E. coli DH5α was used for cloning and was cultivated in Luria-Bertani medium. M. smegmatis mc^2–155 was cultured in Middlebrook 7H9 liquid medium supplemented with 0.2% glycerol and 0.05% Tween-80, or in Middlebrook 7H9 solid medium containing 0.2% glycerol. Antibiotics or inducers were used at the following concentrations when required: kanamycin (50 μg/mL for E. coli, 25 μg/mL for M. smegmatis), hygromycin (150 μg/mL for E. coli, 75 μg/mL for M. smegmatis), anhydrotetracycline (aTc, 100 ng/mL for M. smegmatis) and acetamide (ACE, 28 mM for M. smegmatis). Plasmids, strains, and primers are listed in Supplementary Table S1. THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin at 37°C with 5% CO₂.

Growth curves of wild-type and mutant M. smegmatis strains were determined by spectrophotometry (UV-VIS spectrophotometer, Varian Cary 50). Briefly, overnight cultures grown to mid-late exponential phase were harvested by centrifugation at 6,000 rpm for 10 min, washed twice with 1×phosphate-buffered saline (PBS), and adjusted to an optical density at 600 nm (OD₆₀₀) of 0.8 (approximately 1 × 10⁸ cells mL^-1). A 1% (v/v) inoculum was transferred into fresh 7H9 medium containing either 0.25 mM MG (Macklin) or no additive (untreated control). Cultures were incubated at 37°C with shaking (110 rpm). Where indicated, GSH was added to a final concentration of 0.25 mM at 12 h post-inoculation. The OD₆₀₀ was measured every 4 h for 48 h.

Cultures were prepared as in section 2.3. Cells were then diluted 100−fold in fresh 7H9 medium (10 mL final volume), and treated with 0, 0.5, 1 or 2 mM MG in triplicate. These concentrations were selected based on pilot experiments that revealed differential tolerance between Rv0801 recombinant strains and ΔMSMEG_5827 strains. To evaluate protection against MG toxicity, GSH was added to 7H9 agar plates at equimolar concentrations relative to MG, reflecting the 1:1 stoichiometry of the spontaneous MG-GSH reaction. At indicated time points (4, 8, 12 or 24 h post-treatment), 1 mL of culture was collected, washed with 1×PBS and serially diluted (10^-1 to 10^-5) onto plates with or without GSH. Viability was determined by counting colony−forming units (CFUs) after 3 days of incubation at 37°C.

Bacterial cultures were prepared as in section 2.3 and then exposed to 0.07 mM H₂O₂ in 7H9 agar plates, while control cultures were left untreated. This concentration was selected based on a previous study (32) and further optimized in pilot experiments. Cell viability was assessed by performing serial dilutions, followed by spotting 10 μL aliquots (in triplicate) onto 7H9 agar plates. Viability was determined by CFUs after 3 days of incubation at 37°C.

Bacterial cultures were prepared as in section 2.3 and the cells were then resuspended and normalized to an OD₆₀₀ of 0.8 using 7H9 liquid medium that had been pre-adjusted to pH 4.5, 5.5, 6.5, or 7.5, with a final volume of 10 mL per condition. Cultures were incubated at 37°C with shaking (110 rpm). At 3, 6 or 9 h post-treatment, 1 mL aliquots were collected, washed, and resuspended in 1 mL of fresh PBS. Bacterial viability was assessed by preparing 10-fold serial dilutions in PBS and spotting 10 μL aliquots onto 7H9 agar plates. Viability was determined by CFUs after 3 days of incubation at 37°C. All experiments were performed independently at least three times.

Bacteria or cultured cells were grown under standard conditions (e.g., 7H9 liquid medium for bacteria, RPMI 1640 medium for THP-1 cells) until they reached the logarithmic growth phase (OD₆₀₀ of 0.6 for bacteria; 70-80% confluence for adherent cells). Bacterial strains were then treated with 2 mM MG for 3 h at 37°C prior to analysis. THP-1 cells were infected by different strains for 24 h. Intracellular ROS levels were quantified using the Reactive Oxygen Species Assay Kit (Solarbio), following the manufacturer’s protocol based on the fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA).

Bacterial cultures were prepared as in section 2.3. Antibiotic sensitivity was assessed by a spot dilution assay in which serial 10−fold dilutions of the cultures were spotted onto 7H9 agar plates containing 10 μg/mL rifampicin. This concentration was selected based on our experimental system (33). Plates were incubated at 37°C for 3–5 days, and viability was determined by CFUs. Each experiment was performed independently at least three times to ensure reproducibility.

Cultures were prepared as in section 2.3 and then diluted 10−fold in 7H9 liquid medium (10 mL final volume) containing either 1 mM MG or no additive (untreated control), followed by incubation at 37°C with shaking (110 rpm). After 4 h post-treatment, 1 mL aliquots were collected, centrifuged (4000 rpm, 5 min), and the pellet was washed twice with 1×PBS. Cells were then resuspended in 1 mL of fresh 1×PBS and subjected to 10−fold serial dilutions. A 10 μL aliquot from each dilution was spotted onto 7H9 agar plates supplemented with or without 5 μg/mL rifampicin. Plates were incubated at 37°C for 3-4 days, and bacterial viability was assessed by enumerating CFUs.

THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin at 37°C with 5% CO₂. Cells were seeded at a density of 1 × 10⁶ cells/mL in 12-well plates and differentiated using 100 ng/mL phorbol 12-myristate 13-acetate (PMA). Before infection, differentiated macrophages were pre-treated as follow: (i) with 0.1 mM MG or 25 mM glucose (Macklin) for 4 h; or (ii) with 5 μM CBR−470−1, 5 mM 2-deoxy-D-glucose (2−DG), or 2 mM N−acetylcysteine (NAC) (MedChemExpress) for 18 h. Cells were then infected with MS_pAL or MS_Rv0801 at a multiplicity of infection (MOI) of 10. At 4 h post-infection, cells were washed with PBS, and incubated with complete medium containing gentamicin (100 μg/mL) to eliminate extracellular bacteria. To evaluate the effect of exogenous GSH, 0.1 mM GSH was added to designated wells immediately after the washing step. For assessment of intracellular bacterial survival, infected THP-1 cells were lysed at 6, 12, 24, 36 and 48 h post-infection. Cells were washed three times with PBS, lysed in 1 mL of 0.025% sodium dodecyl sulfate (SDS), serially diluted, and plated onto 7H9 agar plates. Bacterial viability was determined by counting CFUs after 3 days of incubation at 37°C.

THP-1 cells were seeded in a 96-well plate at a density of 1×10⁴ cells per well in 100 µL of complete medium and cultured for 48 h. Cells were then infected separately with either MS_Rv0801 or MS_pAL at a MOI of 10 for 24 hours. Following infection, cell viability was assessed using a Cell Counting Kit-8 (CCK-8). Briefly, after pre-warming the microplate reader for 30 minutes, 10 μL of CCK-8 solution was added to each well and the plate was incubated for an additional 2 h at 37°C. The absorbance at 450 nm was then measured using the microplate reader. Cells viability was calculated relative to uninfected control cells.

THP-1 cells were collected at 24 h post-infection. Intracellular GSH content was determined using a Reduced Glutathione Content Assay Kit (Solarbio), following the manufacturer’s instructions.

To assess the mRNA levels of MSMEG_5827, MshA, MshB, MshC, and MshD under MG exposure, overnight cultures of M. smegmatis were grown to mid-exponential phase. Cells were harvested, washed twice with PBS, and resuspended in 7H9 medium (containing 0.05% Tween 80) to an OD₆₀₀ of 1.0. WT_MS was then treated with 2 mM MG or left untreated for 3 h at 37°C. For infection assays, PMA-differentiated THP-1 cells were pre-treated under the following conditions prior to infection: with 0.1 mM MG for 4 h; with 5 μM CBR−470−1 or 5 mM 2−DG for 18 h; or left untreated as a control. Subsequently, cells were infected with either MS_pAL or MS_Rv0801 at a MOI of 10 for 24 h. Total RNA from all samples was isolated using an RNA extraction kit (Promega), and cDNA was synthesized with a reverse transcription kit (Takara). All samples were prepared in three biological replicates. Quantitative real-time PCR was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad) using SYBR Green Master Mix (Takara). For bacterial samples, gene expression was normalized to rpoB (MSMEG_1367). For THP-1 cell samples, gene expression was normalized to β-actin. Relative mRNA expression for all samples was calculated using the 2^−ΔΔCt method (34). Primer sequences are provided in Supplementary Table S1.

THP-1 cells were infected with different bacterial strains at a MOI of 10. After 24 h of infection, cells were washed three times with PBS, and lysed using RIPA lysis buffer to collect total protein. The protein was separated by 12% SDS-PAGE and transferred onto a nitrocellulose (NC) membrane at 15 V for 1-2 h, with the transfer duration adjusted according to protein size. Membranes were blocked with 5% bovine serum albumin (BSA) in TBST (Tris-buffered saline with Tween-20) for 2 hours at room temperature on a shaker. Then membranes were incubated overnight at 4°C with primary antibodies against β-Actin, Nqo1 and Nrf2 (Abmart). After five washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Beyotime Biotechnology) for 2 hours at room temperature, then were washed five times with TBST. Protein bands were visualized using an ECL chemiluminescence detection kit (Vazyme). Quantitative analysis of the bands was carried out with ImageJ.

Data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical comparisons between two groups were performed using the two-tailed Student’s t-test. Data visualization and statistical analysis were conducted using GraphPad Prism 9.3. Differences were considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.

Bacterial glyoxalase system primarily consists of GloA and GloB. MG spontaneously reacts with GSH to form a hemithioacetal, which is converted by GloA into S-D-lactoylglutathione. This intermediate can regulate bacterial potassium ion efflux pumps, inducing K⁺ efflux and concurrent H⁺/Na⁺ influx, leading to cytoplasmic acidification (35). Intracellular acidification is proposed as a key response mechanism against MG toxicity, potentially triggering activation of DNA damage repair pathways. S-D-lactoylglutathione is further hydrolyzed to D-lactate by GloB, regenerating reduced GSH. Lactate is then oxidized to pyruvate via lactate dehydrogenase (Figure 1A).

To investigate the function of Rv0801, we first predicted its three-dimensional structure using SWISS-MODEL with a Bacillus cereus glyoxalase template (SMTL ID: 1ss4.1.A) (Figure 1B). The model revealed a conserved oxygen-binding domain and a glyoxalase-like domain, with sequence alignment confirming high conservation across Mycobacterium species (Figure 1C). To further explore its function, we heterologously expressed Rv0801 in M. smegmatis (Supplementary Figure S1A), whose ortholog is MSMEG_5827 (Figure 1D). Functional comparisons of both genes were then performed to assess their roles in MG detoxification.

To determine whether MSMEG_5827 response to MG stress, we assessed transcriptional dynamics of wild-type M. smegmatis exposed to 2 mM MG for 3 h. Results revealed a significant upregulation of MSMEG_5827 (Figure 1E). To dissect the function of Rv0801, we expressed it in M. smegmatis, and monitored growth over 48 h in 7H9 medium containing 0.25 mM MG. While the empty vector control strains exhibited clear growth inhibition during the logarithmic phase, the Rv0801 recombinant strains grew normally (Figure 1F). Conversely, the MSMEG_5827 knockout strains (Supplementary Figure S1B) displayed pronounced growth delay upon MG exposure compared to wild-type, with no defect in standard medium (Figure 1G). We further quantified bacterial survival after MG challenge using CFU counting assays. Strains were exposed to 0.5 or 2 mM MG in 7H9 liquid culture for 24 h, then plated for viability counts. The Rv0801 recombinant strains showed enhanced MG tolerance, while the MSMEG_5827 knockout strains exhibited increased sensitivity (Figures 1H, I). Together, these findings establish Rv0801 and its homolog MSMEG_5827 as key components of the mycobacterial MG-detoxification machinery, with direct implications for bacterial fitness under carbonyl stress.

The canonical glyoxalase pathway relies on GSH as a cofactor for detoxifying MG. However, in GSH-deficient Gram-positive Firmicutes such as Bacillus subtilis, MG detoxification depends on the unique bacillithiol (BSH) (36). Similarly, Mycobacteria utilize mycothiol (MSH) as their primary redox buffer against oxidative stress (37), implying a potential role for MSH in MG detoxification.

To assess the role of GSH in mycobacterial MG detoxification, we monitored bacterial growth under MG stress with or without GSH supplementation. The Rv0801 recombinant strains showed no alteration in growth kinetics upon addition of 0.25 mM GSH at 12 h during a 0.25 mM MG challenge (Figure 2A). In contrast, the empty vector control strains exhibited growth arrest in GSH-supplemented medium, while cultures without added GSH demonstrated initial growth inhibition followed by gradual recovery (Figure 2A). However, this does not exclude the effect of the plasmid itself on bacterial growth. For the MSMEG_5827 knockout strains, MG exposure caused early growth retardation but permitted eventual recovery after 36 h (Figure 2B), suggesting that this gene product contributes quantitatively but not absolutely to detoxification capacity. Notably, both wild-type and complemented strains exposed to combined MG and GSH showed transient early growth inhibition, but recovered to levels comparable to those treated with MG alone by 36 h (Figure 2B). This implies functional independence from GSH for Rv0801- and MSMEG_5827-mediated MG detoxification pathways.

To further test this model, we performed CFU counting assays in liquid cultures treated with 1 mM or 2 mM MG, measuring viability after 4, 8 and 12 h. When exposed to GSH-supplemented solid media, no mitigation of MG-induced CFU counts disparities was observed between empty vector controls and Rv0801 recombinant strains (Figure 2C). A similar trend was observed in the MSMEG_5827 knockout strains (Figure 2D). Collectively, these findings support that mycobacterial glyoxalase system operates through non-GSH-dependent mechanisms.

Previous studies revealed that exogenous GSH disrupts MSH-dominated redox homeostasis in mycobacteria, thereby inducing cytotoxicity (38). We therefore hypothesized that GSH may impose reductive stress that synergizes with MG-induced oxidative damage to inhibit bacterial proliferation. To investigate whether MSH is required for mycobacteria MG detoxification, we first examined the transcriptional levels of MSH biosynthetic genes (MshA, MshB, MshC, and MshD) in wild-type M. smegmatis after exposure to 2 mM MG for 3 h. All four genes were significantly upregulated following MG induction (Figure 2E), suggesting MG-driven transcriptional activation of the MSH biosynthetic pathway. To further dissect the functional role of MSH in this process, we generated an MshA knockdown strain (validated by RT-qPCR, Supplementary Figure S2A). When challenged with 1 mM MG, the knockdown strains showed markedly reduced survival compared to the wild-type in CFU assays at both 4 h and 12 h (Figure 2F), establishing MSH’s protective role against MG toxicity.

To clarify whether the MG detoxification pathways mediated by Rv0801 and its homolog MSMEG_5827 depend on MSH, we constructed a double mutant ΔMSMEG_5827(MshA-KD) (Supplementary Figure S2B), and compared its survival with the corresponding single mutants under 2 mM MG exposure for 4 h. Both the single mutant ΔMSMEG_5827 and the double mutant ΔMSMEG_5827(MshA-KD) exhibited significantly lower CFUs than MshA-KD strain (Figure 2G). Notably, the double mutant did not exhibit increased sensitivity compared to the ΔMSMEG_5827 single mutant (Figure 2G), suggesting that disruption of MSH biosynthesis does not further sensitize cells already deficient in MSMEG_5827. Collectively, these findings uncover a non-canonical glyoxalase system in mycobacteria where MSH acts as a critical cofactor synergizing with Rv0801 or MSMEG_5827 to detoxify MG, underscoring mycobacteria’s evolutionary adaptation to employ unique thiol-dependent redox defense mechanisms.

MG accumulation typically elevates intracellular reactive oxygen species (ROS) levels, causing oxidative damage (39). To determine whether Rv0801 confers resistance to oxidative stress beyond MG detoxification, we compared the survival of empty vector controls and Rv0801 recombinant strains under 0.07 mM hydrogen peroxide (H₂O₂) exposure by performing CFU counting assays. Rv0801 recombinant strains exhibited a significant but moderate survival advantage under H₂O₂-induced oxidative stress (Figure 3A), while the MSMEG_5827 knockout strains displayed increased H₂O₂ sensitivity (Figure 3B). These results establish functional conservation between Rv0801 and its homolog MSMEG_5827 in mediating mycobacterial resistance to diverse oxidative insults.

Acidic conditions trigger bacterial starvation responses that restrict carbon utilization and counteract reduction-related stress, which often overlaps with oxidative stress (40). To assess the role of Rv0801 in reductive stress resistance, we analyzed M. smegmatis survival across varying pH levels at 3 h, 6 h and 9 h by CFU counting assays. Notably, Rv0801 recombinant strains exhibited significantly higher CFUs than controls after 6 h of acidic exposure (Figure 3C). This suggests Rv0801 enhances bacterial resilience under adverse environmental conditions. To mechanistically link Rv0801 activity to ROS regulation under oxidative stress, intracellular ROS levels were quantified 3 h after 2mM MG treatment. Rv0801 recombinant strains demonstrated markedly lower ROS levels compared to controls (Figure 3D), indicating that Rv0801 activity mitigates oxidative damage by limiting ROS accumulation.

Given MG’s genotoxicity via DNA mutations (41–43) and ROS-driven DNA damage (44), which can drive rpoB mutations and rifampicin resistance (45), we next investigated Rv0801’s impact on antibiotic tolerance. Strikingly, Rv0801 recombinant strains displayed significantly higher susceptibility to rifampicin (10 μg/mL) than controls (Figure 3E). When challenged concurrently with MG (1 mM) and rifampicin (5 μg/mL), rifampicin-mediated killing was potentiated in both strains (Figure 3F). Notably, the survival benefit conferred by Rv0801 was compromised under these conditions (Figure 3F), suggesting that MG stress exacerbates rifampicin toxicity in this strain. These results indicate that Rv0801 is critical for genomic stability under metabolic stress through coordinated redox regulation.

The metabolic shift to aerobic glycolysis in Mtb−infected macrophages elevates MG (8, 46), presenting a key challenge for intracellular survival. We therefore investigated whether the putative glyoxalase Rv0801 is required for Mtb to withstand this endogenous stress by performing macrophage infection assays. We found Rv0801 significantly enhanced bacterial survival in infected cells (Figure 4B). To dissect the link between MG detoxification and intracellular fitness, macrophages were pre-treated with 0.1 mM MG for 4 h to mimic a carbonyl−stressed host environment (Figure 4A). Under exogenous MG exposure, the survival advantage of the Rv0801 recombinant strains was further enhanced (Figure 4B). However, pre−treatment with 25 mM glucose—which elevates endogenous MG via glycolysis, did not widen the strain disparity (Figures 4A, B).

We next tested whether Rv0801 helps bacteria cope with host−derived MG produced during infection. Pre-treatment of THP-1 cells with 5 μM CBR-470–1 for 18 h (Figure 4C), a protein kinase G (PKG) inhibitor that promotes glycolytic flux and endogenous MG accumulation (13), resulted in a sustained survival advantage for the Rv0801 recombinant strains at 48 h (Figure 4D). Conversely, inhibiting glycolysis with 2-deoxy-D-glucose (2-DG, 5 mM) markedly reduced the Rv0801-conferred survival benefit compared to the untreated group (Figures 4C, E).

The homeostasis of MG is tightly regulated by host cellular antioxidant defenses. To further determine whether Rv0801−mediated survival involves perturbing this balance, we pre-treated THP−1 macrophages with the ROS inhibitor N−acetylcysteine (NAC, 2 mM) for 18 h (Figure 4C) or exogenously supplemented with 0.1 mM GSH after 4 h infection. Both treatments abolished the intracellular survival advantage of the Rv0801 recombinant strains (Figures 4F, G), confirming its fitness benefit depends on altering host redox homeostasis.

The KEAP1-NRF2 pathway is a master regulator of cellular antioxidant defense. However, excessive NRF2 signaling suppresses expression of critical microbicidal effector molecules in bone marrow-derived macrophages (BMMs), impairing mycobacterial clearance (47).

To determine whether Rv0801 influences the KEAP1-NRF2 axis, we first analyzed Nrf2 transcriptional activity in THP-1 macrophages infected with Rv0801 recombinant strains. Compared to empty vector controls, Rv0801 significantly suppressed Nrf2 mRNA expression (Figure 5A), accompanied by reduced transcription of antioxidant genes nqo1, gclc, and txnrd1 (Figure 5B). Pre-treatment with 0.1 mM MG or 5 μM CBR-470-1 further depressed their transcriptional output in Rv0801 recombinant strains-infected cells (Figure 5B), implying carbonyl stress synergizes with Rv0801 activity to dampen the host antioxidant response. Conversely, inhibition of glycolysis with 5 mM 2−DG abolished this suppression (Figure 5C). At the protein level, infection with the Rv0801 recombinant strains reduced NRF2 and its downstream target Nqo1 (Figure 5D). However, in cells pre-treated with CBR−470−1, the difference in Nqo1 protein levels between the Rv0801 recombinant and control strains was no longer observed (Figure 5D). This absence of a difference likely results from CBR−470−1 pretreatment fundamentally rewiring host cell metabolism, thereby obscuring the strain−specific effect. Next, we found out infection with MSMEG_5827 deletion mutants reduced intracellular ROS levels versus vector controls (Figure 5E). This indicates a role for MSMEG_5827 in modulating the host redox environment. Consistent with a perturbed redox state, Rv0801 recombinant strains infection also significantly decreased intracellular GSH pools (Figure 5F).

To evaluate the effect of Rv0801 on host cell viability, we assessed cell survival rates after 24 h infection. Results show Rv0801 recombinant bacteria induced cytotoxicity in THP-1 macrophages (Figure 5G) and suppressed mRNA expression of pro-inflammatory cytokines tnf-α, il-1b, il-6 and the oxidative stress sensor park7, at 24 h post-infection (Figure 5H). Collectively, these findings indicate that by detoxifying host−derived MG, Rv0801 helps maintain KEAP1−mediated repression of NRF2, thereby blunting the antioxidant response. This suppression, coupled with diminished GSH and pro−inflammatory signaling, creates an immunosuppressive niche that favors mycobacterial intracellular persistence.