Bacterial culture media including 7H9 Middlebrook broth and 7H10 Middlebrook agar supplemented with oleic acid-albumin-dextrose-catalase (OADC) were purchased from BD Biosciences (San Diego, CA, USA). Tween 80 and glycerol were obtained from Sigma-Aldrich (St. Louis, MO, USA). RPMI1640 medium, fetal bovine serum (FBS) and Penicillin-Streptomycin Liquid were purchased from Gibco (Waltham, MA, USA). Primary antibodies against LC3, beclin1 and GAPDH were purchased from Abcam (Cambridge, MA, USA). FITC-conjugated goat anti-rabbit IgG (H+L) secondary antibody and 180kd protein molecular weight markers were obtained from Beyotime Biotechnology (Shanghai, China).

The recombinant HBHA-expressing Mycobacterium smegmatis (MS) strain mc²155(rHBHA-MS) was constructed in our previous study (18). Mycobacterium bovis bacillus Calmette-Guérin (BCG) was obtained from ATCC (No.35734). rHBHA-MS and BCG were grown in 7H9 Middlebrook broth supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.05% Tween 80 and 0.2% glycerol, or on 7H10 Middlebrook agar supplemented with 10% OADC. The murine macrophage cell line RAW264.7 was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). ECSIT knockdown RAW264.7 (ECSIT^KD RAW264.7) was generated by transduction of shRNA in our previous research. RAW264.7 and ECSIT^KD RAW264.7 were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin.

The recombinant HBHA protein was expressed and purified as previously described (18). RAW264.7 and ECSIT^KD RAW264.7, either subjected to starvation induction or infected with BCG (MOI = 10:1), were treated with HBHA at final concentrations of 5 μg/mL and 10 μg/mL. Autophagic activity was subsequently assessed to evaluate the inhibitory effects of HBHA-ECSIT interaction on macrophage autophagy.

RAW264.7 were infected or non-infected with BCG (MOI = 10:1) in the presence of varying concentrations (0-10 μg/mL) of HBHA for 48 hours. Cells were washed once with ice-cold 1× PBS and lysed with RIPA lysis buffer (containing 1 mM PMSF). After 10 min incubation on ice, complete cell disruption was achieved through repetitive pipetting. Lysates were centrifuged at 14,000 × g for 10 min at 4 °C, and supernatants were collected for following steps. Protein A magnetic beads were resuspended in lysis buffer, vortexed, and subjected to magnetic separation (15 sec) until clear supernatant was achieved. Cleared lysates (200 μL per sample) were incubated with anti-ECSIT polyclonal antibody (1:100 dilution) under continuous rotation at 4 °C overnight. Antigen-antibody complexes were then captured by adding 20 μL pre-washed magnetic beads, followed by 20 min rotary incubation at room temperature. Bead-bound complexes underwent five stringent washes with 500 μL ice-cold lysis buffer. Immunoprecipitates were eluted in 20-40 μL 4×Loading buffer through 10 min boiling at 95-100 °C. After magnetic separation, eluates were collected for subsequent western blot analysis.

Protein lysates were quantified via BCA assay and equal amounts were resolved on 12% SDS-PAGE gels. Electrophoretically separated proteins were transferred onto 0.45 μm PVDF membranes (Merck Millipore, USA). Membranes were blocked with 5% (w/v) non-fat dry milk (BD Biosciences, USA) in TBST buffer for 1 hour at room temperature (RT), followed by incubation with primary antibodies overnight at 4 °C. Subsequently, membranes were probed with HRP-conjugated secondary antibodies for 2 hours at RT. Detection was performed using an ECL method.

Cells were seeded onto sterile glass coverslips placed in 12-well plates and cultured until reaching 80% confluence. Following experimental treatments, cells were washed three times with PBS to remove residual medium. Cells were fixed with 4% paraformaldehyde (PFA) for 10 min at RT, followed by three PBS washes. Permeabilization was performed using 0.2% Triton X-100 in PBS for 10 min at RT, with subsequent PBS rinses. Non-specific binding was blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour at RT. After three PBS washes, cells were incubated overnight at 4 °C with primary anti-LC3 antibody. Following three PBS washes, FITC-conjugated secondary antibodies were applied for 1–2 hour at RT under light-protected conditions. After final PBS washes, nuclei were counterstained with DAPI in PBS for 15 min at RT. Coverslips were mounted onto glass slides using 20 μL anti-fade mounting medium and imaged using a laser scanning confocal microscope (Olympus FV4000) with 63× oil immersion objective.

RAW264.7 and ECSIT^KD RAW264.7 maintained in RPMI-1640 medium supplemented with 10% FBS were infected with BCG at MOI = 10:1 in the presence of varying concentrations of HBHA. Following different hours bacterial internalization, extracellular BCG was eliminated by 50 μg/mL gentamicin treatment for 1 h. Before lysis, the monolayer cells were gently washed twice with PBS to remove detached dead cells and residual extracellular debris. Washed monolayer cells were lysed with 0.02% SDS for 15 min at 37°C. Lysates were centrifuged at 12,000 × g for 30 min. Pelleted bacilli were resuspended in PBS, serially diluted (10^-² to 10^-4), and plated on Middlebrook 7H10 agar supplemented with OADC enrichment. Colonies were counted after 3 weeks incubation at 37 °C.

All values were expressed as mean ± SD. Data were analyzed by one-way ANOVA with Bonferroni correction for multiple comparisons. The follow-up least significant difference test was used for post hoc comparison with assess differences between groups. Differences with p values <0.05 considered to be statistically significant.

Previous studies have established that the assembly of the TRAF6-ECSIT complex, which facilitates ECSIT ubiquitination, plays a critical role in TLR4-mediated NF-κB activation required for intracellular bacterial clearance in macrophages (22, 24). Building on our initial discovery of direct HBHA-ECSIT interaction through HuProt™ human proteome microarray screening, we performed co-IP assays in RAW264.7 to investigate whether HBHA binding modulates ECSIT-TRAF6 complex formation. RAW264.7 cells were infected with BCG (MOI = 10:1) in the presence of varying concentrations of HBHA for 48 hours. Subsequent immunoprecipitation with anti-ECSIT antibody revealed dose-dependent HBHA co-precipitation with ECSIT, particularly enhanced in BCG-infected groups ( Figure 1 ). Conversely, TRAF6 interaction with ECSIT showed progressive reduction with increasing HBHA concentrations, demonstrating effective disruption of the TRAF6-ECSIT complex by HBHA binding. This disruption of interaction directly correlated with diminished ECSIT ubiquitination levels in macrophages ( Figure 1 ).

Our findings establish a novel mechanism whereby mycobacterial HBHA subverts host defenses through direct interaction with ECSIT. This pathogen-host protein interaction effectively inhibits both ECSIT ubiquitination and subsequent downstream signaling pathways, representing the evidence of mycobacterial HBHA directly targeting the ECSIT complex during macrophage invasion.

Macrophages play a pivotal role in host defense against Mtb infection (13). According to our prior discovery that HBHA inhibits macrophage autophagy (18), we investigated whether ECSIT mediates this suppression. RAW264.7 and ECSIT^KD RAW264.7 were exposed to varying concentrations of HBHA, and autophagic activity was assessed by monitoring LC3-II conversion (a hallmark of autophagosome formation) and Beclin-1 expression (a key regulator of autophagosome nucleation).

In RAW264.7, starvation significantly increased LC3-II levels compared to untreated controls (p < 0.005), while the autophagy inhibitor 3-MA reduced LC3-II accumulation (p < 0.01). Strikingly, HBHA treatment significantly attenuated starvation-induced LC3-II elevation ( Figures 2A, B ). Moreover, starvation upregulated Beclin-1 expression, which was suppressed by both 3-MA and HBHA ( Figures 2A, C ). Intriguingly, genetic ablation of ECSIT abolished the autophagy-inhibitory effects of HBHA. In ECSIT^KD RAW264.7, starvation-induced LC3-II levels remained unchanged upon HBHA treatment, converse results observed with 3-MA ( Figures 2D, E ). Similarly, Beclin-1 expression in ECSIT^KD RAW264.7 exhibited no significant differences between starvation and HBHA-treated groups, whereas 3-MA retained its inhibitory effect (p < 0.01, Figures 2D, F ).

To further validate the role of HBHA-ECSIT interaction in autophagy suppression, we observed autophagosome formation through immunofluorescence staining of LC3 in RAW264.7 and ECSIT^KD RAW264.7 ( Figure 3 ). In RAW264.7, nutrient deprivation significantly elevated LC3 puncta formation compared to untreated controls (p < 0.005), which was attenuated by 3-MA treatment (p < 0.01), and HBHA dose-dependently suppressed starvation-induced autophagosome formation ( Figures 3A, B ). Remarkably, while 3-MA further reduced LC3 puncta in ECSIT^KD RAW264.7 (p < 0.005), HBHA treatment showed no inhibitory effect ( Figures 3C, D ).

Therefore, all these findings demonstrate that Mtb virulence factor HBHA specifically targets ECSIT to impair autophagic flux, revealing a molecular pathway that could be exploited for developing autophagy-modulating therapies against LTBI.

To elucidate how HBHA-ECSIT interaction modulates macrophage autophagy during mycobacterial infection, we established a time-course infection model using M. bovis BCG (MOI = 10) in RAW264.7. Western blot analysis revealed time-dependent modulation of autophagy marker LC3-II in RAW264.7: Significant elevation compared to uninfected controls (p<0.01) at 6 h post-infection, peaking at 12 h (p<0.005), followed by gradual decline at 18/24 h while remaining above baseline ( Figures 4A, B ). This kinetic profile identified 12 h as the optimal infection duration for subsequent experiments. Following mechanistic investigations demonstrated that HBHA treatment significantly attenuated BCG-induced LC3-II and Beclin-1 expression compared to infection alone ( Figures 4C–E ). Strikingly, LC3-II/Beclin-1 expression in ECSIT^KD RAW264.7 were not significantly different (p>0.05) among uninfected, BCG-infected, or HBHA-treated groups ( Figures 4F–H ), suggesting that ECSIT deficiency may constitutively activate autophagic pathways and HBHA specifically inhibits BCG-triggered macrophage autophagy through ECSIT targeting.

Based on these observations, we performed immunofluorescence microscopy to visualize LC3 puncta formation in BCG-infected macrophages. In RAW264.7, co-localization analysis of DAPI-stained nuclei (blue) and FITC-labeled LC3 (green) revealed that BCG infection (MOI = 10, 12 h) significantly increased LC3-positive puncta compared to uninfected controls (p<0.005), whereas HBHA treatment reduced puncta density substantially versus infection alone ( Figures 5A, B ). Notably, there was no statistically significant difference (p>0.05) in LC3 fluorescence intensity among uninfected, BCG-infected, or HBHA-treated groups in ECSIT_KD RAW264.7 ( Figures 5C, D ), which consistent with western blot results.

Collectively, these findings provide conclusive evidence that HBHA exploits ECSIT to subvert mycobacterial infection-induced autophagy, and this regulatory axis strictly dependent on intact ECSIT expression, which reveals a novel immune evasion strategy employed by mycobacteria.

To investigate the functional consequences of autophagy suppression by HBHA-ECSIT interaction on mycobacterial persistence, we performed intracellular bacterial survival assays in RAW264.7 and ECSIT^KD RAW264.7. Cells were infected with BCG at a MOI of 10 in the presence of varying concentrations (0-10 μg/mL) of HBHA. CFU enumeration was performed at 6, 24, and 48 h post-treatment through serial dilution plating on 7H10 Middlebrook agar.

As shown in Figures 6A, B , initial infection efficiency analysis revealed comparable intracellular BCG loads across all groups at 6 h post-infection, confirming that HBHA does not influence bacterial adherence or invasion into macrophages. By 24 h, while overall CFU counts decreased in all groups due to experimental clearance of non-viable cells, HBHA-treated macrophages retained significantly higher bacterial loads compared to untreated controls. This survival advantage still was pronounced at 48 h, with HBHA-treated macrophages sustaining higher CFUs than untreated controls. However, genetic ablation of ECSIT completely abrogated HBHA’s pro-survival effects ( Figures 6C, D ). No significant differences in CFU counts were observed between HBHA-treated and untreated groups at any timepoint, demonstrating absolute ECSIT-dependence of HBHA-mediated bacterial persistence through autophagy suppression. The loss of HBHA-mediated bacterial persistence in ECSIT^KD macrophages directly correlates with our earlier findings of restored autophagic flux, confirming that ECSIT is indispensable for HBHA’s autophagy-subverting activity.