Abstract: Significance: Heme is an important prosthetic group required in wide array of functions including respiration, photosynthesis, metabolism, O<sub>2</sub> transport, xenobiotic detoxification, and peroxide production and destruction, and is an essential cofactor in proteins such as catalases, peroxidases and members of the cytochrome P450 superfamily. Importantly, bacterial heme-based sensor proteins exploit the redox chemistry of heme to sense environmental gases and the intracellular redox state of the cell. Recent Advances: The bacterial proteins FixL (<i>Rhizobium ssp.</i>), CooA (<i>Rhodospirillum rubrum</i>), EcDos (<i>E. coli</i>), RcoM (<i>Burkholderia xenovorans</i>) and particularly <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) DosS and DosT have emerged as model paradigms of environmental heme-based sensors capable of detecting multiple gases including NO, CO and O<sub>2</sub>. Critical issues: How the diatomic gases NO, CO or O<sub>2</sub> bind to heme iron to generate Fe-NO, Fe-CO and Fe-O<sub>2</sub> bonds respectively, and how the oxidation of heme iron by O<sub>2</sub> serves as a sensing mechanism that controls the activity of key proteins is complex and largely unclear. This is particularly important as many bacterial pathogens, including <i>Mtb</i> encounters three overlapping host gases (NO, CO and O<sub>2</sub>) during human infection. Future directions: Heme is an important prosthetic group that monitors the microbe's internal and external surroundings to alter signal transduction or enzymatic activation. Modern expression, metabolomic and biochemical technologies combined with <i>in vivo</i> pathogenesis studies should provide fresh insights into the mechanism of action of heme-based redox sensors.
Abstract: 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 HO 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 of M. avium infected HO-1(-/-) mice and CCR2 expression was higher in HO-1(-/-) alveolar macrophages when compared with 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.Laboratory Investigation advance online publication, 10 September 2012; doi:10.1038/labinvest.2012.125.
Abstract: Understanding the evolutionary and genomic mechanisms responsible for turning the soil-derived saprophytic mycobacteria into lethal intracellular pathogens is a critical step towards the development of strategies for the control of mycobacterial diseases. In this context, Mycobacterium indicus pranii (MIP) is of specific interest because of its unique immunological and evolutionary significance. Evolutionarily, it is the progenitor of opportunistic pathogens belonging to M. avium complex and is endowed with features that place it between saprophytic and pathogenic species. Herein, we have sequenced the complete MIP genome to understand its unique life style, basis of immunomodulation and habitat diversification in mycobacteria. As a case of massive gene acquisitions, 50.5% of MIP open reading frames (ORFs) are laterally acquired. We show, for the first time for Mycobacterium, that MIP genome has mosaic architecture. These gene acquisitions have led to the enrichment of selected gene families critical to MIP physiology. Comparative genomic analysis indicates a higher antigenic potential of MIP imparting it a unique ability for immunomodulation. Besides, it also suggests an important role of genomic fluidity in habitat diversification within mycobacteria and provides a unique view of evolutionary divergence and putative bottlenecks that might have eventually led to intracellular survival and pathogenic attributes in mycobacteria.
Abstract: 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.
Abstract: <u>Significance</u>: <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), the causative agent of tuberculosis (TB), can persist in a latent state for decades without causing overt disease. Since latent <i>Mtb</i> is refractory to current antimycobacterial drugs, the discovery and characterization of the biological mechanisms controlling the entry, maintenance and emergence from latent infection is critical to the development of novel clinical therapies. <u>Recent Advances</u>: Recently, <i>Mtb</i> WhiB3, a member of the family of intracellular iron-sulfur (Fe-S) cluster proteins has emerged as a redox sensor and effector molecule controlling several aspects of <i>Mtb</i> virulence. WhiB3 was shown to contain a 4Fe-4S cluster that specifically reacts with important host gases (O<sub>2</sub> and NO), and exogenous and endogenous metabolic signals to maintain redox balance. Notably, the concept of reductive stress emerged from studies on WhiB3. <u>Critical issues</u>: The detailed mechanism of how WhiB3 functions as an intracellular redox sensor is unknown. Sustaining <i>Mtb</i> redox balance is particularly important since the bacilli encounters a large number of redox stressors during infection, and because several antimycobacterial prodrugs are effective only upon bioreductive activation in the mycobacterial cytoplasm. <u>Future directions</u>: How <i>Mtb</i> WhiB3 monitor its internal and external surroundings and modulate endogenous oxido-reductive pathways which in turn alter <i>Mtb</i> signal transduction, nucleic acid and protein synthesis, and enzymatic activation is mostly unexplored. Modern expression, metabolomic and proteomic technologies should provide fresh insights into these yet unanswered questions.
Abstract: 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 FINDINGS: The comparative analysis of 16S rRNA sequence of MIP by using BLAST algorithm at NCBI (nr database) revealed a similarity of > or =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 > or =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. CONCLUSION: The 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.
Abstract: BACKGROUND: Evolutionary dynamics plays a central role in facilitating the mechanisms of species divergence among pathogenic and saprophytic mycobacteria. The ability of mycobacteria to colonize hosts, to proliferate and to cause diseases has evolved due to its predisposition to various evolutionary forces acting over a period of time. Mycobacterium indicus pranii (MIP), a taxonomically unknown 'generalist' mycobacterium, acts as an immunotherapeutic against leprosy and is approved for use as a vaccine against it. The large-scale field trials of this MIP based leprosy vaccine coupled with its demonstrated immunomodulatory and adjuvant property has led to human clinical evaluations of MIP in interventions against HIV-AIDS, psoriasis and bladder cancer. MIP, commercially available as 'Immuvac', is currently the focus of advanced phase III clinical trials for its antituberculosis efficacy. Thus a comprehensive analysis of MIP vis-Ã -vis evolutionary path, underpinning its immanent immunomodulating properties is of the highest desiderata. PRINCIPAL FINDINGS: Genome wide comparisons together with molecular phylogenetic analyses by fluorescent amplified fragment length polymorphism (FAFLP), enterobacterial repetitive intergenic consensus (ERIC) based genotyping and candidate orthologues sequencing revealed that MIP has been the predecessor of highly pathogenic Mycobacterium avium intracellulare complex (MAIC) that did not resort to parasitic adaptation by reductional gene evolution and therefore, preferred a free living life-style. Further analysis suggested a shared aquatic phase of MAIC bacilli with the early pathogenic forms of Mycobacterium, well before the latter diverged as 'specialists'. CONCLUSIONS/SIGNIFICANCE: This evolutionary paradigm possibly affirms to marshall our understanding about the acquisition and optimization of virulence in mycobacteria and determinants of boundaries therein.
Notes: Niyaz Ahmed and Vikram Saini - Joint First Authors
