^+/+ and Rab14^-/- Hela cells (Top). Cells were transfected with GFP-V0a1 (green) for 24 hours and were then fixed and stained with the indicated antibodies (red), followed by subjecting to confocal microscopy analysis. Scale bars: 10 μm. Bottom, Pearson’s coefficients of V0a1 with the indicated organelles. Fifty cells were counted and analyzed for each biological replicate. f Representative images showing the colocalization of V0a1 with LAMP1 in Rab14^+/+ and Rab14^-/- cells uninfected or infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV (Top). Cells transfected with GFP-V0a1 were uninfected or infected with M. smegmatis (MOI = 10), L. monocytogenes (MOI = 5), HSV-1 (MOI = 1), or SeV (MOI = 1) for 6 hours and were then fixed and stained with the antibody against LAMP1 (red). Scale bars: 5 µm. Bottom, Pearson’s coefficients of V0a1 with LAMP1. Fifty cells were counted and analyzed for each biological replicate. Box-whisker plot indicates the interquartile range (box), the median value (line within the box), and the maximum and minimum value (whiskers). Statistical significance was determined using unpaired two-sided Student’s t-test (a–e) and two-way ANOVA with Tukey’s post-hoc test (f). Source data are provided as a Source Data file.

To verify the regulatory role of Rab14 on the lysosomal localization of V0a1 during pathogen infections, we detected the colocalization of V0a1 and lysosomes in Rab14^+/+ and Rab14^-/- cells infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV. The results showed that the above pathogen infections could increase the trafficking of V0a1 to lysosomes in Rab14^+/+ cells, while this effect is largely reduced in Rab14^-/- cells (Fig. 2f; Supplementary Movies 1, 2). Analysis of V0a1 levels in purified lysosomes also demonstrated a significantly higher enrichment of V0a1 on lysosomes from Rab14^+/+ cells than those from Rab14^-/- cells (Supplementary Fig. 6a). To further confirm whether Rab14 promotes lysosomal acidification in a manner dependent on V0a1, we employed siRNA to knock down V0a1 genes in Rab14^+/+ and Rab14^-/- cells (Supplementary Fig. 6b) and then examined the fluorescence intensity of LysoTracker. We found that V0a1 knockdown eliminated the effects of Rab14 promoting lysosomal acidification during pathogen infections (Supplementary Fig. 6c). Finally, pathogen intracellular survival analysis also confirmed that Rab14 promotes pathogen clearance depending on V0a1 (Supplementary Fig. 6d–g). Altogether, these results indicate that Rab14 facilitates V-ATPase lysosomal targeting to promote lysosomal acidification and pathogen clearance.

Rab14 suppresses CAMK2D-mediated V0a1 phosphorylation to promote V0a1 trafficking to lysosomes

Rab GTPases usually regulate protein trafficking by direct interaction²⁹. Thus, we firstly examined whether Rab14 can interact with V0a1 to promote its transport to lysosomes. The co-immunoprecipitation analysis showed that there is no interaction between Rab14 and V0a1 (Supplementary Fig. 7a), indicating that Rab14 modulates V0a1 trafficking in a way independent of their interaction. Studies have reported that the protein post-translational modification (PTM) of V0a1 is important for its trafficking and localization. For example, the glycosylation of V0a1 is essential for its maturation and the ensuing ER-to-lysosome delivery³⁰, while our data showed no molecular weight differences in V0a1 between Rab14^+/+ and Rab14^-/- cells that would indicate altered glycosylation (Fig. 3a). Furthermore, V0a1 requires palmitoylation for its trafficking from the sorting endosome to the late endosomal/lysosomal membrane³¹, while Rab14 mainly modulates the delivery of V0a1 from ER to Golgi (Fig. 2a), which is a different trafficking way independent of the endosomal sorting pathway. Consistently, the palmitoylation assay demonstrated no significant difference in V0a1 palmitoylation levels between Rab14^+/+ and Rab14^-/- cells (Supplementary Fig. 7b). Taken together, these results suggest that there might be unrecognized PTMs of V0a1 modulated by Rab14 to govern V0a1 trafficking.

Fig. 3. Rab14 deficiency enhances CAMK2D-catalyzed V0a1 phosphorylation and impairs the ER-to-Golgi transport of V0a1.

a Immunoblotting analysis of levels of phosphor-Serine (p-Ser) and phosphor-Threonine (p-Thr) of V0a1 in Rab14^+/+ and Rab14^-/- cells. IgG control or V0a1 was immunoprecipitated from Rab14^+/+ and Rab14^-/- cells, and the immunoprecipitates were subjected to immunoblotting analysis with antibodies against p-Ser, p-Thr, and V0a1. GAPDH was used as the loading control. The ratio of (V0a1) p-Ser or (V0a1) p-Thr to V0a1 in immunoprecipitation samples was calculated for quantification. b Competitive binding assay of GFP-V0a1 (13 μg each) and GST-Rab14 (2 μg or 5 μg each) to His-CAMK2D (5 μg each). Quantification was performed by measuring the relative intensity of the bands. c Immunoblotting analysis of V0a1 (10 μg each) phosphorylation by CAMK2D (5 μg each) in the presence of GST (2 μg each) or GST-Rab14 (5 μg each). Quantification was performed by measuring the relative intensity of the bands. d Immunoblotting analysis of V0a1 phosphorylation in Rab14^+/+, Rab14^-/-, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- cells. Cells were transfected with GFP or GFP-V0a1 for 24 hours and were then lysed for immunoprecipitating GFP-tagged proteins using anti-GFP-Nanoab-Agarose. The immunoprecipitates were subjected to immunoblotting analysis with antibodies against p-Ser and GFP. The ratio of (V0a1) p-Ser to GFP-tagged proteins in immunoprecipitation samples was calculated for quantification. Representative images showing the colocalization of V0a1 with calnexin (e) or LAMP1(f) in Rab14^+/+, Rab14^-/-, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- Hela cells (Left). Cells were transfected with GFP-V0a1 (green) for 24 hours and were then fixed and stained with the antibody against calnexin or LAMP1 (red), followed by subjecting to confocal microscopy analysis. Scale bars: 10 μm. Right, Pearson’s coefficients of V0a1 with ER or lysosomes. Data are mean ± s.e.m. with each symbol representing a biological replicate [n = 3 in (e, f)]. Statistical significance was determined using one-way ANOVA with Tukey’s post-hoc test (e, f). Results are representative of at least three independent experiments. Source data are provided as a Source Data file.

Given that Rab14 lacks enzymatic activity for PTMs, we speculated that Rab14 might recruit other proteins to modulate V0a1 PTM. By conducting immunoprecipitation-mass spectrometry, we obtained 43 proteins potentially interacted with Rab14 (Supplementary Fig. 7c; Supplementary Data 2). Among them, TRIM21 is an E3 ubiquitin ligase mediating protein ubiquitination, and CAMK2D is a serine/threonine kinase catalyzing protein phosphorylation^32,33. Then, we detected whether Rab14 modulates the ubiquitination and phosphorylation of V0a1. In vivo ubiquitination assays showed that V0a1 was ubiquitinated at comparable levels in Rab14^+/+ and Rab14^-/- cells (Supplementary Fig. 7d), indicating that Rab14 does not modulate the ubiquitination of V0a1. However, we interestingly observed a higher serine, but not threonine, phosphorylation level of V0a1 in Rab14^-/- cells than that in Rab14^+/+ cells (Fig. 3a), indicating that Rab14 suppresses the phosphorylation of V0a1. To further investigate whether CAMK2D is involved in the Rab14-mediated suppression of V0a1 phosphorylation, we firstly detected the interactions among Rab14, CAMK2D, and V0a1. We found that Rab14 could interact with CAMK2D (Supplementary Fig. 7e). Interestingly, CAMK2D specifically bound to V0a1, but not V0a2 or V0a3 (Supplementary Fig. 8a). We then conducted molecular docking analysis and revealed that CAMK2D bound to the Arg 179 and Arg 198 sites of V0a1, two residues that are not conserved in V0a2 or V0a3 (Supplementary Fig. 8b, c). Moreover, the V0a1-CAMK2D affinity was stronger in Rab14^-/- cells than that in Rab14^+/+ cells, indicating a competition between Rab14 and V0a1 for CAMK2D (Supplementary Fig. 8d). Through further conducting the in vitro competition binding assay, we verified that Rab14 competitively inhibits the interaction between V0a1 and CAMK2D (Fig. 3b). We then explored the molecular mechanism by which Rab14 and V0a1 competitively bind to CAMK2D. Through constructing the truncated variants of CAMK2D, we found that both Rab14 and V0a1 interacted with the kinase domain of CAMK2D (Supplementary Fig. 8e, f), which is the critical region mediating substrate phosphorylation³⁴. Furthermore, the in vitro phosphorylation assay showed that V0a1, but not V0a2 or V0a3, was phosphorylated by CAMK2D (Supplementary Fig. 8g), while this phenomenon was largely abolished by Rab14 (Fig. 3c). Moreover, CAMK2D deficiency resulted in the loss of V0a1 phosphorylation in Rab14^-/- cells (Fig. 3d). Collectively, these results suggest that Rab14 competitively interacts with CAMK2D to suppress CAMK2D-mediated V0a1 phosphorylation. Next, we explored the role of CAMK2D-mediated V0a1 phosphorylation in Rab14-governed V0a1 trafficking. We found that the Rab14 knockout-caused high colocalization of V0a1 with ER and low colocalization of V0a1 with lysosomes could be reversed by CAMK2D deletion (Fig. 3e, f), indicating that CAMK2D functions as a negative regulator in Rab14-promoted V0a1 trafficking to lysosomes. Altogether, Rab14 promotes V-ATPase lysosomal delivery by antagonizing CAMK2D-mediated V0a1 phosphorylation.

Rab14-mediated inhibition of V0a1 phosphorylation enhances its binding to the COPⅡ complex

Next, we investigated the detailed mechanism by which enhanced V0a1 phosphorylation impairs its transport from ER to Golgi and then to lysosomes in Rab14^-/- cells. Through examining the subcellular localization of CAMK2D by confocal microscopy analysis, we found that CAMK2D exhibits a diffuse cytoplasmic distribution, and this localization pattern remains unchanged in Rab14^-/- cells (Supplementary Fig. 9a), indicating that Rab14 modulates V0a1 trafficking independent of CAMK2D localization. Studies reported that the coat protein complex Ⅱ (COPⅡ), which is composed of secretion-associated Ras-related GTPase 1 (Sar1), Sec23/Sec24, and Sec13/Sec31 subunits, plays a crucial role in the delivery of V0 complex from ER to Golgi³⁵. Since S-palmitoylation is essential for COPII-mediated anterograde ER-to-Golgi trafficking³⁶, we thus examined whether Rab14 deficiency alters this modification. Our results showed that the S-palmitoylation levels of COPII-associated proteins were comparable between Rab14^+/+ and Rab14^-/- cells (Supplementary Fig. 9b), indicating that Rab14 is not involved in regulating the palmitoylation of the COPII complex. Moreover, within the COPII coat, Sec24 is primarily responsible for cargo selection through directly recognizing and binding cargo proteins³⁷. Among the four isoforms of Sec24, including Sec24A, Sec24B, Sec24C, and Sec24D, Sec24C was indicated to be involved in the ER-to-Golgi transport of V0 subunits³⁸. Based on the above evidence, we hypothesized that whether V0a1 phosphorylation influences its interaction with Sec24C. Through conducting the coimmunoprecipitation assay of V0a1 and Sec24C, we demonstrated a specific interaction of these two proteins in Rab14^+/+ cells, but not in Rab14^-/- cells. Intriguingly, the deletion of CAMK2D, which is responsible for mediating the phosphorylation of V0a1, restored the interaction between V0a1 and Sec24C (Fig. 4a), indicating an inhibitory effect of V0a1 phosphorylation on its binding to Sec24C. To further confirm this result, we analyzed the specific phosphorylation site of V0a1 to determine whether its mutation could reverse the V0a1-Sec24C interaction. Through using the GPS 5.0 database (https://gps.biocuckoo.cn/)³⁹, we identified a total of four potential phosphorylation sites on V0a1, including serine 19 (S19), threonine 182 (T182), threonine 371 (T371), and serine 432 (S432) (Supplementary Fig. 9c). We subsequently mutated these residues to alanine (A), and found that while the threonine phosphorylation level of V0a1 was unaffected by CAMK2D, the S19A mutation abolished CAMK2D-mediated serine phosphorylation of V0a1 (Supplementary Fig. 9d). Consistently, the phosphorylation of V0a1 in Rab14^-/- cells was also abrogated by the S19A mutation (Supplementary Fig. 9e). Thus, Rab14 inhibits CAMK2D-mediated V0a1 phosphorylation at the S19 site. To this end, we generated a phosphospecific antibody against the V0a1 S19 site. Through in vitro and in vivo phosphorylation assays using this antibody, we confirmed that CAMK2D phosphorylates V0a1 at S19, while Rab14 inhibits this process (Supplementary Fig. 9f, g). Then, we examined the binding of V0a1 or its S19A mutant to Sec24C in Rab14^+/+ and Rab14^-/- cells, respectively. The result showed that the S19A mutation restored the interaction between V0a1 and Sec24C in Rab14^-/- cells (Fig. 4b). Likewise, the colocalization analysis also demonstrated that the V0a1^S19A mutant exhibited a higher colocalization efficiency with Sec24C than the wild-type V0a1 in Rab14^-/- cells (Fig. 4c). Accordingly, mutating the S19 residue in V0a1 increased the localization of V0a1 in lysosomes, while decreasing its retention in ER in Rab14^-/- cells (Fig. 4d, e). Collectively, Rab14 inhibits CAMK2D-mediated V0a1 phosphorylation to enhance V0a1 binding to COPⅡ complex, thereby eliciting V0a1 trafficking to lysosomes.

Fig. 4. Rab14-mediated suppression of V0a1 phosphorylation enhances V0a1-COPII complex interaction.

a Immunoblotting analysis of the interaction between V0a1 and Sec24C in Rab14^+/+, Rab14^-/-, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- cells. Cells were transfected with GFP or GFP-V0a1 and Myc-Sec24C for 24 hours and were then lysed for immunoprecipitating GFP-tagged proteins using anti-GFP-Nanoab-Agarose, followed by subjecting to immunoblotting analysis with antibodies against Myc and GFP. b Immunoblotting analysis of the interaction between V0a1 or its S19A mutant and Sec24C in Rab14^+/+ and Rab14^-/- cells. c Representative images showing the colocalization of V0a1 or V0a1^S19A mutant with Sec24C in Rab14^+/+ and Rab14^-/- Hela cells (Top). Cells were transfected with GFP-V0a1 or GFP-V0a1^S19A (green) and Myc-Sec24C for 24 hours and were then fixed and stained with the antibody against Myc (red), followed by subjecting to confocal microscopy analysis. Scale bars: 10 μm. Bottom, Pearson’s coefficients of V0a1 or V0a1^S19A with Sec24C. Representative images showing the colocalization of V0a1 or V0a1^S19A mutant with calnexin (d) or LAMP1 (e) in Rab14^+/+ and Rab14^-/- Hela cells (Left). Cells were transfected with GFP-V0a1 or GFP-V0a1^S19A (green) for 24 hours and were then fixed and stained with the antibody against calnexin or LAMP1 (red), followed by subjecting to confocal microscopy analysis. Scale bars: 10 μm. Right, Pearson’s coefficients of V0a1 or V0a1^S19A with ER or lysosomes. Data are mean ± s.e.m. with each symbol representing a biological replicate [n = 3 in (c–e)]. Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test (c–e). Results are representative of at least three independent experiments. Source data are provided as a Source Data file.

Pathogen infection upregulates GTP-bound Rab14 to inhibit CAMK2D-mediated V0a1 phosphorylation

Next, we explored the regulatory dynamics of the Rab14-CAMK2D-V0a1 axis during pathogen infections. As reported, Rab proteins cycle between their active GTP-bound and inactive GDP-bound states, with the GTP-bound conformation typically being crucial for pathogen infections^40,41. Through immunoprecipitating the active GTP-bound Rab14 using RUFY1, which specifically recognizes the constitutively active GTP-locked mutant of Rab14⁴², we observed increased GTP-bound Rab14 levels following infection with M. smegmatis, L. monocytogenes, HSV-1, or SeV (Fig. 5a–d). Given that Rab proteins in their GTP-bound state recruit various effector proteins to facilitate membrane trafficking, we hypothesized that increased GTP-bound Rab14 could enhance the interaction between Rab14 and CAMK2D during pathogen infections. Firstly, we detected the affinity between CAMK2D and Rab14 active or inactive mutant, and the result showed that CAMK2D preferred to bind to the GTP-bound Rab14 (Supplementary Fig. 10a). Then, we consistently observed increased Rab14-CAMK2D interactions during pathogen infections (Fig. 5e–h). Based on the findings that Rab14 competitively binds to CAMK2D to suppress CAMK2D-mediated V0a1 phosphorylation and thus promote V0a1 interacting with Sec24C (Figs. 3, 4), we proceeded to investigate the phosphorylation status of V0a1 and its interaction with Sec24C during infection with different pathogens in Rab14^+/+, Rab14^-/-, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- cells. Strikingly, V0a1 was significantly phosphorylated in Rab14^-/- cells as compared to that in Rab14^+/+ cells, and the low baseline phosphorylation of V0a1 in Rab14^+/+ cells progressively declined over the course of infection (Supplementary Fig. 10b–e). Moreover, CAMK2D knockout resulted in a complete loss of V0a1 phosphorylation in both Rab14^+/+ and Rab14^-/- cells (Supplementary Fig. 10b–e). Conversely, Rab14^-/- cells showed loss of V0a1-Sec24C interaction, whereas their binding increased progressively during infection in Rab14^+/+ cells and remained strong in Rab14^+/+Camk2d^-/- and Rab14^-/-Camk2d^-/- cells (Supplementary Fig. 10b–e). Thus, these results indicate that Rab14 suppresses CAMK2D-mediated V0a1 phosphorylation to facilitate V0a1-Sec24C complex formation during pathogen infections. Finally, quantitative imaging analysis for V0a1-lysosome colocalization revealed that Rab14 promotes the trafficking of V0a1 to lysosomes in a manner dependent on CAMK2D (Fig. 5i). It should be pointed out that Rab14-promoted lysosomal localization of V0a1 was observed not only under infection conditions, but also in non-infection conditions, indicating that Rab14 also regulates the baseline level of lysosomal acidification. Altogether, our data suggest that upon pathogen infections, the active GTP-bound Rab14 increases to interact with CAMK2D, thus suppressing CAMK2D-mediated V0a1 phosphorylation to enhance V0a1 interaction with the COPⅡ complex, ultimately promoting V0a1 trafficking to lysosomes.

Fig. 5. Pathogen infection-induced GTP-bound Rab14 interacts with CAMK2D to promote lysosomal trafficking of V0a1.

Immunoblotting analysis of the active GTP-bound Rab14 immunoprecipitated by GST-RUFY1 in Rab14^+/+ RAW264.7 cells infected with M. smegmatis (a), L. monocytogenes (b), HSV-1 (c), or SeV (d). Cells were uninfected or infected with M. smegmatis (MOI = 10), L. monocytogenes (MOI = 5), HSV-1 (MOI = 1), or SeV (MOI = 1) for 6–12 hours and were then lysed and incubated with GST-RUFY1 (10 μg each) for 4 hours to immunoprecipitate GTP-bound Rab14 using Glutathione Sepharose 4B. The immunoprecipitates were subjected to immunoblotting analysis with antibodies against Rab14 and GST. Quantification was performed by measuring the relative intensity of the bands. Immunoblotting analysis of the interaction between Rab14 and CAMK2D in Rab14^+/+ RAW264.7 cells infected with M. smegmatis (e), L. monocytogenes (f), HSV-1 (g), or SeV (h). Cells were infected as in (a–d) and were then lysed for immunoprecipitating Rab14-interacting proteins using IgG- or Rab14-conjugated protein A/G agarose. The immunoprecipitates were subjected to immunoblotting analysis with antibodies against CAMK2D and Rab14. Quantification was performed by measuring the relative intensity of the bands. i Representative images showing the colocalization of V0a1 with LAMP1 in Rab14^+/+, Rab14^-/-, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- cells uninfected or infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV (Top). Cells transfected with GFP-V0a1 were uninfected or infected with the indicated pathogens as in (a–d) and were then fixed and stained with the antibody against LAMP1 (red). Scale bars: 5 µm. Bottom, Pearson’s coefficients of V0a1 with LAMP1. Data are mean ± s.e.m. with each symbol representing a biological replicate [n = 3 in (i)]. Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test (i). Results are representative of at least three independent experiments. Source data are provided as a Source Data file.

Rab14 promotes lysosomal acidification and pathogen clearance through CAMK2D

Given that Rab14 interacts with CAMK2D to facilitate the lysosomal targeting of V0a1, we then further investigated the role of CAMK2D in Rab14-promoted lysosomal acidification. Through examining the fluorescence intensity of LysoTracker in Rab14^+/+, Rab14^-/-, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- cells infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV, we found that upon pathogen infections, Rab14^-/- cells exhibited a lower LysoTracker fluorescence intensity compared to Rab14^+/+ cells, while CAMK2D knockout enhanced the LysoTracker fluorescence in Rab14^-/- cells, eliminating the phenotypic disparity between Rab14^+/+ and Rab14^-/- cells (Fig. 6a). Consistently, the lysosomal protease CTSD maturation analysis and CTSD activity assay revealed that Rab14 promotes the maturation and activation of CTSD in a manner dependent on CAMK2D (Fig. 6b–e, Supplementary Fig. 11). Furthermore, we monitored the dynamic autophagy flux in the above four types of cell lines by using the mCherry-GFP-LC3 tandem reporter system, which manifests as red puncta within autolysosomes and yellow puncta in immature autophagosomes⁴³. The results showed that although the number of the total LC3 puncta per cell was comparable in all cell lines upon pathogen infections, the ratio of yellow:total LC3 puncta was significantly increased in Rab14^-/- cells than that in Rab14^+/+, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- cells, indicating that Rab14 depends on CAMK2D to enhance autophagy flux during pathogen infections (Supplementary Fig. 12). A recent study reported that Rab14 promotes autophagy flux by facilitating homotypic fusion and protein sorting (HOPS) complex-mediated autophagosome-lysosome fusion in MDCK epithelial cells¹⁹. To determine whether the Rab14–CAMK2D axis regulates this process in macrophages, we examined the colocalization of LC3 with LAMP1 in Rab14^+/+, Rab14^-/-, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- macrophages under uninfected or infected conditions, and our results showed that the Rab14-CAMK2D axis did not affect the fusion between autophagosomes and lysosomes (Supplementary Fig. 13), suggesting that Rab14-CAMK2D axis promotes autophagy flux through facilitating lysosomal acidification without affecting the autophagosome-lysosome fusion. Finally, the pathogen intracellular survival assay demonstrated that Rab14 promotes the clearance of pathogens depending on CAMK2D (Fig. 6f). Collectively, CAMK2D is critical for Rab14-promoted lysosomal acidification and pathogen clearance.

Fig. 6. Rab14 promotes lysosomal acidification and pathogen clearance depending on CAMK2D.

a Representative images showing the LysoTracker staining in Rab14^+/+, Rab14^-/-, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- cells (Left). Cells were uninfected or infected with M. smegmatis (MOI = 10), L. monocytogenes (MOI = 5), HSV-1 (MOI = 1), or SeV (MOI = 1) for 6 hours and were then incubated with 75 nM LysoTracker (red) for an additional 30 minutes. Nuclei were stained with DAPI (blue). Scale bars: 5 µm. Right, Quantitation of LysoTracker fluorescent intensity. Immunoblotting analysis of CTSD in Rab14^+/+, Rab14^-/-, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- cells infected with M. smegmatis (b), L. monocytogenes (c), HSV-1 (d), or SeV (e). Cells were infected as in (a) and were then lysed for immunoblotting analysis with antibodies against CTSD and cleaved CTSD LC. TUBA1A was used as the loading control. The ratio of mature CTSD heavy chain or light chain to TUBA1A was calculated for quantification. f The intracellular survival of pathogens in Rab14^+/+, Rab14^-/-, Rab14^+/+Camk2d^-/-, and Rab14^-/-Camk2d^-/- cells infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV. Data are mean ± s.e.m. with each symbol representing a biological replicate [n = 3 in (a, f)]. Statistical significance was determined using one-way ANOVA with Tukey’s post-hoc test (a) and two-way ANOVA with Tukey’s post-hoc test (f). Source data are provided as a Source Data file.

To further verify whether CAMK2D participates in the modulation of Rab14-mediated lysosomal acidification and pathogen clearance depending on its kinase activity, we detected the LysoTracker fluorescence intensity and pathogen survival in Rab14^+/+ and Rab14^-/- cells with or without the treatment of KN-93 Phosphate (KN-93-P), which is a robust and specific inhibitor towards to CAMKⅡ family⁴⁴. As expected, inhibiting the kinase activity of CAMK2D using KN-93-P increased the fluorescence intensity of LysoTracker and improved the clearance of pathogens in Rab14^-/- cells, which levels were comparable to those in Rab14^+/+ cells (Supplementary Fig. 14). Altogether, these results indicate that Rab14 targets CAMK2D kinase activity to promote lysosomal acidification and pathogen clearance.

Rab14-mediated inhibition of CAMK2D-catalyzed V0a1 phosphorylation is required for pathogen clearance in vivo

To further determine the physiological relevance of Rab14-mediated inhibition of CAMK2D-catalyzed V0a1 phosphorylation in pathogen clearance in vivo, we challenged Rab14^flox/flox (Rab14^fl/fl) and rab14^fl/fl-Lyz2-Cre (rab14^lyz2) mice with M. smegmatis, L. monocytogenes, HSV-1, or SeV in the absence or presence of CMK2D kinase activity inhibitor KN-93-P. Through analyzing the pathogen burdens in different organs during infections, we found that rab14^lyz2 mice displayed higher bacterial loads or virus titers than Rab14^fl/fl mice, while KN-93-P treatment markedly reduced pathogen burdens in rab14^lyz2 mice, restoring them to levels comparable to those observed in Rab14^fl/fl mice (Fig. 7a–d). Consistently, histopathological analysis demonstrated that Rab14 deficiency led to increased inflammatory cell infiltration in the lungs of mice infected with M. smegmatis, HSV-1, or SeV, as well as in the livers of mice infected with L. monocytogenes, but the administration of KN-93-P attenuated these inflammatory responses (Fig. 7e–h). Thus, these results indicate that Rab14 functions as a critical host restriction factor that broadly defends against bacterial and viral infections through suppressing CAMK2D kinase activity towards V0a1.

Fig. 7. Rab14 contributes to pathogen clearance in vivo by targeting CAMK2D.

a Bacterial loads of M. smegmatis in the lungs and spleens of Rab14^fl/fl and rab14^lyz2 mice. Mice were intratracheally infected with 1 × 10⁸ CFU of M. smegmatis for 7 days, concurrently with 5 mg/kg KN-93 Phosphate (KN-93-P) treatment via intraperitoneal injection once every 3 days. Thereafter, the lungs and spleens were collected for CFU counting. b Bacterial loads of L. monocytogenes in the livers and spleens of Rab14^fl/fl and rab14^lyz2 mice. Mice were intraperitoneally infected with 2.5 × 10⁵ CFU of L. monocytogenes for 3 days with the treatment of KN-93-P, and the livers and spleens were collected for CFU counting. c HSV-1 titers in the lungs and spleens of Rab14^fl/fl and rab14^lyz2 mice. Mice were intravenously infected with 2 × 10⁷ PFU of HSV-1 for 2 days with the treatment of KN-93-P, and lungs and spleens were homogenized for detecting virus titers. d SeV titers in the lungs and spleens of Rab14^fl/fl and rab14^lyz2 mice. Mice were intravenously infected with 2 × 10⁷ PFU of SeV for 2 days with the treatment of KN-93-P, and lungs and spleens were homogenized for detecting virus titers. e Histopathology of lung sections from Rab14^fl/fl and rab14^lyz2 mice infected with M. smegmatis. Scale bars: 200 µm. f Histopathology of liver sections from Rab14^fl/fl and rab14^lyz2 mice infected with L. monocytogenes. Scale bars: 200 µm. Histopathology of lung sections from Rab14^fl/fl and rab14^lyz2 mice infected with HSV-1 (g) or SeV (h). Scale bars: 200 µm. Data are mean ± s.e.m. with each symbol representing a biological replicate [n = 4 in (a–d)]. Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test (a–d). Source data are provided as a Source Data file.

In summary, our study demonstrates that upon pathogen infections, the active GTP-bound Rab14 increases and binds to CAMK2D, disrupting the CAMK2D-V0a1 interaction and CAMK2D-mediated phosphorylation of V0a1 at Ser19, thereby enabling V0a1 to engage with Sec24C in the COPII complex to facilitate the anterograde transport of the V0 complex from the ER to the Golgi apparatus and then to lysosomes. Ultimately, the V0 complex assembles with the V1 complex to form a functional V-ATPase for mediating lysosomal acidification and pathogen clearance (Supplementary Fig. 15).

Discussion

Host restriction factors are essential components of the intrinsic immune system that provide a natural defense against infections. While viral restriction factors like TRIM family proteins have been extensively studied^45,46, host restriction factors against bacterial pathogens, particularly those with broad-spectrum activity toward both bacterial and viral pathogens, remain poorly understood. In this study, we identify Rab14 as a unique host restriction factor with dual antiviral and antibacterial functions through promoting lysosomal acidification, broadening the repertoire of restriction factors with broad-spectrum activity. Moreover, it should be mentioned that the role of restriction factors is profoundly shaped by pathogen-host interactions. Some pathogens have evolved strategies to counteract or exploit these host defenses. For example, TRIM5α restricts HIV-1 by recognizing viral capsid proteins, while HIV-1 could modulate capsid surface dynamics to impair TRIM5α recruitment, thereby promoting virus replication⁴⁷. Likewise, TRIM27 functions as a potential restriction factor against mycobacteria, while the pathogen can exploit its tyrosine phosphatase PtpA to antagonize TRIM27 functions⁴⁸. Consistently, Rab14 is also hijacked by several pathogens including Mycobacterium tuberculosis, which employs its effectors like eukaryotic-type protein kinase G to interact with Rab14 to maintain the sustained over-activation of Rab14, thus suppressing phagosome/autophagosome fusion with the late endosomes and lysosomes to eliminate the pathogen^49,50. It should also be mentioned that the role of Rab14 is cell-type dependent. While a recent study reported that Rab14 facilitates autophagosome-lysosome fusion through HOPS complex in MDCK epithelial cells¹⁹, our work demonstrates that in infected macrophages, the Rab14-CAMK2D axis does not regulate this fusion process. Together, Rab14 is a critical host restriction factor whose activity is fine-tuned by cell-type-specific mechanisms, which process could also be exploited by certain pathogens for their immune evasion.

Lysosomal acidification is the basis for the proper lysosomal functions including the elimination of pathogens, and V-ATPase is the central regulator responsible for maintaining an acidic luminal pH. Precise targeting to the specific organelle is crucial for V-ATPase to mediate organelle acidification, yet the trafficking mechanisms underlying its interorganellar transport remain largely unclear. Here, we elucidate a Rab14-mediated trafficking mechanism of V-ATPase from the ER to lysosomes. Specifically, Rab14 facilitates the loading of V0a1 onto Sec24C within the COPⅡ complex through interacting with CAMK2D to inhibit CAMK2D-catalyzed phosphorylation of V0a1, thus promoting the trafficking of the V0 complex from the ERs to the Golgi apparatus and then to lysosomes, where it assembles with the V1 complex to mediate lysosomal acidification. This finding identifies a previously uncharacterized PTM (phosphorylation) that controls V-ATPase trafficking, expanding the previously characterized mechanisms such as glycosylation and palmitoylation^30,31. It is worth mentioning that unlike glycosylation and palmitoylation that facilitate V0a1 trafficking to lysosomes, phosphorylation of V0a1 acts as a negative regulator inhibiting this process, suggesting that phosphorylation serves as a molecular brake to prevent ectopic protein localization and ensure spatiotemporal precision in cellular processes. Similar regulatory paradigms are also observed in other trafficking systems, for instance, GSK-3-mediated phosphorylation of CREB-H, a transcription factor regulating secretion in metabolic pathways, suppresses the ER-Golgi transport and nuclear stabilization of CREB-H, thereby controlling the metabolic homeostasis⁵¹. In the context of V-ATPase, this inhibitory phosphorylation may serve as a safeguard against premature over-acidification of endolysosomal compartments under quiescent conditions, since excessive V-ATPase activation is linked to lysosomal storage disorders⁵². Conversely, under certain circumstances such as pathogen infections, Rab14 undergoes GTP/GDP cycling to relieve phosphorylation-mediated inhibition of V-ATPase, enabling V-ATPase trafficking to lysosomes to mediate lysosomal acidification and pathogen clearance. Furthermore, our study expands the functional repertoire of CAMK2D, a kinase historically linked to cardiac anomalies and neurodevelopment^53,54, by identifying V0a1 as a newly characterized substrate and uncovering its previously unrecognized roles in regulating V-ATPase trafficking and lysosomal acidification during infections.

Traditional antibiotics and antivirals primarily target pathogen-specific components, such as cell walls and viral proteases, but their efficacy is increasingly threatened by the rapid emergence of drug-resistant pathogens, a crisis projected to claim 10 million lives annually by 2050 if unaddressed⁵⁵. Against this backdrop, HDT has gained traction as a paradigm-shifting strategy, which has the potential to provide broad-spectrum protection against a wide range of infectious agents by targeting conserved host factors such as restriction factors³. Through functional and mechanistic studies including adopting the CAMK2D kinase activity inhibitor KN-93-P to block V0a1 phosphorylation, we confirmed that Rab14 serves as a previously unrecognized host restriction factor against infections through interacting with CAMK2D to facilitate V-ATPase trafficking and lysosomal acidification. Based on our findings, we speculate that the Rab14-CAMK2D-V-ATPase axis might be a promising therapeutic target for HDT treating multiple infections. Moreover, considering that dysfunction of lysosomal acidification is also implicated in the pathogenesis of neuroinflammation and neurodegeneration⁵⁶, thus targeting the Rab14-CAMK2D-V-ATPase axis represents a potential dual-purpose strategy capable of addressing both infectious diseases and their comorbidities such as neuroinflammatory/neurodegenerative disorders. In summary, our study advances the understanding of host intrinsic immunity by unveiling a critical role for the restriction factor Rab14 in facilitating V-ATPase trafficking and lysosomal acidification, thereby highlighting a promising HDT strategy for infectious diseases through specifically targeting the Rab14-CAMK2D-V-ATPase axis.

Methods

Research ethics board

All animal breeding and procedures were performed in accordance with the instructional guidelines of the China Council on Animal Care, and were approved by the Biomedical Research Ethics Committee of the Institute of Microbiology, Chinese Academy of Sciences, and the Beijing Chest Hospital, Capital Medical University (HP-SQIMCAS2025159).

Mouse models

Rab14^fl/fl mice and rab14^fl/fl-Lyz2-Cre (rab14^lyz2) mice with Rab14 deficiency in myeloid cell lineage (monocytes, mature macrophages, and granulocytes) were generated as described previously⁴⁹. Briefly, Rab14^fl/fl mice and Lyz2-Cre mice were obtained from Biocytogen (Beijing, China) and Jackson Laboratory, respectively. Rab14^fl/fl mice were crossed with Lyz2-Cre mice to generate F1 rab14^fl/+-Lyz2-Cre mice, followed by crossing with Rab14^fl/fl mice to generate rab14^lyz2 mice. All mice were housed and bred in a specific pathogen-free (SPF) facility using standard humane animal husbandry protocols (12 h light/dark cycle, 50% relative humidity, at 25–27 °C, free access to food and tap water), which were approved by the animal care and use committee of the Institute of Microbiology (Chinese Academy of Sciences).

Rab14^fl/fl mice and rab14^lyz2 mice aged 6–8 weeks and sex-matched are infected with the indicated pathogens. For M. smegmatis infection, mice were intratracheally infected with 1 × 10⁸ CFU of M. smegmatis mc²155 strain per mouse, concurrently with 5 mg/kg KN-93-P treatment via intraperitoneal injection once every 3 days. After 7 days, lungs and spleens were homogenized with a FastPrep-24 System (MP Biomedicals) for CFU counting, and lungs were fixed in 10% formalin and embedded in paraffin for hematoxylin and eosin staining. The tissue slides were scanned with Aperio CS2 (Leica Biosystems), and the histopathology of tissues was analyzed by ImageScope (v12.3.3.7014) software. For L. monocytogenes infection, mice were intraperitoneally infected with 2.5 × 10⁵ CFU of L. monocytogenes per mouse and treated with KN-93-P as above. After 3 days of infection, livers and spleens were homogenized for assessing bacterial numbers in organs, and livers were subjected to section for pathological analysis. For HSV-1 and SeV infection, mice were intravenously infected with 2 × 10⁷ PFU of each virus per mouse and treated with KN-93-P as above. After 2 days of infection, lungs and spleens were homogenized for detecting virus titers, and lungs were subjected to section for pathological analysis.

Bacterial strains, viruses, and plasmids

Mycobacterium smegmatis mc²155 (ATCC, 700084) was grown in Middlebrook 7H9 medium (BD, 271310) supplemented with 10% oleic acid-albumin-dexrose-catalase (OADC) and 0.05% Tween-80 (G-CLONE, CS9029), or on Middlebrook 7H10 agar (BD, 262710) supplemented with 10% OADC. Listeria monocytogenes (ATCC, 19115) was cultured in liquid brain heart infusion (BHI; ELITE-MEDIA, m330-02) or BHI agar plates overnight at 37 °C. HSV-1 was a kind gift of Xin Ye (Institute of Microbiology, Chinese Academy of Sciences, Beijing) and was propagated in Vero cells (ATCC, CCL-81). SeV was a kind gift of George Fu Gao (Institute of Microbiology, Chinese Academy of Sciences, Beijing) and was propagated in 10-day-old SPF chicken embryos.

For protein expression in mammalian cells, Rab14 was cloned into pcDNA6A (with MYC-tag) (Invitrogen, V22120), pEGFP-C1 (with GFP-tag) (Clontech, V012024), or p3xFlag-CMV14 (with Flag-tag) vector (Sigma-Aldrich, E7908); CAMK2D and its truncated mutants were cloned into p3xFlag-CMV14 (with Flag-tag) or pEGFP-C1 (with GFP-tag) vector; V0a1 and its mutants, V0a2, and V0a3 were cloned into pEGFP-C1 (with GFP-tag) vector. For protein expression in bacteria, Rab14 or RUFY1 was cloned into pGEX-6p-1 (with GST-tag) vector (GE Healthcare, 28-9546-48), CAMK2D was cloned into pET30a (with His₆-tag) vector (Novagen, 69909). All the plasmids were sequenced at the Beijing Genomics Institute (BGI) for verification. All of the plasmids and primers used in this study were detailed in Supplementary Data 3.

Antibodies and reagents

All antibodies were used according to the manufacturers’ instructions. The commercial antibodies used in this study were as follows: anti-Rab14 (Santa Cruz Biotechnology, sc-271401, 1:100 for immunoprecipitation, 1:1,000 for immunoblotting), anti-CTSD (Abclonal, A13292, 1:2,000 for immunoblotting), anti-cleaved CTSD LC (Immunoway, YC0038, 1:2,000 for immunoblotting), anti-LC3B (Sigma-Aldrich, L7543, 1:3,000 for immunoblotting), anti-calnexin (Santa Cruz Biotechnology, sc-23954, 1:200 for immunofluorescence), anti-GM130 (Abcam, ab52649, 1:200 for immunofluorescence), anti-Rab5 (Abcam, ab218624, 1:100 for immunofluorescence), anti-Rab7 (Abcam, ab137029, 1:100 for immunofluorescence), anti-LAMP1 (Cell Signaling Technology, 99437, 1:100 for immunofluorescence), anti-p-Ser (Abcam, ab9332, 1:1,000 for immunoblotting), anti-p-Thr (Cell Signaling Technology, 9386, 1:1,000 for immunoblotting), anti-V0a1 (Proteintech, 13828-1-AP, 1:100 for immunoprecipitation, 1:1,000 for immunoblotting), anti-CAMK2D (Abclonal, A9196, 1:2,000 for immunoblotting), anti-Sar1 (Immunoway, YN0749, 1:2,000 for immunoblotting), anti-Sec23A (Immunoway, YN3350, 1:2,000 for immunoblotting), anti-Sec24A (Immunoway, YN6048, 1:2,000 for immunoblotting), anti-Sec24C (Abclonal, A10797, 1:2,000 for immunoblotting), anti-Sec31A (Immunoway, YN5979, 1:2,000 for immunoblotting), anti-TUBA1A (Sigma-Aldrich, T6199, 1:5,000 for immunoblotting), anti-GAPDH (Santa Cruz Biotechnology, sc-25778, 1:5,000 for immunoblotting), anti-Myc (MBL International, M047-7, 1:4,000 for immunoblotting), anti-Myc (Cell Signaling Technologies, 2276S, 1:200 for immunofluorescence), anti-Flag (MBL International, PM020-7, 1:4,000 for immunoblotting), anti-GFP (MBL International, 598-7, 1:4,000 for immunoblotting), anti-GST (MBL International, PM013-7, 1:4,000 for immunoblotting), anti-His (Abclonal, AE003, 1:4,000 for immunoblotting), HRP-labeled goat anti-rabbit IgG (ZSGB Biotech, ZB-2306, 1:10,000 for immunoblotting), HRP-labeled goat anti-mouse IgG (ZSGB Biotech, ZB-2305, 1:10,000 for immunoblotting), Alexa Fluor 594-labeled goat anti-rabbit IgG (ZSGB Biotech, ZF0516, 1:200 for immunofluorescence), Alexa Fluor 594-labeled goat anti-mouse IgG (ZSGB Biotech, ZF0513, 1:200 for immunofluorescence), and Alexa Fluor 488-labeled goat anti-rabbit IgG (CHEJETER, ZF-0511, 1:200 for immunofluorescence). The phosphospecific antibody against p-V0a1 (Ser19) was produced as described previously^57,58. Briefly, a 16-amino acid peptide encompassing phospho-Ser19 was synthesized and conjugated to keyhole limpet hemocyanin (KLH) for immunization in rabbits. Thereafter, the antibody was affinity-purified from the serum using the same phospho-peptide. The purified antibody was validated by immunoblotting at a working concentration of 5 µg/mL, demonstrating a specific signal against GFP-V0a1, but not the GFP-V0a1^S19A mutant.

The commercial reagents used in this study were as follows: Bafilomycin A1 (Baf; Selleck, S1413), KN-93 phosphate (Selleck, S7423), LysoTracker (Invitrogen, L7528), and LysoSensor Yellow/Blue probe (Invitrogen, L7545).

Cell culture and transfection

HEK293T cells (ATCC, CRL-3216), HeLa cells (ATCC, CCL-2), Vero cells (ATCC, CCL-81), and RAW264.7 cells (ATCC, TIB-71) were obtained from the American type culture collection (ATCC). The above cell lines were all cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, C11995500BT) with 10% fetal bovine serum (FBS; Gibco, 16000-044) at 37 °C in a humidified 5% CO₂ incubator. In related experiments, the transfection of HEK293T or HeLa cells was carried out with KunGre™ Liposomal Transfection Reagent (Greact, GT201-03) according to the manufacturers’ instructions.

Recombinant protein expression and purification

For prokaryotic expression of GST, GST-Rab14, GST-RUFY1, and His-CAMK2D, E. coli BL21 (DE3) strains harboring GST or His derivatives were grown in LB medium supplemented with the indicated antibiotics at 37 °C until the OD₆₀₀ = 0.6. Then, 0.1 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the cultures to induce the expression of proteins at 16 °C for 16 hours. Bacteria were then harvested by centrifugation at 6,500 ×g for 10 minutes and suspended in a buffer containing 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. Subsequently, cells were broken using Low-temperature Ultra-high Pressure Continuous Flow Cell Disrupters (JNBIO) for collecting the supernatants. Glutathione Sepharose 4B (GE Healthcare, 17-0756-05) and Ni-NTA Agarose (Qiagen, 30230) were added to the supernatants for purification of GST- or His-tagged proteins, respectively. For eukaryotic expression and purification of GFP-tagged V0a1 or its mutants, Hela cells were transfected with the indicated plasmids for 24 hours and were then lysed in the RIPA lysis buffer (Beyotime Biotechnology, P0013D) supplemented with 1% protease inhibitor cocktail (Bimake, B14001) at 4 °C for 10 minutes. The cell lysates were then incubated with anti-GFP-Nanoab-Agarose (LABLEAD, GNA-50-1000) at 4 °C for 4 hours. After extensively washing with the lysis buffer, the GFP-tagged proteins were eluted by 50 μL elution buffer (0.2 mM glycine, adjust pH to 2.5 by HCl) and neutralized by 5 μL neutralization buffer (1.0 M Tris-HCl, pH 10.4). Protein concentrations were determined by the BCA protein assay (Beyotime, P0009) and protein purity was examined by SDS-PAGE analysis.

Establishment of knockout and knockdown cell lines

RAW264.7 cells and Hela cells with Rab14 or CAMK2D knocking out were generated using the CRISPR/Cas9 gene-editing system. Specifically, the sgRNA sequences targeting Rab14 or CAMK2D (see Supplementary Data 3) were predicted by the CRISPR-Gold (https://crisprgold.mdc-berlin.de/) and were then cloned into the pSpCas9(BB)-2A-GFP vector (Addgene, 48138). Next, RAW264.7 cells and Hela cells were transfected with the specific recombinant plasmid for 24 hours. The single cell with green fluorescence was sorted with BD FACSAria III cell sorter (BD Biosciences) and was then identified by immunoblotting analysis with the antibody against Rab14 or CAMK2D.

For knocking down Rab genes in RAW264.7 cells, the siRNA sequences targeting the specific Rab (see Supplementary Data 3) were designed by BLOCK-iT™ RNAi Designer. Then, 40 pM siRNAs targeting each Rab gene were transfected into RAW264.7 cells by using KunGre^TM Liposomal Transfection Reagent (Greact, GT201-03) according to the manufacturer’s instructions. After 12 hours, cells were re-transfected with the sgRNAs and were then further incubated for another 12 hours. The knocking down efficiency of Rab genes was verified by the quantitative real-time polymerase chain reaction (qPCR) analysis. V0a1-knocking down Rab14^+/+ and Rab14^-/- RAW264.7 cells were generated in the same as above and were then identified by immunoblotting analysis with the antibody against V0a1.

qPCR analysis

RAW264.7 cells transfected with Rab siRNAs were collected for total RNA extraction using the Total RNA Extraction Kit (Dongsheng, R1061). Then, the RNA was reverse-transcribed into cDNA using the Hifair First Strand cDNA Synthesis SuperMix (YEASEN, 11141ES60). The cDNA was then analyzed by qPCR using Hieff qPCR SYBR Green Master Mix (YEASEN, 11202ES08) on an ABI 7500 system (Applied Biosystems). Quantitative expression of the targeted genes was normalized to Gapdh and analyzed by the 2^−ΔΔCT method. All qPCR primers are listed in Supplementary Data 3.

Establishment of RAW264.7 cells stably expressing mCherry-GFP-LC3

RAW264.7 cells stably expressing mCherry-GFP-LC3 were generated as described previously⁴⁹. Briefly, PMSCV puro-mCherry-GFP-LC3 and the two packaging plasmids including pVSV-G and pCMV-Gag-Pol were co-transfected to HEK293T cells for obtaining lentivirus. Then, the lentiviral supernatant was collected at 48 hours post-transfection, filtered with a 0.45-μm filter, and concentrated by Lentivirus Concentration Solution (YEASEN, 41101ES50). The medium containing lentivirus was used to infect RAW264.7 cells, and the cells stably expressing mCherry-GFP-LC3 were selected using 2 μg/mL puromycin (Selleck, s7417). The expression of mCherry-GFP-LC3 was verified by immunofluorescence microscopy.

Measurement of phagolysosomal pH

The pH of pathogen-containing phagolysosomes was detected as described previously⁵⁹. Briefly, the model pathogen M. smegmatis was first labeled with 20 μM pHrodo Red succinimidyl ester (Invitrogen, P36600) at 37 °C for 1 hour. Next, the labelled bacteria were washed three times with Middlebrook 7H9 media containing 0.05% Tween-80 and were then labeled with 25 μg/mL Alexa Fluor 488 succinimidyl ester (Invitrogen, A20000) at 37 °C for 1 hour. After washing again, bacterial pellets were resuspended in DMEM containing 0.05% Tween-80. Thereafter, RAW264.7 cells were infected with the double-labeled M. smegmatis at a multiplicity of infection (MOI) of 10 for 2 hours and were then washed with 1 × PBS to remove any non-internalized bacteria. The infection was incubated for another 2 hours at 37 °C with 5% CO₂, and the fluorescence intensity in the RAW264.7 cells was analyzed by Flow cytometer (BD LSRFortessa SORP) and FlowJo (v10) software. The phagolysosomal pH was calculated by the mean fluorescence ratios of pHrodo and Alexa Fluor 488.

Pathogen infections of macrophages

RAW264.7 cells were seeded at 1 × 10⁶ cells per well in 6-well plates and cultured at 37 °C with 5% CO₂ overnight. For M. smegmatis infection, frozen bacteria that have grown to mid-logarithmic phase were thawed and were then centrifuged and resuspended in DMEM medium with 0.05% Tween-80 to infect RAW264.7 cells at a MOI of 10. After 2 hours of infection, cells were washed three times with 1 × PBS to exclude non-internalized bacteria and were then incubated with the fresh DMEM medium supplemented with 10 μg/mL gentamicin to kill non-washed extracellular bacteria. At the indicated time points, cells were harvested for colony-forming unit (CFU) counting analysis, immunoblotting analysis, and immunofluorescence analysis. For L. monocytogenes infection, bacteria were grown in BHI broth to mid-log phase and were then resuspended in DMEM medium to infect RAW264.7 cells at a MOI of 5. After 1 hour of infection, cells were washed with 1 × PBS to exclude non-internalized bacteria and were then incubated with the fresh DMEM medium supplemented with 10 μg/mL gentamicin until the indicated time points for further CFU, immunoblotting, and immunofluorescence analysis. For viral infection, the DNA virus HSV-1 and RNA virus SeV were both used to infect RAW264.7 cells at a MOI of 1. At the indicated time points, the cells were collected for virus copy number, immunoblotting, and immunofluorescence analysis. For inhibition of CAMK2D or autophagy flux, cells were treated with 10 μM KN-93-P or 50 nM Baf, respectively, and the control groups were treated with equal volume of dimethyl sulfoxide.

CTSD activity assay

CTSD activity was measured using a commercial assay kit (Abcam, ab65302). Briefly, harvested cells were lysed in chilled buffer, and the lysates were incubated with the substrate-containing reaction mix at 37 °C for 1–2 hours, protected from light. Fluorescence was measured at Ex/Em = 328/460 nm using a microplate reader. Enzyme activity was determined based on the fold change in relative fluorescence units (RFU) of treated samples compared to control samples.

CFU counting analysis of bacteria and plaque assay of virus

For CFU counting analysis of M. smegmatis or L. monocytogenes, infected macrophages or tissues were lysed in 1 × PBS containing 0.05% SDS. Then, the cell lysates were plated by serial dilution on 7H10 agar plates for 72 hours or on BHI agar plates for 24 hours, respectively. For plaque assay of virus, the homogenates of lungs or spleens were harvested and diluted to infect confluent Vero cells. At 2 hours post infection, the supernatant was removed and the infected Vero cells were overlaid with a mixed medium of 2% low melting point agarose (YEASEN, 10214ES08) and 2% FBS at a ratio of 1:1. After 72 hours post infection, the overlay was removed and the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, 441244) for 15 minutes and stained with 0.1% crystal violetin (Huaaobio, 01-174) for 30 minutes. Then, the plaques were counted, averaged, and multiplied by the dilution factor to determine viral titers as PFU/mL. Virus copy numbers were determined by qPCR using the corresponding primers (Supplementary Data 3).

ER and lysosome extraction

ER and lysosomal fractions were isolated using the Endoplasmic Reticulum Extraction Kit (Solarbio, EX1370) and the Lysosome Enrichment Kit (Thermo Scientific, 89839), respectively. Briefly, cells were homogenized using a Dounce homogenizer, followed by ER enrichment through differential centrifugation and lysosome purification via density gradient separation according to the manufacturers’ instructions. All procedures were performed at 4 °C with pre-cooled reagents.

Immunoprecipitation

For immunoprecipitation of V0a1 or Rab14 from RAW264.7 cells, cells were lysed with the RIPA lysis buffer supplemented with 1% protease inhibitor cocktail at 4 °C for 10 minutes. Then, cell lysates were incubated with the antibody against V0a1 or Rab14 and protein A/G agarose (Santa Cruz Biotechnology, sc-2003) at 4 °C, followed by washing with the RIPA lysis buffer for three times. For co-immunoprecipitation of two proteins co-overexpressed in HEK293T cells, cells were lysed in the RIPA lysis buffer at 4 °C and were then incubated with anti-Flag M2 Affinity Gel (Sigma-Aldrich, A2220), anti-Myc Agarose (Santa Cruz Biotechnology, sc-40AC), or anti-GFP-Nanoab-Agarose (LABLEAD, GNA-50-1000) at 4 °C for 4 hours. Thereafter, the cells were washed with the RIPA lysis buffer for three times. The immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies.

In vitro precipitation assay

For competitive binding analysis, 5 μg of His-CAMK2D was incubated with 20 μL of Ni-NTA Agarose in 500 μL binding buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM DTT, and 0.1% NP-40) containing 1% protease inhibitor cocktail for 1 hour at 4 °C. Then, the agaroses carrying His-CAMK2D were collected and washed three times with the binding buffer, followed by incubating with 13 μg of purified GFP-V0a1 in the presence or absence of 1 μg, 2 μg of GST or 2 μg, 5 μg of GST-Rab14 in the binding buffer for 4 hours at 4 °C. After three washes, the bound protein complexes were finally subjected to immunoblotting analysis.

Acyl-Rac assay

The acyl-Rac assay was conducted to determine the S-palmitoylation of proteins as described previously^60,61. Briefly, proteins from cell lysates were incubated with blocking buffer (100 mM HEPES, 1 mM EDTA, 2.5 % SDS, 50 mM N- ethylmaleimide, pH 7.5) at 50°C for 1 hour, followed by precipitation with three volumes of ice-cold acetone at -20°C for 20 minutes. After centrifugation at 5,000 ×g for 10 minutes, the pellet was washed four times with 70% acetone. Next, the pellet was resuspended in binding buffer (100 mM HEPES, 1.0 mM EDTA, 1 % SDS, pH 7.5) and split into two aliquots. One aliquot was treated with 2 M NH₂OH (freshly prepared, pH 7.5) to specifically cleave thioester bonds and expose nascent thiol groups, while the other was treated with PBS as a negative control. The newly exposed thiols were then biotinylated using EZ-Link™ HPDP-Biotin (Thermo Scientific, A35390). Biotinylated proteins were affinity-purified with Pierce™ streptavidin coated magnetic beads (Thermo Scientific, 89891) at room temperature for 2 hours. After three washes, the beads were mixed with loading buffer and subjected to immunoblotting analysis.

Immunoblotting analysis

Proteins were separated by SDS-PAGE gels and transferred to the polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010). The membranes were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) solution for 1 hour at room temperature and were then incubated with specific primary antibodies overnight at 4 °C. Thereafter, the membranes were washed with TBST for three times and were then incubated with secondary antibodies for 1 hour at room temperature followed by three washes with TBST. Finally, the membranes were developed by Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500) and exposed to X-ray film. For quantifications, densitometry was performed using ImageJ (v1.8.0) to evaluate the intensity of the immunoblotting band signals. The uncropped and unprocessed scans of all blots are provided in the Source Data file.

Mass spectrometry (MS) analysis

For identifying Rab14-interacting proteins by MS analysis, immunoprecipitates of Flag or Flag-Rab14 were separated by SDS-PAGE. Then, the gels were stained with the Coomassie brilliant blue and the indicated bands were excised. Thereafter, the bands were rinsed with 100 mM ammonium bicarbonate, dried, and reduced using 10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate at 56 °C and were then alkylated with 55 mM iodoacetamide. Proteins were digested with 12.5 ng/μL trypsin overnight at room temperature, and the peptide mixture was extracted by 60% acetonitrile. Next, samples were desalted on a C18 ZipTip pipette tip (Millipore) and were separated via Nano LC-MS/MS analysis on a NanoAcquity system (Waters, Milford, MA) connected to an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The MS raw files were processed using Proteome Discoverer (Version 1.4.0.288, Thermo Fischer Scientific) and were then searched against the Uniprot_human_2018 database by using the SEQUEST engine. The differential proteins with at least five unique peptides in the Flag-Rab14 group compared with the Flag group were considered the potential interactive proteins of Rab14, and the protein-protein interaction network diagram of Rab14-interacting proteins was edited by utilizing Adobe Illustrator CS6 (AI) software.

Immunofluorescence microscopy

Cells were seeded on cover glasses in 6-well cell culture plates and were then transfected with the indicated plasmids or infected with the indicated pathogens. Specifically, for detecting the co-localization of proteins with subcellular organelles, GFP-V0a1, GFP-V0a2, GFP-V0a3, or GFP-LC3 was transfected to Hela cells for 24 hours. Then, cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100 (Solarbio, T8200) for 15 minutes, and blocked with 5% BSA (Solarbio, A8010) for 30 minutes at room temperature. Primary antibodies against subcellular organelles including ER (anti-calnexin), Golgi (anti-GM130), early endosomes (anti-Rab5), late endosomes (anti-Rab7), and lysosomes (anti-LAMP1) were incubated with the cells for 2 hours at room temperature. After washing three times with 1 × PBS, the fluorophore-conjugated secondary antibodies were applied for 1 hour. After washing again, slides were mounted with medium containing 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, C1002). For detecting lysosomal acidification during pathogen infections, RAW264.7 cells were infected with the indicated pathogen as described above and were then incubated with 75 nM LysoTracker for an additional 30 minutes. Next, cells were fixed and stained with DAPI. For detecting autophagy flux during pathogen infections, RAW264.7 cells stably expressing mCherry-GFP-LC3 were infected with the indicated pathogen as described above and were then fixed with 4% paraformaldehyde. For microscopy analysis, confocal images were taken with the Leica SP8 confocal microscope (Leica Microsystems) and analyzed by the Leica Application Suite Las X (v2.0.1.14392) software. For the image quantifications, approximately 50 cells were analyzed with ImageJ (v1.8.0) for each biological replicate, and the data was shown as the mean ± SEM of three independent experiments.

Live-cell imaging

For detecting V0a1 subcellular localization, Rab14^+/+ and Rab14^-/- RAW264.7 cells were plated on Glass Bottom Culture Dishes (NEST, 801002) and were then transfected with GFP-V0a1 for 24 hours. Thereafter, cells were infected with M. smegmatis prestained with Alexa Flour^TM405 (Invitrogen, A30000) at a MOI of 10 for 2 hours and were then incubated with 75 nM LysoTracker for 30 minutes. Next, imaging was performed using a Leica SP8 confocal microscope equipped with an environmental control chamber providing 37 °C, 5% CO₂, and 20–30% humidity. Images were acquired in 5 minutes intervals over a time frame of 4 hours. For examining lysosomal acidification, the LysoSensor Yellow/Blue probe was added at 2 μM to cells and incubated at 37 °C for 30 minutes. After being washed twice with pre-warmed growth medium, the cells were immediately visualized using a Leica SP8 confocal microscope. The fluorescence intensities in the blue and yellow channels were quantified using ImageJ (v1.8.0).

Molecular docking and multiple sequence alignment analysis

Molecular docking analysis of V0a1 and CAMK2D was performed as described previously⁶². Briefly, the structure of V0a1 (AlphaFold DB: AF-Q93050-F1) was predicted using AlphaFold 3, and the solved crystal structure of CAMK2D (PDB: 5VLO) was obtained from the RCSB PDB database. Both structures were then prepared by removing water molecules, heteroatoms, and co-crystallized ligands, followed by the addition of polar hydrogen atoms. The protein–protein molecular docking of V0a1 and CAMK2D was performed by ClusPro web server (https://cluspro.org). Interface analysis was performed using the PISA web server (https://www.ebi.ac.uk/pdbe/pisa/). Visualization and superimposition of docking poses and crystallographic structures were done using PyMOL (v.2.5.7). Additionally, multiple sequence alignment of V0a1, V0a2, and V0a3 was performed using ESPript 3.0 server (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Conserved residues are marked with the red background.

In vitro and in vivo phosphorylation assay

In vitro phosphorylation assay detecting CAMK2D-mediated V0a1 phosphorylation was performed as described previously⁶³. Briefly, 13 μg purified GFP-V0a1 or its mutants was incubated with 5 μg His-CAMK2D in the presence or absence of 5 μg GST-Rab14 in 200 μL kinase reaction buffer (10 mM HEPES, pH 7.2, 1 mM EGTA, 5 mM MgCl₂, 2 mM CaCl₂, and 0.4 mM ATP) supplemented with 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail. After incubation at 30 °C for 30 minutes, the reaction was terminated by the addition of SDS loading buffer and subjected to SDS-PAGE analysis. V0a2 and V0a3 phosphorylation was examined using the same method. For in vivo phosphorylation assay, V0a1 or its mutant was immunoprecipitated from Rab14^+/+ and Rab14^-/- cells and was then subjected to immunoblotting with the antibody against p-Ser or p-Thr.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0 software (https://www.graphpad.com/). All data were analyzed using unpaired two-sided Student’s t-test, one-way ANOVA, or two-way ANOVA analysis followed by multiple comparisons as indicated in the corresponding figure legends. Data are shown as mean and standard error of mean (s.e.m.). Unless otherwise indicated, at least two biological replicates were included in all experiments. Additional details about the statistical analysis of experiments and number of biological replicates (n) are indicated in the corresponding figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

C.H.L. conceived the study and carried out the project administration. C.H.L., J.W., L.Z., and B.L. managed funding acquisition. Z.Lei., L.Q., and J.W. performed the experiments and functional analyses. P.G., Y.Q., T.S., Q.C., Y.Wang., S.L., C.Q., M.Z., Z.Z., Y.Wu., X.Z., Y.Z., and B.L. assisted with experiments. Z.Lu. performed the bioinformatic analyses. C.H.L., J.W., L.Z., Z.Lei., and L.Q. wrote and revised the manuscript. All authors read and approved the final version of the manuscript.

Peer review

Peer review information

Nature Communications thanks Meixin Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

All data are available in the main text and the Supplementary Materials. The plasmids and cell lines generated in this study are available from the corresponding author. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-026-70258-w.