Inhibition of ABCG2 efflux pumps renders the Mycobacterium tuberculosis hiding in mesenchymal stem cells responsive to antibiotic treatment.

The lengthy TB chemotherapeutic regimen, resulting in the emergence of drug resistance strains, poses a serious problem in the cure of the disease. Further, one-quarter of the world's population is infected with dormant M.tb, which creates a lifetime risk of reactivation. M.tb has a remarkable tendency to escape the host immune responses by hiding in unconventional niches. Recent studies have shown that bone-marrow mesenchymal stem cells (BM-MSCs) can serve as a reservoir of the pathogen and have been suggested to keep them beyond the reach of anti-TB drugs. In this study, we have shown that M.tb infects and grows inside BM-MSCs and were unresponsive to the anti-TB drugs rifampicin and isoniazid when compared to the pathogen residing inside THP-1 macrophages. It was further shown that the ABCG2 efflux pumps of the BM-MSCs were upregulated upon exposure to rifampicin, which may be the contributing factor for the antibiotic unresponsiveness of the bacteria inside these cells. Subsequently, it was shown that inhibition of ABCG2 efflux pumps along with administration of anti-TB drugs led to an increased susceptibility and consequently an enhanced killing of the M.tb inside BM-MSCs. These findings for the first time show that the MIC99 values of anti-TB drugs increase many folds for the M.tb residing in BM-MSCs as compared to M.tb residing inside macrophages and the involvement of ABCG2 efflux pumps in this phenomenon. Our study substantiates that these BM-MSCs acts as a useful niche for M.tb wherein they can survive by escaping the antibiotic assault that can be attributed to the host ABCG2 efflux pumps. Inhibiting these efflux pumps can be an attractive adjunctive chemotherapy to eliminate the bacteria from this protective niche.

Unraveling the role of the transcriptional regulator VirS in low pH-induced responses of Mycobacterium tuberculosis and identification of VirS inhibitors.

The ability of Mycobacterium tuberculosis to respond and adapt to various stresses such as oxygen/nitrogen radicals and low pH inside macrophages is critical for the persistence of this human pathogen inside its host. We have previously shown that an AraC/XylS-type transcriptional regulator, VirS, which is induced in low pH, is involved in remodeling the architecture of the bacterial cell envelope. However, how VirS influences gene expression to coordinate these pH responses remains unclear. Here, using a genetic biosensor of cytoplasmic pH, we demonstrate that VirS is required for the intracellular pH maintenance in response to acidic stress and inside acidified macrophages. Furthermore, we observed that VirS plays an important role in blocking phagosomal-lysosomal fusions. Transcriptomics experiments revealed that VirS affects the expression of genes encoding metabolic enzymes, cell-wall envelope proteins, efflux pumps, ion transporters, detoxification enzymes, and transcriptional regulators expressed under low-pH stress. Employing electrophoretic mobility-shift assays, DNA footprinting, and in silico analysis, we identified a DNA sequence to which VirS binds and key residues in VirS required for its interaction with DNA. A significant role of VirS in M. tuberculosis survival in adverse conditions suggested it as a potential anti-mycobacterial drug target. To that end, we identified VirS inhibitors in a virtual screen; the top hit compounds inhibited its DNA-binding activity and also M. tuberculosis growth in vitro and inside macrophages. Our findings establish that VirS mediates M. tuberculosis responses to acidic stress and identify VirS-inhibiting compounds that may form the basis for developing more effective anti-mycobacterial agents.

Effect of Heavy Mass Ion (Gold) and Light Mass Ion (Boron) Irradiation on Microstructure of Tungsten.

The difference in the defect structures produced by different ion masses in a tungsten lattice is investigated using 80 MeV Au7+ ions and 10 MeV B3+ ions. The details of the defects produced by ions in recrystallized tungsten foil samples are studied using transmission electron microscopy. Dislocations of type b = 1/2[111] and [001] were observed in the analysis. While highly energetic gold ion produced small clusters of defects with very few dislocation lines, boron has produced large and sparse clusters with numerous dislocation lines. The difference in the defect structures could be due to the difference in separation between primary knock-on atoms produced by gold and boron ions.

Identification of Mycobacterium tuberculosis BioA inhibitors by using structure-based virtual screening.

BACKGROUND: 7,8-Diaminopelargonic acid synthase (BioA), an enzyme of biotin biosynthesis pathway, is a well-known promising target for anti-tubercular drug development. METHODS: In this study, structure-based virtual screening was employed against the active site of BioA to identify new chemical entities for BioA inhibition and top ranking compounds were evaluated for their ability to inhibit BioA enzymatic activity. RESULTS: Seven compounds inhibited BioA enzymatic activity by greater than 60% at 100 µg/mL with most potent compounds being A36, A35 and A65, displaying IC50 values of 10.48 µg/mL (28.94 µM), 33.36 µg/mL (88.16 µM) and 39.17 µg/mL (114.42 µM), respectively. Compounds A65 and A35 inhibited Mycobacterium tuberculosis (M. tuberculosis) growth with MIC90 of 20 µg/mL and 80 µg/mL, respectively, whereas compound A36 exhibited relatively weak inhibition of M. tuberculosis growth (83% inhibition at 200 µg/mL). Compound A65 emerged as the most potent compound identified in our study that inhibited BioA enzymatic activity and growth of the pathogen and possessed drug-like properties. CONCLUSION: Our study has identified a few hit molecules against M. tuberculosis BioA that can act as potential candidates for further development of potent anti-tubercular therapeutic agents.

An attenuated quadruple gene mutant of Mycobacterium tuberculosis imparts protection against tuberculosis in guinea pigs.

Previously we had developed a triple gene mutant of Mycobacterium tuberculosis (MtbΔmms) harboring disruption in three genes, namely mptpA, mptpB and sapM Though vaccination with MtbΔmms strain induced protection in the lungs of guinea pigs, the mutant strain failed to control the hematogenous spread of the challenge strain to the spleen. Additionally, inoculation with MtbΔmms resulted in some pathological damage to the spleens in the early phase of infection. In order to generate a strain that overcomes the pathology caused by MtbΔmms in spleen of guinea pigs and controls dissemination of the challenge strain, MtbΔmms was genetically modified by disrupting bioA gene to generate MtbΔmmsb strain. Further, in vivo attenuation of MtbΔmmsb was evaluated and its protective efficacy was assessed against virulent M. tuberculosis challenge in guinea pigs. MtbΔmmsb mutant strain was highly attenuated for growth and virulence in guinea pigs. Vaccination with MtbΔmmsb mutant generated significant protection in comparison to sham-immunized animals at 4 and 12 weeks post-infection in lungs and spleen of infected animals. However, the protection imparted by MtbΔmmsb was significantly less in comparison to BCG immunized animals. This study indicates the importance of attenuated multiple gene deletion mutants of M. tuberculosis for generating protection against tuberculosis.

Boosting with recombinant MVA expressing M. tuberculosis α-crystallin antigen augments the protection imparted by BCG against tuberculosis in guinea pigs.

Tuberculosis (TB) is one of the major causes of mortality all over the globe. BCG, the only vaccine available against this disease has been successful in preventing the severe forms of childhood TB. However, the unsatisfactory performance of BCG in controlling the adult pulmonary tuberculosis has made the development of an effective vaccine against M. tuberculosis a prime objective of the TB research. In this study, a genetically stable, marker-free recombinant MVA expressing α-crystallin of M. tuberculosis (rMVA.acr) was generated which was further evaluated for its ability to impart protection as a booster vaccine against tuberculosis in a heterologous prime boost approach. Our results demonstrated that intradermal delivery of rMVA.acr was able to efficiently boost the BCG induced protection against M. tuberculosis infection in guinea pigs by significantly reducing the pulmonary bacillary load (1.27 log10 fewer bacilli) in comparison to BCG vaccination alone. In addition, boosting BCG vaccinated animals with intramuscular delivery of rMVA.acr resulted in significantly superior protective efficacy in both lungs and spleen with 0.83 log10 and 0.74 log10 CFU fewer bacilli, respectively, when compared to animals vaccinated with BCG only. These findings establish the promise of this prime-boost strategy involving rMVA.acr in enhancing the efficacy of BCG.

Virtual Screening, pharmacophore development and structure based similarity search to identify inhibitors against IdeR, a transcription factor of Mycobacterium tuberculosis.

ideR, an essential gene of Mycobacterium tuberculosis, is an attractive drug target as its conditional knockout displayed attenuated growth phenotype in vitro and in vivo. To the best of our knowledge, no inhibitors of IdeR are identified. We carried out virtual screening of NCI database against the IdeR DNA binding domain followed by inhibition studies using EMSA. Nine compounds exhibited potent inhibition with NSC 281033 (I-20) and NSC 12453 (I-42) exhibiting IC50 values of 2 µg/ml and 1 µg/ml, respectively. We then attempted to optimize the leads firstly by structure based similarity search resulting in a class of inhibitors based on I-42 containing benzene sulfonic acid, 4-hydroxy-3-[(2-hydroxy-1-naphthalenyl) azo] scaffold with 4 molecules exhibiting IC50 ≤ 10 µg/ml. Secondly, optimization included development of energy based pharmacophore and screening of ZINC database followed by docking studies, yielding a molecule with IC50 of 60 µg/ml. More importantly, a five-point pharmacophore model provided insight into the features essential for IdeR inhibition. Five molecules with promising IC50 values also inhibited M. tuberculosis growth in broth culture with MIC90 ranging from 17.5 µg/ml to 100 µg/ml and negligible cytotoxicity in various cell lines. We believe our work opens up avenues for further optimization studies.

bioA mutant of Mycobacterium tuberculosis shows severe growth defect and imparts protection against tuberculosis in guinea pigs.

Owing to the devastation caused by tuberculosis along with the unsatisfactory performance of the Bacillus Calmette-Guérin (BCG) vaccine, a more efficient vaccine than BCG is required for the global control of tuberculosis. A number of studies have demonstrated an essential role of biotin biosynthesis in the growth and survival of several microorganisms, including mycobacteria, through deletion of the genes involved in de novo biotin biosynthesis. In this study, we demonstrate that a bioA mutant of Mycobacterium tuberculosis (MtbΔbioA) is highly attenuated in the guinea pig model of tuberculosis when administered aerogenically as well as intradermally. Immunization with MtbΔbioA conferred significant protection in guinea pigs against an aerosol challenge with virulent M. tuberculosis, when compared with the unvaccinated animals. Booster immunization with MtbΔbioA offered no advantage over a single immunization. These experiments demonstrate the vaccinogenic potential of the attenuated M. tuberculosis bioA mutant against tuberculosis.

Differential Roles of Iron Storage Proteins in Maintaining the Iron Homeostasis in Mycobacterium tuberculosis.

Ferritins and bacterioferritins are iron storage proteins that represent key players in iron homeostasis. Several organisms possess both forms of ferritins, however, their relative physiological roles are less understood. Mycobacterium tuberculosis possesses both ferritin (BfrB) and bacterioferritin (BfrA), playing an essential role in its pathogenesis as reported by us earlier. This study provides insights into the role of these two proteins in iron homeostasis by employing M. tuberculosis bfr mutants. Our data suggests that BfrA is required for efficient utilization of stored iron under low iron conditions while BfrB plays a crucial role as the major defense protein under excessive iron conditions. We show that these two proteins provide protection against oxidative stress and hypoxia. Iron incorporation study showed that BfrB has higher capacity for storing iron than BfrA, which augurs well for efficient iron quenching under iron excess conditions. Moreover, iron release assay demonstrated that BfrA has 3 times superior ability to release stored iron emphasizing its requirement for efficient iron release under low iron conditions, facilitated by the presence of heme. Thus, for the first time, our observations suggest that the importance of BfrA or BfrB separately might vary depending upon the iron situation faced by the cell.

Differential proteomics approach to identify putative protective antigens of Mycobacterium tuberculosis presented during early stages of macrophage infection and their evaluation as DNA vaccines.

Unsatisfactory performance of the existing BCG vaccines, especially against the adult pulmonary disease, has urged the need for an effective vaccine against tuberculosis (TB). In this study, we employed differential proteomics to obtain a list of antigens as potential vaccine candidates. Bacterial epitopes being presented at early stages on MHC class I and class II molecules of macrophages infected with Mycobacterium tuberculosis (M. tb) were identified using iTRAQ labelling and reverse phase LC-MS/MS. The putative vaccine candidates, thus identified, were tested as plasmid DNA vaccines in mice to ascertain their protective efficacy against the aerosolized M. tb challenge, based on their ability to reduce the bacterial load in the lungs of infected mice. Here, we observed that 4 out of the 17 selected antigens imparted significant protection against the challenge of M. tb. The four shortlisted antigens were further assessed in a more stringent guinea pig model, where too, they demonstrated.significant protection. It concludes that combining a proteomics approach with the in vivo assessment of vaccine candidates in animal models can be valuable in identifying new potential candidates to expand the antigenic repertoire for novel vaccines against TB.

Comparative analyses of nonpathogenic, opportunistic, and totally pathogenic mycobacteria reveal genomic and biochemical variabilities and highlight the survival attributes of Mycobacterium tuberculosis.

UNLABELLED: Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size--their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets. IMPORTANCE: The complete sequence analysis of Mycobacterium indicus pranii, a novel species of Mycobacterium shown earlier to have strong immunomodulatory properties and currently in use for the treatment of leprosy, places it evolutionarily at the point of transition to pathogenicity. With the purpose of establishing the importance of M. indicus pranii in providing insight into the virulence mechanism of tuberculous and nontuberculous mycobacteria, we carried out comparative genomic and proteomic analyses of 44 mycobacterial species representing nonpathogenic (NP), opportunistic (OP), and totally pathogenic (TP) mycobacteria. Our results clearly placed M. indicus pranii as an ancestor of the M. avium complex. Analyses of comparative metabolic pathways between M. indicus pranii (NP), M. tuberculosis (TP), and M. intracellulare (OP) pointed to the presence of novel alternative pathways in M. tuberculosis with implications for pathogenesis and survival in the human host and identification of new drug targets.

Gene cooption in mycobacteria and search for virulence attributes: comparative proteomic analyses of Mycobacterium tuberculosis, Mycobacterium indicus pranii and other mycobacteria.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a leading infectious disease taking one human life every 15s globally. Mycobacterium undergoes reductive evolution; the ancestors have bigger genome size and rich in metabolic pathways. Mycobacterium indicus pranii (MIP) is placed much above Mycobacterium tuberculosis (M.tb) in evolutionary scale and is a non-pathogenic, saprophytic mycobacterium. Our in silico comparative proteomic analyses of virulence factors of M.tb and their homologs in 12 different Mycobacterial species, including MIP, point toward gene cooption as an important mechanism in evolution of mycobacteria. We propose that adaptive changes in niche factors of non-pathogenic mycobacterium, together with novel gene acquisitions, are key players in the evolution of pathogenicity. Antigenic analyses between M.tb and MIP highlighted the importance of PE/PPE family in host immunomodulation, further supporting the likely potential of MIP as an effective vaccine against TB.

Synthesis of novel 1,2,3-triazole derivatives of isoniazid and their in vitro and in vivo antimycobacterial activity evaluation.

We report herein the synthesis and antimycobacterial activity of 1,2,3-triazole derivatives of isoniazid. Most of the compounds exhibited potent activity against Mycobacterium tuberculosis H37Rv strain with MIC99 values ranging from 0.195 to 1.56 µM in vitro. One compound showed better in vitro activity than the reference, whereas five compounds were equally potent to the reference compound isoniazid. The cytotoxicity of these compounds was studied against THP-1 cell line and no toxicity was observed even at 50 µM concentration. The compound with most potent in vitro activity was evaluated for in vivo in murine model of tuberculosis and significantly reduced bacillary load in both lungs and spleen at 10 weeks post-treatment. However this clearance effect was more pronounced in the case of spleen. Molecular docking and molecular dynamics simulations have been performed using two targets 2IDZ 1 (wild type Enoyl-acyl-carrier-protein reductase) and 4DQU 2 (mutant type Enoyl-acyl-carrier-protein reductase) to study the binding orientation and stability of the compound 47. Docking studies proved compound 47 fit well into the binding pocket of both the targets. Molecular dynamic simulations concluded that the highest active compound 47 in complex with 4DQU was more stable when compared to the 2IDZ. We believe that further optimization of these molecules may lead to potent anti-tubercular agents.

Apurinic/apyrimidinic endonucleases of Mycobacterium tuberculosis protect against DNA damage but are dispensable for the growth of the pathogen in guinea pigs.

In host cells, Mycobacterium tuberculosis encounters an array of reactive molecules capable of damaging its genome. Non-bulky DNA lesions are the most common damages produced on the exposure of the pathogen to reactive species and base excision repair (BER) pathway is involved in the repair of such damage. During BER, apurinic/apyrimidinic (AP) endonuclease enzymes repair the abasic sites that are generated after spontaneous DNA base loss or by the action of DNA glycosylases, which if left unrepaired lead to inhibition of replication and transcription. However, the role of AP endonucleases in imparting protection against DNA damage and in the growth and pathogenesis of M.tuberculosis has not yet been elucidated. To demonstrate the biological significance of these enzymes in M.tuberculosis, it would be desirable to disrupt the relevant genes and evaluate the resulting mutants for their ability to grow in the host and cause disease. In this study, we have generated M.tuberculosis mutants of the base excision repair (BER) system, disrupted in either one (MtbΔend or MtbΔxthA) or both the AP endonucleases (MtbΔendΔxthA). We demonstrate that these genes are crucial for bacteria to withstand alkylation and oxidative stress in vitro. In addition, the mutant disrupted in both the AP endonucleases (MtbΔendΔxthA) exhibited a significant reduction in its ability to survive inside human macrophages. However, infection of guinea pigs with either MtbΔend or MtbΔxthA or MtbΔendΔxthA resulted in the similar bacillary load and pathological damage in the organs as observed in the case of infection with wild-type M.tuberculosis. The implications of these observations are discussed.