Mycobacterium tuberculosis carbon and nitrogen metabolic fluxes

Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens causing tuberculosis (TB), a devastating infectious disease responsible for the highest human mortality and morbidity. The emergence of drug-resistant strains of the pathogen has increased the burden of TB tremendously and new therapeutics to overcome the problem of drug resistance are urgently needed. Metabolism of Mtb and its interactions with the host is important for its survival and virulence; this is an important topic of research where there is growing interest in developing new therapies and drugs that target these interactions and metabolism of the pathogen during infection. Mtb adapts its metabolism in its intracellular niche and acquires multiple nutrient sources from the host cell. Carbon metabolic pathways and fluxes of Mtb has been extensively researched for over a decade and is well-defined. Recently, there has been investigations and efforts to measure metabolism of nitrogen, which is another important nutrient for Mtb during infection. This review discusses our current understanding of the central carbon and nitrogen metabolism, and metabolic fluxes that are important for the survival of the TB pathogen.     

Protein kinase and phosphatase signaling in Mycobacterium tuberculosis physiology and pathogenesis

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), evades the antimicrobial defenses of the host and survives within the infected individual through a complex set of strategies. These include active prevention of host cellular killing processes as well as overwhelming adaptive gene expression. In the past decade, we have gained an increased understanding of how mycobacteria not only have the ability to adapt to a changing host environment but also actively interfere with the signaling machinery within the host cell to counteract or inhibit parts of the killing apparatus employed by the macrophage. Mtb is able to sense its environment via a set of phospho-signaling proteins which mediate its response and interaction with the host in a coordinated manner. In this review, we summarize the current knowledge about selected Mtb serine, threonine, and tyrosine kinase and phosphatase signaling proteins, focusing on the protein kinases, PknG and PtkA, and the protein phosphatase, PtpA.     

Phosphodiesterase-4 inhibition alters gene expression and improves isoniazid-mediated clearance of Mycobacterium tuberculosis in rabbit lungs

Tuberculosis (TB) treatment is hampered by the long duration of antibiotic therapy required to achieve cure. This indolent response has been partly attributed to the ability of subpopulations of less metabolically active Mycobacterium tuberculosis (Mtb) to withstand killing by current anti-TB drugs. We have used immune modulation with a phosphodiesterase-4 (PDE4) inhibitor, CC-3052, that reduces tumor necrosis factor alpha (TNF-α) production by increasing intracellular cAMP in macrophages, to examine the crosstalk between host and pathogen in rabbits with pulmonary TB during treatment with isoniazid (INH). Based on DNA microarray, changes in host gene expression during CC-3052 treatment of Mtb infected rabbits support a link between PDE4 inhibition and specific down-regulation of the innate immune response. The overall pattern of host gene expression in the lungs of infected rabbits treated with CC-3052, compared to untreated rabbits, was similar to that described in vitro in resting Mtb infected macrophages, suggesting suboptimal macrophage activation. These alterations in host immunity were associated with corresponding down-regulation of a number of Mtb genes that have been associated with a metabolic shift towards dormancy. Moreover, treatment with CC-3052 and INH resulted in reduced expression of those genes associated with the bacterial response to INH. Importantly, CC-3052 treatment of infected rabbits was associated with reduced ability of Mtb to withstand INH killing, shown by improved bacillary clearance, from the lungs of co-treated animals compared to rabbits treated with INH alone. The results of our study suggest that changes in Mtb gene expression, in response to changes in the host immune response, can alter the responsiveness of the bacteria to antimicrobial agents. These findings provide a basis for exploring the potential use of adjunctive immune modulation with PDE4 inhibitors to enhance the efficacy of existing anti-TB treatment.     

The structural insights of 16.3 kDa heat shock protein (HSP16.3) from Mycobacterium tuberculosis via in silico study

Mycobacterium tuberculosis (Mtb) is capable of surviving in dormancy before developing to tuberculosis (TB). One of the major challenges of TB management is the identification of patients, making TB diagnosis critical for disease management. This study focuses on the 16 kDa heat shock protein (HSP16.3; a potential biomarker for latent TB infection) that is expressed during the latent phase of Mtb growth. In order to explore the dynamics and interactions of HSP16.3, the 3-D structure of HSP16.3 was built via comparative modelling. The predicted structure shows a predominantly beta-sheet dodecamer with alpha-helical folds at its N-terminal. A known protein-hydrophobic probe (1,1′-Bi(4-anilino)naphthalene-5,5′-disulfonic acid; bisANS) was docked to the HSP16.3 model. Interacting residues predicted from docking and MD simulations are in good accordance with experimental data reported in the literature. MMPBSA calculation from MD simulation also showed favourable binding free energy of ?29.90 kcal/mol, driven mainly by van der waals and non-polar solvation energies. The statistical evaluation and results from the computational study on HSP16.3 indicate the reliability of the built model, which is potentially useful for further structural studies of HSP16.3 for latent TB diagnostics.     

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.     

Importance of differential identification of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains for understanding differences in their prevalence,treatment efficacy,and vaccine development

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a serious global health problem in the 21^st century because of its high mortality. Mtb is an extremely successful human-adapted pathogen that displays a multifactorial ability to control the host immune response and to evade killing by drugs, resulting in the breakdown of BCG vaccine-conferred anti-TB immunity and development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb. Although genetic components of the genomes of the Mtb complex strains are highly conserved, showing over 99% similarity to other bacterial genera, recently accumulated evidence suggests that the genetic diversity of the Mtb complex strains has implications for treatment outcomes, development of MDR/XDR Mtb, BCG vaccine efficacy, transmissibility, and epidemiological outbreaks. Thus, new insights into the pathophysiological features of the Mtb complex strains are required for development of novel vaccines and for control of MDR/XDR Mtb infection, eventually leading to refinement of treatment regimens and the health care system. Many studies have focused on the differential identification of Mtb complex strains belonging to different lineages because of differences in their virulence and geographical dominance. In this review, we discuss the impact of differing genetic characteristics among Mtb complex strains on vaccine efficacy, treatment outcome, development of MDR/XDR Mtb strains, and epidemiological outbreaks by focusing on the best-adapted human Mtb lineages. We further explore the rationale for differential identification of Mtb strains for more effective control of TB in clinical and laboratory settings by scrutinizing current diagnostic methods.     

MiR‐342 controls Mycobacterium tuberculosis susceptibility by modulating inflammation and cell death

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that places a heavy strain on public health. Host susceptibility to Mtb is modulated by macrophages, which regulate the balance between cell apoptosis and necrosis. However, the role of molecular switches that modulate apoptosis and necrosis during Mtb infection remains unclear. Here, we show that Mtb‐susceptible mice and TB patients have relatively low miR‐342‐3p expression, while mice with miR‐342‐3p overexpression are more resistant to Mtb. We demonstrate that the miR‐342‐3p/SOCS6 axis regulates anti‐Mtb immunity by increasing the production of inflammatory cytokines and chemokines. Most importantly, the miR‐342‐3p/SOCS6 axis participates in the switching between Mtb‐induced apoptosis and necrosis through A20‐mediated K48‐linked ubiquitination and RIPK3 degradation. Our findings reveal several strategies by which the host innate immune system controls intracellular Mtb growth via the miRNA‐mRNA network and pave the way for host‐directed therapies targeting these pathways.     

Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages

Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.     

Genetics-directed drug discovery for combating Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis (TB), is one of the most infectious bacteria in the world. The traditional strategy to combat TB involves targeting the pathogen directly; however, the rapid evolution of drug resistance lessens the efficiency of this anti-TB method. Therefore, in recent years, some researchers have turned to an alternative anti-TB strategy, which hinders Mtb infection through targeting host genes. In this work, using a theoretical genetic analysis, we identified 170 Mtb infection-associated genes from human genetic variations related to Mtb infection. Then, the agents targeting these genes were identified to have high potential as anti-TB drugs. In particular, the agents that can target multiple Mtb infection-associated genes are more druggable than the single-target counterparts. These potential anti-TB agents were further screened by gene expression data derived from connectivity map. As a result, some agents were revealed to have high interest for experimental evaluation. This study not only has important implications for anti-TB drug discovery, but also provides inspirations for streamlining the pipeline of modern drug discovery.     

Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis

The success of Mycobacterium tuberculosis (Mtb) as a pathogen rests upon its ability to grow intracellularly in macrophages. Interferon-gamma (IFN-γ) is critical in host defense against Mtb and stimulates macrophage clearance of Mtb through an autophagy pathway. Here we show that the host protein ubiquilin 1 (UBQLN1) promotes IFN-γ-mediated autophagic clearance of Mtb. Ubiquilin family members have previously been shown to recognize proteins that aggregate in neurodegenerative disorders. We find that UBQLN1 can interact with Mtb surface proteins and associates with the bacilli in vitro. In IFN-γ activated macrophages, UBQLN1 co-localizes with Mtb and promotes the anti-mycobacterial activity of IFN-γ. The association of UBQLN1 with Mtb depends upon the secreted bacterial protein, EsxA, which is involved in permeabilizing host phagosomes. In autophagy-deficient macrophages, UBQLN1 accumulates around Mtb, consistent with the idea that it marks bacilli that traffic through the autophagy pathway. Moreover, UBQLN1 promotes ubiquitin, p62, and LC3 accumulation around Mtb, acting independently of the E3 ligase parkin. In summary, we propose a model in which UBQLN1 recognizes Mtb and in turn recruits the autophagy machinery thereby promoting intracellular control of Mtb. Thus, polymorphisms in ubiquilins, which are known to influence susceptibility to neurodegenerative illnesses, might also play a role in host defense against Mtb.     

A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren

Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multi-drug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904–1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0–7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904–1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904–1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug.     

Effect of Mycobacterium tuberculosis infection on adipocyte physiology

Tuberculosis (TB) remains as a major threat to human health worldwide despite of the availability of standardized antibiotic therapy. One of the characteristic of pathogenic Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis is its ability to persist in the host in a dormant state and develop latent infection without clinical signs of active disease. However, the mechanisms involved in bacterial persistence and the establishment of latency is not well understood. Adipose tissue is emerging as an important niche that favors actively replicating as well as dormant Mtb during acute and latent infection. This also suggests that Mtb can disseminate from the lungs to adipose tissue during aerosol infection and/or from adipose tissue to lungs during reactivation of latent infection. In this study, we report the interplay between key adipokine levels and the dynamics of Mtb pathogenesis in the lungs and adipose tissue using a rabbit model of pulmonary infection with two clinical isolates that produce divergent outcome in disease progression. Results show that markers of adipocyte physiology and function were significantly altered during Mtb infection and distinct patterns of adipokine expression were noted between adipose tissue and the lungs. Moreover, these markers were differentially expressed between active disease and latent infection. Thus, this study highlights the importance of targeting adipocyte function as potential target for developing better TB intervention strategies.     

基于蛋白质组芯片的结核分枝杆菌系统生物学研究进展

近些年全球结核病疫情愈发严重,耐药性结核病使其雪上加霜。一个重要原因是结核病新药的匮乏以及结核分枝杆菌相关基础研究的不足。因此迫切需要开发新的技术以促进结核病系统生物学基础研究,并在此基础上研究新机制,发现新靶标,开发新药物。结核分枝杆菌功能蛋白质组芯片的出现旨在促进结核病相关研究工作。考虑到结核分枝杆菌高毒力、复制周期长和需要在生物安全三级实验室中开展研究等特点和难点,该工具为结核病相关研究人员提供了一个强有力的武器。目前这一技术手段的应用已经使我们对结核分枝杆菌-宿主相互作用、小分子-蛋白结合以及抗生素耐药性机制等关键生物过程有了更深入的了解。为了更好地帮助同行了解这一有效的工具,本文综述了结核分枝杆菌功能蛋白组芯片的几种主要应用,期望同行专家能更好地将其应用于结核病相关的基础研究中。     

Bedaquiline: Fallible Hope Against Drug Resistant Tuberculosis

Tuberculosis (TB) is a deadly bacterial infectious disease caused by intra-cellular pathogen Mycobacterium tuberculosis (Mtb). There were an estimated 1.4 million TB deaths in 2015 and an additional 0.4 million deaths resulting from TB among individuals with HIV. Drug-discovery for its cure is very slow in comparison with the causative organism’s fast pace of mutations conferring drug resistance. Moreover, the field of drug-discovery of anti-TB drugs is constantly being challenged by the drug resistant strains of Mtb. Several molecules/inhibitors are being tested across the pharmaceutical industry and research centres for their suitability as drug candidate. It takes immense effort, high costs and a whole lot of screening to bring a single molecule to the clinics for patient cure. In last 60 years, hundreds of molecules have been patented for their probable use to develop drug for treatment of TB. However, only one drug has been successfully approved that is bedaquiline (1-(6-bromo-2 -methoxy-quinolin-3-yl)-4-dimethylamino-2-naphtalen-1-yl-1-phenyl-butan-2-ol). This is a brief review about bedaquiline (BDQ), the only drug in last 45 years approved for curing drug-resistant pulmonary TB, its development, action mechanism and development of resistance against it.     

Role of mycobacteria effectors in phagosome maturation blockage and new drug targets discovery

Tuberculosis remains a serious global health threat with nearly 10 million new cases and 1.7 million deaths every year. The emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) further complicates this problem. It is pressing to find new ways to combat Mtb. The success of Mtb is largely attributed to its ability to persist within macrophages by arresting phagosomal maturation. The bacterial proteins and lipids play important roles in this inhibition which involves several aspects of phagosomal maturation, including both fusion and fission events and recruitment of V-ATPases allowing acidification. Understanding the interaction between the pathogen and host macrophage is essential to eradicate or control tuberculosis. This review focuses on the mechanism of phagolysosome formation, the pivotal event for the fates of infection participants and abundance of novel drug targets.     

NAD hydrolysis by the tuberculosis necrotizing toxin induces lethal oxidative stress in macrophages

Mycobacterium tuberculosis (Mtb) kills infected macrophages through necroptosis, a programmed cell death that enhances mycobacterial replication and dissemination. The tuberculosis necrotizing toxin (TNT) is the major cytotoxicity factor of Mtb in macrophages and induces necroptosis by NAD⁺ hydrolysis. Here, we show that the catalytic activity of TNT triggers the production of reactive oxygen species (ROS) in Mtb‐infected macrophages causing cell death and promoting mycobacterial replication. TNT induces ROS formation both by activating necroptosis and by a necroptosis‐independent mechanism. Most of the detected ROS originate in mitochondria as a consequence of opening the mitochondrial permeability transition pore. However, a significant part of ROS is produced by mechanisms independent of TNT and necroptosis. Expressing only the tnt gene in Jurkat T‐cells also induces lethal ROS formation indicating that these molecular mechanisms are not restricted to macrophages. Both the antioxidant N‐acetyl‐cysteine and replenishment of NAD⁺ by providing nicotinamide reduce ROS levels in Mtb‐infected macrophages, protect them from cell death, and restrict mycobacterial replication. Our results indicate that a host‐directed therapy combining replenishment of NAD⁺ with inhibition of necroptosis and/or antioxidants might improve the health status of TB patients and augment antibacterial TB chemotherapy.     

Structural analysis reveals DNA binding properties of Rv2827c, a hypothetical protein from Mycobacterium tuberculosis

Tuberculosis (TB) is a major global health threat caused by Mycobacterium tuberculosis (Mtb). It is further fueled by the HIV pandemic and by increasing incidences of multidrug resistant Mtb-strains. Rv2827c, a hypothetical protein from Mtb, has been implicated in the survival of Mtb in the macrophages of the host. The three-dimensional structure of Rv2827c has been determined by the three-wavelength anomalous diffraction technique using bromide-derivatized crystals and refined to a resolution of 1.93 Å. The asymmetric unit of the orthorhombic crystals contains two independent protein molecules related by a non-crystallographic translation. The tertiary structure of Rv2827c comprises two domains: an N-terminal domain displaying a winged helix topology and a C-terminal domain, which appears to constitute a new and unique fold. Based on structural homology considerations and additional biochemical evidence, it could be established that Rv2827c is a DNA-binding protein. Once the understanding of the structure-function relationship of Rv2827c extends to the function of Rv2827c in vivo, new clues for the rational design of novel intervention strategies may be obtained.