Rv0102 is involved in copper ions transfer
To generate an adequate number of single mutants, we created a mutant library of M. smegmatis mc2 155 with 20,000 transposon insertions using the Tn7 transposon system. The library was screened on 7H10 medium with high copper ion concentration (20 µmol/L CuSO4), leading to the identification of a copper-tolerant mutant X674. This mutant had a Tn7 transposon insertion between the 375th and 376th positions of M. smegmatis MSMEG_4702, confirmed by High-Tail RCR and BLAST. To validate the phenotype, we deleted the MSMEG_4702 gene in M. smegmatis mc2 155 via homologous chromosome exchange (Fig. 1B) and complemented it with the Mtb homolog gene Rv0102, which corresponds to MSMEG_4702 in Mtb (Supplementary Fig. 1). Multiple-sequence alignment analysis showed high conservation of the MSMEG_4702 amino acid sequence across various mycobacterial species (Fig. 1D). There is also significant homology between the DNA and amino acid sequences (Supplementary Fig. 2).
The Rv0102 gene in Mtb is annotated as an integral membrane protein containing 661 amino acids (1986bp), while MSMEG_4702 in M. smegmatis is annotated as an ABC-type transporter, indicating that Rv0102 might function as a transporter in the cytoplasmic membrane. Protein immunoblotting brought about the localization of the Rv0102 protein, showing its exclusive presence in the cell wall/cell membrane (CW/CM) fraction while being absent in the cytoplasmic (CP) fraction (Fig. 1C), indicating that Rv0102 is a protein associated with the cell envelope. According to the analysis results of the PPM server 20, Rv0102 has 16 transmembrane segments (TMS), with potential Cu-binding motifs in TMS14 to TMS15. InterProScan results suggested a conserved homeodomain in TMS3 to TMS6, akin to CopD, and cytochrome oxidase characteristics in TMS11 to TMS16 (Fig. 1E). CopD, a cytoplasmic copper transfer protein with no Mycobacterium homologs, hints at Mtb Rv0102 possibly having a copper uptake role like CopD. Protein structure predictions via I-TASSER and PyMOL indicated potential copper ion binding sites with features of divalent cation transport (Fig. 1F). The absence of MSMEG_4702 in M. smegmatis confers high copper tolerance, suggesting MSMEG_4702's crucial involvement in facilitating divalent copper ion transport in Mtb.
The deficiency of MSMEG_4702 confers copper tolerance to M. smegmatis
Copper ions are essential for Mycobacterium virulence 21. In contrast to the wild-type (WT) strain, the deletion mutant ΔMSMEG_4702 strain exhibited growth defects in Middlebrook 7H9 liquid medium supplemented with 0.05% Tween80 and 0.2% glycerol (Fig. 2A). The knockout MSMEG_4702 strain resulted in a delayed growth of the ΔMSMEG_4702 strain compared to the WT strain after 8 hours of culture. However, after an extended culture (64 hours), the bacterial densities of both the ΔMSMEG_4702 and WT strains tended to reach a similar level. To eliminate the potential effects of copper present in the commercial Middlebrook 7H9 medium, a copper-free 7H9 medium was prepared for a growth comparison. As anticipated, the growth defect of the ΔMSMEG_4702 strain became significantly more severe after 64 hours of cultivation in the copper-free 7H9 medium, exhibiting a much greater delay compared to the WT strain (Fig. 2B). The observed phenotype provides strong evidence for the involvement of MSMEG_4702 in copper ion utilization. Surprisingly, both the wild-type and complemented strains were capable of growth in copper-free Middlebrook 7H9 media, suggests that copper's fate within bacteria involves its binding to copper-storing proteins such as MymT. These proteins play a crucial role in mitigating copper toxicity under excessive copper conditions and can also serve as a source of copper nutrition during copper scarcity. Additionally, MSMEG_4702 is not the only protein involved in copper uptake, as CtpB also plays a significant role in the copper uptake pathway 6.
When copper ion is excessive, copper could be transferred into the cytoplasm through specific copper transport systems in the membrane, such as the major facilitator superfamily (MSF) transporters 22. CuSO4 of different concentrations (37.8 µΜ and 100 µΜ respectively) were added into the copper-free 7H9 medium and bacterial growth was evaluated again. Interestingly, the growth curves of WT and ΔMSMEG_4702 strains were essentially identical in the presence of 37.8 µmol/L CuSO4 (Fig. 2C). However, when the concentration of CuSO4 reached 100 µmol/L, both WT and complemented strains entered the decline phase, while the ΔMSMEG_4702 strain remained in the logarithmic phase (Fig. 2D). This indicates that M. smegmatis MSMEG_4702 is indeed involved in the utilization of copper ions, and the reduction in copper utilization resulting from the deletion of MSMEG_4702 enables the mutant strain to survive in high concentrations of exogenous copper.
We investigated the impact of copper on bacterial growth on solid medium lacking copper ions and observed a significant growth rate impairment in the ΔMSMEG_4702 strain (Fig. 2E). CuSO4 at concentrations of 6.3 µmol/L, 37.8 µmol/L, and 63 µmol/L were added to copper-free 7H9 solid medium, inoculated with 10 µl of diluted bacterial solution. Results showed that increasing CuSO4 concentration notably inhibited WT strain growth. Conversely, ΔMSMEG_4702 growth was enhanced with rising CuSO4 levels (Fig. 2E). At 37.8 µmol/L CuSO4, both WT and ΔMSMEG_4702 strains exhibited similar growth levels. Moreover, at 63 µmol/L CuSO4, ΔMSMEG_4702 strain displayed normal growth while WT could not survive. These findings suggest that the protein encoded by M. smegmatis MSMEG_4702 is crucial for copper ion utilization.
MSMEG_4702 deficiency impairs the growth of M. smegmatis by reducing intracellular copper concentrations and inhibiting cell division
Using the Cuprizone microplate method, we assessed the intracellular copper ion levels of both WT and ΔMSMEG_4702 strains. WT M. smegmatis maintained stable intracellular copper ion concentrations around 0.08 µmol/L, unaffected by external CuSO4 concentration changes (Fig. 3A). However, at 37.8 µmol/L CuSO4, WT intracellular copper levels rose, suggesting M. smegmatis can regulate copper homeostasis to support growth. In cases of excessive extracellular copper reaching bactericidal levels, M. smegmatis may encounter homeostasis failure, leading to a sharp intracellular copper increase hindering bacterial growth. Notably, ΔMSMEG_4702 strain showed significantly lower intracellular copper content compared to WT, independent of external CuSO4 levels (Fig. 3A). Conversely, very high CuSO4 concentrations (63 µmol/L) resulted in six times higher intracellular copper levels in WT vs. ΔMSMEG_4702. Rv0102 complementation successfully restored ΔMSMEG_4702 intracellular copper levels, emphasizing Rv0102's vital role in copper ion regulation. Zinc ion addition did not yield significant differences (Supplementary Fig. 3A). Overall, our data suggest ΔMSMEG_4702 markedly impairs copper ion utilization in M. smegmatis.
Interestingly, despite the significant intracellular copper content difference, no distinct growth variation was observed between WT and ΔMSMEG_4702 strains on solid medium (Fig. 2E) or liquid medium (Fig. 2C) at 37.8 µM CuSO4 concentration. WT intracellular copper ion concentration was four times higher than ΔMSMEG_4702, restorable by Rv0102 complementation (Fig. 3A). Our data indicated a critical transition threshold of copper from growth promoter to inhibitor in M. smegmatis lying between 0.1 µmol/L and 0.2 µmol/L. At this pivotal copper concentration, ΔMSMEG_4702 growth was boosted while WT strain growth was notably hindered. Despite the growth behavior contrast, both strains ultimately reached similar growth levels. In summary, these findings highlight MSMEG_4702's crucial role in mediating environmental copper ion utilization, maintaining copper homeostasis, and fostering M. smegmatis growth.
To explore the mechanism behind copper's impact on bacterial proliferation and assess its influence on ΔMSMEG_4702 cell growth or division, we conducted experiments to examine the effects of varied copper concentrations on bacterial growth and colony morphology. At optimal CuSO4 levels, wild-type M. smegmatis displayed distinctive colony morphology with large, rounded single colonies (Supplementary Fig. 3B). Supplementing copper-free 7H9 culture medium with different copper concentrations (0 µmol/L, 6.3 µmol/L, 37.8 µmol/L, and 126 µmol/L) revealed reduced growth rates and smaller colony sizes for ΔMSMEG_4702 at 0 µmol/L or 6.3 µmol/L CuSO4, while WT growth rate and colony size remained normal. Under 37.8 µmol/L CuSO4, both WT and ΔMSMEG_4702 exhibited comparable growth rates and colony sizes. Interestingly, exposure to 126 µmol/L CuSO4 led to WT growth failure but allowed ΔMSMEG_4702 growth (Fig. 3B). Disparities in single colonies suggested individual bacteria underwent changes, indicating WT strain susceptibility to copper inhibition.
Copper deficiency leads to the loss of function of several bacterial growth enzymes, likely due to the role of copper ions as cofactors for enzymes that are essential for bacterial growth 23. When cultured with 6.3 µmol/L CuSO4, ΔMSMEG_4702 displayed slightly elongated and more dispersed morphology (Fig. 3C), whereas the wild-type strain exhibited uniform length. TUNEL assay results indicated that ΔMSMEG_4702 accumulated higher DNA damage than WT, implying inefficient copper ion utilization in ΔMSMEG_4702 might result in increased DNA damage and impede cellular division (Fig. 3D).
Bathocuproine (BCS) is a bidentate copper chelator known to form a 1:2 tetrahedral complex with monovalent copper ions (CuI), effectively removing CuI from the medium. The supplementation of BCS restored the growth of WT M. smegmatis under high copper concentrations (Fig. 3E). When added to a copper-free medium, BCS reduced copper utilization by M. smegmatis, leading to decreased growth (Supplementary Fig. 4). These results suggest that Cu(I) ion plays a significant role in the growth disparity between WT and ΔMSMEG_4702.
MMAR_0267 deletion enables M. marinum to inhibit mycobacteria-induced macrophage apoptosis by interfering with phagolysosome acidification
Transition metals such as copper, iron, zinc, and manganese are essential trace nutrients for Mtb and other pathogens during host infection 3. However, it is generally observed that an elevation in copper concentration is employed to regulate the virulence of pathogens within phagosomes. Mtb in guinea pig macrophages can enhance virulence by accelerating copper efflux or upregulating copper-binding protein 24. To investigate the impact of reduced copper utilization on Mycobacteria within macrophages, we disrupted MMAR_0267, the equivalent of the Rv0102 gene in M. marinum. Subsequently, THP-1 macrophages were infected with both WT and ΔMMAR_0267 strains, and their intracellular survival was evaluated by measuring the colony-forming units (CFU) of viable bacteria. Upon infection of THP-1 macrophages, the ΔMMAR_0267 strain demonstrated a significantly higher intracellular survival rate compared to the WT strain at 2 and 3 days post-infection (Fig. 4A). Furthermore, the elimination of internalized ΔMMAR_0267 by THP-1 macrophages was notably delayed (Fig. 4B). These results suggest that the ΔMMAR_0267 mutant displays an enhanced survival rate within macrophages.
In macrophages, pathogens often prevent host cell apoptosis to secure their differentiation, survival, and replication 25,26. The increased intracellular viability of ΔMMAR_0267 may be due to abnormal programmed cell death. The CCK8 fluorescence assay indicated that, at 4 hours (Fig. 4C) and 24 hours (Fig. 4D) post-infection, the number of viable THP-1 cells was significantly higher in cells infected with ΔMMAR_0267 compared to those infected with the WT and complemented strains. These results suggest that ΔMMAR_0267 inhibits programmed cell death in infected cells. To verify if the ΔMMAR_0267 mutant modulates host cell apoptosis and enhances its survival within macrophages, Annexin V with PI co-staining, along with fluorescence microscopy and flow cytometry, were used to evaluate the effects of WT and ΔMMAR_0267 on host cell apoptosis. ΔMMAR_0267 demonstrated a notable reduction in THP-1 apoptosis compared to WT (Fig. 4E-fluorescence microscopy, Fig. 4F-flow cytometry). Interestingly, the levels of pro-inflammatory cytokines, including IL-1β, IL-10, and TNF-α, produced by THP-1 cells infected with ΔMMAR_0267 did not show significant changes (Supplementary Fig. 5). The expression of apoptosis-promoting proteins BAX, P53, and Cas9 was down-regulated by ΔMMAR_0267, while the anti-apoptotic protein Bcl2 was up-regulated (Fig. 5A). These findings indicate that ΔMMAR_0267 can inhibit host cell apoptosis. Immunoblotting analysis also revealed no significant difference in the levels of IL-1, IL-6, and IL-12 among THP-1 cells infected with ΔMMAR_0267, WT, and the complemented strain. However, the levels of P53 and Cas9 were decreased 24 hours post-infection (Fig. 5B). The results suggest that ΔMMAR_0267 enhances the intracellular survival of M. marinum by inhibiting macrophage programmed cell death, primarily by suppressing apoptosis rather than altering pro-inflammatory cytokine levels.
Generally, apoptosis is induced by Mtb and is associated with better killing of mycobacterial cells and protection of the host 27. The suppression of the apoptosis process by ΔMMAR_0267 suggests a potential increase in virulence, enabling evasion of the host's innate immune defenses. Mtb infection is known to cause membrane damage and induce necrosis, with the lysosome-mediated membrane repair pathway playing a crucial role in Mtb protection 28. To investigate whether the ΔMMAR_0267 can hinder lysosomal acidification and LMP, THP-1 cells were infected with GFP-labeled WT, ΔMMAR_0267, and complemented strains. LysoTracker-Blue was employed to visualize and track the acidic endosomes and lysosomes. ΔMMAR_0267 co-localization with lysosomes was significantly higher compared to the WT strain, while the complement strain showed a normal level of lysosomal co-localization (Fig. 5C and Fig. 5D). These findings suggest that infection with ΔMMAR_0267 enhances lysosome fusion but inhibits LMP. In summary, the ΔMMAR_0267 mutant can effectively inhibit macrophage apoptosis, thereby promoting macrophage survival, potentially through interference with normal phagolysosome acidification.
MMAR_0267 deficiency increases zebrafish mortality upon M. marinum infection
To examine how reduced copper utilization impacts the virulence of M. marinum in zebrafish, we intraperitoneally infected a total of thirty healthy adult wild-type zebrafish with 10 µl of 2*105 CFU/mL of either wild-type M. marinum, ΔMMAR_0267, or MMAR_0267 complemented strains. The zebrafish infected with wild-type M. marinum succumbed on the seventh day following infection, whereas those infected with ΔMMAR_0267 died much earlier, on the third day after the infection. Notably, all zebrafish in the ΔMMAR_0267 infection group died 11 days earlier than those in the wild-type infection group (Fig. 6A). These results suggest that the deletion of MMAR_0267 can enhance the virulence of M. marinum. Based on the longtitudinal survival counts, we infected zebrafish with the three strains and dissected all infected fish on the third day. The data demonstrated that zebrafish infected with ΔMMAR_0267 exhibited significantly more severe symptoms of congestion, bleeding, and ulcers compared to those infected with the WT and complemented strains (Fig. 6B). The CFU analysis of zebrafish tissues on the third day post-infection revealed that the bacterial load of ΔMMAR_0267 was significantly higher in the liver and skin (Fig. 6C). Histopathological examination of the infected tissues demonstrated that compared to the WT or complemented strains, zebrafish infected with the ΔMMAR_0267 strain exhibited increased neutrophil infiltration and necrotic areas in the liver and skin (Fig. 6D). The results indicate that decreased copper utilization plays a role in facilitating M. marinum's resistance to the host's innate immunity, thereby enhancing its intracellular growth and virulence during infection.
MMAR_0267 deficiency enables M. marinum to escape host immunity by dampening the macrophage STING-TBK1-IRF3 signaling
The MMAR_0267 deletion mutant revealed the capacity of M. marinum to inhibit zebrafish macrophages apoptosis for promoting survival. In order to delve deeper into the underlying mechanism behind the heightened virulence of ΔMMAR_0267 M. marinum towards zebrafish, we conducted a comprehensive analysis of the transcriptome and metabolome of infected zebrafish. GSEA (Gene Set Enrichment Analysis) analysis of the transcriptome revealed that the deletion of MMAR_0267 activated the zebrafish glycolysis / gluconeogenesis, RIG-I-like receptor signaling, mTOR signaling, and IgA immune signaling (Fig. 7A). Furthermore, transcriptome heat maps revealed varying degrees of differential gene expression associated with the mTOR signaling pathway (TBK1, IRF3, etc.) in zebrafish infected with ΔMMAR_0267 M. marinum to varying degrees. Metabolome analysis also identified increased levels of mTOR signaling pathway-related metabolites (L-Arginine, L-Leucine, etc.) in the ΔMMAR_0267 infection group (Fig. 7B). Metabolomic analysis simultaneously revealed that infection with ΔMMAR_0267 M. marinum resulted in an accumulation of alpha-ketoglutarate and a decrease in lactate levels in infected zebrafish, providing further evidence for the activation of the glycolysis and tricarboxylic acid (TCA) cycle (Supplementary Fig. 6). The results obtained from the omics analysis demonstrate the activation of mTOR signaling played a crucial role in this process, as (1) mTOR complex 1 (mTORC1) can enhance the mitochondrial energy metabolism of infected macrophages through glycolysis to protect them from mycobacteria-induced death 29,30, (2) the mTOR signaling is regulated by early secreted antigenic target 6 (ESAT6) upon Bacillus Calmette-Guérin (BCG) vaccination 31. Previous studies have demonstrated that the activation of TBK1 can inhibit the activity of mTORC1, leading to suppressed protein synthesis and enhanced autophagy 32. The phosphorylation level of STING-TBK1-IRF3 has been shown to impact the activation of the mTOR signaling pathway. In line with this hypothesis, quantitative real-time PCR (qRT-PCR) analysis revealed a significant decrease in the mRNA levels of TBK1 and IRF3 in zebrafish infected with ΔMMAR_0267 M. marinum (Fig. 7C), suggesting that the inhibition of apoptosis is occurring, thereby potentially enhancing the survival of the bacteria within infected macrophages. We observed a noteworthy reduction in phosphorylated TBK1 in THP-1 cells during early infection with ΔMMAR_0267 M. marinum (Fig. 7D). This finding indicates that the STING-TBK1-IRF3 axis may directly modulate the mTOR signaling pathway. Activation of the STING-TBK1-IRF3 axis has been linked to the production of type I interferons (IFN-α/β) and promote cell apoptosis 33,34. Our findings demonstrated a notable decrease in the levels of IFN-β in THP-1 cells infected with MMAR_0267-deficient M. marinum, and this reduction was further inhibited by C176 (a potent and covalent STING inhibitor), while IFN-α showed no significant changes (Fig. 7E). This suggested that ΔMMAR_0267 M. marinum primarily enhances its virulence by suppressing the expression of host type I interferon IFN-β through inhibition of the STING-TBK1-IRF3 axis, possibly by modulating the phosphorylation of TBK1. Additionally, we have observed significant activation of the mTOR signaling pathway. Previous studies have shown that the secreted effectors of the Mtb ESAT-6 secretion system-1 (ESX-1) can regulate the mTOR signaling in infected cells. Hence, we aimed to elucidate which effector among the four secreted antigens (CFP-10, ESAT-6, MM1553, and Mh3881c) of Esx-1 substrates is involved in this process 35. We observed a significant reduction in the expression of the CFP-10 antigen in MMAR_0267-deficient M. marinum under normal copper conditions (6.3 µmol/L), while this expression was increased when copper levels were elevated (63 µmol/L) (Fig. 7F). Indicating that the Esx-1 secretion system may be involved in the inhibition of the STING-TBK1-IRF3 axis. To further investigate this, a double knockout strain of MSMEG_4702 and CFP-10 was constructed and employed it for infecting THP-1 macrophages. Following treatment with 63µmol/L CuSO4, only the ΔMSMEG_4702 M. marinum strain exhibited an increase in TBK1 phosphorylation, while the double knockout strain showed no effect on TBK1 phosphorylation (Supplementary Fig. 7). These findings imply that CFP-10 (Rv3874) may serve as the virulence factor responsible for activating the STING-TBK1-IRF3 axis.