Replication Of Mycobacterium Tuberculosis Research Articles

Mathematical modeling suggests heterogeneous replication of Mycobacterium tuberculosis in rabbits.

How quickly Mycobacterium tuberculosis ( Mtb ) replicates and dies in lungs of infected individuals is likely to determine the outcome of the infection - either control/clearance of the bacteria by the host immune response or progression to active disease, tuberculosis ( TB ). And yet, only a few studies, primarily in mice, rigorously estimated the rates of Mtb replication and death during an in vivo infection. We infected rabbits with a novel clinical isolate of Mtb carrying an unstable, "replication clock" plasmid and followed the dynamics of bacteria over time. Interestingly, previous methods developed to estimate Mtb replication and death rates using similar data for Mtb infection of mice failed to describe our novel data on Mtb dynamics in rabbits; we showed that heterogeneous dynamics of Mtb in semi-independent subpopulations in lungs of Mtb-infected rabbits may be one explanation of this failure of the method. Our results highlight potential differences in Mtb dynamics in different mammalian hosts and suggest ways to evaluate heterogeneity of Mtb replication and death rates in vivo.

Heterogeneity in immune cell composition is associated with Mycobacterium tuberculosis replication at the granuloma level.

The control of bacterial growth is key to the prevention and treatment of tuberculosis (TB). Granulomas represent independent foci of the host immune response that present heterogeneous capacity for control of bacterial growth. At the whole tissue level, B cells and CD4 or CD8 T cells have an established role in immune protection against TB. Immune cells interact within each granuloma response, but the impact of granuloma immune composition on bacterial replication remains unknown. Here we investigate the associations between immune cell composition, including B cell, CD4, and CD8 T cells, and the state of replicating Mycobacterium tuberculosis (Mtb) within the granuloma. A measure of ribosomal RNA synthesis, the RS ratio®, represents a proxy measure of Mtb replication at the whole tissue level. We adapted the RS ratio through use of in situ hybridization, to identify replicating and non-replicating Mtb within each designated granuloma. We applied a regression model to characterize the associations between immune cell populations and the state of Mtb replication within each respective granuloma. In the evaluation of nearly 200 granulomas, we identified heterogeneity in both immune cell composition and proportion of replicating bacteria. We found clear evidence of directional associations between immune cell composition and replicating Mtb. Controlling for vaccination status and endpoint post-infection, granulomas with lower CD4 or higher CD8 cell counts are associated with a higher percent of replicating Mtb. Conversely, changes in B cell proportions were associated with little change in Mtb replication. This study establishes heterogeneity across granulomas, demonstrating that certain immune cell types are differentially associated with control of Mtb replication. These data suggest that evaluation at the granuloma level may be imperative to identifying correlates of immune protection.

A host-directed oxadiazole compound potentiates antituberculosis treatment via zinc poisoning in human macrophages and in a mouse model of infection.

Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

The role of transcriptional regulators in metal ion homeostasis of Mycobacterium tuberculosis.

Metal ions are essential trace elements for all living organisms and play critical catalytic, structural, and allosteric roles in many enzymes and transcription factors. Mycobacterium tuberculosis (MTB), as an intracellular pathogen, is usually found in host macrophages, where the bacterium can survive and replicate. One of the reasons why Tuberculosis (TB) is so difficult to eradicate is the continuous adaptation of its pathogen. It is capable of adapting to a wide range of harsh environmental stresses, including metal ion toxicity in the host macrophages. Altering the concentration of metal ions is the common host strategy to limit MTB replication and persistence. This review mainly focuses on transcriptional regulatory proteins in MTB that are involved in the regulation of metal ions such as iron, copper and zinc. The aim is to offer novel insights and strategies for screening targets for TB treatment, as well as for the development and design of new therapeutic interventions.

RegX3-dependent transcriptional activation of kdpDE and repression of rv0500A are linked to potassium homeostasis in Mycobacterium tuberculosis.

Ionic homeostasis is essential for the survival and replication of Mycobacterium tuberculosis within its host. Low potassium ion concentrations trigger a transition of M. tuberculosis into dormancy. Our current knowledge of the transcriptional regulation mechanisms governing genes involved in potassium homeostasis remains limited. Potassium transport is regulated by the constitutive Trk system and the inducible Kdp system in M. tuberculosis. The two-component system KdpDE (also known as KdpD/KdpE) activates expression of the kdpFABC operon, encoding the four protein subunits of the Kdp potassium uptake system (KdpFABC). We show that, under potassium deficiency, expression of the two-component system senX3/regX3 is upregulated, and bacterial survival is compromised in a regX3-inactivated mutant, ΔregX3. Electrophoretic mobility shift assays (EMSAs), promoter reporter assays and chromatin immunoprecipitation (ChIP) show that RegX3 binds to the kdpDE promoter and activates it under potassium deficiency, whereas RegX3 (K204A), a DNA binding-deficient mutant, fails to bind to the promoter. Mutation of the RegX3 binding motifs on the kdpDE promoter abrogates RegX3 binding. In addition, EMSAs and ChIP assays show that RegX3 represses Rv0500A, a repressor of kdpFABC, by binding to consensus RegX3 binding motifs on the rv0500A promoter. Our findings provide important insight into two converging pathways regulated by RegX3; one in which it activates an activator of kdpFABC, and the other in which it represses a repressor of kdpFABC, during potassium insufficiency. This culminates in increased expression of the potassium uptake system encoded by kdpFABC, enabling bacterial survival. These results further expand the growing transcriptional network in which RegX3 serves as a central node to enable bacterial survival under stress.

Identification of Rv1133c (MetE) as a marker of Mycobacterium tuberculosis replication and as a highly immunogenic antigen with potential immunodiagnostic power.

The immunization of mice with the sterile culture medium supernatants of Mycobacterium tuberculosis (Mtb) H37Rv permitted the production of several monoclonal antibodies (mAbs) specific for secreted and/or released antigens. Two mAbs bound and immunoprecipitated an 80-kDa protein that was identified by mass spectrometry as Rv1133c, the methionine synthase MetE. The protein MetE is ubiquitous among prokaryota and shows a significant sequence homology in many bacteria. We produced both the full-length recombinant MetE and its N-terminal fragment, whose sequence is more conserved among mycobacteria, to select mAbs recognizing an Mtb-specific region of MetE. Finally, we produced and selected eight mAbs that specifically detect the MetE protein in the supernatant and cell lysate of Mtb and BCG, but not other bacteria such as non-tuberculous mycobacteria (NTM), Streptococcus pneumoniae, Staphylococcus aureus, Acinetobacter baumanii, or Escherichia coli. Taking advantage of our mAbs, we studied (i) the vitamin B12 dependence for the synthesis of MetE in Mtb and NTM and (ii) the kinetics of MetE production and secretion in supernatants during the in vitro reproduced replicative, dormant, and resuscitation cycle of Mtb. Our data demonstrate that dormant Mtb, which are assumed to be prevalent in latent infections, as well as NTM do not produce and secrete MetE. Results indicate an unexpected specificity for Mtb of our anti-MetE mAbs and encourage the use of rMetE and our mAbs as tools for the immunodiagnosis of TB and its stages.

TRIMming down Mycobacterium tuberculosis replication: TRIM32 is required for bacterial ubiquitination and autophagy induction in macrophages

TRIMming down Mycobacterium tuberculosis replication: TRIM32 is required for bacterial ubiquitination and autophagy induction in macrophages

Mathematical modeling suggests heterogeneous replication of Mycobacterium tuberculosis in rabbits

Abstract Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb), remains the major health problem. We performed experiments with rabbits infected with a novel, virulent clinical Mtb isolate of the Beijing lineage HN878 carrying an unstable plasmid pBP10, allowing us to quantify how quickly HN878 replicates and dies in lungs of rabbits. In our in vitro experiments we found that pBP10 is more stable in HN878 strain than in a more commonly used laboratory-adapted H37Rv strain (the segregation coefficient being s=0.10 in HN878 vs. s=0.18 in H37Rv). Interestingly, the kinetics of the frequency of plasmid-bearing bacteria in lungs of Mtb-infected rabbits did not follow an expected monotonic decline; the percent of Mtb-bearing cells increased between 4 and 8 weeks post-infection and remained stable between 12 and 18 weeks post-infection despite a large increase in bacterial numbers in the lung at these time points. Mathematical modeling suggested that such a non-monotonic change in the percent of plasmid-bearing cells can be explained if the lung Mtb population consists of several (at least 2) sub-populations with different replication kinetics: one major population expanding early and being controlled, while another, a smaller population expanding at later times causing a counterintuitive increase in the percent of plasmid-bearing cells. Given that HN878 forms well circumscribed granulomas in rabbits, our results suggest independent bacterial dynamics in subsets of such granulomas. Our model predictions can be tested in experiments in which HN878-pBP10 dynamics in individual granulomas is followed over time. 1R01AI158963-01A1

ATG7 and ATG14 restrict cytosolic and phagosomal Mycobacterium tuberculosis replication in human macrophages

Autophagy is a cellular innate-immune defence mechanism against intracellular microorganisms, including Mycobacterium tuberculosis (Mtb). How canonical and non-canonical autophagy function to control Mtb infection in phagosomes and the cytosol remains unresolved. Macrophages are the main host cell in humans for Mtb. Here we studied the contributions of canonical and non-canonical autophagy in the genetically tractable human induced pluripotent stem cell-derived macrophages (iPSDM), using a set of Mtb mutants generated in the same genetic background of the common lab strain H37Rv. We monitored replication of Mtb mutants that are either unable to trigger canonical autophagy (Mtb ΔesxBA) or reportedly unable to block non-canonical autophagy (Mtb ΔcpsA) in iPSDM lacking either ATG7 or ATG14 using single-cell high-content imaging. We report that deletion of ATG7 by CRISPR–Cas9 in iPSDM resulted in increased replication of wild-type Mtb but not of Mtb ΔesxBA or Mtb ΔcpsA. We show that deletion of ATG14 resulted in increased replication of both Mtb wild type and the mutant Mtb ΔesxBA. Using Mtb reporters and quantitative imaging, we identified a role for ATG14 in regulating fusion of phagosomes containing Mtb with lysosomes, thereby enabling intracellular bacteria restriction. We conclude that ATG7 and ATG14 are both required for restricting Mtb replication in human macrophages.

Impact of Vitamin D in Prophylaxis and Treatment in Tuberculosis Patients.

Vitamin D plays a crucial role in many infectious diseases, such as tuberculosis (TB), that remains one of the world’s top infectious killers with 1.5 million deaths from TB in 2021. Vitamin D suppresses the replication of Mycobacterium tuberculosis in vitro and showed a promising role in TB management as a result of its connection with oxidative balance. Our review encourages the possible in vivo benefit of a joint administration with other vitamins, such as vitamin A, which share a known antimycobacterial action with vitamin D. However, considering the low incidence of side effects even at high dosages and its low cost, it would be advisable to assess vitamin D level both in patients with active TB and high-risk groups and administer it, at least to reach sufficiency levels.

Dissecting the Mycobacterium bovis BCG Response to Macrophage Infection to Help Prioritize Targets for Anti-Tuberculosis Drug and Vaccine Discovery.

New strategies are required to reduce the worldwide burden of tuberculosis. Intracellular survival and replication of Mycobacterium tuberculosis after macrophage phagocytosis is a fundamental step in the complex host–pathogen interactions that lead to granuloma formation and disease. Greater understanding of how the bacterium survives and thrives in these environments will inform novel drug and vaccine discovery programs. Here, we use in-depth RNA sequencing of Mycobacterium bovis BCG from human THP-1 macrophages to describe the mycobacterial adaptations to the intracellular environment. We identify 329 significantly differentially regulated genes, highlighting cholesterol catabolism, the methylcitrate cycle and iron homeostasis as important for mycobacteria inside macrophages. Examination of multi-functional gene families revealed that 35 PE/PPE genes and five cytochrome P450 genes were upregulated 24 h after infection, highlighting pathways of potential significance. Comparison of the intracellular transcriptome to gene essentiality and immunogenicity studies identified 15 potential targets that are both required for intracellular survival and induced on infection, and eight upregulated genes that have been demonstrated to be immunogenic in TB patients. Further insight into these new and established targets will support drug and vaccine development efforts.

Identifying innate immune regulators of Mycobacterium tuberculosis pathogenesis in macrophages

Abstract Tuberculosis (TB) is a leading infectious disease that contributes to approximately 1.4 million deaths annually worldwide. In the present situation of the COVID-19 pandemic and disrupted healthcare services, mathematical modelling predicts that there will be an additional 6.3 million new cases and 1.4 million more TB deaths by the end of 2025. Innate immune cells like macrophages are the frontline responders in host defense against Mycobacterium tuberculosis (MTB) and may also play a major role in disease establishment. A major roadblock in developing effective strategies against TB is the inability of current anti-TB drugs to target both replicating and non-replicating bacteria. We have found that lack of specific components of the host innate sensing machinery has a major impact on intracellular bacterial growth and the production of IL-1β and IFN-β during MTB infection of macrophages; the latter cytokines play major roles in host-protective and host-detrimental outcomes of infection respectively. RNA seq reveals that these innate immune modules differentially control the expression of genes implicated in host protein synthesis and MTB dissemination and escape from the granuloma. Ongoing work is utilizing Path-Seq for quantitative profiling of the MTB transcriptome within infected macrophages and investigating macrophage-dependent mechanisms of MTB restriction and persistence in vivo. Our study will potentially identify immune regulators that make the macrophage environment either permissive or restrictive for intracellular replication of MTB with important implications for developing combinatorial targeting approaches for host-directed treatment of TB.

Downregulation of GPR183 on infection restricts the early infection and intracellular replication of mycobacterium tuberculosis in macrophage

GPR183/EBI2 is a key chemotactic receptor for the positioning of B cells in lymphoid organs, and also for the migration of T cells and other immune cells. Here, we demonstrate that the downregulation of GPR183 in macrophage induced during Mtb infection restrains the bacterial early infection and intracellular replication. Overexpression of GPR183 or stimulation with its natural ligand favors Mtb replication in macrophage, while treatment with its antagonist represses both Mtb early infection and intracellular replication. With mutational analysis, we find that substitution of Asp-73, Arg-83, Tyr-112, Tyr-256 abolished the promotive effect of GPR183 on Mtb early infection and replication in macrophage. In conclusion, we demonstrated that beside the known role of chemotaxis receptor, GPR183 also functions directly in the interaction between macrophage and Mtb in a cell-autonomous way.

Modulatory effect of Tim-3/Galectin-9 axis on T-cell-mediated immunity in pulmonary tuberculosis

Patients affected by pulmonary tuberculosis (PTB) manifest deficiencies in innate cellular immunity. The Tim3/Galectin-9 axis is an important regulator of Th1 cell immunity, leading to Th1 cell apoptosis. Herein, this study aims to clarify the underlying roles of the Tim-3/Galectin-9 axis in T-cell immunity in PTB. Peripheral blood mononuclear cells (PBMCs) were extracted from subjects with and without PTB to examine the expression of CD4, CD8, CD25, and Tim-3 under the stimulation of Mycobacterium tuberculosis (MTB) and purified protein derivative (PPD). In addition, the expression of Tim-3 and Galectin-9 in PBMCs was determined. The Tim-3/Galectin-9 axis in the PBMCs was activated or blocked to detect the secreted levels of IFN-γ, TNF-α, IL-2, and IL-22. MTB stimulation increased the expression of CD4, CD8, CD25, Tim-3, and Galectin-9 in PBMCs. The blockade of Tim-3/Galectin-9 axis resulted in reduced secretion of IFN-γ, TNF-α, IL-2, and IL-22 from T-cells. Moreover, Tim-3+CD4+T, Tim-3+CD8+, and Tim-3+CD25+T cells exhibited a greater ability to inhibit the replication of MTB in macrophages. Taken conjointly, the blockade of Tim-3/ Galectin-9 axis inhibits the secretion of inflammatory cytokines in T-cells to regulate the T-cell immunity in PTB.

Cytoplasmic RNA Sensor Pathways and Nitazoxanide Broadly Inhibit Intracellular Mycobacterium tuberculosis Growth.

SummaryTo establish stable infection, Mycobacterium tuberculosis (MTb) must overcome host innate immune mechanisms, including those that sense pathogen-derived nucleic acids. Here, we show that the host cytosolic RNA sensing molecules RIG-I-like receptor (RLR) signaling proteins RIG-I and MDA5, their common adaptor protein MAVS, and the RNA-dependent kinase PKR each independently inhibit MTb growth in human cells. Furthermore, we show that MTb broadly stimulates RIG-I, MDA5, MAVS, and PKR gene expression and their biological activities. We also show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits intracellular MTb growth and amplifies MTb-stimulated RNA sensor gene expression and activity. This study establishes prototypic cytoplasmic RNA sensors as innate restriction factors for MTb growth in human cells and it shows that targeting this pathway is a potential host-directed approach to treat tuberculosis disease.

Human Monocytic Suppressive Cells Promote Replication of Mycobacterium tuberculosis and Alter Stability of in vitro Generated Granulomas.

Tuberculosis (TB) has tremendous public health relevance. It most frequently affects the lung and is characterized by the development of unique tissue lesions, termed granulomas. These lesions encompass various immune populations, with macrophages being most extensively investigated. Myeloid derived suppressor cells (MDSCs) have been recently identified in TB patients, both in the circulation and at the site of infection, however their interactions with Mycobacterium tuberculosis (Mtb) and their impact on granulomas remain undefined. We generated human monocytic MDSCs and observed that their suppressive capacities are retained upon Mtb infection. We employed an in vitro granuloma model, which mimics human TB lesions to some extent, with the aim of analyzing the roles of MDSCs within granulomas. MDSCs altered the structure of and affected bacterial containment within granuloma-like structures. These effects were partly controlled through highly abundant secreted IL-10. Compared to macrophages, MDSCs activated primarily the NF-κB and MAPK pathways and the latter largely contributed to the release of IL-10 and replication of bacteria within in vitro generated granulomas. Moreover, MDSCs upregulated PD-L1 and suppressed proliferation of lymphocytes, albeit with negligible effects on Mtb replication. Further comprehensive characterization of MDSCs in TB will contribute to a better understanding of disease pathogenesis and facilitate the design of novel immune-based interventions for this deadly infection.

Transglutaminase type 2 plays a key role in the pathogenesis of Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (MTB), the aetiological agent of tuberculosis (TB), is capable of interfering with the phagosome maturation pathway, by inhibiting phagosome-lysosome fusion and the autophagic process to ensure survival and replication in macrophages. Thus, it has been proposed that the modulation of autophagy may represent a therapeutic approach to reduce MTB viability by enhancing its clearance. The aim of this study was to investigate whether transglutaminase type 2 (TG2) is involved in the pathogenesis of MTB. We have shown that either genetic or pharmacological inhibition of TG2 leads to a marked reduction in MTB replicative capacity. Infection of TG2 knockout mice demonstrated that TG2 is required for MTB intracellular survival in macrophages and host tissues. The same inhibitory effect can be reproduced in vitro using Z-DON, a specific inhibitor of the transamidating activity of TG2. Massive cell death observed in macrophages that properly express TG2 is hampered by the absence of the enzyme and can be largely reduced by the treatment of wild-type macrophages with the TG2 inhibitor. Our data suggest that reduced MTB replication in cells lacking TG2 is due to the impairment of LC3/autophagy homeostasis. Finally, we have shown that treatment of MTB-infected murine and human primary macrophages with cystamine, a TG2 inhibitor already tested in clinical studies, causes a reduction in intracellular colony-forming units in human macrophages similar to that achieved by the anti-TB drug capreomycin. These results suggest that inhibition of TG2 activity is a potential novel approach for the treatment of TB.

Advancing host-directed therapy for tuberculosis.

Tuberculosis (TB) drug-development strategies, a wide range of candidate host-directed therapies (HDT)s-including new and repurposed drugs, biologics, and cellular therapies-have been proposed to accelerate eradication of infection and overcome the problems associated with current treatment regimens. By investigating the interaction between macrophages and the intracellular parasite Toxoplasma gondii (T. gondii), we uncovered that infection-induced signaling pathways suggest possibilities for the development of novel therapeutic modalities for TB that target the intracellular signaling pathways permitting the replication of Mycobacterium tuberculosis (MTB).

PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis.

ABSTRACTIron is essential for replication of Mycobacterium tuberculosis, but iron is efficiently sequestered in the human host during infection. Heme constitutes the largest iron reservoir in the human body and is utilized by many bacterial pathogens as an iron source. While heme acquisition is well studied in other bacterial pathogens, little is known in M. tuberculosis. To identify proteins involved in heme utilization by M. tuberculosis, a transposon mutant library was screened for resistance to the toxic heme analog gallium(III)-porphyrin (Ga-PIX). Inactivation of the ppe36, ppe62, and rv0265c genes resulted in resistance to Ga-PIX. Growth experiments using isogenic M. tuberculosis deletion mutants showed that PPE36 is essential for heme utilization by M. tuberculosis, while the functions of PPE62 and Rv0265c are partially redundant. None of the genes restored growth of the heterologous M. tuberculosis mutants, indicating that the proteins encoded by the genes have separate functions. PPE36, PPE62, and Rv0265c bind heme as shown by surface plasmon resonance spectroscopy and are associated with membranes. Both PPE36 and PPE62 proteins are cell surface accessible, while the Rv0265c protein is probably located in the periplasm. PPE36 and PPE62 are, to our knowledge, the first proline-proline-glutamate (PPE) proteins of M. tuberculosis that bind small molecules and are involved in nutrient acquisition. The absence of a virulence defect of the ppe36 deletion mutant indicates that the different iron acquisition pathways of M. tuberculosis may substitute for each other during growth and persistence in mice. The emerging model of heme utilization by M. tuberculosis as derived from this study is substantially different from those of other bacteria.

The COX- inhibitor indomethacin reduces Th1 effector and T regulatory cells in vitro in Mycobacterium tuberculosis infection.

BackgroundTuberculosis (TB) causes a major burden on global health with long and cumbersome TB treatment regimens. Host-directed immune modulating therapies have been suggested as adjunctive treatment to TB antibiotics. Upregulated cyclooxygenase-2 (COX-2)-prostaglandin E2 (PGE2) signaling pathway may cause a dysfunctional immune response that favors survival and replication of Mycobacterium tuberculosis (Mtb).MethodsBlood samples were obtained from patients with latent TB (n = 9) and active TB (n = 33) before initiation of anti-TB chemotherapy. COX-2 expression in monocytes and ESAT-6 and Ag85 specific T cell cytokine responses (TNF-α, IFN-γ, IL-2), proliferation (carboxyfluorescein succinimidyl ester staining) and regulation (FOXP3+ T regulatory cells) were analysed by flow cytometry and the in vitro effects of the COX-1/2 inhibitor indomethacin were measured.ResultsWe demonstrate that indomethacin significantly down-regulates the fraction of Mtb specific FOXP3+ T regulatory cells (ESAT-6; p = 0.004 and Ag85; p < 0.001) with a concomitant reduction of Mtb specific cytokine responses and T cell proliferation in active TB. Although active TB tend to have higher levels, there are no significant differences in COX-2 expression between unstimulated monocytes from patients with active TB compared to latent infection. Monocytes in both TB groups respond with a significant upregulation of COX-2 after in vitro stimulation.ConclusionsTaken together, our in vitro data indicate a modulation of the Th1 effector and T regulatory cells in Mtb infection in response to the COX-1/2 inhibitor indomethacin. The potential role as adjunctive host-directed therapy in TB disease should be further evaluated in both animal studies and in human clinical trials.