Tuberculosis (TB) is a global pandemic, partially due to the failure of vaccination approaches. Novel anti-TB vaccines are therefore urgently required. Here we show that aerosol immunization of macaques with the Mtb mutant in SigH (MtbΔsigH) results in significant recruitment of inducible bronchus-associated lymphoid tissue as well as CD4+ and CD8+ T cells expressing activation and proliferation markers to the lungs. Further, the findings indicate that pulmonary vaccination with MtbΔsigH elicited strong central memory CD4+ and CD8+ T cell responses in the lung. Vaccination with MtbΔsigH results in significant protection against a lethal TB challenge, as evidenced by a ~three log reduction in bacterial burdens, significantly diminished clinical manifestations and granulomatous pathology and characterized by the presence of profound iBALT. This highly protective response is virtually absent in unvaccinated and BCG-vaccinated animals after challenge. These results suggest that future TB vaccine candidates can be developed based on MtbΔsigH.

Delayed adaptive responses, a hallmark of M. tuberculosis infection, not only lead to persistence but also interfere with the development of effective antituberculosis vaccines. The DosR regulon therefore modulates both the magnitude and the timing of adaptive immune responses in response to hypoxia in vivo, resulting in persistent infection. Hence, DosR regulates key aspects of the M. tuberculosis life cycle and limits lung pathology.

SignificanceMycobacterium tuberculosis induces the expression of the indoleamine 2,3-dioxygenase (IDO) enzyme, which catabolizes tryptophan. Tryptophan metabolites potently suppress host immunity. The present study demonstrates that blockade of IDO activity reduces both clinical manifestations of tuberculosis (TB) as well as microbial and pathological correlates of the human TB syndrome in macaques. In granulomas, T cells localize in the periphery, and are unable to access the core, where bacilli persist. Inhibiting IDO activity altered granuloma organization such that more T cells translocated to the lesion core and exhibited highly proliferative signatures. Our results identify a highly efficient immunosuppressive mechanism at play in the granuloma environment that aids in M. tuberculosis persistence. The ability to modulate this pathway with safe and approved compounds could, however, facilitate chemotherapy-adjunctive host-directed therapy approaches for the control of TB.

Mycobacterium tuberculosis (Mtb) must counter hypoxia within granulomas to persist. DosR, in concert with sensor kinases DosS and DosT, regulates the response to hypoxia. Yet Mtb lacking functional DosR colonize the lungs of C57Bl/6 mice, presumably owing to the lack of organized lesions with sufficient hypoxia in that model. We compared the phenotype of the D-dosR, D-dosS, and D-dosT mutants to Mtb using C3HeB/FeJ mice, an alternate mouse model where lesions develop hypoxia. C3HeB/FeJ mice were infected via aerosol. The progression of infection was analyzed by tissue bacterial burden and histopathology. A measure of the comparative global immune responses was also analyzed. Although D-dosR and D-dosT grew comparably to wild-type Mtb, D-dosS exhibited a significant defect in bacterial burden and pathology in vivo, accompanied by ablated proinflammatory response. D-dosS retained the ability to induce DosR. The D-dosS mutant was also attenuated in murine macrophages ex vivo, with evidence of reduced expression of the proinflammatory signature. Our results show that DosS, but not DosR and DosT, is required by Mtb to survive in C3HeB/FeJ mice.The attenuation of D-dosS is not due to its inability to induce the DosR regulon, nor is it a result of the accumulation of hypoxia. That the in vivo growth restriction of D-dosS could be mimicked ex vivo suggested sensitivity to macrophage oxidative burst. Anoxic caseous centers within tuberculosis lesions eventually progress to cavities. Our results provide greater insight into the molecular mechanisms of Mtb persistence within host lungs.Keywords: Mycobacterium tuberculosis; hypoxia; response regulator; sensor kinase; latency Clinical RelevanceOur research indicates that the C3HeB/FeJ mouse model is appropriate for the study of Mycobacterium tuberculosis pathogenesis because it reveals attenuation in the D-dosS mutation. It appears that this attenuation is not the result of hypoxia in lung granulomas.

The DevR/DosR regulator is believed to play a key role in dormancy adaptation mechanisms of Mycobacterium tuberculosis in response to a multitude of gaseous stresses, including hypoxia, which prevails within granulomas. DevR activates transcription by binding to target promoters containing a minimum of two binding sites. The proximal site overlaps with the SigA ؊35 element, suggesting that DevR-SigA interaction is required for activating transcription. We evaluated the roles of 14 charged residues of DevR in transcriptional activation under hypoxic stress. Seven of the 14 alanine substitution mutants were defective in regulon activation, of which K191A, R197A, and K179A؉K168A (designated K179A*) mutants were significantly or completely compromised in DNA binding. Four mutants, namely, E154A, R155A, E178A, and K208A, were activation defective in spite of binding to DNA and were classified as positive-control (pc) mutants. The SigA interaction defect of the E154A and E178A proteins was established by in vitro and in vivo assays and implies that these substitutions lead to an activation defect because they disrupt an interaction(s) with SigA. The relevance of DevR interaction to the transcriptional machinery was further established by the hypoxia survival phenotype displayed by SigA interaction-defective mutants. Our findings demonstrate the role of DevRSigA interaction in the activation mechanism and in bacterial survival under hypoxia and establish the housekeeping sigma factor SigA as a molecular target of DevR. The interaction of DevR and RNA polymerase suggests a new and novel interceptable molecular interface for future antidormancy strategies for Mycobacterium tuberculosis.

Although it is accepted that the environment within the granuloma profoundly affects Mycobacterium tuberculosis (Mtb) and infection outcome, our ability to understand Mtb gene expression in these niches has been limited. We determined intragranulomatous gene expression in human-like lung lesions derived from nonhuman primates with both active tuberculosis (ATB) and latent TB infection (LTBI). We employed a non-laser-based approach to microdissect individual lung lesions and interrogate the global transcriptome of Mtb within granulomas. Mtb genes expressed in classical granulomas with central, caseous necrosis, as well as within the caseum itself, were identified and compared with other Mtb lesions in animals with ATB (n = 7) or LTBI (n = 7). Results were validated using both an oligonucleotide approach and RT-PCR on macaque samples and by using human TB samples. We detected approximately 2,900 and 1,850 statistically significant genes in ATB and LTBI lesions, respectively (linear models for microarray analysis, Bonferroni corrected, P < 0.05). Of these genes, the expression of approximately 1,300 (ATB) and 900 (LTBI) was positively induced. We identified the induction of key regulons and compared our results to genes previously determined to be required for Mtb growth. Our results indicate pathways that Mtb uses to ensure its survival in a highly stressful environment in vivo. A large number of genes is commonly expressed in granulomas with ATB and LTBI. In addition, the enhanced expression of the dormancy survival regulon was a key feature of lesions in animals with LTBI, stressing its importance in the persistence of Mtb during the chronic phase of infection.

Mycobacterium tuberculosis (Mtb) persists within lung granulomas, despite being subjected to diverse stress conditions, including hypoxia. We hypothesized that the response of host phagocytes to Mtb experiencing hypoxia is radically altered and designed in vitro experiment to study this phenomenon. Hypoxia-stressed (Mtb-H) and aerobically grown Mtb (Mtb-A) were used to infect Rhesus Macaque Bone Marrow Derived Macrophages (Rh-BMDMs) and the comparative host response to Mtb infection studied. Mechanistic insights were gained by employing RNAi. Mtb-H accumulated significantly lower bacterial burden during growth in Rh-BMDMs, concomitantly generating a drastically different host transcriptional profile (with only <2% of all genes perturbed by either infection being shared between the two groups). A key component of this signature was significantly higher TNF and apopotosis in Mtb-H- compared to Mtb-A-infected Rh-BMDMs. Silencing of TNF by RNAi reversed the significant control of Mtb replication. These results indicate a potential mechanism for the rapid clearance of hypoxia-conditioned bacilli by phagocytes. In conclusion, hypoxia-conditioned Mtb undergo significantly different interactions with host macrophages compared to Mtb grown in normoxia. These interactions result in the induction of the TNF signaling pathway, activation of apoptosis, and DNA-damage stress response. Our results show that Mtb-H bacilli are particularly susceptible to killing governed by TNF.

BackgroundDevR (also called as DosR) is a two-domain response regulator of the NarL subfamily that controls dormancy adaptation of Mycobacterium tuberculosis (M. tb). In response to inducing signals such as hypoxia and ascorbic acid, the N-terminal receiver domain of DevR (DevRN) is phosphorylated at Asp54. This results in DevR binding to DNA via its C-terminal domain (DevRC) and subsequent induction of the DevR regulon. The mechanism of phosphorylation-mediated activation is not known. The present study was designed to understand the role of the N- and C-terminal domains of DevR in DevR regulon genes activation.Methodology/Principal FindingsTowards deciphering the activation mechanism of DevR, we compared the DNA binding properties of DevRC and DevR and correlated the findings with their ability to activate gene expression. We show that isolated DevRC can interact with DNA, but only with the high affinity site of a representative target promoter. Therefore, one role of DevRN is to mask the intrinsic DNA binding function of DevRC. However, unlike phosphorylated DevR, isolated DevRC does not interact with the adjacent low affinity binding site suggesting that a second role of DevRN is in cooperative binding to the secondary site. Transcriptional analysis shows that consistent with unmasking of its DNA binding property, DevRC supports the aerobic induction, albeit feebly, of DevR regulon genes but is unable to sustain gene activation during hypoxia.Conclusions/SignificanceDevR is a unique response regulator that employs a dual activation mechanism including relief of inhibition and cooperative interaction with binding sites. Importantly, both these functions reside outside the C-terminal domain. DevRN is also essential for stabilizing DevR and sustaining autoregulation under hypoxia. Hence, both domains of DevR are required for robust transcription activation.