MAB_0676c-induced enhanced IL-10 production inhibits the autophagic flux via the MTOR/RUBCN pathway

ABSTRACT

Mycobacterium abscessus subsp. abscessus (M.abs) is a nontuberculous mycobacterium that can infect human lung macrophages, which poses a public health concern. Understanding its mechanism is crucial for developing strategies to combat M.abs infections. M.abs survives within host cells by inhibiting autophagy, a defense mechanism used against intracellular pathogens; therefore, we investigated the mechanism underlying autophagy inhibition and human lung macrophage infection by M.abs. This study focuses on the M.abs UC22 strain, which exhibits stronger inhibition of autophagic flux compared to the M.abs ATCC 19,977 strain. Central to this study is MAB_0676c, a protein secreted by M.abs UC22, and its effects on autophagic flux and the innate immune response, particularly its role in enhancing IL-10 production, a known autophagy regulator. Experiments showed that MAB_0676c expression stabilizes autophagy-related proteins while reducing LC3-LAMP2 co-localization in macrophages, thereby inhibiting autophagy and promoting bacterial growth. Furthermore, blocking IL-10 reduced both autophagy-related protein levels and the intracellular growth of MAB_0676c-expressing bacteria. Therefore, M.abs UC22 mediates intracellular survival by inhibiting autophagy through IL-10 production. Our study reveals bacterial immune-evasion tactics and identifies a potential therapeutic target for treating infectious diseases caused by nontuberculous mycobacteria.

Introduction

Mycobacteria are a diverse group of bacteria that includes tuberculosis-causing, leprosy-causing, and non-tuberculous mycobacteria (NTM) [1,2]. Pulmonary diseases caused by Mycobacterium avium and Mycobacterium abscessus complexes are the most prevalent NTM-associated diseases [1,3]. Within the M. abscessus complex, M. abscessus subsp. abscessus (M.abs) is a fast-growing NTM that causes pulmonary infections in immunocompromised patients with cystic fibrosis and soft tissue infection [4]. Based on the presence or absence of surface-associated glycopeptidolipids (GPLs), M.abs can be classified into two distinct morphotypes – the rough (R) and the smooth (S) variant; of the two, the R variant is considered more virulent [5,6]. However, the mechanisms underlying the increased virulence of the R variant and its ability to evade the immune system, particularly in patients with cystic fibrosis, remain unclear.

Autophagy is a conserved cellular degradation pathway involved in pathogen clearance and the immune response [7]. Cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10), play important roles in autophagy regulation [8,9]. TNF-α activates autophagy through the Jun kinase signaling pathway and upregulation of autophagy-associated genes, whereas IL-10 inhibits autophagy by activating IL-10 receptor/STAT and PI3K/MTORC1 signaling pathways [10,11]. Autophagy participates in innate and adaptive immune responses by clearing pathogens and modulating inflammatory processes [12]. However, certain intracellular pathogens, such as Mycobacterium tuberculosis (Mtb) have evolved strategies to evade autophagy [13]. Mtb proteins, such as PE/PPE, PknG, and Eis, suppress autophagy through various mechanisms [14,15].

The run domain Beclin-1-interacting and cysteine-rich domain-containing protein (Rubicon, hereafter referred to as RUBCN) is involved in several steps of autophagy. It acts as a negative regulator by inhibiting PI3K (phosphoinositide 3-kinase) activity and hinders autophagosomal maturation by binding to the UVRAG complex [16,17]. In addition, RUBCN plays a role in LC3-associated phagocytosis (LAP) and contributes to reactive oxygen species (ROS) production by activating and stabilizing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [18,19]. During Mtb infection, leucine-rich repeat kinase 2 negatively regulates phagosome maturation by recruitment of RUBCN to phagosome [20]. However, the function of RUBCN in M.abs infections has not yet been elucidated.

Given that M.abs UC22, a clinical R variant that we previously isolated from patients with pulmonary diseases, survives in macrophages by effectively inhibiting autophagic flux compared to M.abs ATCC 19,977 (S variant) [6], in this study, we investigated the mechanism underlying autophagy inhibition by M.abs UC22. We focused on understanding how M.abs UC22 manipulates autophagy-related pathways and the roles of specific bacterial proteins. By elucidating the strategies employed by M.abs UC22 to evade host immune responses via autophagy inhibition, this study aims to improve our understanding of bacterial pathogenesis and contribute to the development of novel therapeutic approaches.

Materials and methods

Cell culture

RAW 264.7 cells (ATCC, TIB-71) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; WELGENE) supplemented with 10% fetal bovine serum (FBS; WELGENE). THP-1 cells (ATCC, TIB-202) were cultured in RPMI 1640 medium (WELGENE) supplemented with 10% FBS. THP-1 cells were differentiated into macrophages for 24 h in a medium containing 100 ng/mL phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich).

Differentiation into bone marrow-derived macrophages

Bone marrow macrophages (BMDMs) were isolated from the femurs and tibias of 6-week-old C57BL/6 mice (Narabiotec) under sterile conditions. The bones were flushed with sterile DMEM using a 25-gauge needle, and the collected cells were passed through a 70 μm cell strainer to obtain a single-cell suspension. The cells were then cultured in DMEM supplemented with 10% FBS and 25 ng/mL recombinant murine macrophage colony-stimulating factor (M-CSF; R&D Systems) in non-tissue culture-treated Petri dishes.

Culture and infection of M.abs strain

M.abs ATCC 19,977 and M.abs UC22 were grown till the mid-log phase in Middlebrook 7H9 medium (BD Biosciences) containing 10% oleic albumin dextrose catalase supplement (Navi Biotech) and 0.05% polysorbate 80 (Thermo Fisher Scientific). The bacteria were washed, resuspended in Dulbecco’s phosphate-buffered saline (DPBS; WELGENE), and frozen at − 80°C for future use. BMDMs and RAW 264.7 cells were infected with M.abs strains at the indicated multiplicity of infection (MOI). After 2 h, the infected cells were washed three times with DPBS and incubated in the medium containing 200 μg/mL amikacin (Sigma-Aldrich). Following incubation, the cells were washed thrice and lysed with 0.25% Triton X-100 for 20 min. Intracellular bacteria were serially diluted with DPBS and spread onto Middlebrook 7H10 plates for enumeration.

Purification of recombinant proteins

The target genes were amplified via PCR using the EmeraldAmp GT PCR Master Mix (Takara Bio). The amplified genes were cloned between BamHI and HindIII sites of pET22b to generate an expression plasmid for the target protein with a C-terminal His-tag. Following sequence confirmation, the constructs were transformed into E. coli BL21. The cells harboring recombinant plasmids were cultured in LB medium (Sigma-Aldrich) containing ampicillin (Sigma-Aldrich) at 37°C until an optical density of approximately 0.6 at 600 nm (OD₆₀₀) was achieved. Production of the recombinant proteins was induced with the addition of 1 mM IPTG (Sigma-Aldrich) and cultured at 37°C for 6 h. Recombinant proteins were purified using nickel-nitrilotriacetic acid (Invitrogen) affinity chromatography. The primers used were listed in Table S1.

Transformation of recombinant M. smegmatis strain

The MAB_0676c was amplified and cloned between BamHI and HindIII sites of pVV16 vector. The preparation of M. smegmatis-competent cells and electroporation procedures have been described previously [21]. The pVV16 or pVV16_MAB0676c plasmid was transformed into M. smegmatis and the recombinant strains were grown on Middlebrook 7H10 plate (BD biosciences) containing 50 μg/mL kanamycin (Sigma-Aldrich). The primers used were listed in Table S1.

Immunoblotting

Cells were harvested and lysed in RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Thermo Fisher Scientific) on ice for 30 min. Equal amounts of protein were loaded and separated using SDS-PAGE and then transferred onto a PVDF membrane (Merck Millipore). Membranes with proteins were then blocked with EZBlock Chemi (ATTO) and then incubated overnight with primary antibodies at 4°C. The following primary antibodies were used for western blotting: anti-LC3 A/B (Cell Signaling Technology), anti-RUBCN (Thermo Fisher Scientific), anti-SQSTM1/p62 (Cell Signaling Technology), anti-SOCS3 (Abcam), anti-STAT3 (Cell Signaling Technology), anti-phospho-STAT3 (Cell Signaling Technology), anti-phospho-MTOR (Cell Signaling Technology), anti-Galectin 3 (Invitrogen), and anti-β-Actin (Cell Signaling Technology). The following day, the membranes were blotted with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich) for 1 h. The blots were developed using WesternBright ECL (Advansta). The ChemiDoc XRS+ system (Bio-Rad) was used to detect the bands, and Image Lab software was used to quantify the bands.

Confocal microscopy and co-localization analysis

M.abs and M. smegmatis strains were stained by carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) as previously described [22]. Cells were seeded onto glass coverslips and infected with CFSE-stained M.abs or M. smegmatis strains. The infected cells were washed three times and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min. The coverslips were incubated with 0.1% Triton X-100 (Sigma-Aldrich) in DPBS for 15 min, blocked with 3% BSA (Sigma-Aldrich) for 1 h, and then incubated overnight with anti-LC3 A/B (Cell Signaling Technology) and anti-LAMP2 (Thermo Fisher Scientific) at 4°C. The following day, the coverslips were washed three times and incubated with Alexa Fluor 647 conjugated IgG (Invitrogen) and Alexa Fluor 594 conjugated IgG (Invitrogen) for 1 h. Each coverslip was mounted with VECTASHIELD Antifade Mounting Medium and DAPI (Vector Laboratories), and fluorescence was observed using Leica TCS SP8 (Leica Microsystems). To analyze co-localization, Pearson’s correlation coefficient was calculated by using the PLUGIN: BIOP JACoP of ImageJ software as previously described [23]. This coefficient serves as a quantitative index for co-localization, ranging from − 1.0 to + 1.0. A value of + 1.0 indicates perfect correlation, 0 indicates no correlation, and − 1.0 indicates perfect anti-correlation. In each experiment, at least 10 visual fields containing 5–10 cells were counted.

Mouse infection model

Pathogen-free, six-week-old female C57BL/6 mice were purchased from Narabiotec and randomly divided into two groups (total 10 mice, 5 mice per group). All animal experiments were approved by the Institutional Research and Ethics Committee of Chungnam National University (CNU-202006A-CNU-088) and conducted in accordance with the guidelines of the Korean Food and Drug Administration. This study was carried out in compliance with the ARRIVE guideline. Each group of C57BL/6 mice (n = 5) was anesthetized with 2,2,2-tribromoethanol (125 mg/kg, Sigma-Aldrich, IP) and intratracheally infected with M. smegmatis strains (either MAB_0676c-expressing M. smegmatis or vector control, with each strain administered at 2 × 10⁶ CFU/mouse). The mice were monitored until the effects of anesthesia wore off and, upon awakening, were returned to their cages. Mice were sacrificed on the first- and fifth-days post-infection to measure the bacterial burden in the lungs. Lung samples were collected and homogenized, after which the bacteria were serially diluted with DPBS and spread onto Middlebrook 7H10 plates for enumeration. Three different investigators were involved in the experiment: the first investigator administered the anesthesia and was the only individual aware of the infection groups. The other investigators, who were blinded to the infection group, carried out the infection procedure and performed the bacterial counts. The infection experiments were conducted in duplicate. In the first experiment (total 10 mice, 5 mice per group), mice were observed for a week post-infection to assess the toxicity of the recombinant M. smegmatis strain. In the second experiment (total 10 mice, 5 mice per group), the bacterial load in the lungs was measured. Mice for sacrifice were randomly selected, and one mouse that died (n = 1) was excluded from the bacterial count in the second experiment. Data from the infection experiments were analyzed using an unpaired t-test in GraphPad Prism 5 (version 5.01) to determine the effect of MAB_0676c on M. smegmatis survival.

Transfection experiments

For siRNA knockdown, BMDMs and RAW 264.7 cells were transfected with mouse Rubcn siRNA (Bioneer) or a negative control (Bioneer) using Lipofectamine 3000 (Invitrogen) at an siRNA: Lipofectamine reagent ratio of 1:1.5 according to the manufacturer’s instructions.

Identification of mycobacterial proteins

M.abs ATCC 19,977 and M.abs UC22 were grown till the mid-log phase in Sauton’s medium. The bacteria were removed using centrifugation and filtered through a 3-kDa NMW Ultrafiltration Discs (Merck Millipore). The CFPs of M.abs ATCC 19,977 and M.abs UC22 were precipitated using 80% ammonium sulfate (Sigma-Aldrich). CFPs were identified at the Yonsei Proteomics Research Center using two-dimensional electrophoresis (2-DE) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS). 2-DE was used to determine protein expression in M.abs strains, as previously outlined [24,25]. Peptide sequences were identified using MASCOT software.

Sandwich enzyme-linked immunosorbent assay (ELISA)

Cell-free supernatant was collected and stored at − 80°C until assessed for cytokine concentrations using ELISA kits for IL-10 (Invitrogen), TNF-α (Invitrogen), IL-6 (Invitrogen), and IL-12p70 (Invitrogen) according to the manufacturer’s protocols. The optical density of the color reactions was measured using a SpectraMax ABS microplate reader (Molecular Devices) at 450 and 570 nm.

Statistical analysis

All experiments were performed at least thrice. All data were analyzed using an unpaired t-test or one-way ANOVA, followed by Tukey’s test using GraphPad Prism 5 (version 5.01). The data are expressed as the mean values ± SEM. The statistical significance was set at p < 0.05.

Results

M.abs UC22 evaded autophagy by inducing RUBCN expression

We have previously demonstrated that M.abs UC22 eludes host defense by inhibiting autophagy [6]. To investigate the intracellular survival capabilities of different M.abs strains in macrophages, we infected RAW 264.7, THP-1, and BMDM cells with M.abs UC22 (R variant), M.abs ATCC 19,977 (S variant), or M. smegmatis, and quantified the number of intracellular bacteria. At 2 h post-infection, no significant differences were noted in the intracellular bacterial counts among the NTM strains (Figure 1a). However, M.abs UC22 exhibited significantly higher intracellular survival across all macrophage types compared to the other NTM strains, whereas M.abs ATCC 19,977 showed sustained survival.

Figure 1.

M.Abs UC22 inhibits autophagic flux by enhancing RUBCN expression. (a) The number of intracellular bacteria in macrophages. Different cell lines (THP-1, RAW 264.7, and BMDM) were infected with different NTMs. Intracellular bacteria were measured using lysates of infected cells at the indicated times. (b) Western blotting of autophagy-related proteins obtained from the M.Abs-infected BMDMs. Target protein levels were normalized to those of β-actin and are presented as the mean ± SEM from three independent experiments. (c) BMDMs were infected with M.Abs strains for 24 h. Infected cells were stained and visualized using confocal microscopy to detect bacteria (blue), LC3 (green), LAMP2 (red), and DAPI (nuclei; white gray). Scale bars, 2 μm. (d) Quantification of LC3-LAMP2 co-localization based on the images shown in (c). Statistical significance was determined using the one-tailed unpaired t-test. Data are shown as mean ± SEM from three independent experiments. Statistically significant differences are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant. M.Abs, Mycobacterium abscessus subsp. abscessus; BMDM, bone marrow-derived macrophage; NTM, non-tuberculous mycobacteria; DAPI, 4',6-diamidino-2-phenylindole.

To assess the autophagy signaling during M.abs infection, BMDMs were infected with both M.abs strains, and autophagy-related proteins were monitored using western blot analysis. The accumulation of SQSTM1/p62 and LC3 II was observed in M.abs UC22-infected BMDMs for up to 48 h (Figure 1b), suggesting impaired autophagic flux. Confocal microscopy showed that M.abs ATCC 19,977 co-localized with LC3, an autophagosome marker, and Lysotracker, which labels acidic organelles. In contrast, M.abs UC22 partially co-localized with both LC3 and Lysotracker (Figure S1). Notably, autophagosome co-localization with LAMP2 (a lysosomal membrane protein) was significantly reduced in M.abs UC22-infected BMDMs compared to M. abs ATCC 19,977-infected BMDMs (Figure 1c,d). Collectively, these findings suggest that M.abs UC22 evades elimination in the macrophages by inhibiting the autophagic flux.

We hypothesized that M.abs UC22 disrupts autophagic flux by interfering with proteins involved in autophagosome-lysosome fusion. To investigate this, the expressions of genes associated with autophagosome-lysosome fusion, including those associated with the SNARE complex and tether proteins, were investigated in NTM-infected RAW 264.7 cells. Most of the examined genes did not differ significantly between M.abs ATCC 19,977- and M.abs UC22-infected cells, except for a 3-fold increase in Rubcn expression particularly in M.abs UC22-infected RAW 264.7 cells (Figure S2). Furthermore, RUBCN protein expression was elevated in the M.abs UC22-infected BMDMs (Figure 1b). The observed impairments in autophagy signaling, autophagosome-lysosome fusion, and elevated RUBCN expression may be involved in the survival strategies of M.abs UC22 within macrophages.

Knockdown of Rubcn enhanced autophagic flux in M.abs UC22 infection

As shown in Figure 1, the expression of RUBCN considerably increased at both gene and protein levels in M.abs UC22-infected macrophages. RUBCN has an inhibitory effect on the autophagic flux by suppressing the class III phosphatidylinositol 3-kinase complex 2 (PI3KC3-C2) in the autophagy pathway [19,26]. This led us to question whether the impaired autophagic flux observed during M.abs UC22 infection could be attributed to the elevated expression of RUBCN. To investigate this, macrophages were transfected with either a negative control or Rubcn siRNA, followed by an infection with M.abs UC22 to assess the intracellular bacterial load. Remarkably, the intracellular survival of M.abs UC22 was significantly reduced in Rubcn siRNA-transfected BMDMs and RAW 264.7 cells 24 h post-infection (Figure 2a), suggesting a correlation between RUBCN expression and the intracellular survival of M.abs UC22.

Figure 2.

Knockdown of Rubcn enhances autophagosomal maturation in M.Abs UC22-infected BMDMs. BMDMs were transfected with either the negative control or Rubcn siRNA for 24 h and then infected with M.Abs UC22 at an MOI of 1. (a) Intracellular survival of M.Abs UC22 in transfected BMDMs or RAW 264.7 cells. Data are shown as mean ± SEM from three independent experiments. (b) Western blotting of the autophagy-related proteins in Rubcn knockdown-BMDMs at the indicated time. Target protein levels were normalized to those of β-actin and are presented as mean ± SEM of three independent experiments. (c) siRubcn- or negative control-transfected BMDMs were infected with M.Abs UC22 at an MOI of 1. Infected cells were stained and visualized under a confocal microscope to detect bacteria (blue), LC3 (green), LAMP2 (red), and DAPI (nuclei; white gray). Scale bars, 2 μm. (d) Quantification of LC3-LAMP2 co-localization based on the images shown in (c). Data are shown as mean ± SEM from three independent experiments. Statistically significant differences are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant. MOI, multiplicity of infection; M.Abs, Mycobacterium abscessus subsp. abscessus; BMDM, bone marrow-derived macrophage; NC, siRNA negative control; DAPI, 4',6-diamidino-2-phenylindole.

The impact of Rubcn knockdown on autophagy signaling during M.abs UC22 infection was examined. The levels of Galectin-3 did not significantly differ between Rubcn siRNA-transfected cells and the negative control during an M.abs UC22 infection. However, the levels of phospho-MTOR, SQSTM1/p62 and LC3 II were decreased in Rubcn siRNA-transfected cells (Figure 2b). To further investigate the role of RUBCN in autophagosome-lysosome fusion, confocal microscopy was performed. Notably, the co-localization of LC3 and LAMP2 was significantly higher in Rubcn siRNA-transfected cells than in negative control-transfected cells (Figure 2c,d). Additionally, M.abs UC22 was found to fuse with LC3 and Lysotracker-positive compartments in Rubcn siRNA-transfected cells (Figure S3). These findings collectively indicate that although RUBCN plays a critical role in facilitating autophagosome-lysosome fusion. Overall, these results demonstrate that M.abs UC22 promotes its survival within macrophages by upregulating RUBCN expression, which in turn negatively modulates autophagosome maturation.

MAB_0676c modulated RUBCN expression

Increasing evidence supports the mycobacterial protein-based inhibition of autophagic flux [14]. Consequently, the identification of specific bacterial proteins that stimulate RUBCN expression is crucial. To address this, culture filtrate proteins (CFPs) from M.abs ATCC 19,977 and M.abs UC22 were collected using ammonium sulfate precipitation. We constructed a library targeting bacterial proteins of M.abs UC22 that were more abundantly expressed compared to those in the CFPs of M.abs ATCC19977. Thereupon, MAB_0676c and MAB_4702c proteins were identified and purified using an E. coli expression system (Figure S4a-f). Notably, RUBCN expression was increased to a greater extent in MAB_0676c-treated BMDMs (Figure 3a). Hence, we further investigated the role of MAB_0676c in autophagy.

Figure 3.

Identification of MAB_0676c as an inducer of RUBCN expression. (a) Western blot showing RUBCN expression in BMDMs treated with recombinant MAB_0676c and MAB_4702 (5 μg/mL). (b) Western blot showing autophagy-related protein expression in BMDMs treated with recombinant MAB_0676c. BMDMs were incubated with recombinant MAB_0676c protein, heat-inactivated MAB_0676c protein, or LPS for 24 h. (c) Cytotoxicity of MAB_0676c. BMDMs were incubated with recombinant MAB_0676c, STS, or LPS for 24 h. Data are shown as mean ± SEM from three independent experiments. Statistically significant differences are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant. M.Abs, Mycobacterium abscessus subsp. abscessus; BMDM, bone marrow-derived macrophage; LPS, lipopolysaccharide; STS, staurosporine.

BMDMs were treated with LPS (0.1 μg/mL), MAB_0676c (5 μg/mL), or heat-inactivated MAB_0676c (5 μg/mL), and autophagy-related proteins were monitored using western blotting. MAB_0676c-treated BMDMs exhibited an increased expression of phospho-MTOR and RUBCN. Additionally, LC3 II and SQSTM1/p62 levels were elevated in MAB_0676c-treated BMDMs. In contrast, these changes were not observed in BMDMs stimulated with heat-inactivated MAB_0676c (Figure 3b). The concentration of MAB_0676c used in this study did not affect cell viability (Figure 3c). These results demonstrate the critical role of the bacterial protein MAB_0676c in inhibiting autophagic flux.

MAB_0676c inhibited autophagic flux during M. smegmatis infection

To determine the role of MAB_0676c in bacterial infection, MAB_0676c-expressing M. smegmatis strains were generated. MAB_0676c is annotated as a hypothetical HIT-like (Histidine Triad) protein (UniProt ID #B1MHV1). However, there is currently no functional information specific to MAB_0676c beyond this annotation. To evaluate whether MAB_0676c affects bacterial physiology, we examined the growth curves and colony morphology of MAB_0676c-expressing M. smegmatis. Our results showed no significant differences in either growth kinetics or colony appearance compared to the control strain. These findings suggest that MAB_0676c expression does not alter the general growth characteristics of M. smegmatis under the tested conditions (Figure S5a,b).

We investigated the effects of MAB_0676c on the intracellular survival of M. smegmatis. BMDMs were infected with either MAB_0676c-expressing M. smegmatis or vector control, and the number of intracellular bacteria was counted. Remarkably, MAB_0676c-expressing M. smegmatis had a significantly higher bacterial burden than the vector control 72 h post-infection (Figure 4a).

Figure 4.

MAB_0676c expression inhibits autophagic flux in M. smegmatis infection. (a) Intracellular survival of recombinant M. smegmatis strains in BMDMs. (b) Western blot showing autophagy-related protein expression in infected BMDMs. Target protein levels were normalized to those of β-actin and are presented as mean ± SEM from three independent experiments. (c) Infected cells were stained and visualized under a confocal microscope to detect bacteria (blue), LC3 (green), LAMP2 (red), and DAPI (nuclei; white gray). Scale bars, 2 μm. (c) Representative confocal microscopy images. (d) Quantification of LC3-LAMP2 co-localization in (c). (e, f) Mice were infected with recombinant M. smegmatis strains. (e) Survival rate of C57/BL6 mice. Data are from a representative experiment with five mice per group. (f) Bacterial burden in the lungs of infected mice. Each time point represents the mean value two mice per group. (g) Representative lung lesions in mice infected with recombinant M. smegmatis. Scale bars, 600 μm. Data are shown as mean ± SEM. Statistically significant differences are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant. BMDM, bone marrow-derived macrophage; DAPI, 4',6-diamidino-2-phenylindole; Ms_VC, M. smegmatis vector control; Ms_MAB0676c, MAB_0676c-expressing M. smegmatis.

We investigated whether MAB_0676c-expressing M. smegmatis inhibits autophagic flux by regulating RUBCN expression. To this end, BMDMs were infected with either MAB_0676c-expressing M. smegmatis or vector control, and autophagy-related protein levels were analyzed through western blotting. Notably, BMDMs infected with MAB_0676c-expressing M. smegmatis had increased levels of phospho-MTOR and RUBCN as well as higher levels of LC3 II and SQSTM1/p62 than those in the vector control (Figure 4b). To further investigate the effect of MAB_0676c on autophagosome-lysosome fusion during M. smegmatis infection, we used confocal microscopy to evaluate the co-localization of LAMP2 and LC3. In MAB_0676c-expressing M. smegmatis-infected BMDMs, a significant reduction in LAMP2 and LC3 co-localization was observed compared to the vector control (Figure 4c,d). Interestingly, MAB_0676c-expressing M. smegmatis was absent from LC3 and Lysotracker-positive compartments (Figure S6). These findings suggest that MAB_0676c contributes to bacterial survival by inhibiting autophagic flux in M. smegmatis infection models, similar to that seen during M.abs UC22 infection.

We investigated the role of MAB_0676c in a mouse model of infection, in which C57BL/6 mice were intratracheally infected with MAB_0676c-expressing M. smegmatis or vector control. Consistent results were observed in the mouse model of infection. Two mice infected with MAB_0676c-expressing M. smegmatis succumbed to the infection, whereas all mice infected with the vector control strain survived (Figure 4e). Additionally, five days post-infection, the bacterial burden in the lungs of mice infected with MAB_0676c-expressing M. smegmatis was higher than that in the mice infected with the vector control (Figure 4f). An inflammatory response with massive immune cell infiltration was observed in the lungs of mice infected with MAB_0676c-expressing M. smegmatis. In contrast, only mild infiltration was observed in the lungs of mice infected with the vector control strain (Figure 4g). These results indicate that MAB_0676c contributed to bacterial survival in both in vitro and in vivo M. smegmatis infection models. Taken together, our findings suggest that MAB_0676c is a critical virulence factor that contributes to bacterial survival by inhibiting autophagic flux via RUBCN expression.

Rubcn knockdown abrogated the effect of MAB_0676c on M. smegmatis infection

We conducted further experiments to determine whether Rubcn knockdown counteracted the effects of MAB_0676c during M. smegmatis infection. BMDMs were transfected with either negative control or Rubcn siRNA, followed by infection with MAB_0676c-expressing M. smegmatis. Thereafter, the number of intracellular bacteria was quantified. Rubcn knockdown was found to significantly decrease the intracellular survival of MAB_0676c-expressing M. smegmatis in BMDMs 24 h post-infection (Figure 5a).

Figure 5.

Rubcn knockdown enhances autophagic flux in BMDMs infected with MAB_0676c-expressing M. smegmatis. BMDMs were transfected with either the negative control or Rubcn siRNA for 24 h. Transfected BMDMs were infected with the recombinant M. smegmatis strains at an MOI of 1. (a) Intracellular survival of MAB_0676c-expressing M. smegmatis in siRubcn-transfected BMDMs. (b) Western blot showing autophagy-related protein expression at the indicated time points. Target protein levels were normalized to those of β-actin and are presented as mean ± SEM from three independent experiments. (c) Infected cells were stained and visualized under a confocal microscope to detect bacteria (blue), LC3 (green), LAMP2 (red), and DAPI (nuclei; white gray). Scale bars, 2 μm. (d) Quantification of LC3-LAMP2 co-localization based on the images shown in (c). Data are shown as mean ± SEM from three independent experiments. Statistically significant differences are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant. MOI, multiplicity of infection; BMDM, bone marrow-derived macrophage; NTM, non-tuberculous mycobacteria; DAPI, 4',6-diamidino-2-phenylindole; NC, siRNA negative control; Ms_VC, M. smegmatis vector control; Ms_MAB0676c, MAB_0676c-expressing M. smegmatis.

To elucidate the relationship between MAB_0676c and autophagy during a bacterial infection, the expression levels of autophagy-related proteins were determined. Notably, the levels of phospho-MTOR, SQSTM1/p62 and LC3 II were decreased in Rubcn siRNA-transfected cells (Figure 5b). Moreover, confocal microscopy revealed a substantial increase in LC3 and LAMP2 co-localization in Rubcn siRNA-transfected BMDMs during MAB_0676c-expressing M. smegmatis infection (Figure 5c,d). In Rubcn siRNA-transfected cells, MAB_0676c-expressing M. smegmatis was fused with LC3 and Lysotracker-positive compartment (Figure S7). These findings underscore the importance of the interaction between MAB_0676c and RUBCN in the survival of M.abs UC22.

IL-10 receptor blockade reversed MAB_0676c-induced suppression of autophagic flux

Building on previous findings highlighting the critical role of the MAB_0676c and RUBCN interaction as a survival strategy for M.abs UC22, we investigated the influence of MAB_0676c on macrophage antibacterial activity by evaluating cytokine production. Initially, cytokine levels in BMDMs infected with different M.abs strains were measured, revealing a remarkable increase in IL-10 and IL-6 production in M.abs UC22-infected BMDMs (Figure S8a). Furthermore, the effects of MAB_0676c on IL-10 production were inspected after treating BMDMs with either LPS or MAB_0676c for 24 h. Notably, the levels of IL-10 were elevated in MAB_0676c-treated BMDMs (Figure S8b), indicating the contribution of MAB_0676c to the increased IL-10 production observed during M.abs UC22 infection. To further validate our hypothesis that MAB_0676c-expressing M. smegmatis could induce IL-10 expression similar to M.abs UC22 infection, BMDMs were infected with MAB_0676c-expressing M. smegmatis or vector control, and the expression of IL-10 and IL-6 was measured. Notably, MAB_0676c-expressing M. smegmatis-infected BMDMs exhibited IL-10 and IL-6 secretion (Figure S8c). These findings suggest that MAB_0676c hinders macrophage antimicrobial activity by promoting IL-10 production.

Increasing evidence suggests that IL-10 inhibits autophagy via the STAT3 signaling pathway [9,27]. We investigated the effects of IL-10 on MAB_0676c-induced autophagy. To elucidate the relationship between IL-10 signaling and autophagy, we used an IL-10 receptor-blocking antibody to block IL-10 signaling in BMDMs treated with either LPS or MAB_0676c. Both MAB_0676c and LPS induced phospho-STAT3 expression, which was effectively suppressed in the presence of the IL-10 receptor-blocking antibody (Figure 6a), indicating that MAB_0676c activates the IL-10 signaling pathway.

Figure 6.

MAB_0676c inhibits autophagy through the IL-10 and STAT3 pathways. (a) Western blot showing STAT3 pathway protein expression in BMDMs stimulated with MAB_0676c. BMDMs were treated with native MAB_0676c, recombinant MAB_0676c, heat-inactivated MAB_0676c, or LPS in the presence or absence of anti-IL10R for 24 h. The level of phopho-STAT3 was normalized to those of β-actin and presented as mean ± SEM of three independent experiments. (b) Western blot showing STAT3 pathway protein expression in BMDMs incubated with MAB_0676c and each STAT3 inhibitors. BMDMs were incubated with anti-IL10R (25 μg/mL), cryptotanshinone (CTS; 20 μM), or S3I–201 (S3I; 75 μM), and then treated with MAB_0676c (5 μg/mL) for 24 h. Target protein levels were normalized to those of β-actin and presented as mean ± SEM of three independent experiments. (c) Western blot showing autophagy-related protein expression in BMDMs treated with recombinant MAB_0676c and anti-IL10R. BMDMs were treated with recombinant MAB_0676c proteins in the presence or absence of anti-IL-10 R for 24 h. Target protein levels were normalized to those of β-actin and presented as mean ± SEM of three independent experiments. (d, e, f) BMDMs were infected with M.Abs UC22 or MAB_0676c-expressing M. smegmatis at an MOI of 1 with or without anti-IL10R. Infected cells were stained and visualized under a confocal microscope to detect bacteria (blue), LC3 (green), LAMP2 (red), and DAPI (nuclei; white gray). Scale bars, 2 μm. (d) Representative confocal microscopy images. (e, f) Quantification of LC3-LAMP2 co-localization based on the images shown in (d). (g, h) Intracellular survival of M.Abs UC22 or MAB_0676c-expressing M. smegmatis in the presence of anti-IL10R. Data are shown as mean ± SEM from three independent experiments. Statistically significant differences are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant. MOI, multiplicity of infection; M.Abs, Mycobacterium abscessus subsp. abscessus; Ms_MAB0676c, MAB_0676c-expressing M. smegmatis; BMDM, bone marrow-derived macrophage; anti-IL10R, IL-10 receptor blocking antibody; CTS, cryptotanshinone; DAPI, 4',6-diamidino-2-phenylindole.

To further verify the involvement of IL-10 signaling in autophagic flux, we used STAT3 inhibitors, including an IL-10 receptor-blocking antibody (Anti-IL 10 R), cryptotanshinone (CTS), and S3I–201 (S3I). BMDMs were first incubated with the IL-10 receptor-blocking antibody (25 μg/mL), CTS (20 μM), or S3I (75 μM), and subsequently treated with MAB_0676c (5 μg/mL). Remarkably, the MAB_0676c-induced expression of phospho-MTOR and RUBCN was significantly reduced in the presence of the IL-10 receptor-blocking antibody and CTS, but not with S3I (Figure 6b). Importantly, the blockade of IL-10 signaling abrogated phospho-MTOR, and RUBCN expression. Moreover, LC3 II and SQSTM1/p62 accumulation was inhibited by the IL-10 receptor-blocking antibody at 24 h postinfection (Figure 6c). The effects of IL-10 signaling on autophagosome-lysosome fusion were also determined by first infecting BMDMs with either M.abs UC22 or MAB_0676c-expressing M. smegmatis, followed by incubation with an IL-10 receptor-blocking antibody. Confocal microscopy revealed that IL-10 signaling blockade enhanced the co-localization of LC3 and LAMP2 in BMDMs infected with M.abs UC22 or MAB_0676c-expressing M. smegmatis (Figure 6d–f). In the presence of the IL-10 receptor-blocking antibody, both M.abs UC22 and MAB_0676c-expressing M. smegmatis were found to co-localize with Lysotracker-positive compartments (Figure S9, S10).

Figure 6.

(Continued).

Consistent with the previous data, the blockade of IL-10 signaling increased the autophagic flux. Therefore, we examined the effect of IL-10 on the intracellular survival of M.abs UC22 and MAB_0676c-expressing M. smegmatis. The IL-10 receptor-blocking antibody significantly decreased the intracellular survival of M.abs UC22 and MAB_0676c-expressing M. smegmatis (Figure 6g,h). Collectively, these findings suggest that the inhibition of autophagic flux by MAB_0676c is mediated by the activation of IL-10 signaling.

Discussion

The cumbersome treatment of the M.abs infections, which is attributed to their biological properties such as antibiotic resistance, necessitates a comprehensive understanding of M.abs pathogenicity for the development of effective therapeutic targets and strategies. In our previous study, we demonstrated the inhibition of autophagosome-lysosome fusion by M.abs clinical isolates of UC22 [6], although the underlying mechanism of this inhibition remained unclear. In this study, we investigated the role of MAB_0676c, which is abundantly secreted by M.abs UC22, in autophagy and its effect on the intracellular survival of M.abs UC22 in macrophages.

Galectin-3 is a well-known marker of ruptured phagosomal membranes during M.abs R variant infection [28]. In our study, we confirmed that BMDMs infected with M.abs UC22 (R variant) exhibited higher Galectin-3 expression compared to those infected with M.abs ATCC 19,977 (S variant). This suggests that M.abs UC22 May inhibit autophagy by inducing damage to phagosomal membranes. We further demonstrated an increase in LC3 and LAMP2 co-localization in cells either transfected with siRubcn or treated with an IL-10 receptor-blocking antibody. However, Galectin-3 expression remained unaffected by RUBCN or IL-10 levels. Our study leaves unanswered the question of how suppressed RUBCN and IL-10 signaling leads to autophagy despite the presence of damaged phagosomal membranes. A prior study by Chauhan et al. [29], reported that the interaction between TRIM16 and Galectin-3 helps protect cells from lysosomal damage and Mycobacterium tuberculosis invasion. Another study by Kim et al [30]. reported that the rough variant (R variant) of M.abs induces cGAS-STING-dependent type I interferon production and NLRP3 inflammasome-mediated IL-1β secretion via bacterial phagosome escape. The binding of cGAS to Beclin-1 leads to the release of RUBCN from the Beclin-1 complex. This interaction activates the class III phosphatidylinositol 3-kinase complex and promotes autophagy to eliminate cytosolic bacterial DNA [31]. These studies suggest that intracellular bacteria can be cleared through alternative mechanisms – such as the cGAS – STING pathway or TRIM16–Galectin-3 signaling – even when RUBCN is inhibited. Further research is needed to explore the relationship between TRIM16, Galectin-3, cGAS-STING pathway, and RUBCN during M.abs UC22 infection to address this issue.

Previous studies [32,33] have demonstrated the critical roles of the bacteria-induced IL-10 and STAT3 pathways in inhibition of autophagic flux. These findings support our observations regarding the inhibition of autophagosome-lysosome fusion by STAT3-activated MTOR signaling in MAB_0676c-treated BMDMs. IL-10 is a potent negative feedback regulator that controls inflammation by inhibiting immune cell activity and destabilizing mRNA levels of various inflammatory cytokines, thereby preventing tissue death and organ dysfunction. IL-10 also prevents the overproduction of IL-4, IL-5, and IL-13 during mycobacterial infections, thereby preventing severe fibrosis [34,35]. However, excessive IL-10 production may compromise pathogen control and hinder autophagosomal maturation during Mtb infection [36,37]. In our study, mice infected with MAB_0676c-expressing M. smegmatis exhibited higher mortality, inflammatory response and bacterial burden than those in the vector control group. Although the IL-10 production in MAB_0676c-expressing M. smegmatis-infected mice was not directly confirmed, we observed an upregulation in the IL-10 production in MAB_0676c-treated cells, activating the STAT3/MTOR signaling pathway, which negatively regulates autophagy. A previous study suggested that administration of an anti-IL-10 R antibody reduced the bacterial loads in the lungs and spleens of mice infected with Rv2145c-expressing M. smegmatis [38]. This finding supports our results, which show that blocking IL-10 signaling reduced the intracellular survival of M.abs UC22 and MAB_0676c-expressing M. smegmatis by enhancing autophagic flux. Therefore, IL-10 production may be a key mechanism of action of MAB_0676c in M.abs UC22 infection.

To investigate the role of MAB_0676c in bacterial infection, we utilized the M. smegmatis expression system. Unlike M.abs infection, M. smegmatis infection is associated with an active autophagy process, characterized by decreased expression of SQSTM1/p62 and increased lysosomal co-localization. This observation is consistent with a previous study by Feng et al. [39], which reported distinct autophagy responses in macrophages infected with Mabs and M. smegmatis, supporting our findings. Although the M. smegmatis expression system is a limited model for infection due to its differing autophagy response compared to Mabs, we observed impaired autophagic flux in BMDMs infected with MAB_0676c-expressing M. smegmatis, as indicated by sustained levels of phospho-MTOR, LC3, SQSTM1/p62, and RUBCN. Furthermore, under Rubcn knockdown conditions, both bacterial infections showed reduced SQSTM1/p62 expression and enhanced phagosome maturation. These results strongly support our hypothesis that MAB_0676c suppresses autophagic flux by upregulating RUBCN expression.

To elucidate the mechanism by which MAB_0676c modulates RUBCN expression, we explored the effect of MAB_0676c on RUBCN regulation. Sunahara et al. [40]. have reported that rapamycin decreases the gene and protein levels of RUBCN in a mouse model of cecal ligation and puncture sepsis. Therefore, we investigated whether RUBCN expression is regulated by the MTOR pathway. To address this issue, BMDMs were treated with MAB_0676c and rapamycin. Our experimental data revealed that MAB_0676c-induced MTOR and p70 S6K phosphorylation were diminished in the presence of rapamycin. However, the RUBCN induction by MAB_0676c was unaffected in the presence of rapamycin (Figure S10). These findings suggest an alternative mechanism that independently regulates RUBCN expression, distinct from the MAB_0676c-activated MTOR signaling pathway. Moreover, we examined the effect of CTS, a known inhibitor of STAT3 signaling, on both the MTOR signaling pathway and RUBCN expression in the presence of MAB_0676c. CTS administration abrogated both the MTOR signaling pathway and RUBCN expression, despite the presence of MAB_0676c. Remarkably, we also observed that the MTOR signaling pathway and RUBCN expression were abolished after treatment with an IL-10 receptor-blocking antibody. These results suggest that IL-10/STAT3 signaling plays a crucial role in the inhibition of autophagy mediated by MAB_0676c. Additionally, our study revealed that MAB_0676c activated the STAT3 signaling pathway via IL-10 production. STAT3 is activated by various cytokines, including IL-10, IL-6, fibroblast growth factor, insulin-like growth factor, and epidermal growth factor [41]. Further investigation into the relationship between these molecules and autophagy could yield valuable insights into its complex regulatory mechanisms.

In summary, our study revealed a sequence of events through which MAB_0676c inhibited autophagosome maturation in M.abs UC22-infected macrophages. These events involve (1) MAB_0676c secretion-induced IL-10 expression, (2) IL-10/STAT3 signaling pathway activation by enhanced IL-10 production, (3) RUBCN expression and MTOR phosphorylation induction by activated STAT3, (4) inhibition of autophagy initiation by phosphorylated MTOR, and (5) blocking of autophagosome maturation by RUBCN. Importantly, we established that MAB_0676c induced IL-10 production, leading to the RUBCN-mediated inhibition of autophagy through the STAT3 signaling pathway. To the best of our knowledge, this is the first report of the inhibition of RUBCN-mediated autophagy via IL-10 and the MTOR/STAT3 signaling pathway induced by MAB_0676c. This pathogenic mechanism of MAB_0676c provides insights into the immune-evasion strategies employed by various bacteria and identifies a potential therapeutic target for the treatment of NTM-caused infectious diseases.

Funding Statement

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education [RS-2023-00273828 to Dong Ho Kim]. This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea [RS-2024-00336984 and HR22C1734 to Chul Hee Choi]. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) [RS-2024-00406568 to Chul Hee Choi].

Abbreviations

BMDM,

Bone marrow-derived macrophage;

CFPs,

Culture filtrate proteins;

CFSE,

carboxyfluorescein diacetate succinimidyl ester;

CTS,

Cryptotanshinone;

DAPI,

4',6-diamidino-2-phenylindole;

IL-10,

Interleukin-10;

LAMP2

Lysosomal-associated membrane protein 2;

LC3,

Microtubule-associated protein 1 light chain 3;

LPS,

Lipopolysaccharide;

STS,

Staurosporine; M.abs, Mycobacterium abscessus subsp. abscessus;

MOI,

multiplicity of infection;

Mtb,

Mycobacterium tuberculosis;

MTOR,

Mechanistic target of rapamycin kinase;

NTM,

non-tuberculous mycobacteria;

RUBCN,

Run domain Beclin-1-interacting and cysteine-rich domain-containing protein;

SQSTM1,

Sequestosome 1;

STAT3,

Signal transducer and activator of transcription 3;

S3I,

S3I–201;

TLR,

Toll-like receptor;

TNF-α,

Tumor necrosis factor-α.

Author contributions

Dong Ho Kim and Chul Hee Choi conceived and designed the study. Dong Ho Kim, Kyungho Woo, Ho-Sung Park, Hye-Soo Park performed the experiments and analyzed the data. Dong Ho Kim and Chul Hee Choi wrote the manuscript. Hwa-Jung Kim and Chul Hee Choi reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data generated during the study are available at the repository fi gshare at https://doi.org/10.6084/m9.fi gshare.27248187.v3

ARRIVE guidelines compliance statement

We adhered to the ARRIVE guidelines and have uploaded the completed checklist at https://doi.org/10.6084/m9.fi gshare.27248187.v3

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2025.2529493