Mtb Growth Research Articles

Ethnicity based comprehensive evaluation of polymorphism in interferon-gamma gene and its association with pulmonary and extra-pulmonary tuberculosis risk: An updated trial sequential meta-analysis.

Host genetic background plays an important role in susceptibility to intracellular infectious pathogens like Mycobacterium tuberculosis (Mtb). Cellular immune response activation is vital for protection to these pathogens. Interferon-gamma (IFN-γ) plays a crucial role in this activation and preventing the intracellular growth of Mtb. A mutation in the IFN-γ gene, therefore, may lead to increased susceptibility to tuberculosis (TB) that may vary in different ethnic groups and its consequence also varies in pulmonary and extra-pulmonary TB (EPTB). Several IFN-γ gene polymorphisms are investigated for susceptibility to TB, but their associations are not always consistent as its impact may vary from one ethnicity to the other as well as with the type of TB. Hence, we performed a meta-analysis to overcome this problem. The present study involves comprehensive meta-analysis of + 874T/A polymorphism in the IFN-γ gene based on type of TB within five different ethnic groups to show its association with increased susceptibility to TB. Using PubMed and Google Scholar databases, a total of 50 case-control studies were retrieved having 8152 cases and 9755 controls in this meta-analysis. Thirty-eight studies of + 874T/A polymorphism of IFN-γ gene were correlated for Pooled odds ratios with 95% confidence intervals. The polymorphism was analyzed for six genetic models for five major ethnic groups accounting for heterogeneity among studies. Moreover, the sub-group analysis was based on the type of TB within each ethnic group. Trial sequential analysis was also performed for all the sub-groups to estimate the statistical consistency. IFN-γ +874 T/A polymorphism analysis clearly confirmed the increased association of + 874AA genotype with increased TB risk. This polymorphism also showed significant association in East Asian, European, American, and African ethnic groups whereas no such association was found in Asians. Patients with pulmonary TB (PTB) confirmed the association in East Asians, Africans, and Americans, whereas patients with EPTB showed association in Asian and East Asian populations only. This study reaffirms the association of IFN-γ+874 T/A polymorphism with TB risk. It specifically confirms that IFN-γ+874 T/A polymorphism increases the susceptibility of pulmonary infection in Africans and Americans, while the East Asian population is more susceptible to both, pulmonary and EPTB.

Mycobacterium tuberculosis Rv2145c Promotes Intracellular Survival by STAT3 and IL-10 Receptor Signaling

Although Mycobacterium tuberculosis (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in Mycobacterium smegmatis (M. smegmatis) led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing M. smegmatis in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing M. smegmatis died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.

MCherry-expressing Mycobacterium tuberculosis gives insight into host cell tropism and immune pressure at the single bacterium level in macaques with tuberculosis

Abstract Tuberculosis is caused by Mycobacterium tuberculosis (Mtb) and is associated with granuloma formation in the lungs and other tissues. These lesions can limit bacterial replication but also contain host cells for Mtb and support bacterial persistence and dissemination in poorly controlled disease. Macrophages are often viewed as primary host cells for Mtb but work in mice demonstrates that other cell types host Mtb in acute infection. Less is known about host cell tropism in human TB, especially in the early stages of infection but these early interactions may have implications for later stages of disease. To identify which cells are infected in early and established TB, we infected two cynomolgus macaques with an mCherry-expressing Mtb strain and used flow cytometry and microscopy to identify and phenotype the infected cells at four- and ten-weeks post infection. This approach also allowed us to compare bacterial mCherry expression and size to determine if granulomas exert different levels of pressure on Mtb that vary by timepoint or lesion type. We found that granulomas contained numerous Mtb-infected neutrophils and macrophages at four weeks post infection but fewer infected neutrophils at ten weeks post infection. When the fluorescence and size of individual bacteria were compared across granulomas and timepoints, we found that bacteria in high Mtb burden granulomas were more fluorescent than bacteria in granulomas that contained fewer bacteria, and the bacteria in the four-week granulomas were longer, suggesting that early granulomas are better at supporting Mtb growth than granulomas in established disease. These results provide new insights into Mtb-host cell dynamics in a nonhuman primate model that replicates human TB.

Tpl2 Signaling Regulates Dendritic Cell Activation and Responding T Cell Differentiation Following Mycobacterium tuberculosis Infection

Abstract Mycobacterium tuberculosis (Mtb) utilizes a number of immune evasion mechanisms in order to persist inside of host antigen-presenting cells. Dendritic cells (DCs) are important in restricting Mtb growth by migrating to draining lymph nodes and activating antigen-specific T cell responses, but the roles of DCs in Mtb infection require further study. This study investigated DC activation and functional outcomes following Mtb H37Ra-driven tumor progression locus 2 (Tpl2) signaling. The role of Tpl2 was interrogated genetically, utilizing bone marrow-derived DCs from Tpl2−/− mice. We assessed cytokine production via ELISA, mRNA levels via qRT-PCR, and expression of cell surface molecules via flow cytometry. In Mtb-treated DCs, genetic depletion of Tpl2 increased production of pro-inflammatory cytokines such as IL-12p40 and IL-6. Loss of Tpl2 in Mtb-treated DCs also led to decreased E-cadherin expression, and increased expression of Icam-1 (Cd54) and Mmp2, which are molecules involved in DC transmigration. Expression of Ccr7 and Ccr4 was also enhanced in Mtb-treated Tpl2−/− DCs, which correlated with improved migration of Tpl2−/− DCs towards CCL19 and CCL21 in vitro (assessed using a trans-well assay). When antigen-specific CD4+ T cells were co-cultured with Mtb-infected DCs, deletion of Tpl2−/− in the DCs resulted in increased T cell production of IFNγ and IL-2, as well as increased Tbet expression, indicative of enhanced Th1 polarization. Together, these data indicate that Mtb-induced Tpl2 signaling suppresses certain aspects of DC activation, leading to blunted Th1 responses against the pathogen.

Uncovering the underlying immune perturbations that determine long-term severity of chronic virus and Mycobacterium tuberculosis coinfection

Abstract Chronic viral infections increase severity of Mycobacterium tuberculosis (Mtb) coinfection, yet how they alter the pulmonary microenvironment to foster coinfection and worsen disease severity is unclear. We developed a coinfection model in mice with chronic lymphocytic choriomeningitis virus and Mtb coinfection that recapitulated the central clinical manifestations of coinfection, including increased Mtb burden, extra-pulmonary dissemination and heightened mortality. These long-term disease consequences were not due to chronic virus-induced immunosuppression or exhaustion, but instead were determined by early alterations in immune surveillance of Mtb coinfection. Mechanistically, increased chronic virus induced TNFα production initially arrested pulmonary Mtb growth, impeding dendritic cell mediated antigen transportation to the lung-draining lymph nodes (LNs) and allowing bacterial sanctuary. The inhibited antigen arrival to LNs delayed CD4 T cell priming, allowing Mtb to replicate to higher set-points before T cell mediated control could be initiated. Once primed, Mtb-specific CD4 T cell differentiation skewed away from the Th1 responses associated with Mtb control, and instead toward Th17 differentiation. The elevated IL17 increased pulmonary neutrophil influx that decreased the long-term survival of coinfected mice. Therapeutically correcting the timing of CD4 T cell priming re-established CD4 Th1 over Th17 dominance, diminished pulmonary neutrophilia, and enabled enhanced Mtb control in the presence of chronic viral coinfection. Thus, Mtb co-opts TNFα from the chronic inflammatory environment to subvert immune-surveillance, avert early immune function and foster long-term coinfection.

Sacubitril‐Based Urea and Thiourea Derivatives as Novel Inhibitors for Anti‐Tubercular against Dormant Tuberculosis

AbstractIn the present study, we have attempted to identify Sacubitril derivatives as lead compounds for dormant tuberculosis. A total of twenty‐one compounds belongs to a series of the Sacubitril derivatives 5(a–u) were synthesized using (2R,4S)‐5‐([1,1′‐biphenyl]‐4‐yl)‐4‐(amino)‐2‐methylpentanoic acid ethyl ester hydrochloride with different isocyanates and isothiocyanates. All the compounds structures were determined by 1H & 13C NMR spectroscopy, mass spectrometry, and CHN analysis. Compound, 5 r, the structure confirmed by single‐crystal X‐ray diffraction analysis (SXRD). The newly synthesized compounds were screened for their in vitro antituberculosis activity (anti‐TB) against dormant Mycobacterium tuberculosis H37Rv (ATCC27294). Among the twenty‐one compounds, 5 p and 5 q were exhibited good potent anti‐TB activity compared to the standard drug Ethambutol. Further, the anti‐TB activities of the compounds (5 p and 5 q) were evaluated against M. tuberculosis (Mtb) using the nutrient starvation model (NSM). Moreover, these two compounds, 5 p and 5 q have shown significant inhibition of growth of Mtb as compared to the control. To determine the toxicity nature, the potent anti‐TB active compounds were evaluated against RAW 264.7 cells. Further, the anti‐TB activities of all these compounds have shown a good correlation to their in‐silico molecular docking analysis by exhibiting strong interactions with the inhibitor Mur‐B.

Regulatory Mechanisms of Autophagy-Targeted Antimicrobial Therapeutics Against Mycobacterial Infection.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen causing human tuberculosis, an infectious disease that still remains as a global health problem. Autophagy, a lysosomal degradative process, has emerged as a critical pathway to restrict intracellular Mtb growth through enhancement of phagosomal maturation. Indeed, several autophagy-modulating agents show promise as host-directed therapeutics for Mtb infection. In this Review, we discuss recent progress in our understanding the molecular mechanisms underlying the action of autophagy-modulating agents to overcome the immune escape strategies mediated by Mtb. The factors and pathways that govern such mechanisms include adenosine 5′-monophosphate-activated protein kinase, Akt/mammalian TOR kinase, Wnt signaling, transcription factor EB, cathelicidins, inflammation, endoplasmic reticulum stress, and autophagy-related genes. A further understanding of these mechanisms will facilitate the development of host-directed therapies against tuberculosis as well as infections with other intracellular bacteria targeted by autophagic degradation.

Subunit Vaccine ESAT-6:c-di-AMP Delivered by Intranasal Route Elicits Immune Responses and Protects Against Mycobacterium tuberculosis Infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, remains the most common cause of death from a single infectious disease. More safe and effective vaccines are necessary for preventing the prevalence of TB. In this study, a subunit vaccine of ESAT-6 formulated with c-di-AMP (ESAT-6:c-di-AMP) promoted mucosal and systemic immune responses in spleen and lung. ESAT-6:c-di-AMP inhibited the differentiations of CD8+ T cells as well as macrophages, but promoted the differentiations of ILCs in lung. The co-stimulation also enhanced inflammatory cytokines production in MH-S cells. It was first revealed that ESAT-6 and c-di-AMP regulated autophagy of macrophages in different stages, which together resulted in the inhibition of Mtb growth in macrophages during early infection. After Mtb infection, the level of ESAT-6-specific immune responses induced by ESAT-6:c-di-AMP dropped sharply. Finally, inoculation of ESAT-6:c-di-AMP led to significant reduction of bacterial burdens in lungs and spleens of immunized mice. Our results demonstrated that subunit vaccine ESAT-6:c-di-AMP could elicit innate and adaptive immune responses which provided protection against Mtb challenge, and c-di-AMP as a mucosal adjuvant could enhance immunogenicity of antigen, especially for innate immunity, which might be used for new mucosal vaccine against TB.

Covalent Inactivation of Mycobacterium tuberculosis Isocitrate Lyase by cis-2,3-Epoxy-Succinic Acid.

The isocitrate lyases (ICL1/2) are essential enzymes of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. At present, no ICL1/2 inhibitors have progressed to clinical evaluation, despite extensive drug discovery efforts. Herein, we surveyed succinate analogs against ICL1 and found that dicarboxylic acids constrained in their synperiplanar conformations, such as maleic acid, comprise uncompetitive inhibitors of ICL1 and inhibit more potently than their trans-isomers. From this, we identified cis-2,3 epoxysuccinic acid (cis-EpS) as a selective, irreversible covalent inactivator of Mtb ICL1 (kinact/Kinact= (5.0 ± 1.4) × 104 M-1 s-1; Kinact = 200 ± 50 nM), the most potent inactivator of ICL1 yet characterized. Crystallographic and mass spectrometric analysis demonstrated that Cys191 of ICL1 was S-malylated by cis-EpS, and a crystallographic "snapshot" of inactivation lent insight into the chemical mechanism of this inactivation. Proteomic analysis of E. coli lysates showed that cis-EpS selectively labeled plasmid-expressed Mtb ICL1. Consistently, cis-EpS, but not its trans-isomer, inhibited the growth of Mtb under conditions in which ICL function is essential. These findings encourage the development of analogs of cis-2,3-epoxysuccinate as antituberculosis agents.

A CD4+CD161+ T-Cell Subset Present in Unexposed Humans, Not Tb Patients, Are Fast Acting Cells That Inhibit the Growth of Intracellular Mycobacteria Involving CD161 Pathway, Perforin, and IFN-γ/Autophagy

It remains undefined whether a subset of CD4+ T cells can function as fast-acting cells to control Mycobacterium tuberculosis (Mtb) infection. Here we show that the primary CD4+CD161+ T-cell subset, not CD4+CD161-, in unexposed healthy humans fast acted as unconventional T cells capable of inhibiting intracellular Mtb and BCG growth upon exposure to infected autologous and allogeneic macrophages or lung epithelial A549 cells. Such inhibition coincided with the ability of primary CD4+CD161+ T cells to rapidly express/secrete anti-TB cytokines including IFN-γ, TNF-α, IL-17, and perforin upon exposure to Mtb. Mechanistically, blockades of CD161 pathway, perforin or IFN-γ by blocking mAbs abrogated the ability of CD4+CD161+ T cells to inhibit intracellular mycobacterial growth. Pre-treatment of infected macrophages with inhibitors of autophagy also blocked the CD4+CD161+ T cell-mediated growth inhibition of mycobacteria. Furthermore, adoptive transfer of human CD4+CD161+ T cells conferred protective immunity against mycobacterial infection in SCID mice. Surprisingly, CD4+CD161+ T cells in TB patients exhibited a loss or reduction of their capabilities to produce perforin/IFN-γ and to inhibit intracellular growth of mycobacteria in infected macrophages. These immune dysfunctions were consistent with PD1/Tim3 up-regulation on CD4+CD161+ T cells in active tuberculosis patients, and the blockade of PD1/Tim3 on this subset cells enhanced the inhibition of intracellular mycobacteria survival. Thus, these findings suggest that a fast-acting primary CD4+CD161+T-cell subset in unexposed humans employs the CD161 pathway, perforin, and IFN-γ/autophagy to inhibit the growth of intracellular mycobacteria, thereby distinguishing them from the slow adaptive responses of conventional CD4+ T cells. The presence of fast-acting CD4+CD161+ T-cell that inhibit mycobacterial growth in unexposed humans but not TB patients also implicates the role of these cells in protective immunity against initial Mtb infection.

Biosynthetic Interrogation of Soil Metagenomes Reveals Metamarin, an Uncommon Cyclomarin Congener with Activity against Mycobacterium tuberculosis.

Tuberculosis (TB) remains one of the deadliest infectious diseases. Unfortunately, the development of antibiotic resistance threatens our current therapeutic arsenal, which has necessitated the discovery and development of novel antibiotics against drug-resistant Mycobacterium tuberculosis (Mtb). Cyclomarin A and rufomycin I are structurally related cyclic heptapeptides assembled by nonribosomal peptide synthetases (NRPSs), which show potent anti-Mtb activity with a new cellular target, the caseinolytic protein ClpC1. An NRPS adenylation domain survey using DNA extracted from ∼2000 ecologically diverse soils found low cyclomarin/rufomycin biosynthetic diversity. In this survey, a family of cyclomarin/rufomycin-like biosynthetic gene clusters (BGC) that encode metamarin, an uncommon cyclomarin congener with potent activity against both Mtb H37Rv and multidrug-resistant Mtb clinical isolates was identified. Metamarin effectively inhibits Mtb growth in murine macrophages and increases the activities of ClpC1 ATPase and the associated ClpC1/P1/P2 protease complex, thus causing cell death by uncontrolled protein degradation.

The Cholinergic System Contributes to the Immunopathological Progression of Experimental Pulmonary Tuberculosis.

The cholinergic system is present in both bacteria and mammals and regulates inflammation during bacterial respiratory infections through neuronal and non-neuronal production of acetylcholine (ACh) and its receptors. However, the presence of this system during the immunopathogenesis of pulmonary tuberculosis (TB) in vivo and in its causative agent Mycobacterium tuberculosis (Mtb) has not been studied. Therefore, we used an experimental model of progressive pulmonary TB in BALB/c mice to quantify pulmonary ACh using high-performance liquid chromatography during the course of the disease. In addition, we performed immunohistochemistry in lung tissue to determine the cellular expression of cholinergic system components, and then administered nicotinic receptor (nAChR) antagonists to validate their effect on lung bacterial burden, inflammation, and pro-inflammatory cytokines. Finally, we subjected Mtb cultures to colorimetric analysis to reveal the production of ACh and the effect of ACh and nAChR antagonists on Mtb growth. Our results show high concentrations of ACh and expression of its synthesizing enzyme choline acetyltransferase (ChAT) during early infection in lung epithelial cells and macrophages. During late progressive TB, lung ACh upregulation was even higher and coincided with ChAT and α7 nAChR subunit expression in immune cells. Moreover, the administration of nAChR antagonists increased pro-inflammatory cytokines, reduced bacillary loads and synergized with antibiotic therapy in multidrug resistant TB. Finally, in vitro studies revealed that the bacteria is capable of producing nanomolar concentrations of ACh in liquid culture. In addition, the administration of ACh and nicotinic antagonists to Mtb cultures induced or inhibited bacterial proliferation, respectively. These results suggest that Mtb possesses a cholinergic system and upregulates the lung non-neuronal cholinergic system, particularly during late progressive TB. The upregulation of the cholinergic system during infection could aid both bacterial growth and immunomodulation within the lung to favor disease progression. Furthermore, the therapeutic efficacy of modulating this system suggests that it could be a target for treating the disease.

HDAC3 inhibitor RGFP966 controls bacterial growth and modulates macrophage signaling during Mycobacterium tuberculosis infection

RationaleHost-directed therapeutics for Mycobacterium tuberculosis (Mtb) offer potential strategies for combatting antibiotic resistance and for killing non-replicating bacilli. Phenylbutyrate, a partially selective histone-deacetylase (HDAC) inhibitor, was previously shown to control Mtb growth and alter macrophage inflammatory pathways at 2–4 mM concentrations. ObjectiveTo identify a more potent and selective HDAC inhibitor that modulates macrophage responses to mycobacteria and has direct antibacterial effects against Mtb. MethodsWe used cellular approaches to characterize the role of pharmacologic inhibition of HDAC3 on Mtb growth and Mtb-induced peripheral and alveolar macrophage immune functions. Measurements and main resultsRGFP966, an HDAC3 inhibitor, controlled Mtb, BCG and M. avium growth directly in broth culture and in human peripheral blood monocyte-derived and alveolar macrophages with an MIC50 of approximately 5–10 μM. In contrast, RGFP966 did not inhibit growth of several other intracellular and extracellular bacteria. We also found that RGFP966 modulated macrophage pro-inflammatory cytokine secretion in response to Mtb infection with decreased IL6 and TNF secretion. ConclusionsWe identified a potent and selective small molecule inhibitor of HDAC3 with direct antimicrobial activity against Mtb and modulation of macrophage signaling pathways.

Mycobacterium smegmatis GlnR Regulates the Glyoxylate Cycle and the Methylcitrate Cycle on Fatty Acid Metabolism by Repressing icl Transcription.

Mycobacterium smegmatis (Msm), along with its pathogenic counterpart Mycobacterium tuberculosis (Mtb), utilizes fatty acids and cholesterol as important carbon and energy sources during the persistence within host cells. As a dual-functional enzyme in the glyoxylate cycle and the methylcitrate cycle, isocitrate lyase (ICL, encoded by icl or MSMEG_0911) is indispensable for the growth of Msm and Mtb on short-chain fatty acids. However, regulation of icl in mycobacteria in response to nutrient availability remains largely unknown. Here, we report that the global nitrogen metabolism regulator GlnR represses icl expression by binding to an atypical binding motif in the icl promoter region under nitrogen-limiting conditions. We further show that GlnR competes with PrpR, a transcriptional activator of icl, and dominantly occupies the co-binding motif in the icl promoter region. In the absence of GlnR or in response to the excess nitrogen condition, Msm cells elongate and exhibit robust growth on short-chain fatty acids due to the PrpR-mediated activation of icl, thereby inducing enhanced apoptosis in infected macrophages. Taken together, our findings reveal the GlnR-mediated repression of icl on fatty acid metabolism, which might be a general strategy of nutrient sensing and environmental adaptation employed by mycobacteria.

Early innate and adaptive immune perturbations determine long-term severity of chronic virus and Mycobacterium tuberculosis coinfection

Chronic viral infections increase severity of Mycobacterium tuberculosis (Mtb) coinfection. Here, we examined how chronic viral infections alter the pulmonary microenvironment to foster coinfection and worsen disease severity. We developed a coordinated system of chronic virus and Mtb infection that induced central clinical manifestations of coinfection, including increased Mtb burden, extra-pulmonary dissemination, and heightened mortality. These disease states were not due to chronic virus-induced immunosuppression or exhaustion; rather, increased amounts of the cytokine TNFα initially arrested pulmonary Mtb growth, impeding dendritic cell mediated antigen transportation to the lymph node and subverting immune-surveillance, allowing bacterial sanctuary. The cryptic Mtb replication delayed CD4 Tcell priming, redirecting T helper (Th) 1 toward Th17 differentiation and increasing pulmonary neutrophilia, which diminished long-term survival. Temporally restoring CD4 Tcell induction overcame these diverse disease sequelae to enhance Mtb control. Thus, Mtb co-opts TNFα from the chronic inflammatory environment to subvert immune-surveillance, avert early immune function, and foster long-term coinfection.

Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA-874-3p.

ObjectivesGenetic and epigenetic mechanisms regulate antimicrobial immunity against Mycobacterium tuberculosis (Mtb) infection.MethodsThe present study assessed circular RNA TRAPPC6B (circTRAPPC6B) for antimicrobial immune functions and defined mechanisms wherein circTRAPPC6B regulates Mtb growth, autophagy and microRNA in macrophages.ResultsThe Mtb infection of monocytes/macrophages resulted in a significantly decreased level of circTRAPPC6B that inhibited intracellular Mtb growth in macrophages. Conversely, circTRAPPC6B expression enhanced autophagy or autophagy‐associated protein LC3‐II production in Mtb‐infected macrophages. circTRAPPC6B‐enhanced autophagy aggregation or sequestration was also observed in fluorescence in situ hybridisation (FISH) analysis and confocal imaging. Mechanistically, circTRAPPC6B targets an inhibiting element miR‐874‐3p, as shown by bioinformatics, dual‐luciferase reporter gene analysis and pull‐down assay, respectively. Notably, miR‐874‐3p prohibited autophagy via suppressing autophagy protein ATG16L1 by binding to its 3′‐untranslated region (UTR) in Mtb‐infected macrophages and thus promoting intracellular Mtb growth. Concurrently, circTRAPPC6B enhanced autophagy in Mtb‐infected macrophages by blocking the ability of miR‐874‐3p to inhibit ATG16L1. Thus, circTRAPPC6B antagonises the ability of miR‐874‐3p to suppress ATG16L1 expression and activate and enhance autophagy sequestration to restrict Mtb growth in macrophages.ConclusionThe current findings suggested that both circTRAPPC6B and miR‐874‐3p mechanisms can be explored as potential therapeutics against Mtb infection.

ODELAM: Rapid Sequence-independent Detection of Drug Resistance in Mycobacterium tuberculosis Isolates.

Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths globally each year. Current assays of antimicrobial resistance require knowledge of the mutations that confer drug resistance or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. This protocol describes sample and media preparation and contains instructions for assembling the ODELAM sample chamber. The ODELAM sample chamber is designed to provide a controlled environment to safely observe the growth of Mtb by time-lapse microscopy on an inverted wide-field microscope. A brief description of the ODELAM software is also provided here. ODELAM tracks up to 1500 colony forming units per region of interest and can observe up to 96 regions for up to seven days in a single experiment. This technique allows the quantification of population heterogeneity. ODELAM enables rapid quantitative measurements of growth kinetics in as few as 30 h under a wide variety of environmental conditions. Graphic abstract: Schematic representation of the ODELAM platform.

Helminth Antigen Exposure Enhances Early Immune Control of Mycobacterium tuberculosis in Monocytes and Macrophages

Helminth and Mycobacterium tuberculosis (Mtb) coinfection is common and suggested to influence the risk of developing active tuberculosis (TB). It is known that helminths in contrast to TB induce a strong Th2 response in the host. However, the direct impact of helminth antigen exposure on host immunity against TB is largely unknown. Our aim was to explore the effects of helminth antigen exposure on the early immune control of Mtb in monocytes and macrophages. Ascaris lumbricoides (ASC) and Schistosoma mansoni (SM) protein antigens were used to study the immediate effect of helminth antigen exposure in monocytes, on monocyte-to-macrophage differentiation, or mature macrophages, in the control of virulent Mtb H37Rv. Pre-exposure of peripheral blood mononuclear cells reduced Mtb growth in monocytes, especially with SM, but no Th1/Th2 cytokines or activation markers indicated involvement of T cells. Monocytes exposed before maturing into macrophages reduced Mtb growth in macrophages (ASC), and pre-exposure of mature macrophages reduced (ASC) or kept Mtb growth at control levels (SM). This in vitro model shows how helminth infection directly affects the monocyte-macrophage axis at an early stage before cell-mediated immunity develops. During acute helminth coinfection or when helminth antigen concentration is elevated at the site of Mtb infection, these helminths provide an enhanced control and killing of Mtb owing to the direct stimulatory effect of helminth antigens on phagocytic cells.

Two-Way Regulation of MmpL3 Expression Identifies and Validates Inhibitors of MmpL3 Function in Mycobacterium tuberculosis.

MmpL3, an essential mycolate transporter in the inner membrane of Mycobacterium tuberculosis (Mtb), has been identified as a target of multiple, chemically diverse antitubercular drugs. However, several of these molecules seem to have secondary targets and inhibit bacterial growth by more than one mechanism. Here, we describe a cell-based assay that utilizes two-way regulation of MmpL3 expression to readily identify MmpL3-specific inhibitors. We successfully used this assay to identify a novel guanidine-based MmpL3 inhibitor from a library of 220 compounds that inhibit growth of Mtb by largely unknown mechanisms. We furthermore identified inhibitors of cytochrome bc1-aa3 oxidase as one class of off-target hits in whole-cell screens for MmpL3 inhibitors and report a novel sulfanylacetamide as a potential QcrB inhibitor.

GeneXpert MTB/RIF and Mycobacterium tuberculosis Sputum Culture in Establishing the Diagnosis of Pulmonary Tuberculosis and Rifampicin Resistance in Suspected Childhood Pulmonary Tuberculosis in Soetomo Hospital

The diagnosis of childhood tuberculosis remains a challenge worldwide. The GeneXpert MTB/RIF test, a rapid Mycobacteria tuberculosis diagnostic tool, was recommended for use in children. No pediatric studies of GeneXpert MTB/RIF assessing pulmonary tuberculosis within a hospital setting has been done in Indonesia. We evaluated the performance of the GeneXpert MTB/RIF test compared with sputum culture on Lowenstein-Jensen (LJ) for the diagnosis of childhood pulmonary tuberculosis. This study was conducted in pediatric respirology inpatient and outpatient Dr. Soetomo Hospital, a tertiary care facility in Surabaya between June and August 2015 with a cross-sectional design. We consecutively enrolled 27 children aged 3 months to 14 years who had history of close contact with adult tuberculosis patients and showed symptoms of pulmonary tuberculosis. Sputum collection was performed by induced sputum and three examination methods were performed (microscopic, GeneXpert MTB/RIF and sputum culture) simultaneously followed by a drug sensitivity test for specimens detected with MTB growth. The GeneXpert MTB/RIF test had a sensitivity of 100% (95% CI 100-100) and a specificity of 95% (95% CI 85-100). The positive predictive value for diagnosing pulmonary TB was 89% (95% CI 68-100), the negative predictive value was 100% (95% CI 100-100) and positive likelihood ratio was 20 (95% CI 2.82-128). The GeneXpert MTB/RIF test on one sputum sample rapidly and correctly identified all children with culture-confirmed pulmonary tuberculosis with high specificity. Similar results were obtained between GeneXpert MTB/RIF and sputum culture based on age groups and clinical manifestations. Rifampicin resistance were both detected in GeneXpert MTB/RIF and MTB sensitivity test