Mycobacterium tuberculosis Infection of Dendritic Cells Leads to Partially Caspase-1/11-Independent IL-1β and IL-18 Secretion but Not to Pyroptosis

Background

Interleukin-1β (IL-1β) is important for host resistance against Mycobacterium tuberculosis (Mtb) infections. The response of the dendritic cell inflammasome during Mtb infections has not been investigated in detail.

Methodology/Principal Findings

Here we show that Mtb infection of bone marrow-derived dendritic cells (BMDCs) induces IL-1β secretion and that this induction is dependent upon the presence of functional ASC and NLRP3 but not NLRC4 or NOD2. The analysis of cell death induction in BMDCs derived from these knock-out mice revealed the important induction of host cell apoptosis but not necrosis, pyroptosis or pyronecrosis. Furthermore, NLRP3 inflammasome activation and apoptosis induction were both reduced in BMDCs infected with the esxA deletion mutant of Mtb demonstrating the importance of a functional ESX-1 secretion system. Surprisingly, caspase-1/11-deficient BMDCs still secreted residual levels of IL-1βand IL-18 upon Mtb infection which was abolished in cells infected with the esxA Mtb mutant.

Conclusion

Altogether we demonstrate the partially caspase-1/11-independent, but NLRP3- and ASC- dependent IL-1β secretion in Mtb-infected BMDCs. These findings point towards a potential role of DCs in the host innate immune response to mycobacterial infections via their capacity to induce IL-1β and IL-18 secretion.     

Mycobacterium tuberculosis Infection Interferes with HIV Vaccination in Mice

Tuberculosis (TB) has emerged as the most prominent bacterial disease found in human immunodeficiency virus (HIV)-positive individuals worldwide. Due to high prevalence of asymptomatic Mycobacterium tuberculosis (Mtb) infections, the future HIV vaccine in areas highly endemic for TB will often be administrated to individuals with an ongoing Mtb infection. The impact of concurrent Mtb infection on the immunogenicity of a HIV vaccine candidate, MultiHIV DNA/protein, was investigated in mice. We found that, depending on the vaccination route, mice infected with Mtb before the administration of the HIV vaccine showed impairment in both the magnitude and the quality of antibody and T cell responses to the vaccine components p24Gag and gp160Env. Mice infected with Mtb prior to intranasal HIV vaccination exhibited reduced p24Gag-specific serum IgG and IgA, and suppressed gp160Env-specific serum IgG as compared to respective titers in uninfected HIV-vaccinated controls. Importantly, in Mtb-infected mice that were HIV-vaccinated by the intramuscular route the virus neutralizing activity in serum was significantly decreased, relative to uninfected counterparts. In addition mice concurrently infected with Mtb had fewer p24Gag-specific IFN-γ-expressing T cells and multifunctional T cells in their spleens. These results suggest that Mtb infection might interfere with the outcome of prospective HIV vaccination in humans.     

Aggravated infection in mice co-administered with Mycobacterium tuberculosis and the 27-kDa lipoprotein

We have previously reported that mice immunized with the mycobacterial 27-kDa lipoprotein were more susceptible to Mycobacterium tuberculosis (Mtb) challenge. We also showed that 27-kDa lipoprotein abrogated the protection afforded by the BCG vaccine when administrated together, suggesting that the 27-kDa lipoprotein may modulate the course of experimental mycobacterial infection. In this study, we address the role of the 27-kDa lipoprotein in modulating the immune response to mycobacteria. Our results show that co-administration of BALB/c mice with Mtb and the 27-kDa lipoprotein (Mtb+27kDa), but not its non-acylated form, increases the susceptibility of mice to Mtb infection. Significantly lower DTH reaction and splenocyte proliferation to PPD stimulation were also observed in Mtb+27kDa-infected mice compared to Mtb-infected mice. Furthermore, during infection, splenocytes and purified T cells lost their ability to proliferate in response to concanavalin A stimulation more rapidly in the Mtb+27kDa-infected mice, which was accompanied by high IFN-gamma and NO production, but low TNF-alpha secretion levels. Addition of L-NMMA, anti-IFN-gamma and anti-TNF-alpha antibodies restored in vitro proliferative responses of T cells from Mtb+27kDa-infected mice. Short-term L-NMMA treatment of Mtb+27kDa-infected mice prevented the 27-kDa-mediated immunosuppression and increase in susceptibility to Mtb. Altogether, these data suggest that the 27-kDa lipoprotein plays a role in Mtb infection by inducing increased suppression of the immune response due to elevated NO production.     

IFN-alpha beta released by Mycobacterium tuberculosis-infected human dendritic cells induces the expression of CXCL10: selective recruitment of NK and activated T cells

We recently reported that dendritic cells (DC) infected with Mycobacterium tuberculosis (Mtb) produce Th1/IFN-gamma-inducing cytokines, IFN-alpha beta and IL-12. In the present article, we show that maturing Mtb-infected DC express high levels of CCR7 and they become responsive to its ligand CCL21. Conversely, CCR5 expression was rapidly lost from the cell surface following Mtb infection. High levels of CCL3 and CCL4 were produced within 8 h after infection, which is likely to account for the observed CCR5 down-modulation on Mtb-infected DC. In addition, Mtb infection stimulated the secretion of CXCL9 and CXCL10. Interestingly, the synthesis of CXCL10 was mainly dependent on the Mtb-induced production of IFN-alpha beta. Indeed, IFN-alpha beta neutralization down-regulated CXCL10 expression, whereas the expression of CXCL9 appeared to be unaffected. The chemotactic activity of the Mtb-infected DC supernatants was evaluated by migration assays using activated NK, CD4(+), and CD8(+) cells that expressed both CCR5 and CXCR3. Mtb-induced expression of CCL3, CCL4, CXCL9, and CXCL10 was involved in the stimulation of NK and T cell migration. In accordance with the data on the IFN-alpha beta-induced expression of CXCL10, neutralization of IFN-alpha beta significantly reduced the chemotactic activity of the supernatant from Mtb-infected DC. This indicates that IFN-alpha beta may modulate the immune response through the expression of CXCL10, which along with CXCL9, CCL3, and CCL4 participates in the recruitment and selective homing of activated/effector cells, which are known to accumulate at the site of Mtb infection and take part in the formation of the granulomas.     

Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-alpha, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-gamma-inducing cytokines, IL-12 and IFN-alpha, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-gamma production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-alpha, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-alpha and IL-18 play a significant role in enhancing IFN-gamma synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.     

Control of experimental pulmonary tuberculosis depends more on immunostimulatory leukotrienes than on the absence of immunosuppressive prostaglandins

Prostaglandins (PGs) and leukotrienes (LTs) are produced in Mycobacterium tuberculosis (Mtb)-infected lungs and have immune suppressive and protective effects, respectively. Considering that both of these mediators are produced during mycobacterial infection, we investigated the specific and relative biological importance of each in regulating host response in experimental tuberculosis. Administration of celecoxib, which was found to reduce lung levels of PGE₂ and increase LTB₄, enhanced the 60-day survival of Mtb-infected mice in 14%. However administration of MK-886, which reduced levels of LTB₄ but did not enhance PGE₂, reduced 60-day survival from 86% to 43% in Mtb-infected mice, and increased lung bacterial burden. MK-886 plus celecoxib reduced survival to a lesser extent than MK-886 alone. MK-886- and MK-886 plus celecoxib-treated animals exhibited reduced levels of the protective interleukin-12 and gamma-interferon. Our findings indicate that in this model, the protective effect of LTs dominates over the suppressive effect of PGs.     

Mycobacterium tuberculosis inhibits the NLRP3 inflammasome activation via its phosphokinase PknF

Mycobacterium tuberculosis (Mtb) has evolved to evade host innate immunity by interfering with macrophage functions. Interleukin-1β (IL-1β) is secreted by macrophages after the activation of the inflammasome complex and is crucial for host defense against Mtb infections. We have previously shown that Mtb is able to inhibit activation of the AIM2 inflammasome and subsequent pyroptosis. Here we show that Mtb is also able to inhibit host cell NLRP3 inflammasome activation and pyroptosis. We identified the serine/threonine kinase PknF as one protein of Mtb involved in the NLRP3 inflammasome inhibition, since the pknF deletion mutant of Mtb induces increased production of IL-1β in bone marrow-derived macrophages (BMDMs). The increased production of IL-1β was dependent on NLRP3, the adaptor protein ASC and the protease caspase-1, as revealed by studies performed in gene-deficient BMDMs. Additionally, infection of BMDMs with the pknF deletion mutant resulted in increased pyroptosis, while the IL-6 production remained unchanged compared to Mtb-infected cells, suggesting that the mutant did not affect the priming step of inflammasome activation. In contrast, the activation step was affected since potassium efflux, chloride efflux and the generation of reactive oxygen species played a significant role in inflammasome activation and subsequent pyroptosis mediated by the Mtb pknF mutant strain. In conclusion, we reveal here that the serine/threonine kinase PknF of Mtb plays an important role in innate immune evasion through inhibition of the NLRP3 inflammasome.     

Human Lung Epithelial Cells Contain Mycobacterium tuberculosis in a Late Endosomal Vacuole and Are Efficiently Recognized by CD8+ T Cells

Mycobacterium tuberculosis (Mtb) is transmitted via inhalation of aerosolized particles. While alveolar macrophages are thought to play a central role in the acquisition and control of this infection, Mtb also has ample opportunity to interact with the airway epithelium. In this regard, we have recently shown that the upper airways are enriched with a population of non-classical, MR1-restricted, Mtb-reactive CD8⁺ T cells (MAIT cells). Additionally, we have demonstrated that Mtb-infected epithelial cells lining the upper airways are capable of stimulating IFNγ production by MAIT cells. In this study, we demonstrate that airway epithelial cells efficiently stimulate IFNγ release by MAIT cells as well as HLA-B45 and HLA-E restricted T cell clones. Characterization of the intracellular localization of Mtb in epithelial cells indicates that the vacuole occupied by Mtb in epithelial cells is distinct from DC in that it acquires Rab7 molecules and does not retain markers of early endosomes such as Rab5. The Mtb vacuole is also heterogeneous as there is a varying degree of association with Lamp1 and HLA-I. Although the Mtb vacuole shares markers associated with the late endosome, it does not acidify, and the bacteria are able to replicate within the cell. This work demonstrates that Mtb infected lung epithelial cells are surprisingly efficient at stimulating IFNγ release by CD8⁺ T cells.     

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.     

Pre-Exposure of Mycobacterium tuberculosis-Infected Macrophages to Crystalline Silica Impairs Control of Bacterial Growth by Deregulating the Balance between Apoptosis and Necrosis

Inhalation of crystalline silica (CS) particles increases the risk of pulmonary tuberculosis; however, the precise mechanism through which CS exposure facilitates Mycobacterium tuberculosis (Mtb) infection is unclear. We speculate that macrophage exposure to CS deregulates the cell death pathways that could explain, at least in part, the association observed between exposure to CS and pulmonary tuberculosis. We therefore established an in vitro model in which macrophages were exposed to CS and then infected with Mtb. Expression of surface markers was analyzed by flow cytometry, JNK1/2, ASK1, caspase 9, P-p38, Bcl-2 and Mcl-1 were analyzed by Western blot, and cytokines by ELISA. Our results show that exposure to CS limits macrophage ability to control Mtb growth. Moreover, this exposure reduced the expression of TLR2, Bcl-2 and Mcl-1, but increased that of JNK1 and ASK1 molecules in the macrophages. Finally, when the pre-exposed macrophages were infected with Mtb, the concentrations of TNFα, IL-1β and caspase-9 expression increased. This pro-inflammatory profile of the macrophage unbalanced the apoptosis/necrosis pathway. Taken together, these data suggest that macrophages exposed to CS are sensitized to cell death by MAPK kinase-dependent signaling pathway. Secretion of TNF-α and IL-1β by Mtb-infected macrophages promotes necrosis, and this deregulation of cell death pathways may favor the release of viable bacilli, thus leading to the progression of tuberculosis.     

In vivo depletion of CD11c+ cells delays the CD4+ T cell response to Mycobacterium tuberculosis and exacerbates the outcome of infection

Although dendritic cells (DC) are potent APC that prime T cells against many pathogens, there is no direct evidence that DC are required for immunity to Mycobacterium tuberculosis (Mtb) infection. The requirement for DC to prime the CD4+ T cell response following Mtb infection was investigated using pCD11c-diptheria toxin receptor/GFP transgenic mice, in which DC can be transiently ablated in vivo. We show a critical role for DC in initiation of the CD4+ T cell response to the mycobacterial Ag early secretory Ag of tuberculosis 6. The delay in initiating the Ag-specific T cell response led to impaired control of Mtb replication. Interestingly, DC were not required for the secondary CD4+ T cell response following Mtb infection in peptide-vaccinated mice. Thus, this study shows that DC are essential for the initiation of the adaptive T cell response to the human pathogen Mtb.     

Secreted proteins from Mycobacterium tuberculosis gain access to the cytosolic MHC class-I antigen-processing pathway

CD8+ T cells play an important role in the host response to infection with Mycobacterium tuberculosis (Mtb). Mtb resides in an arrested phagosome that is phenotypically similar to an early endosome. The mechanisms by which Mtb-derived Ags gain access to the HLA-I-processing pathway are incompletely characterized. Studies with CD8+ T cell lines have suggested that Mtb Ags gain access to the HLA-I pathway in an alternate vacuolar pathway that is both brefeldin A (BFA) and TAP independent. To define the requirements of entry of Ag into the HLA-I pathway, we have used human CD8+ T cell clones specific for the secreted Mtb Ag CFP10. Human monocyte-derived dendritic cells were pulsed with CFP10 expressed in a recombinant adenovirus, surface adsorbed to microspheres, or in its native form by Mtb. When delivered by adenovirus, processing and presentation of CFP10 were blocked by both BFA and the proteasomal blocker lactacystin. In contrast, processing of CFP10 adsorbed to the surface of microspheres was not affected by either of these Ag-processing inhibitors. BFA, lactacystin, and TAP inhibition blocked the recognition of Mtb-infected dendritic cells, suggesting that processing was via a cytosolic pathway for this secreted protein Ag. We conclude that secreted proteins from Mtb can be processed in a BFA- and proteasome-dependent manner, consistent with egress of Ag into the cytosol and subsequent loading of proteasomally derived peptides.     

Classically restricted human CD8+ T lymphocytes derived from Mycobacterium tuberculosis-infected cells: definition of antigenic specificity

Previous studies in murine and human models have suggested an important role for HLA Ia-restricted CD8(+) T cells in host defense to Mycobacterium tuberculosis (Mtb). Therefore, understanding the Ags presented via HLA-Ia will be important in understanding the host response to Mtb and in rational vaccine design. We have used monocyte-derived dendritic cells in a limiting dilution analysis to generate Mtb-specific CD8(+) T cells. Two HLA-Ia-restricted CD8(+) T cell clones derived by this method were selected for detailed analysis. One was HLA-B44 restricted, and the other was HLA-B14 restricted. Both were found to react with Mtb-infected, but not bacillus Calmette-Guérin-infected, targets. For both these clones, the Ag was identified as culture filtrate protein 10 (CFP10)/Mtb11, a 10.8-kDa protein not expressed by bacillus Calmette-Guérin. Both clones were inhibited by the anti-class I Ab and anti-HLA-B,C Abs. Using a panel of CFP10/Mtb11-derived 15-aa peptides overlapping by 11 aa, the region containing the epitopes for both clones has been defined. Minimal 10-aa epitopes were defined for both clones. CD8(+) effector cells specific for these two epitopes are present at high frequency in the circulating pool. Moreover, the CD8(+) T cell response to CFP10/Mtb11 can be largely accounted for by the two epitopes defined herein, suggesting that this is the immunodominant response for this purified protein derivative-positive donor. This study represents the first time CD8(+) T cells generated against Mtb-infected APC have been used to elucidate an Mtb-specific CD8(+) T cell Ag.     

Host Defense and Recruitment of Foxp3+ T Regulatory Cells to the Lungs in Chronic Mycobacterium tuberculosis Infection Requires Toll-like Receptor 2

Acute resistance to low dose M. tuberculosis (Mtb) infection is not dependent on Toll-like receptor (TLR) 2. However, whether TLR2 contributes to resistance in chronic Mtb infection has remained uncertain. Here we report that, following low dose aerosol infection with Mtb, mice lacking TLR2 (TLR2KO), in comparison with wild type (WT) mice, exhibit enhanced cellular infiltration and inflammation in the lungs, and fail to stably control bacterial burden during chronic infection. IFNγ and IL-17 was expressed at equivalent levels in the two groups; however, the characteristic accumulation of Foxp3⁺ T regulatory cells (Tregs) in pulmonary granulomas was significantly reduced in TLR2KO mice. Nonetheless, this reduction in Tregs was independent of whether Tregs expressed TLR2 or not. To directly link the reduced number of Tregs to the increased inflammation present in the TLR2KO mice, we used a macrophage adoptive transfer model. At seven weeks post-Mtb infection, TLR2KO mice, which were adoptively transferred with WT macrophages, displayed enhanced accumulation of Tregs in the lungs and a concomitant reduction in inflammation in contrast with control mice that received TLR2KO macrophages. However, the pulmonary bacterial burden between the two groups remained similar indicating that TLR2''s role in modulating immunopathology is functionally distinct from its role in restricting Mtb growth in chronic infection. Together, these findings unequivocally demonstrate that TLR2 contributes to host resistance against chronic Mtb infection and reveal a novel role for TLR2 in mediating the recruitment of Foxp3⁺ Tregs to the lungs to control inflammation.     

Human Mucosal Associated Invariant T Cells Detect Bacterially Infected Cells

Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8⁺ T cells play a unique role. High frequency Mtb-reactive CD8⁺ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8⁺ T cells are universally present, but predominate in Mtb-uninfected individuals. Interestingly, these Mtb-reactive cells expressed the Vα7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an “innate” T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection.     

Membrane TNF confers protection to acute mycobacterial infection

Background

Tumour necrosis factor (TNF) is crucial for the control of mycobacterial infection as TNF deficient (KO) die rapidly of uncontrolled infection with necrotic pneumonia. Here we investigated the role of membrane TNF for host resistance in knock-in mice with a non-cleavable and regulated allele (mem-TNF).

Methods

C57BL/6, TNF KO and mem-TNF mice were infected with M. tuberculosis H37Rv (Mtb at 100 CFU by intranasal administration) and the survival, bacterial load, lung pathology and immunological parameters were investigated. Bone marrow and lymphocytes transfers were used to test the role of membrane TNF to confer resistance to TNF KO mice.

Results

While TNF-KO mice succumbed to infection within 4–5 weeks, mem-TNF mice recruited normally T cells and macrophages, developed mature granuloma in the lung and controlled acute Mtb infection. However, during the chronic phase of infection mem-TNF mice succumbed to disseminated infection with necrotic pneumonia at about 150 days. Reconstitution of irradiated TNF-KO mice with mem-TNF derived bone marrow cells, but not with lymphocytes, conferred host resistance to Mtb infection in TNF-KO mice.

Conclusion

Membrane expressed TNF is sufficient to allow cell-cell signalling and control of acute Mtb infection. Bone marrow cells, but not lymphocytes from mem-TNF mice confer resistance to infection in TNF-KO mice. Long-term infection control with chronic inflammation likely disrupting TNF mediated cell-cell signalling, additionally requires soluble TNF.     

Heme oxygenase-1-derived carbon monoxide induces the Mycobacterium tuberculosis dormancy regulon

The mechanisms that allow Mycobacterium tuberculosis (Mtb) to persist in human tissue for decades and to then abruptly cause disease are not clearly understood. Regulatory elements thought to assist Mtb to enter such a state include the heme two-component sensor kinases DosS and DosT and the cognate response regulator DosR. We have demonstrated previously that O(2), nitric oxide (NO), and carbon monoxide (CO) are regulatory ligands of DosS and DosT. Here, we show that in addition to O(2) and NO, CO induces the complete Mtb dormancy (Dos) regulon. Notably, we demonstrate that CO is primarily sensed through DosS to induce the Dos regulon, whereas DosT plays a less prominent role. We also show that Mtb infection of macrophage cells significantly increases the expression, protein levels, and enzymatic activity of heme oxygenase-1 (HO-1, the enzyme that produces CO), in an NO-independent manner. Furthermore, exploiting HO-1(+/+) and HO-1(-/-) bone marrow-derived macrophages, we demonstrate that physiologically relevant levels of CO induce the Dos regulon. Finally, we demonstrate that increased HO-1 mRNA and protein levels are produced in the lungs of Mtb-infected mice. Our data suggest that during infection, O(2), NO, and CO are being sensed concurrently rather than independently via DosS and DosT. We conclude that CO, a previously unrecognized host factor, is a physiologically relevant Mtb signal capable of inducing the Dos regulon, which introduces a new paradigm for understanding the molecular basis of Mtb persistence.