SUMMARY The mechanisms by which Mycobacterium tuberculosis (Mtb) maintains metabolic equilibrium to survive during infection and upon exposure to antimycobacterial drugs are poorly characterized. Ergothioneine (EGT) and mycothiol (MSH) are the major redox buffers present in Mtb, but the contribution of EGT to Mtb redox homeostasis and virulence remains unknown. We report that Mtb WhiB3, a 4Fe-4S redox sensor protein, regulates EGT production and maintains bioenergetic homeostasis. We show that central carbon metabolism and lipid precursors regulate EGT production and that EGT modulates drug sensitivity. Notably, EGT and MSH are both essential for redox and bioenergetic homeostasis. Transcriptomic analyses of EGT and MSH mutants indicate overlapping, but distinct functions of EGT and MSH. Lastly, we show that EGT is critical for Mtb survival in both macrophages and mice. This study has uncovered a dynamic balance between Mtb redox and bioenergetic homeostasis, which critically influences Mtb drug susceptibility and pathogenicity.
Mycobacterium tuberculosis (Mtb) is a metabolically flexible pathogen that has the extraordinary ability to sense and adapt to the continuously changing host environment experienced during decades of persistent infection. Mtb is continually exposed to endogenous reactive oxygen species (ROS) as part of normal aerobic respiration, as well as exogenous ROS and reactive nitrogen species (RNS) generated by the host immune system in response to infection. The magnitude of tuberculosis (TB) disease is further amplified by exposure to xenobiotics from the environment such as cigarette smoke and air pollution, causing disruption of the intracellular prooxidant–antioxidant balance. Both oxidative and reductive stresses induce redox cascades that alter Mtb signal transduction, DNA and RNA synthesis, protein synthesis and antimycobacterial drug resistance. As reviewed in this article, Mtb has evolved specific mechanisms to protect itself against endogenously produced oxidants, as well as defend against host and environmental oxidants and reductants found specifically within the microenvironments of the lung. Maintaining an appropriate redox balance is critical to the clinical outcome because several antimycobacterial prodrugs are only effective upon bioreductive activation. Proper homeostasis of oxido-reductive systems is essential for Mtb survival, persistence and subsequent reactivation. The progress and remaining deficiencies in understanding Mtb redox homeostasis are also discussed.
Hydrogen sulfide (H 2 S) is involved in numerous pathophysiological processes and shares overlapping functions with CO and •NO. However, the importance of host-derived H 2 S in microbial pathogenesis is unknown. Here we show that Mtb-infected mice deficient in the H 2 S-producing enzyme cystathionine β-synthase (CBS) survive longer with reduced organ burden, and that pharmacological inhibition of CBS reduces Mtb bacillary load in mice. Highresolution respirometry, transcriptomics and mass spectrometry establish that H 2 S stimulates Mtb respiration and bioenergetics predominantly via cytochrome bd oxidase, and that H 2 S reverses •NO-mediated inhibition of Mtb respiration. Further, exposure of Mtb to H 2 S regulates genes involved in sulfur and copper metabolism and the Dos regulon. Our results indicate that Mtb exploits host-derived H 2 S to promote growth and disease, and suggest that host-directed therapies targeting H 2 S production may be potentially useful for the management of tuberculosis and other microbial infections.
Iron is an essential factor for the growth and virulence of Mycobacterium tuberculosis (Mtb). However, little is known about the mechanisms by which the host controls iron availability during infection. Since ferritin heavy chain (FtH) is a major intracellular source of reserve iron in the host, we hypothesized that the lack of FtH would cause dysregulated iron homeostasis to exacerbate TB disease. Therefore, we used knockout mice lacking FtH in myeloid-derived cell populations to study Mtb disease progression. We found that FtH plays a critical role in protecting mice against Mtb, as evidenced by increased organ burden, extrapulmonary dissemination, and decreased survival in Fth−/− mice. Flow cytometry analysis showed that reduced levels of FtH contribute to an excessive inflammatory response to exacerbate disease. Extracellular flux analysis showed that FtH is essential for maintaining bioenergetic homeostasis through oxidative phosphorylation. In support of these findings, RNAseq and mass spectrometry analyses demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo. Further, we show that FtH deficiency leads to iron dysregulation through the hepcidin–ferroportin axis during infection. To assess the clinical significance of our animal studies, we performed a clinicopathological analysis of iron distribution within human TB lung tissue and showed that Mtb severely disrupts iron homeostasis in distinct microanatomic locations of the human lung. We identified hemorrhage as a major source of metabolically inert iron deposition. Importantly, we observed increased iron levels in human TB lung tissue compared to healthy tissue. Overall, these findings advance our understanding of the link between iron-dependent energy metabolism and immunity and provide new insight into iron distribution within the spectrum of human pulmonary TB. These metabolic mechanisms could serve as the foundation for novel host-directed strategies.
Background: Personal Protective Equipment (PPE) is required to safely work with biological agents of bacterial (i.e. Mycobacterium tuberculosis) or viral origin (Ebola and SARS). COVID-19 pandemic especially has created unforeseen public health challenges including a global shortage of PPE needed for the safety of health care workers (HCWs). Although sufficient stocks of PPE are currently available, their critical shortage may develop soon due to increase in demand and depletion of existing supply lines. To empower our HCWs and ensure their continued protection, proactive measures are urgently required to develop procedures to safely decontaminate the PPEs to allow their "selective reuse" during contingency situations. Methods: Herein, we have successfully developed a decontamination method based on vaporized hydrogen peroxide (VHP). We have used a range of concentration of hydrogen peroxide to disinfect PPE (coveralls, face-shields, and N-95 masks). To ensure a proper disinfection, we have evaluated three biological indicators namely Escherichia coli, Mycobacterium smegmatis and spores of Bacillus stearothermophilus, considered as the gold standard for disinfection processes. We next evaluated the impact of repeated VHP treatment on physical features, permeability, and fabric integrity of coveralls and N-95 masks. Next, we performed Scanning Electron Microscopy (SEM) to evaluate microscopic changes in fiber thickness of N-95 masks, melt blown layer or coverall body suits. Considering the fact that any disinfection procedure should be able to meet local requirements, our study included various regionally procured N-95 masks and coveralls available at our institute All India Institute of Medical Sciences (AIIMS), New Delhi, India. Lastly, the practical utility of VHP method developed herein was ascertained by operationalizing a dedicated research facility disinfecting used PPE during COVID-19. Results: Our prototype studies show that a single VHP cycle (7-8% Hydrogen peroxide) could disinfect PPE and PPE housing room of about 1200 cubic feet (length10 ft × breadth 10 ft × height 12 ft) in less than 10 min, as noted by a complete loss of B. stearothermophilus spore revival. The results are consistent and reproducible as tested in over 10 cycles in our settings. Further, repeated VHP treatment did not result in any physical tear, deformity or other
Non-tuberculous mycobacterial (NTM) infections occur in both immunocompromised and immunocompetent hosts and are an increasingly recognized cause of morbidity and mortality. The hallmark of pulmonary mycobacterial infections is the formation of granuloma in the lung. Our study focuses on the role of heme oxygenase-1 (HO-1), a cytoprotective enzyme, in the regulation of granuloma development and maturation following infection with Mycobacterium avium. We examined the role of HO-1 in regulating monocyte chemoattractant protein-1 (MCP-1) and chemokine receptor 2 (CCR2), two molecules involved in monocyte-macrophage cell trafficking after infection. We showed that RAW 264.7 mouse monocytes exposed to M. avium expressed HO-1 and MCP-1. Inhibition of heme oxygenase by zinc protoporphyrin-IX led to inhibition of MCP-1 and increased expression of CCR2, its cognate receptor. HO-1-/- mice did not develop organized granuloma in their lungs, had higher lung colony forming unit of M. avium when infected with intratracheal M. avium, and had loose collections of inflammatory cells in the lung parenchyma. Mycobacteria were found only inside defined granulomas but not outside granuloma in the lungs of HO-1+/+ mice. In HO-1-/- mice, mycobacteria were also found in the liver and spleen and showed increased mortality. Peripheral blood monocytes isolated from GFP+ mice and given intravenously to HO-1+/+ mice localized into tight granulomas, while in HO-1-/- mice they remained diffusely scattered in areas of parenchymal inflammation. Higher MCP-1 levels were found in bronchoalveolar lavage fluid (BALF) of M. avium infected HO-1-/- mice and CCR2 expression was higher in HO-1-/- alveolar macrophages when compared to HO-1+/+ mice. CCR2 expression localized to granuloma in HO-1+/+ mice but not in the HO-1-/- mice. These findings strongly suggest that HO-1 plays a protective role in the control of M. avium infection.
Background Mycobacterium indicus pranii (MIP), popularly known as Mw, is a cultivable, non-pathogenic organism, which, based on its growth and metabolic properties, is classified in Runyon Group IV along with M. fortuitum, M. smegmatis and M. vaccae. The novelty of this bacterium was accredited to its immunological ability to undergo antigen driven blast transformation of leukocytes and delayed hypersensitivity skin test in leprosy patients, a disease endemic in the Indian sub-continent. Consequently, MIP has been extensively evaluated for its biochemical and immunological properties leading to its usage as an immunomodulator in leprosy and tuberculosis patients. However, owing to advances in sequencing and culture techniques, the citing of new strains with almost 100% similarity in the sequences of marker genes like 16S rRNA, has compromised the identity of MIP as a novel species. Hence, to define its precise taxonomic position, we have carried out polyphasic taxonomic studies on MIP that integrate its phenotypic, chemotaxonomic and molecular phylogenetic attributes.Methodology/Principal FindingsThe comparative analysis of 16S rRNA sequence of MIP by using BLAST algorithm at NCBI (nr database) revealed a similarity of ≥99% with M. intracellulare, M. arosiense, M. chimaera, M. seoulense, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis and M. bohemicum. Further analysis with other widely used markers like rpoB and hsp65 could resolve the phylogenetic relationship between MIP and other closely related mycobacteria apart from M. intracellulare and M. chimaera, which shares ≥99% similarity with corresponding MIP orthologues. Molecular phylogenetic analysis, based on the concatenation of candidate orthologues of 16S rRNA, hsp65 and rpoB, also substantiated its distinctiveness from all the related organisms used in the analysis excluding M. intracellulare and M. chimaera with which it exhibited a close proximity. This necessitated further analysis of MIP with more sensitive and segregating parameters to ascertain its precise taxonomic position as a new species. The analysis of MIP and its comparison with other mycobacterial reference strains based on cellular and biochemical features, growth characteristics and chemotaxonomic studies like FAME profiling confirmed that MIP is uniquely endowed with diverse metabolic attributes that effectively distinguishes it from all the closely related mycobacteria including M. intracellulare and M. chimaera.ConclusionThe results presented in this study coupled with the non-pathogenic nature and different biochemical and immunomodulatory properties of MIP affirm it as a distinct species belonging to M. avium complex (MAC). It is further proposed to use an earlier suggested name Mycobacterium indicus pranii for this newly established mycobacterial species. This study also exemplifies the growing need for a uniform, consensus based broader polyphasic frame work for the purpose of taxonomy and speciation, particularly in the genus Mycobacterium.
The control of antimicrobial resistance (AMR) seems to have come to a dead end. The major consequences of the use and abuse of antibacterial drugs are the development of resistant strains due to genetic mutability of both pathogenic and nonpathogenic microorganisms. We, herein, report the synthesis, characterization and biological activities of coumarin-thiazole-pyrazole (CTP) molecular hybrids with an effort to explore and overcome the increasing antimicrobial resistance. The compounds were characterized by analyzing their IR, Mass, 1H and13C NMR spectral data and elemental analysis. The in vitro antimicrobial activity of the synthesized compounds was investigated against various pathogenic strains; the results obtained were further explained with the help of DFT and molecular orbital calculations. Compound 1b and 1f displayed good antimicrobial activity and synergistic effects when used with kanamycin and amphotericin B. Furthermore, in vitro cytotoxicity of compounds 1b and 1f were studied against HeLa cells (cervical cancer cell) and Hek-293 cells. The results of molecular docking study were used to better rationalize the action and prediction of the binding modes of these compounds.
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