Andrés Obregón-Henao

Beijing sublineages of Mycobacterium tuberculosis differ in pathogenicity in the guinea pig.

ABSTRACT The Beijing family of Mycobacterium tuberculosis strains is part of lineage 2 (also known as the East Asian lineage). In clinical studies, we have observed that isolates from the sublineage RD207 of lineage 2 were more readily transmitted among humans. To investigate the basis for this difference, we tested representative strains with the characteristic Beijing spoligotype from four of the five sublineages of lineage 2 in the guinea pig model and subjected these strains to comparative whole-genome sequencing. The results of these studies showed that all of the clinical strains were capable of growing and causing lung pathology in guinea pigs after low-dose aerosol exposure. Differences between the abilities of the four sublineages to grow in the lungs of these animals were not overt, but members of RD207 were significantly more pathogenic, resulting in severe lung damage. The RD207 strains also induced much higher levels of markers associated with regulatory T cells and showed a significant loss of activated T cells in the lungs over the course of the infections. Whole-genome sequencing of the strains revealed mutations specific for RD207 which may explain this difference. Based on these data, we hypothesize that the sublineages of M. tuberculosis are associated with distinct pathological and clinical phenotypes and that these differences influence the transmissibility of particular M. tuberculosis strains in human populations.

Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion

Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell‐based screening of FDA‐approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug‐resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR‐independent pathway controlled by cellular depletion of myo‐inositol. While strain‐specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug‐resistant mycobacterial infection.

Mycobacterium bovis BCG-mediated protection against W-Beijing strains of Mycobacterium tuberculosis is diminished concomitant with the emergence of regulatory T cells.

ABSTRACT Despite issues relating to variable efficacy in the past, the Mycobacterium bovis BCG vaccine remains the basis for new-generation recombinant vaccines currently in clinical trials. To date, vaccines have been tested mostly against laboratory strains and not against the newly emerging clinical strains. In this study, we evaluated the ability of BCG Pasteur to protect mice from aerosol infections with two highly virulent W-Beijing clinical strains, HN878 and SA161. In a conventional 30-day protection assay, BCG was highly protective against both strains, but by day 60 of the assay, this protection was diminished. Histological examination of the lungs of vaccinated animals showed reduced lung consolidation and smaller and more-organized granulomas in the vaccinated mice after 30 days, but in both cases, these tissues demonstrated worsening pathology over time. Effector T cell responses were increased in the vaccinated mice infected with HN878, but these diminished in number after day 30 of the infections concomitant with increased CD4+ Foxp3+ T cells in the lungs, draining lymph nodes, and the spleen. Given the concomitant decrease in effector immunity and continued expansion of regulatory Foxp3+ cells observed here, it is reasonable to hypothesize that downregulation of effector immunity by these cells may be a serious impediment to the efficacy of BCG-based vaccines.

Lipoarabinomannan of Mycobacterium: mannose capping by a multifunctional terminal mannosyltransferase.

Biosynthesis of phosphatidylinositol (PI)-containing lipoarabinomannan (LAM) and lipomannan (LM) of Mycobacterium spp. follows a conserved pathway involving multiple membrane-associated, substrate-specific mannosyltransferases (ManTs) responsible for the sequential addition of α-mannopyranosyl (Manp) units donated by decaprenyl-P-Manp on the periplasmic side of the plasma membrane. Because of their receptor-binding and immunomodulatory properties, the α(1→2)-linked di- and tri-Manp motifs that functionalize the nonreducing arabinan termini of LAM (ManLAM) in Mycobacterium tuberculosis are of crucial importance. We now show that the M. tuberculosis ManT, Rv2181, is required for the addition of these α(1→2)-linked Manp residues but also at other locations of the LAM molecule. Structural analyses of the LM and LAM variants produced by a M. tuberculosis Rv2181 knockout mutant revealed the presence of but a single Manp residue on the nonreducing arabinan termini of LAM and also a complete absence of α(1→2)-linked Man branching on the mannan backbones of LM and LAM. A recombinant strain was constructed in ManLAM-deficient Mycobacterium smegmatis that coexpressed Rv2181 and Rv1635c—the ManT responsible for the addition of the first Manp capping residue of ManLAM. Analysis revealed LAM termini fully capped with di- and tri-Manp motifs in addition to α(1→2)Man branching on the mannan backbones of LM and LAM, confirming the involvement of the α(1→2)ManT Rv2181 in the dual role of Man capping and mannan-core branching, and in the process generated a rapidly growing, ManLAM-containing strain, a tool for the study of the role of ManLAM in the pathogenesis of tuberculosis.

Gr1intCD11b+ Myeloid-Derived Suppressor Cells in Mycobacterium tuberculosis Infection

Background Tuberculosis is one of the world’s leading killers, stealing 1.4 million lives and causing 8.7 million new and relapsed infections in 2011. The only vaccine against tuberculosis is BCG which demonstrates variable efficacy in adults worldwide. Human infection with Mycobacterium tuberculosis results in the influx of inflammatory cells to the lung in an attempt to wall off bacilli by forming a granuloma. Gr1intCD11b+ cells are called myeloid-derived suppressor cells (MDSC) and play a major role in regulation of inflammation in many pathological conditions. Although MDSC have been described primarily in cancer their function in tuberculosis remains unknown. During M. tuberculosis infection it is crucial to understand the function of cells involved in the regulation of inflammation during granuloma formation. Understanding their relative impact on the bacilli and other cellular phenotypes is necessary for future vaccine and drug design. Methodology/Principal Findings We compared the bacterial burden, lung pathology and Gr1intCD11b+ myeloid-derived suppressor cell immune responses in M. tuberculosis infected NOS2-/-, RAG-/-, C3HeB/FeJ and C57/BL6 mice. Gr-1+ cells could be found on the edges of necrotic lung lesions in NOS2-/-, RAG-/-, and C3HeB/FeJ, but were absent in wild-type mice. Both populations of Gr1+CD11b+ cells expressed high levels of arginase-1, and IL-17, additional markers of myeloid derived suppressor cells. We then sorted the Gr1hi and Gr1int populations from M. tuberculosis infected NOS-/- mice and placed the sorted both Gr1int populations at different ratios with naïve or M. tuberculosis infected splenocytes and evaluated their ability to induce activation and proliferation of CD4+T cells. Our results showed that both Gr1hi and Gr1int cells were able to induce activation and proliferation of CD4+ T cells. However this response was reduced as the ratio of CD4+ T to Gr1+ cells increased. Our results illustrate a yet unrecognized interplay between Gr1+ cells and CD4+ T cells in tuberculosis.

Mycobacterium tuberculosis Nucleoside Diphosphate Kinase Inactivates Small GTPases Leading to Evasion of Innate Immunity

Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67phox from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67phox was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67phox interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.

A mouse model of tuberculosis reinfection

Recent clinical observations shows that individuals treated with chemotherapy for tuberculosis who live in endemic areas are four times more likely to develop secondary disease, often as not caused by exogenous reinfection. In a mouse model described here, we show that mice infected with the virulent W-Beijing Mycobacterium tuberculosis strain HN878, then given chemotherapy to clear the infection, were resistant to re-challenge with the same organism thereafter. This resistance, which was mediated by rapid expression of CD4 T cells expressing markers consistent with both central and effector memory immunity, was only transient however. After 20-30 days of the reinfection the numbers of these cells steadily declined, the bacterial load in the lungs surged up, and the lung tissues became increasingly consolidated. No evidence was found for a regulatory T cell response in these mice, but many T cells harvested from the lungs showed evidence of increased PD-1 expression, indicating exhaustion. These data indicate that the memory T cell response to reinfection may not be as stable and long lived as previously thought, a finding with obvious implications for vaccine development.

Therapeutic potential of the Mycobacterium tuberculosis mycolic acid transporter, MmpL3

ABSTRACT In recent years, whole-cell-based screens for novel small molecule inhibitors active against Mycobacterium tuberculosis in culture followed by the whole-genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we have conclusively shown in this study, using conditionally regulated knockdown mutants, that mmpL3 is required for the replication and viability of M. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencing mmpL3 had a rapid bactericidal effect on actively replicating cells in vitro and reduced by 3 to 5 logs in less than 4 weeks the bacterial loads of acutely and chronically infected mouse lungs, respectively. Depletion of MmpL3 further rendered M. tuberculosis hypersusceptible to MmpL3 inhibitors. The exquisite vulnerability of MmpL3 at all stages of the infection establishes this transporter as an attractive new target with the potential to improve and shorten current drug-susceptible and drug-resistant tuberculosis chemotherapies.

Susceptibility of Mycobacterium abscessus to Antimycobacterial Drugs in Preclinical Models

ABSTRACT Over the last 10 years, Mycobacterium abscessus group strains have emerged as important human pathogens, which are associated with significantly higher fatality rates than any other rapidly growing mycobacteria. These opportunistic pathogens are widespread in the environment and can cause a wide range of clinical diseases, including skin, soft tissue, central nervous system, and disseminated infections; by far, the most difficult to treat is the pulmonary form. Infections with M. abscessus are often multidrug-resistant (MDR) and require prolonged treatment with various regimens and, many times, result in high mortality despite maximal therapy. We report here the evaluation of diverse mouse infection models for their ability to produce a progressive high level of infection with M. abscessus. The nude (nu/nu), SCID (severe combined immunodeficiency), gamma interferon knockout (GKO), and granulocyte-macrophage colony-stimulating factor (GMCSF) knockout mice fulfilled the criteria for an optimal model for compound screening. Thus, we set out to assess the antimycobacterial activity of clarithromycin, clofazimine, bedaquiline, and clofazimine-bedaquiline combinations against M. abscessus-infected GKO and SCID murine infection models. Treatment of GKO and SCID mice with a combination of clofazimine and bedaquiline was the most effective in decreasing the M. abscessus organ burden.

Increased Severity of Tuberculosis in Guinea Pigs with Type 2 Diabetes: A Model of Diabetes-Tuberculosis Comorbidity

Impaired glucose tolerance and type 2 diabetes were induced in guinea pigs to model the emerging comorbidity of Mycobacterium tuberculosis infection in diabetic patients. Type 2 diabetes mellitus was induced by low-dose streptozotocin in guinea pigs rendered glucose intolerant by first feeding a high-fat, high-carbohydrate diet before M. tuberculosis exposure. M. tuberculosis infection of diabetic guinea pigs resulted in severe and rapidly progressive tuberculosis (TB) with a shortened survival interval, more severe pulmonary and extrapulmonary pathology, and a higher bacterial burden compared with glucose-intolerant and nondiabetic controls. Compared with nondiabetics, diabetic guinea pigs with TB had an exacerbated proinflammatory response with more severe granulocytic inflammation and higher gene expression for the cytokines/chemokines interferon-γ, IL-17A, IL-8, and IL-10 in the lung and for interferon-γ, tumor necrosis factor-α, IL-8, and monocyte chemoattractant protein-1 in the spleen. TB disease progression in guinea pigs with impaired glucose tolerance was similar to that of nondiabetic controls in the early stages of infection but was more severe by day 90. The guinea pig model of type 2 diabetes-TB comorbidity mimics important features of the naturally occurring disease in humans. This model will be beneficial in understanding the complex pathogenesis of TB in diabetic patients and to test new strategies to improve TB and diabetes control when the two diseases occur together.