Smriti Mehra

Genetic Requirements for the Survival of Tubercle Bacilli in Primates

BACKGROUND Tuberculosis (TB) leads to the death of 1.7 million people annually. The failure of the bacille Calmette-Guérin vaccine, synergy between AIDS and TB, and the emergence of drug resistance have worsened this situation. It is imperative to delineate the mechanisms employed by Mycobacterium tuberculosis to successfully infect and persist in mammalian lungs. METHODS Nonhuman primates (NHPs) are arguably the best animal system to model critical aspects of human TB. We studied genes essential for growth and survival of M. tuberculosis in the lungs of NHPs experimentally exposed to aerosols of an M. tuberculosis transposon mutant library. RESULTS Mutants in 108 M. tuberculosis genes (33.13% of all genes tested) were attenuated for in vivo growth. Comparable studies have reported the attenuation of only approximately 6% of mutants in mice. The M. tuberculosis mutants attenuated for in vivo survival in primates were involved in the transport of various biomolecules, including lipid virulence factors; biosynthesis of cell-wall arabinan and peptidoglycan; DNA repair; sterol metabolism; and mammalian cell entry. CONCLUSIONS Our study highlights the various virulence mechanisms employed by M. tuberculosis to overcome the hostile environment encountered during infection of primates. Prophylactic approaches aimed against bacterial factors that respond to such in vivo stressors have the potential to prevent infection at an early stage, thus likely reducing the extent of transmission of M. tuberculosis.

CXCR5+ T helper cells mediate protective immunity against tuberculosis

One third of the worlds population is infected with Mycobacterium tuberculosis (Mtb). Although most infected people remain asymptomatic, they have a 10% lifetime risk of developing active tuberculosis (TB). Thus, the current challenge is to identify immune parameters that distinguish individuals with latent TB from those with active TB. Using human and experimental models of Mtb infection, we demonstrated that organized ectopic lymphoid structures containing CXCR5+ T cells were present in Mtb-infected lungs. In addition, we found that in experimental Mtb infection models, the presence of CXCR5+ T cells within ectopic lymphoid structures was associated with immune control. Furthermore, in a mouse model of Mtb infection, we showed that activated CD4+CXCR5+ T cells accumulated in Mtb-infected lungs and produced proinflammatory cytokines. Mice deficient in Cxcr5 had increased susceptibility to TB due to defective T cell localization within the lung parenchyma. We demonstrated that CXCR5 expression in T cells mediated correct T cell localization within TB granulomas, promoted efficient macrophage activation, protected against Mtb infection, and facilitated lymphoid follicle formation. These data demonstrate that CD4+CXCR5+ T cells play a protective role in the immune response against TB and highlight their potential use for future TB vaccine design and therapy.

S100A8/A9 Proteins Mediate Neutrophilic Inflammation and Lung Pathology during Tuberculosis

RATIONALE A hallmark of pulmonary tuberculosis (TB) is the formation of granulomas. However, the immune factors that drive the formation of a protective granuloma during latent TB, and the factors that drive the formation of inflammatory granulomas during active TB, are not well defined. OBJECTIVES The objective of this study was to identify the underlying immune mechanisms involved in formation of inflammatory granulomas seen during active TB. METHODS The immune mediators involved in inflammatory granuloma formation during TB were assessed using human samples and experimental models of Mycobacterium tuberculosis infection, using molecular and immunologic techniques. MEASUREMENTS AND MAIN RESULTS We demonstrate that in human patients with active TB and in nonhuman primate models of M. tuberculosis infection, neutrophils producing S100 proteins are dominant within the inflammatory lung granulomas seen during active TB. Using the mouse model of TB, we demonstrate that the exacerbated lung inflammation seen as a result of neutrophilic accumulation is dependent on S100A8/A9 proteins. S100A8/A9 proteins promote neutrophil accumulation by inducing production of proinflammatory chemokines and cytokines, and influencing leukocyte trafficking. Importantly, serum levels of S100A8/A9 proteins along with neutrophil-associated chemokines, such as keratinocyte chemoattractant, can be used as potential surrogate biomarkers to assess lung inflammation and disease severity in human TB. CONCLUSIONS Our results thus show a major pathologic role for S100A8/A9 proteins in mediating neutrophil accumulation and inflammation associated with TB. Thus, targeting specific molecules, such as S100A8/A9 proteins, has the potential to decrease lung tissue damage without impacting protective immunity against TB.

The non‐human primate model of tuberculosis

Non‐human primates (NHPs) are used to model human disease owing to their remarkably similar genomes, physiology, and immune systems. Recently, there has been an increased interest in modeling tuberculosis (TB) in NHPs. Macaques are susceptible to infection with different strains of Mycobacterium tuberculosis (Mtb), producing the full spectrum of disease conditions, including latent infection, chronic progressive infection, and acute TB, depending on the route and dose of infection. Clearly, NHPs are an excellent model of human TB. While the initial aim of the NHP model was to allow preclinical testing of candidate vaccines and drugs, it is now also being used to study pathogenesis and immune correlates of protection. Recent advances in this field are discussed in this review. Key questions such as the effect of hypoxia on the biology of Mtb and the basis of reactivation of latent TB can now be investigated through the use of this model.

A Mycobacterium tuberculosis Sigma Factor Network Responds to Cell-Envelope Damage by the Promising Anti-Mycobacterial Thioridazine

Background Novel therapeutics are urgently needed to control tuberculosis (TB). Thioridazine (THZ) is a candidate for the therapy of multidrug and extensively drug-resistant TB. Methodology/Principal Findings We studied the impact of THZ on Mycobacterium tuberculosis (Mtb) by analyzing gene expression profiles after treatment at the minimal inhibitory (1x MIC) or highly inhibitory (4x MIC) concentrations between 1–6 hours. THZ modulated the expression of genes encoding membrane proteins, efflux pumps, oxido-reductases and enzymes involved in fatty acid metabolism and aerobic respiration. The Rv3160c-Rv3161c operon, a multi-drug transporter and the Rv3614c/3615c/3616c regulon, were highly induced in response to THZ. A significantly high number of Mtb genes co-expressed with σB (the σB regulon) was turned on by THZ treatment. σB has recently been shown to protect Mtb from envelope-damage. We hypothesized that THZ damages the Mtb cell-envelope, turning on the expression of the σB regulon. Consistent with this hypothesis, we present electron-microscopy data which shows that THZ modulates cell-envelope integrity. Moreover, the Mtb mutants in σH and σE, two alternate stress response sigma factors that induce the expression of σB, exhibited higher sensitivity to THZ, indicating that the presence and expression of σB allows Mtb to resist the impact of THZ. Conditional induction of σB levels increased the survival of Mtb in the presence of THZ. Conclusions/Significance THZ targets different pathways and can thus be used as a multi-target inhibitor itself as well as provide strategies for multi-target drug development for combination chemotherapy. Our results show that the Mtb sigma factor network comprising of σH, σE and σB plays a crucial role in protecting the pathogen against cell-envelope damage.

Transcriptional reprogramming in nonhuman primate (rhesus macaque) tuberculosis granulomas.

Background In response to Mtb infection, the host remodels the infection foci into a dense mass of cells known as the granuloma. The key objective of the granuloma is to contain the spread of Mtb into uninfected regions of the lung. However, it appears that Mtb has evolved mechanisms to resist killing in the granuloma. Profiling granuloma transcriptome will identify key immune signaling pathways active during TB infection. Such studies are not possible in human granulomas, due to various confounding factors. Nonhuman Primates (NHPs) infected with Mtb accurately reflect human TB in clinical and pathological contexts. Methodology/Principal Findings We studied transcriptomics of granuloma lesions in the lungs of NHPs exhibiting active TB, during early and late stages of infection. Early TB lesions were characterized by a highly pro-inflammatory environment, expressing high levels of immune signaling pathways involving IFNγ, TNFα, JAK, STAT and C-C/C-X-C chemokines. Late TB lesions, while morphologically similar to the early ones, exhibited an overwhelming silencing of the inflammatory response. Reprogramming of the granuloma transcriptome was highly significant. The expression of ∼ two-thirds of all genes induced in early lesions was later repressed. Conclusions/Significance The transcriptional characteristics of TB granulomas undergo drastic changes during the course of infection. The overwhelming reprogramming of the initial pro-inflammatory surge in late lesions may be a host strategy to limit immunopathology. We propose that these host profiles can predict changes in bacterial replication and physiology, perhaps serving as markers for latency and reactivation.

Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus

Background  Tuberculosis (TB) and AIDS together present a devastating public health challenge. Over 3 million deaths every year are attributed to these twin epidemics. Annually, ∼11 million people are coinfected with HIV and Mycobacterium tuberculosis (Mtb). AIDS is thought to alter the spontaneous rate of latent TB reactivation.

Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis

Tuberculosis (TB) is a global pandaemic, 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 (iBALT) 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 an approximately 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 on the basis of MtbΔsigH.

The Mycobacterium tuberculosis Stress Response Factor SigH Is Required for Bacterial Burden as Well as Immunopathology in Primate Lungs

BACKGROUND Sigma H (sigH) is a major Mycobacterium tuberculosis (Mtb) stress response factor. It is induced in response to heat, oxidative stress, cell wall damage, and hypoxia. Infection of macrophages with the Δ-sigH mutant generates more potent innate immune response than does infection with Mtb. The mutant is attenuated for pathology in mice. METHODS We used a nonhuman primate (NHP) model of acute tuberculosis, to better understand the phenotype of the Δ-sigH mutant in vivo. NHPs were infected with high doses of Mtb or the mutant, and the progression of tuberculosis was analyzed in both groups using clinical, pathological, microbiological, and immunological parameters. RESULTS Animals exposed to Mtb rapidly progressed to acute pulmonary tuberculosis as indicated by worsening clinical correlates, high lung bacterial burden, and granulomatous immunopathology. All the animals rapidly succumbed to tuberculosis. On the other hand, the NHPs exposed to the Mtb:Δ-sigH mutant did not exhibit acute tuberculosis, instead showing significantly blunted disease. These NHPs survived the entire duration of the study. CONCLUSIONS The Mtb:Δ-sigH mutant is completely attenuated for bacterial burden as well as immunopathology in NHPs. SigH and its regulon are required for complete virulence in primates. Further studies are needed to identify the molecular mechanism of this attenuation.

The DosR Regulon Modulates Adaptive Immunity and Is Essential for Mycobacterium tuberculosis Persistence

RATIONALE Hypoxia promotes dormancy by causing physiologic changes to actively replicating Mycobacterium tuberculosis. DosR controls the response of M. tuberculosis to hypoxia. OBJECTIVES To understand DosRs contribution in the persistence of M. tuberculosis, we compared the phenotype of various DosR regulon mutants and a complemented strain to M. tuberculosis in macaques, which faithfully model M. tuberculosis infection. METHODS We measured clinical and microbiologic correlates of infection with M. tuberculosis relative to mutant/complemented strains in the DosR regulon, studied lung pathology and hypoxia, and compared immune responses in lung using transcriptomics and flow cytometry. MEASUREMENTS AND MAIN RESULTS Despite being able to replicate initially, mutants in DosR regulon failed to persist or cause disease. On the contrary, M. tuberculosis and a complemented strain were able to establish infection and tuberculosis. The attenuation of pathogenesis in animals infected with the mutants coincided with the appearance of a Th1 response and organization of hypoxic lesions wherein M. tuberculosis expressed dosR. The lungs of animals infected with the mutants (but not the complemented strain) exhibited early transcriptional signatures of T-cell recruitment, activation, and proliferation associated with an increase of T cells expressing homing and proliferation markers. CONCLUSIONS 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.