The mycobacterial mel2 locus (mycobacterial enhanced infection locus, Rv1936-1941) is Mycobacterium marinum and M. tuberculosis specific, which can withstand reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced stress. A library of over a million compounds was screened using in silico virtual ligand screening (VLS) to identify inhibitors against the modeled structure of MelF protein expressed by melF of mel2 locus so that M. marinum’s ability to withstand ROS/RNS stress could be reduced. The top ranked 1000 compounds were further screened to identify 178 compounds to maximize the scaffold diversity by manually evaluating the interaction of each compound with the target site. M. marinum melF was cloned, expressed and purified as maltose binding protein (MBP)-tagged recombinant protein in Escherichia coli. After establishing the flavin dependent oxidoreductase activity of MelF (~ 84 kDa), the inhibitors were screened for the inhibition of enzyme activity of whole cell lysate (WCL) and the purified MelF. Amongst these, 16 compounds could significantly inhibit the enzyme activity of purified MelF. For the six best inhibitory compounds, the minimal inhibitory concentration (MIC) was determined to be 3.4–19.4 μM and 13.5–38.8 μM for M. marinum and M. tuberculosis, respectively. Similarly, the minimal bactericidal concentration (MBC) was determined to be 6.8–38.8 μM and 27–38.8 μM against M. marinum and M. tuberculosis, respectively. One compound each in combination with isoniazid (INH) also showed synergistic inhibitory effect against M. marinum and M. tuberculosis with no cytotoxicity in HeLa cells. Interestingly, these inhibitors did not display any non-specific protein-structure destabilizing effect. Such inhibitors targeting the anti-ROS/RNS machinery may facilitate the efficient killing of replicating and nonreplicating mycobacteria inside the host cells.
Protein-protein interaction (PPI) network analysis is a powerful strategy to understand M. tuberculosis (Mtb) system level physiology in the identification of hub proteins. In the present study, the PPI network of 79 Mtb toxin-antitoxin (TA) systems comprising of 167 nodes and 234 edges was investigated. The topological properties of PPI network were examined by 'Network analyzer' a cytoscape plugin app and STRING database. The key enriched biological processes and the molecular functions of Mtb TA systems were analyzed by STRING. Manual curation of the PPI data identified four proteins (i.e. Rv2762c, VapB14, VapB42 and VapC42) to possess the highest number of interacting partners. The top 15% hub proteins were identified in the PPI network by employing two statistical measures, i.e. betweenness and radiality by employing cytohubba. Insights gained from the molecular protein models of VapC9 and VapC10 are also documented.
Macrophages produce antimicrobial reactive oxygen species (ROS) and reactive nitrogen species (RNS) through NADPH oxidase (NOX2/gp91 phox ) and inducible nitric oxide synthase in response to mycobacterial infections [1]. In general, mycobacteria are resistant to ROS, but RNS inhibit growth and even kill mycobacteria within the activated macrophages. Mycobacterium tuberculosis is also continually exposed to endogenous ROS including the production of superoxide radicals as part of normal aerobic respiration [2]. In fact, M. tuberculosis has developed several defense mechanisms to counteract ROS stress, for example, lipid enriched cell wall, maintenance of cytosolic redox homeostasis, genes involved in repair and protection of DNA/protein as well as several ROS scavenger genes, including katG (Rv1908c), SodA (Rv3846), sodC (Rv0432), ahpC (Rv2428), ahpE (Rv2238c), melF (Rv1936), etc. [3,4,5]. Notably, global regulator oxyR is inactive in M. tuberculosis, but is active in other pathogenic mycobacterial species including M. marinum. Pathways involved in mycobacterial resistance to RNS include the generation of RNS scavenger genes such as noxR1 (Rv2997), noxR3 (Rv1500), dlaT (Rv2215), msrA (Rv0137c) and cysH (Rv2392), DNA repair as well as protein degradation in the proteasome [3]. Moreover, peroxynitrite is produced by SOD in the presence of H 2 O 2 and nitric oxide, connecting the two important mechanisms of oxidative and nitrosative stress-mediated bacterial cell death. M. tuberculosis possesses inherent resistance to peroxynitrite as compared with less pathogenic mycobacteria [3]. Hence, targeting anti-ROS/RNS machinery of mycobacterial cells would facilitate efficient clearance of bacteria from the invading tissue.The mycobacterial mel2 locus (mycobacterial enhanced infection locus, Rv1936-1941, size 7.9 kb), originally identified by El-Etr et al. [6], is M. marinum and M. tuberculosis (∼98% sequence homology with M. marinum) specific and is absent in other pathogenic and nonpathogenic mycobacterial species, including M. bovis, M. avium, M. leprae and M. smegmatis, which can tolerate ROS and RNS stress responses. In silico analysis of six genes of mel2 locus, in other words melF, melG, melH, etc. revealed them to be close homologs of lux genes present in Vibrio harveyi [3]. Moreover, the melF within this locus shows high similarity to luxA, a monooxygenase gene involved in resistance to ROS in bioluminescent bacteria. The mel2 locus also confers resistance to ROS/RNS stress in vitro and reveals a similar function in activated murine macrophages [3,4]. Moreover, the M. marinum mel2 mutants do not display growth defect in macrophages in presence of ROS scavengers or nitric oxide synthase inhibitors, thus demonstrating that the growth defect is dependent on the production of both ROS and RNS [4]. Interestingly, the mutation in melF of mel2 locus displays a polar effect on the downstream genes of mel2 locus as the enhanced susceptibility of melF mutant to ROS/RNS could only be partially recovered by melF alone and comp...
Various animal models are used to study the immunology, genetics and molecular biology of tuberculosis (TB) as well as for testing the new vaccines and drugs. Mice are widely used to study the immunology of chronic TB infection, while guinea pigs are used for aerosol TB infection and rabbits are used to study the lung cavitations. Cattle are natural host to Mycobacterium bovis infection, which act as a connecting link between the small laboratory animals and human counterparts for testing the vaccine efficacy. By using cattle as an experimental model, the disease outcome is understood through natural infection with M. bovis and a comparison can be made with M. tuberculosis infection. In this manuscript, the utility of cattle in understanding the progression of disease and the immunological correlates to evaluate the protective efficacy of vaccines are described.
We recently identified inhibitors targeting Mycobacterium marinum MelF (Rv1936) by in silico analysis, which exhibited bacteriostatic/bactericidal activity against M. marinum and M. tuberculosis in vitro . Herein, we evaluated the effect of best four inhibitors (# 5175552, # 6513745, # 5255829, # 9125618) obtained from the ChemBridge compound libraries, on intracellular replication and persistence of bacteria within IFN-γ activated murine RAW264.7 and human THP-1 macrophages infected with M. marinum . Inhibitors # 5175552 and # 6513745 significantly reduced ( p < 0.05) the intracellular replication of bacilli during day 7 post-infection (p.i.) within RAW264.7 and THP-1 macrophages infected at multiplicity of infection (MOI) of ~1.0. These observations were substantiated by electron microscopy, which revealed the protective effect of # 5175552 in clearing the bacilli inside murine macrophages. Strikingly, # 6513745 displayed synergism with isoniazid against M. marinum in murine macrophages, whereas # 5175552 significantly suppressed ( p < 0.05) the persistent bacilli during day 10–14 p.i. in infected RAW264.7 and THP-1 macrophages (MOI of ~ 0.1). Moreover, # 5175552 and # 6513745 were non-cytotoxic to host macrophages at both 1X and 5X MIC. Further validation of these inhibitors against M. tuberculosis -infected macrophages and animal models has potential for development as novel anti-tubercular agents.
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