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Sample records for active mycobacterium tuberculosis

  1. Mycobacterium tuberculosis prevents inflammasome activation.

    PubMed

    Master, Sharon S; Rampini, Silvana K; Davis, Alexander S; Keller, Christine; Ehlers, Stefan; Springer, Burkhard; Timmins, Graham S; Sander, Peter; Deretic, Vojo

    2008-04-17

    Mycobacterium tuberculosis (Mtb) parasitizes host macrophages and subverts host innate and adaptive immunity. Several cytokines elicited by Mtb are mediators of mycobacterial clearance or are involved in tuberculosis pathology. Surprisingly, interleukin-1beta (IL-1beta), a major proinflammatory cytokine, has not been implicated in host-Mtb interactions. IL-1beta is activated by processing upon assembly of the inflammasome, a specialized inflammatory caspase-activating protein complex. Here, we show that Mtb prevents inflammasome activation and IL-1beta processing. An Mtb gene, zmp1, which encodes a putative Zn(2+) metalloprotease, is required for this process. Infection of macrophages with zmp1-deleted Mtb triggered activation of the inflammasome, resulting in increased IL-1beta secretion, enhanced maturation of Mtb containing phagosomes, improved mycobacterial clearance by macrophages, and lower bacterial burden in the lungs of aerosol-infected mice. Thus, we uncovered a previously masked role for IL-1beta in the control of Mtb and a mycobacterial system that prevents inflammasome and, therefore, IL-1beta activation.

  2. Oxadiazoles Have Butyrate-Specific Conditional Activity against Mycobacterium tuberculosis

    PubMed Central

    Early, Julie V.; Casey, Allen; Martinez-Grau, Maria Angeles; Gonzalez Valcarcel, Isabel C.; Vieth, Michal; Ollinger, Juliane; Bailey, Mai Ann; Alling, Torey; Files, Megan; Ovechkina, Yulia

    2016-01-01

    Mycobacterium tuberculosis is a global pathogen of huge importance which can adapt to several host niche environments in which carbon source availability is likely to vary. We developed and ran a phenotypic screen using butyrate as the sole carbon source to be more reflective of the host lung environment. We screened a library of ∼87,000 small compounds and identified compounds which demonstrated good antitubercular activity against M. tuberculosis grown with butyrate but not with glucose as the carbon source. Among the hits, we identified an oxadiazole series (six compounds) which had specific activity against M. tuberculosis but which lacked cytotoxicity against mammalian cells. PMID:27044545

  3. Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis

    PubMed Central

    2016-01-01

    We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells. PMID:27144688

  4. A Focused Screen Identifies Antifolates with Activity on Mycobacterium tuberculosis

    PubMed Central

    Kumar, Anuradha; Colmenarejo, Gonzalo; Pérez, Esther; Gonzalez, Ruben R.; Torres, Pedro; Calvo, David; Gómez, Ruben M.; Ortega, Fátima; Jiménez, Elena; Gabarro, Raquel C.; Rullás, Joaquín; Ballell, Lluis

    2015-01-01

    Antifolates are widely used to treat several diseases but are not currently used in the first-line treatment of tuberculosis, despite evidence that some of these molecules can target Mycobacterium tuberculosis (Mtb) bacilli in vitro. To identify new antifolate candidates for animal-model efficacy studies of tuberculosis, we paired knowledge and tools developed in academia with the infrastructure and chemistry resources of a large pharmaceutical company. Together we curated a focused library of 2508 potential antifolates, which were then tested for activity against live Mtb. We identified 210 primary hits, confirmed the on-target activity of potent compounds, and now report the identification and characterization of 5 hit compounds, representative of 5 different chemical scaffolds. These antifolates have potent activity against Mtb and represent good starting points for improvement that could lead to in vivo efficacy studies. PMID:26771003

  5. Indoleamides are active against drug-resistant Mycobacterium tuberculosis

    PubMed Central

    Lun, Shichun; Guo, Haidan; Onajole, Oluseye K.; Pieroni, Marco; Gunosewoyo, Hendra; Chen, Gang; Tipparaju, Suresh K.; Ammerman, Nicole C.; Kozikowski, Alan P.; Bishai, William R.

    2014-01-01

    Responsible for nearly two million deaths each year, the infectious disease tuberculosis remains a serious global health challenge. The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis confounds control efforts, and new drugs with novel molecular targets are desperately needed. Here we describe lead compounds, the indoleamides, with potent activity against both drug-susceptible and drug-resistant strains of M. tuberculosis by targeting the mycolic acid transporter MmpL3. We identify a single mutation in mmpL3 which confers high resistance to the indoleamide class while remaining susceptible to currently used first- and second-line tuberculosis drugs, indicating a lack of cross-resistance. Importantly, an indoleamide derivative exhibits dose-dependent anti-mycobacterial activity when orally administered to M. tuberculosis-infected mice. The bioavailability of the indoleamides, combined with their ability to kill tubercle bacilli, indicates great potential for translational developments of this structure class for the treatment of drug-resistant tuberculosis. PMID:24352433

  6. Anti-Mycobacterium tuberculosis activity of fungus Phomopsis stipata

    PubMed Central

    de Prince, Karina Andrade; Sordi, Renata; Pavan, Fernando Rogério; Barreto Santos, Adolfo Carlos; Araujo, Angela R.; Leite, Sergio R.A.; Leite, Clarice Q. F.

    2012-01-01

    Our purpose was to determine the anti-Mycobacterium tuberculosis activity of the metabolites produced by the endophitic fungus Phomopsis stipata (Lib.) B. Sutton, (Diaporthaceae), cultivated in different media. The antimycobacterial activity was assessed through the Resazurin Microtiter Assay (REMA) and the cytotoxicity test performed on macrophage cell line. The extracts derived from fungi grown on Corn Medium and Potato Dextrose Broth presented the smallest values of Minimum Inhibitory Concentration (MIC) and low cytotoxicity, which implies a high selectivity index. This is the first report on the chemical composition and antitubercular activity of metabolites of P. stipata, as well as the influence of culture medium on these properties. PMID:24031821

  7. Native New Zealand plants with inhibitory activity towards Mycobacterium tuberculosis

    PubMed Central

    2010-01-01

    Background Plants have long been investigated as a source of antibiotics and other bioactives for the treatment of human disease. New Zealand contains a diverse and unique flora, however, few of its endemic plants have been used to treat tuberculosis. One plant, Laurelia novae-zelandiae, was reportedly used by indigenous Maori for the treatment of tubercular lesions. Methods Laurelia novae-zelandiae and 44 other native plants were tested for direct anti-bacterial activity. Plants were extracted with different solvents and extracts screened for inhibition of the surrogate species, Mycobacterium smegmatis. Active plant samples were then tested for bacteriostatic activity towards M. tuberculosis and other clinically-important species. Results Extracts of six native plants were active against M. smegmatis. Many of these were also inhibitory towards M. tuberculosis including Laurelia novae-zelandiae (Pukatea). M. excelsa (Pohutukawa) was the only plant extract tested that was active against Staphylococcus aureus. Conclusions Our data provide support for the traditional use of Pukatea in treating tuberculosis. In addition, our analyses indicate that other native plant species possess antibiotic activity. PMID:20537175

  8. Rv3168 phosphotransferase activity mediates kanamycin resistance in Mycobacterium tuberculosis.

    PubMed

    Ahn, Jae-Woo; Kim, Kyung-Jin

    2013-11-28

    Tuberculosis is a worldwide epidemic disease caused by Mycobacterium tuberculosis, with an estimated one-third of the human population currently affected. Treatment of this disease with aminoglycoside antibiotics has become less effective owing to antibiotic resistance. Recent determination of the crystal structure of the M. tuberculosis Rv3168 protein suggests a structure similar to that of Enterococcus faecalis APH(3')-IIIa, and that this protein may be an aminoglycoside phosphotransferase. To determine whether Rv3168 confers antibiotic resistance against kanamycin, we performed dose-response antibiotic resistance experiments using kanamycin. Expression of the Rv3168 protein in Escherichia coli conferred antibiotic resistance against 100 μM kanamycin, a concentration that effected cell growth arrest in the parental E. coli strain and an E. coli strain expressing the Rv3168(D249A) mutant, in which the catalytic Asp249 residue was mutated to alanine. Furthermore, we detected phosphotransferase activity of Rv3168 against kanamycin as a substrate. Moreover, docking simulation of kanamycin into the Rv3168 structure suggests that kanamycin fits well into the substrate binding pocket of the protein, and that the phosphorylation-hydroxyl-group of kanamycin was located at a position similar to that in E. faecalis APH(3')-IIIa. On the basis of these results, we suggest that the Rv3168 mediates kanamycin resistance in M. tuberculosis, likely through phosphotransferase targeting of kanamycin.

  9. LAG3 expression in active Mycobacterium tuberculosis infections.

    PubMed

    Phillips, Bonnie L; Mehra, Smriti; Ahsan, Muhammad H; Selman, Moises; Khader, Shabaana A; Kaushal, Deepak

    2015-03-01

    Mycobacterium tuberculosis (MTB) is a highly successful pathogen because of its ability to persist in human lungs for long periods of time. MTB modulates several aspects of the host immune response. Lymphocyte-activation gene 3 (LAG3) is a protein with a high affinity for the CD4 receptor and is expressed mainly by regulatory T cells with immunomodulatory functions. To understand the function of LAG3 during MTB infection, a nonhuman primate model of tuberculosis, which recapitulates key aspects of natural human infection in rhesus macaques (Macaca mulatta), was used. We show that the expression of LAG3 is highly induced in the lungs and particularly in the granulomatous lesions of macaques experimentally infected with MTB. Furthermore, we show that LAG3 expression is not induced in the lungs and lung granulomas of animals exhibiting latent tuberculosis infection. However, simian immunodeficiency virus-induced reactivation of latent tuberculosis infection results in an increased expression of LAG3 in the lungs. This response is not observed in nonhuman primates infected with non-MTB bacterial pathogens, nor with simian immunodeficiency virus alone. Our data show that LAG3 was expressed primarily on CD4(+) T cells, presumably by regulatory T cells but also by natural killer cells. The expression of LAG3 coincides with high bacterial burdens and changes in the host type 1 helper T-cell response.

  10. Anaerobic incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis.

    PubMed

    Wade, Mary Margaret; Zhang, Ying

    2004-08-01

    Pyrazinamide (PZA) is an unconventional front line tuberculosis drug characterized by high in vivo sterilizing activity, but poor in vitro activity. This disparity in PZA activity may reflect differences between the in vivo tissue environment and in vitro culture conditions. This study examined the effect of anaerobic conditions, which exist in granulomatous lesions in vivo, on PZA activity in vitro. Low oxygen enhanced the activity of PZA against Mycobacterium tuberculosis, with anaerobic conditions resulting in greater enhancement than microaerobic conditions. ATPase and respiratory chain enzyme inhibitors enhanced PZA activity under normal atmospheric conditions, but not under anaerobic conditions. Furthermore, the inhibitors did not enhance isoniazid or rifampicin activity. Nitrate as an alternative electron acceptor antagonized PZA activity under anaerobic conditions. These findings provide further support for a proposed mechanism of action of PZA in which the active form of PZA (pyrazinoic acid) depletes the membrane energy reserve. They also provide another explanation for the higher sterilizing activity of PZA within in vivo lesions with low oxygen than under in vitro drug susceptibility testing conditions with ambient oxygen.

  11. Mycobacterium tuberculosis 19-kDa lipoprotein promotes neutrophil activation.

    PubMed

    Neufert, C; Pai, R K; Noss, E H; Berger, M; Boom, W H; Harding, C V

    2001-08-01

    Certain microbial substances, e.g., LPS, can activate neutrophils or prime them to enhance their response to other activating agents, e.g., fMLP. We investigated the role of the Mycobacterium tuberculosis (MTB) 19-kDa lipoprotein in activation of human neutrophils. MTB 19-kDa lipoprotein initiated phenotypic changes characteristic of neutrophil activation, including down-regulation of CD62 ligand (L-selectin) and up-regulation of CD35 (CR1) and CD11b/CD18 (CR3, Mac-1). In addition, exposure of neutrophils to MTB 19-kDa lipoprotein enhanced the subsequent oxidative burst in response to fMLP as assessed by oxidation of dihydrorhodamine 123 (determined by flow cytometry). LPS also produced these effects with similar kinetics, but an oligodeoxynucleotide containing a CpG motif failed to induce any priming or activation response. Although the effects of LPS required the presence of serum, neutrophil activation by MTB 19-kDa lipoprotein occurred independently of serum factors, suggesting the involvement of different receptors and signaling mechanisms for LPS and MTB 19-kDa lipoprotein. Thus, MTB 19-kDa lipoprotein serves as a pathogen-associated molecular pattern that promotes neutrophil priming and activation.

  12. RP105 facilitates macrophage activation by Mycobacterium tuberculosis lipoproteins.

    PubMed

    Blumenthal, Antje; Kobayashi, Toshihiko; Pierini, Lynda M; Banaei, Niaz; Ernst, Joel D; Miyake, Kensuke; Ehrt, Sabine

    2009-01-22

    RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins.

  13. In Vitro Activity of a New Isothiazoloquinolone, ACH-702, against Mycobacterium tuberculosis and Other Mycobacteria▿

    PubMed Central

    Molina-Torres, Carmen A.; Ocampo-Candiani, Jorge; Rendón, Adrian; Pucci, Michael J.; Vera-Cabrera, Lucio

    2010-01-01

    In this work, we describe the activity of ACH-702 against clinical isolates of Mycobacterium tuberculosis and six different nontuberculous mycobacteria. The MIC50 and MIC90 of both susceptible and drug-resistant M. tuberculosis strains tested were 0.0625 and 0.125 μg/ml, respectively. The MIC50 and MIC90 values for Mycobacterium fortuitum isolates were 0.0625 μg/ml in both cases; Mycobacterium avium complex isolates showed MIC50 and MIC90 values of 0.25 and 4 μg/ml, respectively. PMID:20231398

  14. Validation of Mycobacterium tuberculosis Rv1681 Protein as a Diagnostic Marker of Active Pulmonary Tuberculosis

    PubMed Central

    Macovei, Lilia; Kanunfre, Kelly; Dhiman, Rakesh; Restrepo, Blanca I.; Zarate, Izelda; Pino, Paula A.; Mora-Guzman, Francisco; Fujiwara, Ricardo T.; Michel, Gerd; Kashino, Suely S.

    2013-01-01

    The development of an accurate antigen detection assay for the diagnosis of active tuberculosis (TB) would represent a major clinical advance. Here, we demonstrate that the Mycobacterium tuberculosis Rv1681 protein is a biomarker for active TB with potential diagnostic utility. We initially identified, by mass spectroscopy, peptides from the Rv1681 protein in urine specimens from 4 patients with untreated active TB. Rabbit IgG anti-recombinant Rv1681 detected Rv1681 protein in lysates and culture filtrates of M. tuberculosis and immunoprecipitated it from pooled urine specimens from two TB patients. An enzyme-linked immunosorbent assay formatted with these antibodies detected Rv1681 protein in unconcentrated urine specimens from 11/25 (44%) TB patients and 1/21 (4.8%) subjects in whom TB was initially clinically suspected but then ruled out by conventional methods. Rv1681 protein was not detected in urine specimens from 10 subjects with Escherichia coli-positive urine cultures, 26 subjects with confirmed non-TB tropical diseases (11 with schistosomiasis, 5 with Chagas' disease, and 10 with cutaneous leishmaniasis), and 14 healthy subjects. These results provide strong validation of Rv1681 protein as a promising biomarker for TB diagnosis. PMID:23390284

  15. Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis.

    PubMed

    Höner Zu Bentrup, K; Miczak, A; Swenson, D L; Russell, D G

    1999-12-01

    Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.

  16. Tetrahdroxysqualene from Rhus taitensis Shows Antimycobacterial Activity Against Mycobacterium tuberculosis

    PubMed Central

    Noro, Jeffrey C.; Barrows, Louis R.; Gideon, Osia G.; Ireland, Chris M.; Koch, Michael; Matainaho, Teatulohi; Piskaut, Pius; Pond, Christopher D.; Bugni, Tim S.

    2010-01-01

    Tuberculosis has become a major health problem, in particular with the emergence of extremely drug resistant tuberculosis (XDRTB). In our search for new therapeutic leads against TB, we isolated a new triterpene (1) from the plant Rhus taitensis collected in Papua New Guinea. Tetrahydroxysqualene (1) was isolated using bioassay-guided fractionation of the methanolic extract of R. taitensis leaves and twigs. The structure of tetrahydroxysqualene (1) was elucidated on the basis of HRESIMS and 1D and 2D NMR spectra. Tetrahydroxysqualene (1) exhibited anti–tuberculosis activity with an MIC of 10.0 μg/mL while showing only modest cytotoxicity. PMID:18710283

  17. Pyrazinamide Is Active against Mycobacterium tuberculosis Cultures at Neutral pH and Low Temperature.

    PubMed

    den Hertog, Alice L; Menting, Sandra; Pfeltz, Richard; Warns, Matthew; Siddiqi, Salman H; Anthony, Richard M

    2016-08-01

    For the past decades, an acidic pH has been used to render Mycobacterium tuberculosis susceptible to pyrazinamide for in vitro testing. Here, we show that at the standard breakpoint concentration and reduced culture temperatures, pyrazinamide (PZA) is active against tuberculosis (TB) at neutral pH. This finding should help unravel the mechanism of action of PZA and allow drug susceptibility testing (DST) methods to be optimized.

  18. Activity against multidrug-resistant Mycobacterium tuberculosis in Mexican plants used to treat respiratory diseases.

    PubMed

    Jimenez-Arellanes, Adelina; Meckes, Mariana; Ramirez, Raquel; Torres, Javier; Luna-Herrera, Julieta

    2003-09-01

    The increase of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) demands the search for alternative antimycobacterial drugs. The aim of this study was to evaluate plants used in Mexican traditional medicine to treat respiratory diseases for activity against MDR-TB. A group of 22 plants was screened for activity against Mycobacterium tuberculosis H37Rv and Mycobacterium avium at concentrations from 50 to 200 microg/mL. The antimycobacterial effect was determined by a microcolorimetric assay with Alamar blue dye. None of the aqueous extracts had antimycobacterial activity. Hexane extracts from Artemisia ludoviciana, Chamaedora tepejilote, Lantana hispida, Juniperus communis and Malva parviflora, and methanol extracts from Artemisia ludoviciana and Juniperus communis inhibited the growth of Mycobacterium tuberculosis. Mycobacterium avium was inhibited by Juniperus communis hexane extract and by Malva parviflora methanol extract. The active extracts were tested against monoresistant variants of Mycobacterium tuberculosis H37Rv (isoniazid, rifampin, streptomycin and ethambutol resistant) and the hexane extract of Lantana hispida showed the best activity. Lantana hispida hexane extract was also active against a group of MDR-TB clinical isolates. In contrast, it did not inhibit the growth of non-tuberculous mycobacteria. The hexane extract of Lantana hispida was fractionated by column chromatography and one of its fractions (FVI) inhibited the growth of all the MDR-TB clinical isolates at concentrations up to 25 microg/mL. This study supports the fact that selecting plants by ethnobotanical criteria enhances the probability of finding species with activity against mycobacteria, and our results point to Lantana hispida as an important source of potential compounds against MDR-TB.

  19. [Development and study of structure-activity relationship of drugs against Mycobacterium tuberculosis].

    PubMed

    Baska, Ferenc; Székely, Edina Rita; Szántai-Kis, Csaba; Bánhegyi, Péter; Hegymegi-Barakonyi, Bálint; Németh, Gábor; Breza, Nóra; Zsákai, Lilian; Greff, Zoltán; Pató, János; Kéri, György; Orfi, Lászlo

    2013-01-01

    Tuberculosis is considered to be one of the major health problem not only in the less developed countries but in the economically developed countries as well. Roughly one third of the world's population are infected with Mycobacterium tuberculosis and a significant part of them are carriers of latent tuberculosis. From ten percent of these latent infections are developing the active TB disease and fifty percent of them die from the illness without appropriate treatment. The drug-resistant Mycobacterium tuberculosis (MDR-TB, XDR-TB) and TB-HIV co-infection attracted attention to the most serious infectious disease. Inhibition of alternative signaling pathways were an important part of the research strategies for cancer and inflammatory diseases in recent years. In case of Mycobacterium tuberculosis such pathways were also identified, for example, three serine-threonine kinases (PknA, PknB, PknG) which are necessary and essential for bacterial growth. In this paper we summarize our best anti-TB active compounds, their biological effects and structure-activity relationships using in silico modeling, biochemical measurements and tests on active bacteria.

  20. Activation of the Wnt Pathway by Mycobacterium tuberculosis: A Wnt–Wnt Situation

    PubMed Central

    Villaseñor, Tomás; Madrid-Paulino, Edgardo; Maldonado-Bravo, Rafael; Urbán-Aragón, Antonio; Pérez-Martínez, Leonor; Pedraza-Alva, Gustavo

    2017-01-01

    Mycobacterium tuberculosis (M. tuberculosis), an intracellular pathogenic Gram-positive bacterium, is the cause of tuberculosis (TB), a major worldwide human infectious disease. The innate immune system is the first host defense against M. tuberculosis. The recognition of this pathogen is mediated by several classes of pattern recognition receptors expressed on the host innate immune cells, including Toll-like receptors, Nod-like receptors, and C-type lectin receptors like Dectin-1, the Mannose receptor, and DC-SIGN. M. tuberculosis interaction with any of these receptors activates multiple signaling pathways among which the protein kinase C, the MAPK, and the NFκB pathways have been widely studied. These pathways have been implicated in macrophage invasion, M. tuberculosis survival, and impaired immune response, thus promoting a successful infection and disease. Interestingly, the Wnt signaling pathway, classically regarded as a pathway involved in the control of cell proliferation, migration, and differentiation in embryonic development, has recently been involved in immunoregulatory mechanisms in infectious and inflammatory diseases, such as TB, sepsis, psoriasis, rheumatoid arthritis, and atherosclerosis. In this review, we present the current knowledge supporting a role for the Wnt signaling pathway during macrophage infection by M. tuberculosis and the regulation of the immune response against M. tuberculosis. Understanding the cross talk between different signaling pathways activated by M. tuberculosis will impact on the search for new therapeutic targets to fuel the rational design of drugs aimed to restore the immunological response against M. tuberculosis. PMID:28203237

  1. Activation of the Wnt Pathway by Mycobacterium tuberculosis: A Wnt-Wnt Situation.

    PubMed

    Villaseñor, Tomás; Madrid-Paulino, Edgardo; Maldonado-Bravo, Rafael; Urbán-Aragón, Antonio; Pérez-Martínez, Leonor; Pedraza-Alva, Gustavo

    2017-01-01

    Mycobacterium tuberculosis (M. tuberculosis), an intracellular pathogenic Gram-positive bacterium, is the cause of tuberculosis (TB), a major worldwide human infectious disease. The innate immune system is the first host defense against M. tuberculosis. The recognition of this pathogen is mediated by several classes of pattern recognition receptors expressed on the host innate immune cells, including Toll-like receptors, Nod-like receptors, and C-type lectin receptors like Dectin-1, the Mannose receptor, and DC-SIGN. M. tuberculosis interaction with any of these receptors activates multiple signaling pathways among which the protein kinase C, the MAPK, and the NFκB pathways have been widely studied. These pathways have been implicated in macrophage invasion, M. tuberculosis survival, and impaired immune response, thus promoting a successful infection and disease. Interestingly, the Wnt signaling pathway, classically regarded as a pathway involved in the control of cell proliferation, migration, and differentiation in embryonic development, has recently been involved in immunoregulatory mechanisms in infectious and inflammatory diseases, such as TB, sepsis, psoriasis, rheumatoid arthritis, and atherosclerosis. In this review, we present the current knowledge supporting a role for the Wnt signaling pathway during macrophage infection by M. tuberculosis and the regulation of the immune response against M. tuberculosis. Understanding the cross talk between different signaling pathways activated by M. tuberculosis will impact on the search for new therapeutic targets to fuel the rational design of drugs aimed to restore the immunological response against M. tuberculosis.

  2. LL-37 Immunomodulatory Activity during Mycobacterium tuberculosis Infection in Macrophages

    PubMed Central

    Torres-Juarez, Flor; Cardenas-Vargas, Albertina; Montoya-Rosales, Alejandra; González-Curiel, Irma; Garcia-Hernandez, Mariana H.; Enciso-Moreno, Jose A.; Hancock, Robert E. W.

    2015-01-01

    Tuberculosis is one of the most important infectious diseases worldwide. The susceptibility to this disease depends to a great extent on the innate immune response against mycobacteria. Host defense peptides (HDP) are one of the first barriers to counteract infection. Cathelicidin (LL-37) is an HDP that has many immunomodulatory effects besides its weak antimicrobial activity. Despite advances in the study of the innate immune response in tuberculosis, the immunological role of LL-37 during M. tuberculosis infection has not been clarified. Monocyte-derived macrophages were infected with M. tuberculosis strain H37Rv and then treated with 1, 5, or 15 μg/ml of exogenous LL-37 for 4, 8, and 24 h. Exogenous LL-37 decreased tumor necrosis factor alpha (TNF-α) and interleukin-17 (IL-17) while inducing anti-inflammatory IL-10 and transforming growth factor β (TGF-β) production. Interestingly, the decreased production of anti-inflammatory cytokines did not reduce antimycobacterial activity. These results are consistent with the concept that LL-37 can modulate the expression of cytokines during mycobacterial infection and this activity was independent of the P2X7 receptor. Thus, LL-37 modulates the response of macrophages during infection, controlling the expression of proinflammatory and anti-inflammatory cytokines. PMID:26351280

  3. Alternative activation deprives macrophages of a coordinated defense program to Mycobacterium tuberculosis.

    PubMed

    Kahnert, Antje; Seiler, Peter; Stein, Maik; Bandermann, Silke; Hahnke, Karin; Mollenkopf, Hans; Kaufmann, Stefan H E

    2006-03-01

    A potent Th1 immune response is critical to the control of tuberculosis. The impact of an additive Th2 response on the course of disease has so far been insufficiently characterized, despite increased morbidity after co-infection with Mycobacterium tuberculosis and Th2-eliciting helminths and possible involvement of Th2 polarization in reactivation of latent tuberculosis. Here, we describe the gene expression profile of murine bone marrow-derived macrophages alternatively activated by IL-4 in response to infection with M. tuberculosis. Comparison of transcriptional profiles of infected IL-4- and IFN-gamma-activated macrophages revealed delayed and partially diminished responses to intracellular bacteria in alternatively activated macrophages, characterized by reduced exposure to nitrosative stress and increased iron availability, respectively. Alternative activation of host macrophages correlated with elevated expression of the M. tuberculosis iron storage protein bacterioferritin as well as reduced expression of the mycobactin synthesis genes mbtI and mbtJ. The extracellular matrix-remodeling enzyme matrix metalloproteinase (MMP)-12 was induced in alternatively activated macrophages in vitro, and MMP-12-expressing macrophages were abundant at late, but not early, stages of tuberculosis in murine lungs. Our findings emphasize that alternative activation deprives macrophages of control mechanisms that limit mycobacterial growth in vivo, thus supporting intracellular persistence of M. tuberculosis.

  4. Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

    PubMed Central

    Brust, Belinda; Lecoufle, Mélanie; Tuaillon, Edouard; Dedieu, Luc; Canaan, Stéphane; Valverde, Viviane; Kremer, Laurent

    2011-01-01

    Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high

  5. The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

    PubMed Central

    Gao, Wei; Kim, Jin-Yong; Anderson, Jeffrey R.; Akopian, Tatos; Hong, Seungpyo; Jin, Ying-Yu; Kandror, Olga; Kim, Jong-Woo; Lee, In-Ae; Lee, Sun-Young; McAlpine, James B.; Mulugeta, Surafel; Sunoqrot, Suhair; Wang, Yuehong; Yang, Seung-Hwan; Yoon, Tae-Mi; Goldberg, Alfred L.; Pauli, Guido F.; Cho, Sanghyun

    2014-01-01

    Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development. PMID:25421483

  6. Intracellular activity of tedizolid phosphate and ACH-702 versus Mycobacterium tuberculosis infected macrophages

    PubMed Central

    2014-01-01

    Background Due to the emergency of multidrug-resistant strains of Mycobacterium tuberculosis, is necessary the evaluation of new compounds. Findings Tedizolid, a novel oxazolidinone, and ACH-702, a new isothiazoloquinolone, were tested against M. tuberculosis infected THP-1 macrophages. These two compounds significantly decreased the number of intracellular mycobacteria at 0.25X, 1X, 4X and 16X the MIC value. The drugs were tested either in nanoparticules or in free solution. Conclusion Tedizolid and ACH-702 have a good intracellular killing activity comparable to that of rifampin or moxifloxacin. PMID:24708819

  7. Mycobacterium tuberculosis enhances human immunodeficiency virus-1 replication by transcriptional activation at the long terminal repeat.

    PubMed Central

    Zhang, Y; Nakata, K; Weiden, M; Rom, W N

    1995-01-01

    Tuberculosis has emerged as an epidemic fueled by the large number of individuals infected with the human immunodeficiency virus, especially those who are injecting drug users. We found a striking increase from 4- to 208-fold in p24 levels in bronchoalveolar lavage fluid from involved sites of Mycobacterium tuberculosis infection vs uninvolved sites in three HIV+ patients. We used an in vitro cell culture model to determine if tuberculosis could activate replication of HIV-1. Mononuclear phagocyte cell lines U937 and THP-1 infected with HIV-1JR-CSF, in vitro and stimulated with live M. tuberculosis H37Ra, had a threefold increase in p24 in culture supernatants. Using the HIV-1 long terminal repeat with a chloramphenicol acetyltransferase (CAT) reporter construct, live M. tuberculosis increased transcription 20-fold in THP-1 cells, and cell wall components stimulated CAT expression to a lesser extent. The nuclear factor-kappa B enhancer element was responsible for the majority of the increased CAT activity although two upstream nuclear factor-IL6 sites may also contribute to enhanced transcription. Antibodies to TNF-alpha and IL-1 inhibited the increase in CAT activity of the HIV-1 long terminal repeat by M. tuberculosis from 21-fold to 8-fold. Stimulation of HIV-1 replication by M. tuberculosis may exacerbate dysfunction of the host immune response in dually infected individuals. Images PMID:7738195

  8. Targeting phenotypically tolerant Mycobacterium tuberculosis

    PubMed Central

    Gold, Ben; Nathan, Carl

    2016-01-01

    While the immune system is credited with averting tuberculosis in billions of individuals exposed to Mycobacterium tuberculosis, the immune system is also culpable for tempering the ability of antibiotics to deliver swift and durable cure of disease. In individuals afflicted with tuberculosis, host immunity produces diverse microenvironmental niches that support suboptimal growth, or complete growth arrest, of M. tuberculosis. The physiological state of nonreplication in bacteria is associated with phenotypic drug tolerance. Many of these host microenvironments, when modeled in vitro by carbon starvation, complete nutrient starvation, stationary phase, acidic pH, reactive nitrogen intermediates, hypoxia, biofilms, and withholding streptomycin from the streptomycin-addicted strain SS18b, render M. tuberculosis profoundly tolerant to many of the antibiotics that are given to tuberculosis patients in a clinical setting. Targeting nonreplicating persisters is anticipated to reduce the duration of antibiotic treatment and rate of post-treatment relapse. Some promising drugs to treat tuberculosis, such as rifampicin and bedaquiline, only kill nonreplicating M. tuberculosis in vitro at concentrations far greater than their minimal inhibitory concentrations against replicating bacilli. There is an urgent demand to identify which of the currently used antibiotics, and which of the molecules in academic and corporate screening collections, have potent bactericidal action on nonreplicating M. tuberculosis. With this goal, we review methods of high throughput screening to target nonreplicating M. tuberculosis and methods to progress candidate molecules. A classification based on structures and putative targets of molecules that have been reported to kill nonreplicating M. tuberculosis revealed a rich diversity in pharmacophores. However, few of these compounds were tested under conditions that would exclude the impact of adsorbed compound acting during the recovery phase of

  9. Stress and host immunity amplify Mycobacterium tuberculosis phenotypic heterogeneity and induce nongrowing metabolically active forms.

    PubMed

    Manina, Giulia; Dhar, Neeraj; McKinney, John D

    2015-01-14

    Nonreplicating and metabolically quiescent bacteria are implicated in latent tuberculosis infections and relapses following "sterilizing" chemotherapy. However, evidence linking bacterial dormancy and persistence in vivo is largely inconclusive. Here we measure the single-cell dynamics of Mycobacterium tuberculosis replication and ribosomal activity using quantitative time-lapse microscopy and a reporter of ribosomal RNA gene expression. Single-cell dynamics exhibit heterogeneity under standard growth conditions, which is amplified by stressful conditions such as nutrient limitation, stationary phase, intracellular replication, and growth in mouse lungs. Additionally, the lungs of chronically infected mice harbor a subpopulation of nongrowing but metabolically active bacteria, which are absent in mice lacking interferon-γ, a cytokine essential for antituberculosis immunity. These cryptic bacterial forms are prominent in mice treated with the antituberculosis drug isoniazid, suggesting a role in postchemotherapeutic relapses. Thus, amplification of bacterial phenotypic heterogeneity in response to host immunity and drug pressure may contribute to tuberculosis persistence.

  10. Bacteriostatic and bactericidal activities of benzoxazinorifamycin KRM-1648 against Mycobacterium tuberculosis and Mycobacterium avium in human macrophages.

    PubMed Central

    Mor, N; Simon, B; Heifets, L

    1996-01-01

    Inhibitory and bactericidal activities of KRM-1648 were determined against Mycobacterium tuberculosis and M. avium residing in human monocyte-derived macrophages and extracellular M. tuberculosis and M. avium. MICs and MBCs of KRM-1648 against intracellular and extracellular bacteria were substantially lower than those of rifampin. The MICs and MBCs of either drug against the intracellular bacteria were only twofold lower than or equal to the values found for extracellular bacteria. The prolonged effect of KRM-1648 found in this study is probably associated with high ratios of intracellular accumulation, which were 50- to 100-fold higher than that found for rifampin. Further studies on intracellular distribution of KRM-1648 and on the sites of actual interaction between the drug and bacteria residing in macrophages are necessary, as well as evaluation of combined effects of KRM-1648 with other drugs in long-term macrophage culture experiments. PMID:8726023

  11. Mycobacterium tuberculosis activates the DNA-dependent cytosolic surveillance pathway within macrophages.

    PubMed

    Manzanillo, Paolo S; Shiloh, Michael U; Portnoy, Daniel A; Cox, Jeffery S

    2012-05-17

    Cytosolic bacterial pathogens activate the cytosolic surveillance pathway (CSP) and induce innate immune responses, but how the host detects vacuolar pathogens like Mycobacterium tuberculosis is poorly understood. We show that M. tuberculosis also initiates the CSP upon macrophage infection via limited perforation of the phagosome membrane mediated by the ESX-1 secretion system. Although the bacterium remains within the phagosome, this permeabilization results in phagosomal and cytoplasmic mixing and allows extracellular mycobacterial DNA to access host cytosolic receptors, thus blurring the distinction between "vacuolar" and "cytosolic" pathogens. Activation of cytosolic receptors induces signaling through the Sting/Tbk1/Irf3 axis, resulting in IFN-β production. Surprisingly, Irf3(-/-) mice, which cannot respond to cytosolic DNA, are resistant to long-term M. tuberculosis infection, suggesting that the CSP promotes M. tuberculosis infection. Thus, cytosolic sensing of mycobacterial DNA plays a key role in M. tuberculosis pathogenesis and likely contributes to the high type I IFN signature in tuberculosis.

  12. Iron-dependent RNA-binding activity of Mycobacterium tuberculosis aconitase.

    PubMed

    Banerjee, Sharmistha; Nandyala, Ashok Kumar; Raviprasad, Podili; Ahmed, Niyaz; Hasnain, Seyed E

    2007-06-01

    Cellular iron levels are closely monitored by iron regulatory and sensor proteins of Mycobacterium tuberculosis for survival inside macrophages. One such class of proteins systematically studied in eukaryotes and reported in a few prokaryotes are the iron-responsive proteins (IRPs). These IRPs bind to iron-responsive elements (IREs) present at untranslated regions (UTRs) of mRNAs and are responsible for posttranscriptional regulation of the expression of proteins involved in iron homeostasis. Amino acid sequence analysis of M. tuberculosis aconitase (Acn), a tricarboxylic acid (TCA) cycle enzyme, showed the presence of the conserved residues of the IRP class of proteins. We demonstrate that M. tuberculosis Acn is bifunctional. It is a monomeric protein that is enzymatically active in converting isocitrate to cis-aconitate at a broad pH range of 7 to 10 (optimum, pH 8). As evident from gel retardation assays, M. tuberculosis Acn also behaves like an IRP by binding to known mammalian IRE-like sequences and to predicted IRE-like sequences present at the 3' UTR of thioredoxin (trxC) and the 5' UTR of the iron-dependent repressor and activator (ideR) of M. tuberculosis. M. tuberculosis Acn when reactivated with Fe(2+) functions as a TCA cycle enzyme, but upon iron depletion by a specific iron chelator, it behaves like an IRP, binding to the selected IREs in vitro. Since iron is required for the Acn activity and inhibits the RNA-binding activity of Acn, the two activities of M. tuberculosis Acn are mutually exclusive. Our results demonstrate the bifunctional nature of M. tuberculosis Acn, pointing to its likely role in iron homeostasis.

  13. Modification of Rifamycin Polyketide Backbone Leads to Improved Drug Activity against Rifampicin-resistant Mycobacterium tuberculosis*

    PubMed Central

    Nigam, Aeshna; Almabruk, Khaled H.; Saxena, Anjali; Yang, Jongtae; Mukherjee, Udita; Kaur, Hardeep; Kohli, Puneet; Kumari, Rashmi; Singh, Priya; Zakharov, Lev N.; Singh, Yogendra; Mahmud, Taifo; Lal, Rup

    2014-01-01

    Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains. PMID:24923585

  14. Modification of rifamycin polyketide backbone leads to improved drug activity against rifampicin-resistant Mycobacterium tuberculosis.

    PubMed

    Nigam, Aeshna; Almabruk, Khaled H; Saxena, Anjali; Yang, Jongtae; Mukherjee, Udita; Kaur, Hardeep; Kohli, Puneet; Kumari, Rashmi; Singh, Priya; Zakharov, Lev N; Singh, Yogendra; Mahmud, Taifo; Lal, Rup

    2014-07-25

    Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.

  15. A Dual Read-Out Assay to Evaluate the Potency of Compounds Active against Mycobacterium tuberculosis

    PubMed Central

    Ollinger, Juliane; Bailey, Mai Ann; Moraski, Garrett C.; Casey, Allen; Florio, Stephanie; Alling, Torey; Miller, Marvin J.; Parish, Tanya

    2013-01-01

    Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors. PMID:23593234

  16. Osteoarticular manifestations of Mycobacterium tuberculosis infection.

    PubMed

    Zychowicz, Michael E

    2010-01-01

    Mycobacterium tuberculosis has affected humans for much of our existence. The incidence of global tuberculosis infection continues to rise, especially in concert with HIV coinfection. Many disease processes, such as diabetes, increase the likelihood of tuberculosis infection. Tuberculosis bacteria can infect any bone, joint, tendon, or bursa; however, the most common musculoskeletal site for infection includes the spine and weight-bearing joints of the hip and knee. Many patients who present with osteoarticular tuberculosis infection will have a gradual onset of pain at the site of infection. Many patients who develop a musculoskeletal tuberculosis infection will have no evidence of a pulmonary tuberculosis infection on x-ray film and many will have very mild symptoms with the initial infection. Healthcare providers must remember that many patients who develop tuberculosis infection do not progress to active tuberculosis disease; however, the latent infection may become active with immune compromise.

  17. Mycobacterium tuberculosis Activates Human Macrophage Peroxisome Proliferator-Activated Receptor γ Linking Mannose Receptor Recognition to Regulation of Immune Responses

    PubMed Central

    Rajaram, Murugesan V. S.; Brooks, Michelle N.; Morris, Jessica D.; Torrelles, Jordi B.; Azad, Abul K.; Schlesinger, Larry S.

    2010-01-01

    Mycobacterium tuberculosis enhances its survival in macrophages by suppressing immune responses in part through its complex cell wall structures. Peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor superfamily member, is a transcriptional factor that regulates inflammation and has high expression in alternatively activated alveolar macrophages and macrophage-derived foam cells, both cell types relevant to tuberculosis pathogenesis. In this study, we show that virulent M. tuberculosis and its cell wall mannose-capped lipoarabinomannan induce PPARγ expression through a macrophage mannose receptor-dependent pathway. When activated, PPARγ promotes IL-8 and cyclooxygenase 2 expression, a process modulated by a PPARγ agonist or antagonist. Upstream, MAPK-p38 mediates cytosolic phospholipase A2 activation, which is required for PPARγ ligand production. The induced IL-8 response mediated by mannose-capped lipoarabinomannan and the mannose receptor is independent of TLR2 and NF-κB activation. In contrast, the attenuated Mycobacterium bovis bacillus Calmette-Guérin induces less PPARγ and preferentially uses the NF-κB–mediated pathway to induce IL-8 production. Finally, PPARγ knockdown in human macrophages enhances TNF production and controls the intracellular growth of M. tuberculosis. These data identify a new molecular pathway that links engagement of the mannose receptor, an important pattern recognition receptor for M. tuberculosis, with PPARγ activation, which regulates the macrophage inflammatory response, thereby playing a role in tuberculosis pathogenesis. PMID:20554962

  18. Fc gamma receptors regulate immune activation and susceptibility during Mycobacterium tuberculosis infection.

    PubMed

    Maglione, Paul J; Xu, Jiayong; Casadevall, Arturo; Chan, John

    2008-03-01

    The critical role of cellular immunity during tuberculosis (TB) has been extensively studied, but the impact of Abs upon this infection remains poorly defined. Previously, we demonstrated that B cells are required for optimal protection in Mycobacterium tuberculosis-infected mice. FcgammaR modulate immunity by engaging Igs produced by B cells. We report that C57BL/6 mice deficient in inhibitory FcgammaRIIB (RIIB-/-) manifested enhanced mycobacterial containment and diminished immunopathology compared with wild-type controls. These findings corresponded with enhanced pulmonary Th1 responses, evidenced by increased IFN-gamma-producing CD4+ T cells, and elevated expression of MHC class II and costimulatory molecules B7-1 and B7-2 in the lungs. Upon M. tuberculosis infection and immune complex engagement, RIIB-/- macrophages produced more of the p40 component of the Th1-promoting cytokine IL-12. These data strongly suggest that FcgammaRIIB engagement can dampen the TB Th1 response by attenuating IL-12p40 production or activation of APCs. Conversely, C57BL/6 mice lacking the gamma-chain shared by activating FcgammaR had enhanced susceptibility and exacerbated immunopathology upon M. tuberculosis challenge, associated with increased production of the immunosuppressive cytokine IL-10. Thus, engagement of distinct FcgammaR can divergently affect cytokine production and susceptibility during M. tuberculosis infection.

  19. IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

    PubMed Central

    Rovetta, Ana I; Peña, Delfina; Hernández Del Pino, Rodrigo E; Recalde, Gabriela M; Pellegrini, Joaquín; Bigi, Fabiana; Musella, Rosa M; Palmero, Domingo J; Gutierrez, Marisa; Colombo, María I; García, Verónica E

    2015-01-01

    Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen. PMID:25426782

  20. Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase.

    PubMed

    Aldred, Katie J; Blower, Tim R; Kerns, Robert J; Berger, James M; Osheroff, Neil

    2016-02-16

    Mycobacterium tuberculosis is a significant source of global morbidity and mortality. Moxifloxacin and other fluoroquinolones are important therapeutic agents for the treatment of tuberculosis, particularly multidrug-resistant infections. To guide the development of new quinolone-based agents, it is critical to understand the basis of drug action against M. tuberculosis gyrase and how mutations in the enzyme cause resistance. Therefore, we characterized interactions of fluoroquinolones and related drugs with WT gyrase and enzymes carrying mutations at GyrA(A90) and GyrA(D94). M. tuberculosis gyrase lacks a conserved serine that anchors a water-metal ion bridge that is critical for quinolone interactions with other bacterial type II topoisomerases. Despite the fact that the serine is replaced by an alanine (i.e., GyrA(A90)) in M. tuberculosis gyrase, the bridge still forms and plays a functional role in mediating quinolone-gyrase interactions. Clinically relevant mutations at GyrA(A90) and GyrA(D94) cause quinolone resistance by disrupting the bridge-enzyme interaction, thereby decreasing drug affinity. Fluoroquinolone activity against WT and resistant enzymes is enhanced by the introduction of specific groups at the C7 and C8 positions. By dissecting fluoroquinolone-enzyme interactions, we determined that an 8-methyl-moxifloxacin derivative induces high levels of stable cleavage complexes with WT gyrase and two common resistant enzymes, GyrA(A90V) and GyrA(D94G). 8-Methyl-moxifloxacin was more potent than moxifloxacin against WT M. tuberculosis gyrase and displayed higher activity against the mutant enzymes than moxifloxacin did against WT gyrase. This chemical biology approach to defining drug-enzyme interactions has the potential to identify novel drugs with improved activity against tuberculosis.

  1. Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase

    PubMed Central

    Aldred, Katie J.; Kerns, Robert J.; Berger, James M.; Osheroff, Neil

    2016-01-01

    Mycobacterium tuberculosis is a significant source of global morbidity and mortality. Moxifloxacin and other fluoroquinolones are important therapeutic agents for the treatment of tuberculosis, particularly multidrug-resistant infections. To guide the development of new quinolone-based agents, it is critical to understand the basis of drug action against M. tuberculosis gyrase and how mutations in the enzyme cause resistance. Therefore, we characterized interactions of fluoroquinolones and related drugs with WT gyrase and enzymes carrying mutations at GyrAA90 and GyrAD94. M. tuberculosis gyrase lacks a conserved serine that anchors a water–metal ion bridge that is critical for quinolone interactions with other bacterial type II topoisomerases. Despite the fact that the serine is replaced by an alanine (i.e., GyrAA90) in M. tuberculosis gyrase, the bridge still forms and plays a functional role in mediating quinolone–gyrase interactions. Clinically relevant mutations at GyrAA90 and GyrAD94 cause quinolone resistance by disrupting the bridge–enzyme interaction, thereby decreasing drug affinity. Fluoroquinolone activity against WT and resistant enzymes is enhanced by the introduction of specific groups at the C7 and C8 positions. By dissecting fluoroquinolone–enzyme interactions, we determined that an 8-methyl-moxifloxacin derivative induces high levels of stable cleavage complexes with WT gyrase and two common resistant enzymes, GyrAA90V and GyrAD94G. 8-Methyl-moxifloxacin was more potent than moxifloxacin against WT M. tuberculosis gyrase and displayed higher activity against the mutant enzymes than moxifloxacin did against WT gyrase. This chemical biology approach to defining drug–enzyme interactions has the potential to identify novel drugs with improved activity against tuberculosis. PMID:26792518

  2. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis.

    PubMed

    Jastrab, Jordan B; Wang, Tong; Murphy, J Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P; Li, Huilin; Darwin, K Heran

    2015-04-07

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens.

  3. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    SciTech Connect

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; Li, Huilin; Darwin, K. Heran

    2015-03-23

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.

  4. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    DOE PAGES

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; ...

    2015-03-23

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and proteinmore » degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.« less

  5. Activity of rifapentine and its metabolite 25-O-desacetylrifapentine compared with rifampicin and rifabutin against Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis and M. bovis BCG.

    PubMed

    Rastogi, N; Goh, K S; Berchel, M; Bryskier, A

    2000-10-01

    The in vitro activity of rifapentine and its metabolite, 25-O:-desacetylrifapentine, as compared with that of rifampicin and rifabutin, was determined against Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis and M. bovis BCG. MICs were determined radiometrically and by the 1% proportional method using Middlebrook 7H11 agar. The bactericidal effect of the drugs was determined in parallel at selected concentrations. For drugsusceptible isolates of M. tuberculosis, the Bactec MICs of rifapentine and 25-O:-desacetylrifapentine were 0.03-0.06 mg/L and 0. 125-0.25 mg/L, respectively. Similar MICs were obtained for M. africanum (0.03-0.125 and 0.125-0.50 mg/L, respectively), and M. bovis (0.063-0.25 and 0.125-1.0 mg/L, respectively), but MICs were considerably lower for M. bovis BCG (0.008-0.063 mg/L for rifapentine and 0.016-0.125 mg/L for its metabolite). In general, MICs determined using 7H11 agar medium were usually one or two dilutions higher than those obtained using Bactec broth. When compared with rifampicin and rifabutin, the inhibitory activity of rifapentine for drug-susceptible isolates was roughly equal to that of rifabutin, and the inhibitory activity of 25-O:-desacetylrifapentine was comparable to that of rifampicin; however, rifapentine was somewhat more bactericidal than rifabutin at equal concentrations. Clinical isolates of M. tuberculosis with a high degree of resistance to rifampicin (MIC >/= 32 mg/L) were also highly resistant to rifabutin, rifapentine and 25-O:-desacetylrifapentine, although the MICs of rifabutin in this case were somewhat lower than the MICs of rifapentine.

  6. Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages

    PubMed Central

    1992-01-01

    Tuberculosis remains one of the major infectious causes of morbidity and mortality in the world, yet the mechanisms by which macrophages defend against Mycobacterium tuberculosis have remained obscure. Results from this study show that murine macrophages, activated by interferon gamma, and lipopolysaccharide or tumor necrosis factor alpha, both growth inhibit and kill M. tuberculosis. This antimycobacterial effect, demonstrable both in murine macrophage cell lines and in peritoneal macrophages of BALB/c mice, is independent of the macrophage capacity to generate reactive oxygen intermediates (ROI). Both the ROI-deficient murine macrophage cell line D9, and its ROI-generating, parental line J774.16, expressed comparable antimycobacterial activity upon activation. In addition, the oxygen radical scavengers superoxide dismutase (SOD), catalase, mannitol, and diazabicyclooctane had no effect on the antimycobacterial activity of macrophages. These findings, together with the results showing the relative resistance of M. tuberculosis to enzymatically generated H2O2, suggest that ROI are unlikely to be significantly involved in killing M. tuberculosis. In contrast, the antimycobacterial activity of these macrophages strongly correlates with the induction of the L-arginine- dependent generation of reactive nitrogen intermediates (RNI). The effector molecule(s) that could participate in mediating this antimycobacterial function are toxic RNI, including NO, NO2, and HNO2, as demonstrated by the mycobacteriocidal effect of acidified NO2. The oxygen radical scavenger SOD adventitiously perturbs RNI production, and cannot be used to discriminate between cytocidal mechanisms involving ROI and RNI. Overall, our results provide support for the view that the L-arginine-dependent production of RNI is the principal effector mechanism in activated murine macrophages responsible for killing and growth inhibiting virulent M. tuberculosis. PMID:1552282

  7. Contribution of efflux activity to isoniazid resistance in the Mycobacterium tuberculosis complex.

    PubMed

    Rodrigues, Liliana; Machado, Diana; Couto, Isabel; Amaral, Leonard; Viveiros, Miguel

    2012-06-01

    Resistance to isoniazid (INH), one of the main drugs used in tuberculosis (TB) therapy, is mostly due to chromosomal mutations in target genes. However, approximately 20-30% of INH resistant Mycobacterium tuberculosis isolates do not have mutations in any of the genes associated with INH resistance. This suggests that other mechanism(s) may be involved, namely efflux pump systems capable of extruding the drug to the exterior of the cell. In a previous work, we have induced clinical INH susceptible M. tuberculosis isolates and the H37Rv reference strain to high-level resistance to INH, by gradual exposure to increasing concentrations of this drug. In the present study, we have characterized these strains and Mycobacterium bovis BCG induced to INH resistance with respect to their efflux activity and its contribution to INH resistance using the following approach: determination of the susceptibility to INH in the presence and absence of the efflux inhibitors (EIs) chlorpromazine, thioridazine and verapamil; evaluation of efflux activity by a semi-automated fluorometric method; and quantification of the expression level of genes coding for efflux pumps by real-time RT-qPCR. The EIs decreased INH resistance in the INH induced strains, in particular verapamil promoted a reversal of resistance in some of the strains tested. The induced strains presented an increased efflux activity that was inhibited by the EIs and showed overexpression of the efflux pump genes efpA, mmpL7, mmr, p55 and the Tap-like gene Rv1258c. Altogether, these results correlate efflux activity with INH resistance and demonstrate that efflux pumps play an important role in acquired INH resistance in M. tuberculosis complex. The development of EIs that can restore the antimicrobial activity of the antibiotic subject to efflux is an approach that can be useful in order to prevent the emergence of this resistance and guide the development of new effective anti-TB therapeutical approaches.

  8. Functional analysis of TPM domain containing Rv2345 of Mycobacterium tuberculosis identifies its phosphatase activity.

    PubMed

    Sinha, Avni; Eniyan, Kandasamy; Sinha, Swati; Lynn, Andrew Michael; Bajpai, Urmi

    2015-07-01

    Mycobacterium tuberculosis (Mtb) is the causal agent of tuberculosis, the second largest infectious disease. With the rise of multi-drug resistant strains of M. tuberculosis, serious challenge lies ahead of us in treating the disease. The availability of complete genome sequence of Mtb has improved the scope for identifying new proteins that would not only further our understanding of biology of the organism but could also serve to discover new drug targets. In this study, Rv2345, a hypothetical membrane protein of M. tuberculosis H37Rv, which is reported to be a putative ortholog of ZipA cell division protein has been assigned function through functional annotation using bioinformatics tools followed by experimental validation. Sequence analysis showed Rv2345 to have a TPM domain at its N-terminal region and predicted it to have phosphatase activity. The TPM domain containing region of Rv2345 was cloned and expressed using pET28a vector in Escherichia coli and purified by Nickel affinity chromatography. The purified TPM domain was tested in vitro and our results confirmed it to have phosphatase activity. The enzyme activity was first checked and optimized with pNPP as substrate, followed by using ATP, which was also found to be used as substrate by the purified protein. Hence sequence analysis followed by in vitro studies characterizes TPM domain of Rv2345 to contain phosphatase activity.

  9. Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis

    PubMed Central

    Roberts, Julia; Ling, Yan; Quezada, Landys Lopez; Glasheen, Jou; Ballinger, Elaine; Somersan-Karakaya, Selin; Warrier, Thulasi; Warren, J. David; Nathan, Carl

    2015-01-01

    The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicating M. tuberculosis may arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days—about 2 weeks sooner than required to count CFU—fluorometry reveals whether M. tuberculosis bacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log10 CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic. PMID:26239979

  10. Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae)

    PubMed Central

    Pires, Claudia Terencio Agostinho; Brenzan, Mislaine Adriana; Scodro, Regiane Bertin de Lima; Cortez, Diógenes Aparício Garcia; Lopes, Luciana Dias Ghiraldi; Siqueira, Vera Lucia Dias; Cardoso, Rosilene Fressatti

    2014-01-01

    We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis. PMID:24676652

  11. Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae).

    PubMed

    Pires, Claudia Terencio Agostinho; Brenzan, Mislaine Adriana; Scodro, Regiane Bertin de Lima; Cortez, Diógenes Aparício Garcia; Lopes, Luciana Dias Ghiraldi; Siqueira, Vera Lucia Dias; Cardoso, Rosilene Fressatti

    2014-06-01

    We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.

  12. Identification and structure-activity relationship study of carvacrol derivatives as Mycobacterium tuberculosis chorismate mutase inhibitors.

    PubMed

    Alokam, Reshma; Jeankumar, Variam Ullas; Sridevi, Jonnalagadda Padma; Matikonda, Siddharth Sai; Peddi, Santosh; Alvala, Mallika; Yogeeswari, Perumal; Sriram, Dharmarajan

    2014-08-01

    In the present study, we identified carvacrol, a major phenolic component of oregano oil as a novel small molecule inhibitor of Mycobacterium tuberculosis (MTB) chorismate mutase (CM) enzyme with IC50 of 1.06 ± 0.4 µM. Virtual screening of the BITS-Pilani in-house database using the crystal structure of the MTB CM bound transition state intermediate (PDB: 2FP2) as framework identified carvacrol as a potential lead. Further various carvacrol derivatives were evaluated in vitro for their ability to inhibit MTB CM enzyme, whole cell MTB and cytotoxicity as steps toward the derivation of structure-activity relationships (SAR) and lead optimization.

  13. Non-transpeptidase binding arylthioether β-lactams active against Mycobacterium tuberculosis and Moraxella catarrhalis.

    PubMed

    Beck, Tim N; Lloyd, Dina; Kuskovsky, Rostislav; Minah, Jeanette; Arora, Kriti; Plotkin, Balbina J; Green, Jacalyn M; Boshoff, Helena I; Barry, Clifton; Deschamps, Jeffrey; Konaklieva, Monika I

    2015-02-01

    The prevalence of drug resistance in both clinical and community settings as a consequence of alterations of biosynthetic pathways, enzymes or cell wall architecture is a persistent threat to human health. We have designed, synthesized, and tested a novel class of non-transpeptidase, β-lactamase resistant monocyclic β-lactams that carry an arylthio group at C4. These thioethers exhibit inhibitory and cidal activity against serine β-lactamase producing Mycobacterium tuberculosis wild type strain (Mtb) and multiple (n=8) β-lactamase producing Moraxella catarrhalis clinical isolates.

  14. Target Prediction for an Open Access Set of Compounds Active against Mycobacterium tuberculosis

    PubMed Central

    Martínez-Jiménez, Francisco; Papadatos, George; Yang, Lun; Wallace, Iain M.; Kumar, Vinod; Pieper, Ursula; Sali, Andrej; Brown, James R.; Overington, John P.; Marti-Renom, Marc A.

    2013-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects an estimated two billion people worldwide and is the leading cause of mortality due to infectious disease. The development of new anti-TB therapeutics is required, because of the emergence of multi-drug resistance strains as well as co-infection with other pathogens, especially HIV. Recently, the pharmaceutical company GlaxoSmithKline published the results of a high-throughput screen (HTS) of their two million compound library for anti-mycobacterial phenotypes. The screen revealed 776 compounds with significant activity against the M. tuberculosis H37Rv strain, including a subset of 177 prioritized compounds with high potency and low in vitro cytotoxicity. The next major challenge is the identification of the target proteins. Here, we use a computational approach that integrates historical bioassay data, chemical properties and structural comparisons of selected compounds to propose their potential targets in M. tuberculosis. We predicted 139 target - compound links, providing a necessary basis for further studies to characterize the mode of action of these compounds. The results from our analysis, including the predicted structural models, are available to the wider scientific community in the open source mode, to encourage further development of novel TB therapeutics. PMID:24098102

  15. In vitro Anti-mycobacterial activity of selected medicinal plants against Mycobacterium tuberculosis and Mycobacterium bovis Strains

    PubMed Central

    2013-01-01

    Background Tuberculosis (TB) is a global burden with one –third of the world’s population infected with the pathogen Mycobacterium tuberculosis complex and annually 1.4 million deaths occur due to the disease. This high incidence of infection and the increased rate of multi-drug resistant and extensively-drug resistant strains of the organism further complicated the problem of TB control and have called for an urgent need to develop new anti-TB drugs from plants. In this study, the in vitro activity of root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis were evaluated against M. tuberculosis and M. bovis strains. Methods Five Ethiopian medicinal plants, root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis used locally for the management of TB. They were investigated for in vitro antimycobacterial activity against M. tuberculosis and M. bovis strains. 80% methanolic extracts of the plant materials were obtained by maceration. The antimycobacterial activity was determined using 96 wells of microplate with the help of visual Resazurin Microtiter Assay. Results The crude 80% methanolic extracts of the root of C. aurea, seeds of O. basilicum, and leaves of A. abyssinica, C. macrostachyus, and E. camaldulensis had anti-mycobacterial activity with minimum inhibitory concentration (MIC) ranging from 6.25–100 μg/mL. The MIC of 80% methanol extracts in the order mentioned above ranged 25-100 μg/ml and 12.5-75 μg/mL, 25–100 μg/mL and 25–50 μg/mL, 6.25-50 μg/mL and 12.5-50 μg/mL, 12.5-100 μg/mL and 18.25-50 μg/mL and 6.25-50 μg/mL and 12.5-50 μg/mL, respectively for M. tuberculosis and M. bovis strains. Conclusions The results support the local use of these plants in the treatment of TB and it is suggested that these plants may have therapeutic value in the treatment of TB. However

  16. In vitro and in vivo activities of the nitroimidazole CGI 17341 against Mycobacterium tuberculosis.

    PubMed Central

    Ashtekar, D R; Costa-Perira, R; Nagrajan, K; Vishvanathan, N; Bhatt, A D; Rittel, W

    1993-01-01

    CGI 17341 (2-ethyl-5-nitro-2,3-dihydro[2-1b]imidazo-oxazole) is a novel orally active representative of the 5-nitroimidazole series of antimicrobial agents. At concentrations ranging from 0.1 to 0.3 micrograms/ml, CGI 17341 inhibited the drug-susceptible and multi-drug-resistant strains of Mycobacterium tuberculosis. CGI 17341 had no cross-resistance with isoniazid, rifampin, streptomycin, or ethambutol. While the in vitro activity of CGI 17341 against M. tuberculosis was comparable to those of isoniazid and rifampin, it was superior to those of streptomycin, ciprofloxacin or norfloxacin, and oxazolidinone DuP 721. The MIC of CGI 17341 was not affected when the pH of the medium was decreased from 6.8 to 5.6, while four- to sixfold increases in the MICs of ciprofloxacin and isoniazid were observed. In mice infected with M. tuberculosis, the 50% effective dose for CGI 17341 was 7.7 mg/kg of body weight (95% confidence limits, 3.5 and 10.27) when administered on days 11 and 12 postinfection. CGI 17341 gave a dose-dependent (r = 0.995) and significant increase in the survival time. Our data indicate that the 5-nitroimidazole CGI 17341 is a promising and novel antituberculosis compound with potent in vitro and in vivo activities. Further investigations on this compound are warranted. PMID:8452346

  17. Role of Metal Ions on the Activity of Mycobacterium tuberculosis Pyrazinamidase

    PubMed Central

    Sheen, Patricia; Ferrer, Patricia; Gilman, Robert H.; Christiansen, Gina; Moreno-Román, Paola; Gutiérrez, Andrés H.; Sotelo, Jun; Evangelista, Wilfredo; Fuentes, Patricia; Rueda, Daniel; Flores, Myra; Olivera, Paula; Solis, José; Pesaresi, Alessandro; Lamba, Doriano; Zimic, Mirko

    2012-01-01

    Pyrazinamidase of Mycobacterium tuberculosis catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in Escherichia coli with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co2+, Mn2+, and Zn2+ restored pyrazinamidase activity, only Co2+ enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu2+, Fe2+, Fe3+, and Mg2+ did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in E. coli most likely contained Zn. In conclusion, this study suggests that M. tuberculosis pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but in vivo, it is more likely to coordinate Zn2+. However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn. PMID:22764307

  18. Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

    SciTech Connect

    Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew; Sadler, Natalie C.; Brown, Robert W.; Smith, Richard D.; Wright, Aaron T.; Grundner, Christoph

    2016-02-01

    The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

  19. Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

    PubMed Central

    2014-01-01

    Background Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified. Results In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. Conclusion We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis. PMID:24975018

  20. Activity against Mycobacterium smegmatis and M. tuberculosis by extract of South African medicinal plants.

    PubMed

    Mativandlela, Sannah Patience Nkami; Meyer, Jacob Jacobus Marion; Hussein, Ahmed A; Houghton, Peter J; Hamilton, Chris J; Lall, Namrita

    2008-06-01

    Seven ethnobotanically selected medicinal plants were screened for their antimycobacterial activity. The minimum inhibitory concentration (MIC) of four plants namely Artemisia afra, Dodonea angustifolia, Drosera capensis and Galenia africana ranged from 0.781 to 6.25 mg/mL against Mycobacterium smegmatis. G. africana showed the best activity exhibiting an MIC of 0.78 mg/mL and a minimum bactericidal concentration (MBC) of 1.56 mg/mL. The MICs of ethanol extracts of D. angustifolia and G. africana against M. tuberculosis were found to be 5.0 and 1.2 mg/mL respectively. The mammalian cytotoxicity IC(50) value of the most active antimycobacterial extract, from G. africana, was found to be 101.3 microg/mL against monkey kidney Vero cells. Since the ethanol G. africana displayed the best antimycobacterial activity, it was subjected to fractionation which led to the isolation of a flavone, 5,7,2'-trihydroxyflavone. The MIC of this compound was found to be 0.031 mg/mL against M. smegmatis and 0.10 mg/mL against M. tuberculosis. This study gives some scientific basis to the traditional use of these plants for TB-related symptoms.

  1. Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates.

    PubMed

    Balganesh, Meenakshi; Dinesh, Neela; Sharma, Sreevalli; Kuruppath, Sanjana; Nair, Anju V; Sharma, Umender

    2012-05-01

    Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms in Mycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil and l-phenylalanyl-l-arginyl-β-naphthylamide (PAβN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded by Rv1218c, and the SMR (small multidrug resistance) class, encoded by Rv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded by Rv0849 and Rv1258c also mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WT M. tuberculosis cells by these compounds in the presence of either verapamil or PAβN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps of M. tuberculosis play a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization of Rv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.

  2. Comparative Proteomics of Activated THP-1 Cells Infected with Mycobacterium tuberculosis Identifies Putative Clearance Biomarkers for Tuberculosis Treatment.

    PubMed

    Kaewseekhao, Benjawan; Naranbhai, Vivek; Roytrakul, Sittiruk; Namwat, Wises; Paemanee, Atchara; Lulitanond, Viraphong; Chaiprasert, Angkana; Faksri, Kiatichai

    2015-01-01

    Biomarkers for determining clearance of Mycobacterium tuberculosis (Mtb) infection during anti-tuberculosis therapy or following exposure could facilitate enhanced monitoring and treatment. We screened for biomarkers indicating clearance of Mtb infection in vitro. A comparative proteomic analysis was performed using GeLC MSI/MS. Intracellular and secreted proteomes from activated THP-1 cells infected with the Mtb H37Rv strain (MOI = 1) and treated with isoniazid and rifampicin for 1 day (infection stage) and 5 days (clearance stage) were analyzed. Host proteins associated with early infection (n = 82), clearance (n = 121), sustained in both conditions (n = 34) and suppressed by infection (n = 46) were elucidated. Of the potential clearance markers, SSFA2 and CAECAM18 showed the highest and lowest protein intensities, respectively. A western blot of CAECAM18 validated the LC MS/MS result. For three clearance markers (SSFA2, PARP14 and PSME4), in vivo clinical validation was concordantly reported in previous patient cohorts. A network analysis revealed that clearance markers were enriched amongst four protein interaction networks centered on: (i) CD44/CCND1, (ii) IFN-β1/NF-κB, (iii) TP53/TGF-β and (iv) IFN-γ/CCL2. After infection, proteins associated with proliferation, and recruitment of immune cells appeared to be enriched possibly reflecting recruitment of defense mechanisms. Counteracting proteins (CASP3 vs. Akt and NF-κB vs. TP53) associated with apoptosis regulation and its networks were enriched among the early and sustained infection biomarkers, indicating host-pathogen competition. The BRCA1/2 network was suppressed during infection, suggesting that cell proliferation suppression is a feature of Mtb survival. Our study provides insights into the mechanisms of host-Mtb interaction by comparing the stages of infection clearance. The identified clearance biomarkers may be useful in monitoring tuberculosis treatment.

  3. Mycobacterium Tuberculosis in Spinal Tuberculosis

    PubMed Central

    Kim, Sung-Sim; Moon, Han-Lim; Kim, Dong-Hyeon

    2017-01-01

    Even in an era of remarkable medical advances, there is an issue of why tuberculosis remains in the list of disastrous diseases, afflicting humans and causing suffering. There has not been a plausible answer to this, and it has been suggested that clinicians and medical scientists could presently not win the war against the tubercle bacilli. With regards to this issue, based on the authors' own clinical and research experiences, in this review, the available literature was revisited in order to address the raised questions and to provide recent information on characteristics of tubercle bacilli and possible ways to more effectively treat tuberculosis. PMID:28243382

  4. Evaluation of the anti-mycobacterium tuberculosis activity and in vivo acute toxicity of Annona sylvatic

    PubMed Central

    2014-01-01

    Background The recent emergence of extensively multidrug-resistant Mycobacterium tuberculosis strains has further complicated the control of tuberculosis. There is an urgent need for the development of new molecular candidates antitubercular drugs. Medicinal plants have been an excellent source of leads for the development of drugs. The aim of this study was to evaluate the in vitro activity of 28 alcoholic extracts and essential oils of native and exotic Brazilian plants against Mycobacterium tuberculosis and to further study these extracts through chemical fractionation, the isolation of their constituents, and an evaluation of the in vivo acute toxicity of the active extracts. To the best of our knowledge this is the first chemical characterization, antituberculosis activity and acute toxicity evaluation of Annona sylvatica. Methods The anti-mycobacterial activity of these extracts and their constituent compounds was evaluated using the resazurin reduction microtiter assay (REMA). To investigate the acute toxicity of these extracts in vivo, female Swiss mice were treated with the extracts at doses of 500, 1000 and 2000 mg · kg-1 of body weight. The extracts were characterized by LC-MS, and the constituents were isolated and identified by chromatographic analysis of spectroscopic data. Results Of the 28 extracts, the methanol extract obtained from the leaves of Annona sylvatica showed anti-mycobacterial activity with an minimal inhibitory concentration (MIC) of 184.33 μg/mL, and the ethyl acetate fraction (EAF) resulting from liquid-liquid partitioning of the A. sylvatica extract showed an MIC of 115.2 μg/mL. The characterization of this extract by LC-MS identified flavonoids and acetogenins as its main constituents. The phytochemical study of the A. sylvatica EAF resulted in the isolation of quercetin, luteolin, and almunequin. Conclusions Among the compounds isolated from the EAF, luteolin and almunequin were the most promising, with MICs of 236.8

  5. High Affinity Inha Inhibitors with Activity Against Drug-Resistant Strains of Mycobacterium Tuberculosis

    SciTech Connect

    Sullivan,T.; Truglio, J.; Boyne, M.; Novichenok, P.; Zhang, X.; Stratton, C.; Li, H.; Kaur, T.; Amin, A.; et al.

    2006-01-01

    Novel chemotherapeutics for treating multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) are required to combat the spread of tuberculosis, a disease that kills more than 2 million people annually. Using structure-based drug design, we have developed a series of alkyl diphenyl ethers that are uncompetitive inhibitors of InhA, the enoyl reductase enzyme in the MTB fatty acid biosynthesis pathway. The most potent compound has a Ki{prime} value of 1 nM for InhA and MIC{sub 99} values of 2-3 {micro}g mL{sup -1} (6-10 {micro}M) for both drug-sensitive and drug-resistant strains of MTB. Overexpression of InhA in MTB results in a 9-12-fold increase in MIC{sub 99}, consistent with the belief that these compounds target InhA within the cell. In addition, transcriptional response studies reveal that the alkyl diphenyl ethers fail to upregulate a putative efflux pump and aromatic dioxygenase, detoxification mechanisms that are triggered by the lead compound triclosan. These diphenyl ether-based InhA inhibitors do not require activation by the mycobacterial KatG enzyme, thereby circumventing the normal mechanism of resistance to the front line drug isoniazid (INH) and thus accounting for their activity against INH-resistant strains of MTB.

  6. Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

    PubMed Central

    Mavrici, Daniela; Marakalala, Mohlopheni J.; Holton, James M.; Prigozhin, Daniil M.; Gee, Christine L.; Zhang, Yanjia J.; Rubin, Eric J.; Alber, Tom

    2014-01-01

    Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis. PMID:24843173

  7. Activation of JAK2/STAT1-alpha-dependent signaling events during Mycobacterium tuberculosis-induced macrophage apoptosis.

    PubMed

    Rojas, Mauricio; Olivier, Martin; García, Luis F

    2002-01-01

    Induction of apoptosis by Mycobacterium tuberculosis in murine macrophage involves TNF-alpha and nitric oxide (NO) production and caspase cascade activation; however, the intracellular signaling pathways implicated remain to be established. Our results indicate that infection of the B10R murine macrophage line with M. tuberculosis induces apoptosis independent of mycobacterial phagocytosis and that M. tuberculosis induces protein tyrosine kinase (PTK) activity, JAK2/STAT1-alpha phosphorylation, and STAT1-alpha nuclear translocation. Inhibitors of PTK (AG-126), or JAK2 (AG-490) inhibited TNF-alpha and NO production, caspase 1 activation and apoptosis, suggesting that M. tuberculosis-induction of these events depends on JAK2/STAT1-alpha activation. In addition, we have obtained evidence that ManLAM capacity to inhibit M. tuberculosis-induced apoptosis involves the activation of the PTP SHP-1. The finding that M. tuberculosis infection activate JAK2/STAT1-alpha pathway suggests that M. tuberculosis might mimic macrophage-activating stimuli.

  8. [Mycobacterium tuberculosis infection following organ transplantation].

    PubMed

    Haas, Charles; Le Jeunne, Claire

    2006-11-01

    In transplant recipients, immunosuppressive treatment affects cell-mediated immunity and increases the risk of tuberculosis. Tuberculosis may be transmitted by the donor organ or occur de novo, but such cases are rare. The vast majority of cases of active tuberculosis in transplant recipients result from reactivation of latent Mycobacterium tuberculosis infection. The incidence varies from one region of the globe to another, from 0.5-1.0% in North America, to 0.36-5.5% in Europe and 7.0-11.8% in India. The incidence of tuberculosis among transplant recipients is much higher than in the general population. Diabetes mellitus, renal impairment, systemic lupus erythematosus, chronic liver disease and AIDS all increase the risk of post-transplant tuberculosis. Extrapulmonary and disseminated forms are frequent in this setting. The diagnosis of tuberculosis in transplant recipients is often difficult, and treatment is frequently delayed. Tuberculosis can be life-threatening in such cases. Treatment is difficult because rifampicin is a cytochrome P450 inducer (leading to reduced levels of cyclosporine), and because the hepatotoxicity of isoniazid, rifampin and pyrazinamide is frequently increased in transplant recipients. Treatment of latent tuberculosis before transplantation markedly reduces the risk of developing active tuberculosis after transplantation.

  9. ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis.

    PubMed

    D'Heygère, François; Schwartz, Annie; Coste, Franck; Castaing, Bertrand; Boudvillain, Marc

    2015-07-13

    The bacterial transcription termination factor Rho-a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity-is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal 'RNA binding' domain and by a non-canonical R-loop residue in its C-terminal 'motor' domain. We also show that the 'motor' domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors.

  10. Activity of Medicinal Plant Extracts on Multiplication of Mycobacterium tuberculosis under Reduced Oxygen Conditions Using Intracellular and Axenic Assays

    PubMed Central

    Bhatter, Purva D.; Gupta, Pooja D.; Birdi, Tannaz J.

    2016-01-01

    Aim. Test the activity of selected medicinal plant extracts on multiplication of Mycobacterium tuberculosis under reduced oxygen concentration which represents nonreplicating conditions. Material and Methods. Acetone, ethanol and aqueous extracts of the plants Acorus calamus L. (rhizome), Ocimum sanctum L. (leaf), Piper nigrum L. (seed), and Pueraria tuberosa DC. (tuber) were tested on Mycobacterium tuberculosis H37Rv intracellularly using an epithelial cell (A549) infection model. The extracts found to be active intracellularly were further studied axenically under reducing oxygen concentrations. Results and Conclusions. Intracellular multiplication was inhibited ≥60% by five of the twelve extracts. Amongst these 5 extracts, in axenic culture, P. nigrum (acetone) was active under aerobic, microaerophilic, and anaerobic conditions indicating presence of multiple components acting at different levels and P. tuberosa (aqueous) showed bactericidal activity under microaerophilic and anaerobic conditions implying the influence of anaerobiosis on its efficacy. P. nigrum (aqueous) and A. calamus (aqueous and ethanol) extracts were not active under axenic conditions but only inhibited intracellular growth of Mycobacterium tuberculosis, suggesting activation of host defense mechanisms to mediate bacterial killing rather than direct bactericidal activity. PMID:26941797

  11. Structure-activity relationships of compounds targeting mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate synthase.

    PubMed

    Mao, Jialin; Eoh, Hyungjin; He, Rong; Wang, Yuehong; Wan, Baojie; Franzblau, Scott G; Crick, Dean C; Kozikowski, Alan P

    2008-10-01

    We report on a target-based approach to identify possible Mycobacterium tuberculosis DXS inhibitors from the structure of a known transketolase inhibitor. A small focused library of analogs was assembled in order to begin elucidating some meaningful structure-activity relationships of 3-(4-chloro-phenyl)-5-benzyl-4H-pyrazolo[1,5-a]pyrimidin-7-one. Ultimately we found that 2-methyl-3-(4-fluorophenyl)-5-(4-methoxy-phenyl)-4H-pyrazolo[1,5-a]pyrimidin-7-one, although still weak, was able to inhibit M. tuberculosis DXS with an IC(50) of 10.6 microM.

  12. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis.

    PubMed

    Bai, Lin; Hu, Kuan; Wang, Tong; Jastrab, Jordan B; Darwin, K Heran; Li, Huilin

    2016-04-05

    The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.

  13. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

    DOE PAGES

    Bai, Lin; Hu, Kuan; Wang, Tong; ...

    2016-03-21

    Here, the human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, themore » truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.« less

  14. Immunological activity of a 38-kilodalton protein purified from Mycobacterium tuberculosis.

    PubMed Central

    Young, D; Kent, L; Rees, A; Lamb, J; Ivanyi, J

    1986-01-01

    A 38-kilodalton (kDa) protein antigen from Mycobacterium tuberculosis was purified by monoclonal antibody TB71-based affinity chromatography. This molecule carries two nonoverlapping epitopes recognized by monoclonal antibodies TB71 and TB72, which are expressed substantially more strongly by M. tuberculosis than by Mycobacterium bovis. However, cross-reactive determinants between these two species were revealed on the 38-kDa protein by a rabbit anti-BCG serum. An immunoradiometric assay based on the TB71 and TB72 antibody pair specifically determined 38-kDa-antigen concentrations in mycobacterial extracts. Antibodies in sera from tuberculosis patients estimated by binding to 38-kDa-antigen-coated microtiter plates were positively correlated with TB72 competing titers. Unlike antibodies, T-cell proliferative responses to the 38-kDa protein were expressed equally by 60% of tuberculosis patients and healthy BCG-vaccinated subjects. Similarly, delayed-type hypersensitivity skin reactions were elicited in both M. tuberculosis- and M. bovis-sensitized guinea pigs. The results suggest the immunodominance of the species-specific B-cell and cross-reactive T-cell stimulatory epitopes. Images PMID:2428751

  15. Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain.

    PubMed

    Garrett, Christopher K; Broadwell, Lindsey J; Hayne, Cassandra K; Neher, Saskia B

    2015-01-01

    Mycobacterium tuberculosis harbors over 160 genes encoding PE/PPE proteins, several of which have roles in the pathogen's virulence. A number of PE/PPE proteins are secreted via Type VII secretion systems known as the ESX secretion systems. One PE protein, LipY, has a triglyceride lipase domain in addition to its PE domain. LipY can regulate intracellular triglyceride levels and is also exported to the cell wall by one of the ESX family members, ESX-5. Upon export, LipY's PE domain is removed by proteolytic cleavage. Studies using cells and crude extracts suggest that LipY's PE domain not only directs its secretion by ESX-5, but also functions to inhibit its enzymatic activity. Here, we attempt to further elucidate the role of LipY's PE domain in the regulation of its enzymatic activity. First, we established an improved purification method for several LipY variants using detergent micelles. We then used enzymatic assays to confirm that the PE domain down-regulates LipY activity. The PE domain must be attached to LipY in order to effectively inhibit it. Finally, we determined that full length LipY and the mature lipase lacking the PE domain (LipYΔPE) have similar melting temperatures. Based on our improved purification strategy and activity-based approach, we concluded that LipY's PE domain down-regulates its enzymatic activity but does not impact the thermal stability of the enzyme.

  16. Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

    PubMed Central

    Vasconcelos, Sidra E. G.; Esteves, Leonardo S.; Gomes, Harrison M.; Almeida da Silva, Pedro; Perdigão, João; Portugal, Isabel; Viveiros, Miguel; McNerney, Ruth; Pain, Arnab; Clark, Taane G.; Rastogi, Nalin; Unis, Gisela; Rossetti, Maria Lucia R.

    2015-01-01

    We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RDRio, and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n = 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers. PMID:26400784

  17. Immune Responses in Cattle Inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare antigen-specific immune responses to varied patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis), colonization without path...

  18. Predominance of modern Mycobacterium tuberculosis strains and active transmission of Beijing sublineage in Jayapura, Indonesia Papua.

    PubMed

    Chaidir, Lidya; Sengstake, Sarah; de Beer, Jessica; Oktavian, Antonius; Krismawati, Hana; Muhapril, Erfin; Kusumadewi, Inri; Annisa, Jessi; Anthony, Richard; van Soolingen, Dick; Achmad, Tri Hanggono; Marzuki, Sangkot; Alisjahbana, Bachti; van Crevel, Reinout

    2016-04-01

    Mycobacterium tuberculosis genotype distribution is different between West and Central Indonesia, but there are no data on the most Eastern part, Papua. We aimed to identify the predominant genotypes of M. tuberculosis responsible for tuberculosis in coastal Papua, their transmission, and the association with patient characteristics. A total of 199 M. tuberculosis isolates were collected. Spoligotyping was applied to describe the population structure of M. tuberculosis, lineage identification was performed using a combination of lineage-specific markers, and genotypic clusters were identified using a combination of 24-locus-MIRU-VNTR and spoligotyping. A high degree of genetic diversity was observed among isolates based on their spoligopatterns. Strains from modern lineage 4 made up almost half of strains (46.9%), being more abundant than the ancient lineage 1 (33.7%), and modern lineage 2 (19.4%). Thirty-five percent of strains belonged to genotypic clusters, especially strains in the Beijing genotype. Previous TB treatment and mutations associated with drug resistance were more common in patients infected with strains of the Beijing genotype. Papua shows a different distribution of M. tuberculosis genotypes compared to other parts of Indonesia. Clustering and drug resistance of modern strains recently introduced to Papua may contribute to the high tuberculosis burden in this region.

  19. The Activity of a Hexameric M17 Metallo-Aminopeptidase Is Associated With Survival of Mycobacterium tuberculosis

    PubMed Central

    Correa, Andre F.; Bastos, Izabela M. D.; Neves, David; Kipnis, Andre; Junqueira-Kipnis, Ana P.; de Santana, Jaime M.

    2017-01-01

    Mycobacterium tuberculosis is one of the most prevalent human pathogens causing millions of deaths in the last years. Moreover, tuberculosis (TB) treatment has become increasingly challenging owing to the emergence of multidrug resistant M. tuberculosis strains. Thus, there is an immediate need for the development of new anti-TB drugs. Proteases appear to be a promising approach and may lead to shortened and effective treatments for drug-resistant TB. Although the M. tuberculosis genome predicts more than 100 genes encoding proteases, only a few of them have been studied. Aminopeptidases constitute a set of proteases that selectively remove amino acids from the N-terminus of proteins and peptides and may act as virulence factors, essential for survival and maintenance of many microbial pathogens. Here, we characterized a leucine aminopeptidase of M. tuberculosis (MtLAP) as a cytosolic oligomeric metallo-aminopeptidase. Molecular and enzymatic properties lead us to classify MtLAP as a typical member of the peptidase family M17. Furthermore, the aminopeptidase inhibitor bestatin strongly inhibited MtLAP activity, in vitro M. tuberculosis growth and macrophage infection. In murine model of TB, bestatin treatment reduced bacterial burden and lesion in the lungs of infected mice. Thus, our data suggest that MtLAP participates in important metabolic pathways of M. tuberculosis necessary for its survival and virulence and consequently may be a promising target for new anti-TB drugs.

  20. Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents.

    PubMed

    Reddi, Ravikumar; Arya, Tarun; Kishor, Chandan; Gumpena, Rajesh; Ganji, Roopa J; Bhukya, Supriya; Addlagatta, Anthony

    2014-09-01

    Methionine aminopeptidases (MetAPs) cleave initiator methionine from ~ 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs.

  1. Target-based identification of whole-cell active inhibitors of biotin biosynthesis in Mycobacterium tuberculosis.

    PubMed

    Park, Sae Woong; Casalena, Dominick E; Wilson, Daniel J; Dai, Ran; Nag, Partha P; Liu, Feng; Boyce, Jim P; Bittker, Joshua A; Schreiber, Stuart L; Finzel, Barry C; Schnappinger, Dirk; Aldrich, Courtney C

    2015-01-22

    Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counterscreen in biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counterscreen proved crucial to filter out compounds whose whole-cell activity was off target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were cocrystallized with BioA to provide a framework for future structure-based drug design efforts.

  2. UvrD2 is essential in Mycobacterium tuberculosis, but its helicase activity is not required.

    PubMed

    Williams, Alan; Güthlein, Carolin; Beresford, Nicola; Böttger, Erik C; Springer, Burkhard; Davis, Elaine O

    2011-09-01

    UvrD is an SF1 family helicase involved in DNA repair that is widely conserved in bacteria. Mycobacterium tuberculosis has two annotated UvrD homologues; here we investigate the role of UvrD2. The uvrD2 gene at its native locus could be knocked out only in the presence of a second copy of the gene, demonstrating that uvrD2 is essential. Analysis of the putative protein domain structure of UvrD2 shows a distinctive domain architecture, with an extended C terminus containing an HRDC domain normally found in SF2 family helicases and a linking domain carrying a tetracysteine motif. Truncated constructs lacking the C-terminal domains of UvrD2 were able to compensate for the loss of the chromosomal copy, showing that these C-terminal domains are not essential. Although UvrD2 is a functional helicase, a mutant form of the protein lacking helicase activity was able to permit deletion of uvrD2 at its native locus. However, a mutant protein unable to hydrolyze ATP or translocate along DNA was not able to compensate for lack of the wild-type protein. Therefore, we concluded that the essential role played by UvrD2 is unlikely to involve its DNA unwinding activity and is more likely to involve DNA translocation and, possibly, protein displacement.

  3. Target-Based Identification of Whole-Cell Active Inhibitors of Biotin Biosynthesis in Mycobacterium tuberculosis

    PubMed Central

    Park, Sae Woong; Casalena, Dominick; Wilson, Daniel; Dai, Ran; Nag, Partha; Liu, Feng; Boyce, Jim P.; Bittker, Joshua; Schreiber, Stuart; Finzel, Barry C.; Schnappinger, Dirk; Aldrich, Courtney C.

    2014-01-01

    SUMMARY Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counter-screen in either biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counter-screen proved crucial to filter out compounds whose whole-cell activity was off-target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were co-crystallized with BioA to provide a framework for future structure-based drug design efforts. PMID:25556942

  4. Virulence factors of the Mycobacterium tuberculosis complex

    PubMed Central

    Forrellad, Marina A.; Klepp, Laura I.; Gioffré, Andrea; Sabio y García, Julia; Morbidoni, Hector R.; Santangelo, María de la Paz; Cataldi, Angel A.; Bigi, Fabiana

    2013-01-01

    The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world. PMID:23076359

  5. Comparison of activities of rifapentine and rifampin against Mycobacterium tuberculosis residing in human macrophages.

    PubMed Central

    Mor, N; Simon, B; Mezo, N; Heifets, L

    1995-01-01

    The activities of rifapentine and rifampin against Mycobacterium tuberculosis residing in human monocyte-derived macrophages were determined. The MICs and MBCs of rifapentine for intracellular bacteria were two- to fourfold lower than those of rifampin. For extracellular bacteria, this difference was less noticeable. Nevertheless, the more favorable pharmacokinetics of rifapentine over rifampin was addressed in other experimental models. These models showed substantial differences after short pulsed exposures of the infected macrophages to the drugs and when the infected macrophages were exposed to changing drug concentrations that imitated the pharmacokinetic curves observed in blood. Once-a-week exposures to rifapentine concentrations equivalent to those attained in blood after one 600-mg dose resulted during the first week in a dramatic decline in the number of bacteria, and this decline was maintained at a minimal level for a period of four weeks. The results of this study have shown the suitability of rifapentine for intermittent-treatment regimens. The prolonged effect of rifapentine found in this study may be associated with high ratios of intracellular accumulation, which were four- to fivefold higher than those found for rifampin. Further studies on the intracellular distribution of rifamycins and on the sites of actual interaction between the drugs and bacteria residing in macrophages are necessary. PMID:8540718

  6. Structure-activity relationships of 2-aminothiazoles effective against Mycobacterium tuberculosis

    PubMed Central

    Meissner, Anja; Boshoff, Helena I.; Vasan, Mahalakshmi; Duckworth, Benjamin P.; Barry, Clifton E.; Aldrich, Courtney C.

    2013-01-01

    A series of 2-aminothiazoles was synthesized based on a HTS scaffold from a whole-cell screen against Mycobacterium tuberculosis (Mtb). The SAR shows the central thiazole moiety and the 2-pyridyl moiety at C-4 of the thiazole are intolerant to modification. However, the N-2 position of the aminothiazole exhibits high flexibility and we successfully improved the antitubercular activity of the initial hit by more than 128-fold through introduction of substituted benzoyl groups at this position. N-(3-Chlorobenzoyl)-4-(2-pyridinyl)-1,3-thiazol-2-amine (55) emerged as one of the most promising analogues with a MIC of 0.024 μM or 0.008 μg/mL in 7H9 media and therapeutic index of nearly ~300. However, 55 is rapidly metabolized by human liver microsomes (t1/2 = 28 min) with metabolism occurring at the invariant aminothiazole moiety and Mtb develops spontaneous resistance with a high frequency of ~10−5. PMID:24075144

  7. Mycobacterium tuberculosis EsxH inhibits ESCRT-dependent CD4(+) T-cell activation.

    PubMed

    Portal-Celhay, Cynthia; Tufariello, JoAnn M; Srivastava, Smita; Zahra, Aleena; Klevorn, Thais; Grace, Patricia S; Mehra, Alka; Park, Heidi S; Ernst, Joel D; Jacobs, William R; Philips, Jennifer A

    2016-12-05

    Mycobacterium tuberculosis (Mtb) establishes a persistent infection, despite inducing antigen-specific T-cell responses. Although T cells arrive at the site of infection, they do not provide sterilizing immunity. The molecular basis of how Mtb impairs T-cell function is not clear. Mtb has been reported to block major histocompatibility complex class II (MHC-II) antigen presentation; however, no bacterial effector or host-cell target mediating this effect has been identified. We recently found that Mtb EsxH, which is secreted by the Esx-3 type VII secretion system, directly inhibits the endosomal sorting complex required for transport (ESCRT) machinery. Here, we showed that ESCRT is required for optimal antigen processing; correspondingly, overexpression and loss-of-function studies demonstrated that EsxH inhibited the ability of macrophages and dendritic cells to activate Mtb antigen-specific CD4(+) T cells. Compared with the wild-type strain, the esxH-deficient strain induced fivefold more antigen-specific CD4(+) T-cell proliferation in the mediastinal lymph nodes of mice. We also found that EsxH undermined the ability of effector CD4(+) T cells to recognize infected macrophages and clear Mtb. These results provide a molecular explanation for how Mtb impairs the adaptive immune response.

  8. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

    The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354

  9. Mycobacterium tuberculosis Rv2882c Protein Induces Activation of Macrophages through TLR4 and Exhibits Vaccine Potential

    PubMed Central

    Back, Yong Woo; Park, Hye-Soo; Bae, Hyun Shik; Choi, Chul Hee; Kim, Hwa-Jung

    2016-01-01

    Macrophages constitute the first line of defense against Mycobacterium tuberculosis and are critical in linking innate and adaptive immunity. Therefore, the identification and characterization of mycobacterial proteins that modulate macrophage function are essential for understanding tuberculosis pathogenesis. In this study, we identified the novel macrophage-activating protein, Rv2882c, from M. tuberculosis culture filtrate proteins. Recombinant Rv2882c protein activated macrophages to secrete pro-inflammatory cytokines and express co-stimulatory and major histocompatibility complex molecules via Toll-like receptor 4, myeloid differentiation primary response protein 88, and Toll/IL-1 receptor-domain-containing adaptor inducing IFN-beta. Mitogen-activated protein kinases and NF-κB signaling pathways were involved in Rv2882c-induced macrophage activation. Further, Rv2882c-treated macrophages induced expansion of the effector/memory T cell population and Th1 immune responses. In addition, boosting Bacillus Calmette-Guerin vaccination with Rv2882c improved protective efficacy against M. tuberculosis in our model system. These results suggest that Rv2882c is an antigen that could be used for tuberculosis vaccine development. PMID:27711141

  10. Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain

    PubMed Central

    Garrett, Christopher K.; Broadwell, Lindsey J.; Hayne, Cassandra K.; Neher, Saskia B.

    2015-01-01

    Mycobacterium tuberculosis harbors over 160 genes encoding PE/PPE proteins, several of which have roles in the pathogen’s virulence. A number of PE/PPE proteins are secreted via Type VII secretion systems known as the ESX secretion systems. One PE protein, LipY, has a triglyceride lipase domain in addition to its PE domain. LipY can regulate intracellular triglyceride levels and is also exported to the cell wall by one of the ESX family members, ESX-5. Upon export, LipY’s PE domain is removed by proteolytic cleavage. Studies using cells and crude extracts suggest that LipY’s PE domain not only directs its secretion by ESX-5, but also functions to inhibit its enzymatic activity. Here, we attempt to further elucidate the role of LipY’s PE domain in the regulation of its enzymatic activity. First, we established an improved purification method for several LipY variants using detergent micelles. We then used enzymatic assays to confirm that the PE domain down-regulates LipY activity. The PE domain must be attached to LipY in order to effectively inhibit it. Finally, we determined that full length LipY and the mature lipase lacking the PE domain (LipYΔPE) have similar melting temperatures. Based on our improved purification strategy and activity-based approach, we concluded that LipY’s PE domain down-regulates its enzymatic activity but does not impact the thermal stability of the enzyme. PMID:26270534

  11. Aminopyrazolo[1,5-a]pyrimidines as potential inhibitors of Mycobacterium tuberculosis: Structure activity relationships and ADME characterization.

    PubMed

    Soares de Melo, Candice; Candice, Soares de Melo; Feng, Tzu-Shean; van der Westhuyzen, Renier; Gessner, Richard K; Street, Leslie J; Morgans, Garreth L; Warner, Digby F; Moosa, Atica; Naran, Krupa; Lawrence, Nina; Boshoff, Helena I M; Barry, Clifton E; Harris, C John; Gordon, Richard; Chibale, Kelly

    2015-11-15

    Whole-cell high-throughput screening of a diverse SoftFocus library against Mycobacterium tuberculosis (Mtb) generated a novel aminopyrazolo[1,5-a]pyrimidine hit series. The synthesis and structure activity relationship studies identified compounds with potent antimycobacterial activity. The SAR of over 140 compounds shows that the 2-pyridylmethylamine moiety at the C-7 position of the pyrazolopyrimidine scaffold was important for Mtb activity, whereas the C-3 position offered a higher degree of flexibility. The series was also profiled for in vitro cytotoxicity and microsomal metabolic stability as well as physicochemical properties. Consequently liabilities to be addressed in a future lead optimization campaign have been identified.

  12. The Cytosolic Sensor cGAS Detects Mycobacterium tuberculosis DNA to Induce Type I Interferons and Activate Autophagy.

    PubMed

    Watson, Robert O; Bell, Samantha L; MacDuff, Donna A; Kimmey, Jacqueline M; Diner, Elie J; Olivas, Joanna; Vance, Russell E; Stallings, Christina L; Virgin, Herbert W; Cox, Jeffery S

    2015-06-10

    Type I interferons (IFNs) are critical mediators of antiviral defense, but their elicitation by bacterial pathogens can be detrimental to hosts. Many intracellular bacterial pathogens, including Mycobacterium tuberculosis, induce type I IFNs following phagosomal membrane perturbations. Cytosolic M. tuberculosis DNA has been implicated as a trigger for IFN production, but the mechanisms remain obscure. We report that the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), is required for activating IFN production via the STING/TBK1/IRF3 pathway during M. tuberculosis and L. pneumophila infection of macrophages, whereas L. monocytogenes short-circuits this pathway by producing the STING agonist, c-di-AMP. Upon sensing cytosolic DNA, cGAS also activates cell-intrinsic antibacterial defenses, promoting autophagic targeting of M. tuberculosis. Importantly, we show that cGAS binds M. tuberculosis DNA during infection, providing direct evidence that this unique host-pathogen interaction occurs in vivo. These data uncover a mechanism by which IFN is likely elicited during active human infections.

  13. Activity of phosphino palladium(II) and platinum(II) complexes against HIV-1 and Mycobacterium tuberculosis.

    PubMed

    Gama, Ntombenhle H; Elkhadir, Afag Y F; Gordhan, Bhavna G; Kana, Bavesh D; Darkwa, James; Meyer, Debra

    2016-08-01

    Treatment of human immunodeficiency virus (HIV) is currently complicated by increased prevalence of co-infection with Mycobacterium tuberculosis. The development of drug candidates that offer the simultaneous management of HIV and tuberculosis (TB) would be of great benefit in the holistic treatment of HIV/AIDS, especially in sub-Saharan Africa which has the highest global prevalence of HIV-TB coinfection. Bis(diphenylphosphino)-2-pyridylpalladium(II) chloride (1), bis(diphenylphosphino)-2-pyridylplatinum(II) chloride (2), bis(diphenylphosphino)-2-ethylpyridylpalladium(II) chloride (3) and bis(diphenylphosphino)-2-ethylpyridylplatinum(II) (4) were investigated for the inhibition of HIV-1 through interactions with the viral protease. The complexes were subsequently assessed for biological potency against Mycobacterium tuberculosis H37Rv by determining the minimal inhibitory concentration (MIC) using broth microdilution. Complex (3) showed the most significant and competitive inhibition of HIV-1 protease (p = 0.014 at 100 µM). Further studies on its in vitro effects on whole virus showed reduced viral infectivity by over 80 % at 63 µM (p < 0.05). In addition, the complex inhibited the growth of Mycobacterium tuberculosis at an MIC of 5 µM and was non-toxic to host cells at all active concentrations (assessed by tetrazolium dye and real time cell electronic sensing). In vitro evidence is provided here for the possibility of utilizing a single metal-based compound for the treatment of HIV/AIDS and TB.

  14. Oral Vaccination with Heat Inactivated Mycobacterium bovis Activates the Complement System to Protect against Tuberculosis

    PubMed Central

    Garrido, Joseba M.; Aranaz, Alicia; Sevilla, Iker; Villar, Margarita; Boadella, Mariana; Galindo, Ruth C.; Pérez de la Lastra, José M.; Moreno-Cid, Juan A.; Fernández de Mera, Isabel G.; Alberdi, Pilar; Santos, Gracia; Ballesteros, Cristina; Lyashchenko, Konstantin P.; Minguijón, Esmeralda; Romero, Beatriz; de Juan, Lucía; Domínguez, Lucas; Juste, Ramón; Gortazar, Christian

    2014-01-01

    Tuberculosis (TB) remains a pandemic affecting billions of people worldwide, thus stressing the need for new vaccines. Defining the correlates of vaccine protection is essential to achieve this goal. In this study, we used the wild boar model for mycobacterial infection and TB to characterize the protective mechanisms elicited by a new heat inactivated Mycobacterium bovis vaccine (IV). Oral vaccination with the IV resulted in significantly lower culture and lesion scores, particularly in the thorax, suggesting that the IV might provide a novel vaccine for TB control with special impact on the prevention of pulmonary disease, which is one of the limitations of current vaccines. Oral vaccination with the IV induced an adaptive antibody response and activation of the innate immune response including the complement component C3 and inflammasome. Mycobacterial DNA/RNA was not involved in inflammasome activation but increased C3 production by a still unknown mechanism. The results also suggested a protective mechanism mediated by the activation of IFN-γ producing CD8+ T cells by MHC I antigen presenting dendritic cells (DCs) in response to vaccination with the IV, without a clear role for Th1 CD4+ T cells. These results support a role for DCs in triggering the immune response to the IV through a mechanism similar to the phagocyte response to PAMPs with a central role for C3 in protection against mycobacterial infection. Higher C3 levels may allow increased opsonophagocytosis and effective bacterial clearance, while interfering with CR3-mediated opsonic and nonopsonic phagocytosis of mycobacteria, a process that could be enhanced by specific antibodies against mycobacterial proteins induced by vaccination with the IV. These results suggest that the IV acts through novel mechanisms to protect against TB in wild boar. PMID:24842853

  15. In vitro activity of rifampicin and verapamil combination in multidrug-resistant mycobacterium tuberculosis.

    PubMed

    Demitto, Fernanda de Oliveira; do Amaral, Renata Claro Ribeiro; Maltempe, Flaviane Granero; Siqueira, Vera Lúcia Dias; Scodro, Regiane Bertin de Lima; Lopes, Mariana Aparecida; Caleffi-Ferracioli, Katiany R; Canezin, Pedro Henrique; Cardoso, Rosilene Fressatti

    2015-01-01

    The aim of the present study was to evaluate the effect of the combination of rifampicin (RIF) and verapamil (VP) against the Mycobacterium tuberculosis H37Rv reference strain and six multidrug-resistant (MDR) M. tuberculosis clinical isolates by determining Time-Kill Curves and the ability to efflux drug by fluorometry. The RIF+VP combination showed synergism in one MDR clinical isolate. For the other five MDR clinical isolates, the drug combination showed no interaction. The MDR clinical isolate had lower ethidium bromide (EtBr) accumulation when exposed to the RIF+VP combination, compared with RIF and VP exposure alone. The other MDR clinical isolates showed no significant difference in EtBr accumulation. These results suggest greater efflux action in one of the MDR clinical isolates compared with the M. tuberculosis H37Rv reference strain. The other five MDR isolates may have additional mechanisms of drug resistance to RIF. The use of the RIF+VP combination made one MDR bacillus more susceptible to RIF probably by inhibiting efflux pumps, and this combination therapy, in some cases, may contribute to a reduction of resistance to RIF in M. tuberculosis.

  16. Mycobacterium tuberculosis inhibits neutrophil apoptosis, leading to delayed activation of naive CD4 T cells.

    PubMed

    Blomgran, Robert; Desvignes, Ludovic; Briken, Volker; Ernst, Joel D

    2012-01-19

    Mycobacterium tuberculosis promotes its replication by inhibiting the apoptosis of infected macrophages. A proapoptotic M. tuberculosis mutant lacking nuoG, a subunit of the type I NADH dehydrogenase complex, exhibits attenuated growth in vivo, indicating that this virulence mechanism is essential. We show that M. tuberculosis also suppresses neutrophil apoptosis. Compared to wild-type, the nuoG mutant spread to a larger number of lung phagocytic cells. Consistent with the shorter lifespan of infected neutrophils, infection with the nuoG mutant resulted in fewer bacteria per infected neutrophil, accelerated bacterial acquisition by dendritic cells, earlier trafficking of these dendritic cells to lymph nodes, and faster CD4 T cell priming. Neutrophil depletion abrogated accelerated CD4 T cell priming by the nuoG mutant, suggesting that inhibiting neutrophil apoptosis delays adaptive immunity in tuberculosis. Thus, pathogen modulation of apoptosis is beneficial at multiple levels, and enhancing phagocyte apoptosis promotes CD4 as well as CD8 T cell responses.

  17. 2-(Quinolin-4-yloxy)acetamides Are Active against Drug-Susceptible and Drug-Resistant Mycobacterium tuberculosis Strains

    PubMed Central

    2016-01-01

    2-(Quinolin-4-yloxy)acetamides have been described as potent in vitro inhibitors of Mycobacterium tuberculosis growth. Herein, additional chemical modifications of lead compounds were carried out, yielding highly potent antitubercular agents with minimum inhibitory concentration (MIC) values as low as 0.05 μM. Further, the synthesized compounds were active against drug-resistant strains and were devoid of apparent toxicity to Vero and HaCat cells (IC50s ≥ 20 μM). In addition, the 2-(quinolin-4-yloxy)acetamides showed intracellular activity against the bacilli in infected macrophages with action similar to rifampin, low risk of drug–drug interactions, and no sign of cardiac toxicity in zebrafish (Danio rerio) at 1 and 5 μM. Therefore, these data indicate that this class of compounds may furnish candidates for future development to, hopefully, provide drug alternatives for tuberculosis treatment. PMID:26985307

  18. Mycobacterium tuberculosis heat shock proteins use diverse Toll-like receptor pathways to activate pro-inflammatory signals.

    PubMed

    Bulut, Yonca; Michelsen, Kathrin S; Hayrapetian, Linda; Naiki, Yoshikazu; Spallek, Ralf; Singh, Mahavir; Arditi, Moshe

    2005-06-03

    Although the Toll-like receptors used by Mycobacterium tuberculosis membrane and secreted factors are known, the pathways activated by M. tuberculosis heat shock proteins are not. An efficient immune response against the intracellular pathogen M. tuberculosis is critically dependent on rapid detection of the invading pathogen by the innate immune system and coordinated activation of the adaptive immune response. Macrophage phagocytosis of M. tuberculosis is accompanied by activation of the transcription factor NF-kappaB and secretion of inflammatory mediators that play an important role in granuloma formation and immune protection during M. tuberculosis infection. The interaction between M. tuberculosis and the various Toll-like receptors is complex, and it appears that distinct mycobacterial components may interact with different members of the Toll-like receptor family. Here we show that recombinant, purified, mycobacterial heat shock proteins 65 and 70 induce NF-kappaB activity in a dose-dependent manner in human endothelial cells. Furthermore, we show that whereas mycobacterial heat shock protein 65 signals exclusively through Toll-like receptor 4, heat shock protein 70 also signals through Toll-like receptor 2. Mycobacterial heat shock protein 65-induced NF-kappaB activation was MyD88-, TIRAP-, TRIF-, and TRAM-dependent and required the presence of MD-2. A better understanding of the recognition of mycobacterial heat shock proteins and their role in the host immune response to the pathogen may open the way to a better understanding of the immunological processes induced by this important human pathogen and the host-pathogen interactions and may help in the rational design of more effective vaccines or vaccine adjuvants.

  19. Strong In Vitro Activities of Two New Rifabutin Analogs against Multidrug-Resistant Mycobacterium tuberculosis ▿ †

    PubMed Central

    García, Ana-Belén; Palacios, Juan J.; Ruiz, María-Jesús; Barluenga, José; Aznar, Fernando; Cabal, María-Paz; García, José María; Díaz, Natalia

    2010-01-01

    Two new rifabutin analogs, RFA-1 and RFA-2, show high in vitro antimycobacterial activities against Mycobacterium tuberculosis. MIC values of RFA-1 and RFA-2 were ≤0.02 μg/ml against rifamycin-susceptible strains and 0.5 μg/ml against a wide selection of multidrug-resistant strains, compared to ≥50 μg/ml for rifampin and 10 μg/ml for rifabutin. Molecular dynamic studies indicate that the compounds may exert tighter binding to mutants of RNA polymerase that have adapted to the rifamycins. PMID:20855731

  20. Mycobacterium tuberculosis Promotes Anti-apoptotic Activity of the Macrophage by PtpA Protein-dependent Dephosphorylation of Host GSK3α*

    PubMed Central

    Poirier, Valérie; Bach, Horacio; Av-Gay, Yossef

    2014-01-01

    Mycobacterium tuberculosis tyrosine phosphatase PtpA inhibits two key cellular events in macrophages required for the elimination of invading organisms, phagosome acidification, and maturation. Kinome analysis revealed multiple PtpA-dependent changes to the phosphorylation status of macrophage proteins upon M. tuberculosis infection. Among those proteins we show that PtpA dephosphorylates GSK3α on amino acid Tyr279, which leads to modulation of GSK3α anti-apoptotic activity, promoting pathogen survival early during infection. PMID:25187516

  1. Characterization of Antibacterial and Hemolytic Activity of Synthetic Pandinin 2 Variants and Their Inhibition against Mycobacterium tuberculosis

    PubMed Central

    Rodríguez, Alexis; Villegas, Elba; Montoya-Rosales, Alejandra; Rivas-Santiago, Bruno; Corzo, Gerardo

    2014-01-01

    The contention and treatment of Mycobacterium tuberculosis and other bacteria that cause infectious diseases require the use of new type of antibiotics. Pandinin 2 (Pin2) is a scorpion venom antimicrobial peptide highly hemolytic that has a central proline residue. This residue forms a structural “kink” linked to its pore-forming activity towards human erythrocytes. In this work, the residue Pro14 of Pin2 was both substituted and flanked using glycine residues (P14G and P14GPG) based on the low hemolytic activities of antimicrobial peptides with structural motifs Gly and GlyProGly such as magainin 2 and ponericin G1, respectively. The two Pin2 variants showed antimicrobial activity against E. coli, S. aureus, and M. tuberculosis. However, Pin2 [GPG] was less hemolytic (30%) than that of Pin2 [G] variant. In addition, based on the primary structure of Pin2 [G] and Pin2 [GPG], two short peptide variants were designed and chemically synthesized keeping attention to their physicochemical properties such as hydrophobicity and propensity to adopt alpha-helical conformations. The aim to design these two short antimicrobial peptides was to avoid the drawback cost associated to the synthesis of peptides with large sequences. The short Pin2 variants named Pin2 [14] and Pin2 [17] showed antibiotic activity against E. coli and M. tuberculosis. Besides, Pin2 [14] presented only 25% of hemolysis toward human erythrocytes at concentrations as high as 100 µM, while the peptide Pin2 [17] did not show any hemolytic effect at the same concentration. Furthermore, these short antimicrobial peptides had better activity at molar concentrations against multidrug resistance M. tuberculosis than that of the conventional antibiotics ethambutol, isoniazid and rifampicin. Therefore, Pin2 [14] and Pin2 [17] have the potential to be used as an alternative antibiotics and anti-tuberculosis agents with reduced hemolytic effects. PMID:25019413

  2. Characterization of antibacterial and hemolytic activity of synthetic pandinin 2 variants and their inhibition against Mycobacterium tuberculosis.

    PubMed

    Rodríguez, Alexis; Villegas, Elba; Montoya-Rosales, Alejandra; Rivas-Santiago, Bruno; Corzo, Gerardo

    2014-01-01

    The contention and treatment of Mycobacterium tuberculosis and other bacteria that cause infectious diseases require the use of new type of antibiotics. Pandinin 2 (Pin2) is a scorpion venom antimicrobial peptide highly hemolytic that has a central proline residue. This residue forms a structural "kink" linked to its pore-forming activity towards human erythrocytes. In this work, the residue Pro14 of Pin2 was both substituted and flanked using glycine residues (P14G and P14GPG) based on the low hemolytic activities of antimicrobial peptides with structural motifs Gly and GlyProGly such as magainin 2 and ponericin G1, respectively. The two Pin2 variants showed antimicrobial activity against E. coli, S. aureus, and M. tuberculosis. However, Pin2 [GPG] was less hemolytic (30%) than that of Pin2 [G] variant. In addition, based on the primary structure of Pin2 [G] and Pin2 [GPG], two short peptide variants were designed and chemically synthesized keeping attention to their physicochemical properties such as hydrophobicity and propensity to adopt alpha-helical conformations. The aim to design these two short antimicrobial peptides was to avoid the drawback cost associated to the synthesis of peptides with large sequences. The short Pin2 variants named Pin2 [14] and Pin2 [17] showed antibiotic activity against E. coli and M. tuberculosis. Besides, Pin2 [14] presented only 25% of hemolysis toward human erythrocytes at concentrations as high as 100 µM, while the peptide Pin2 [17] did not show any hemolytic effect at the same concentration. Furthermore, these short antimicrobial peptides had better activity at molar concentrations against multidrug resistance M. tuberculosis than that of the conventional antibiotics ethambutol, isoniazid and rifampicin. Therefore, Pin2 [14] and Pin2 [17] have the potential to be used as an alternative antibiotics and anti-tuberculosis agents with reduced hemolytic effects.

  3. The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions.

    PubMed

    Backus, Keriann M; Dolan, Michael A; Barry, Conor S; Joe, Maju; McPhie, Peter; Boshoff, Helena I M; Lowary, Todd L; Davis, Benjamin G; Barry, Clifton E

    2014-09-05

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.

  4. Relationship between Pyrazinamide Resistance, Loss of Pyrazinamidase Activity, and Mutations in the pncA Locus in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosis

    PubMed Central

    Mestdagh, M.; Fonteyne, P. A.; Realini, L.; Rossau, R.; Jannes, G.; Mijs, W.; De Smet, K. A. L.; Portaels, F.; Van den Eeckhout, E.

    1999-01-01

    Sixty-two Mycobacterium tuberculosis isolates were tested for pyrazinamidase activity, and their pyrazinamide susceptibility was determined by the radiometric method. Sequencing of pncA genes in the 23 resistant strains revealed mutations in 16 pyrazinamidase-negative strains, 11 of which had not been previously described. Six isolates containing wild-type pncA might possess alternative resistance mechanisms. PMID:10471589

  5. Polymorphisms of twenty regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans or animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and the other members o...

  6. ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis

    PubMed Central

    D'Heygère, François; Schwartz, Annie; Coste, Franck; Castaing, Bertrand; Boudvillain, Marc

    2015-01-01

    The bacterial transcription termination factor Rho—a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity—is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal ‘RNA binding’ domain and by a non-canonical R-loop residue in its C-terminal ‘motor’ domain. We also show that the ‘motor’ domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors. PMID:25999346

  7. Mycobacterium tuberculosis and the host response

    PubMed Central

    Kaufmann, Stefan H.E.; Cole, Stewart T.; Mizrahi, Valerie; Rubin, Eric; Nathan, Carl

    2005-01-01

    Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. Advances reported at a recent international meeting highlight insights and controversies in the genetics of M. tuberculosis and the infected host, the nature of protective immune responses, adaptation of the bacillus to host-imposed stresses, animal models, and new techniques. PMID:15939785

  8. Biochemical characterization of quinolinic acid phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv and inhibition of its activity by pyrazinamide.

    PubMed

    Kim, Hyun; Shibayama, Keigo; Rimbara, Emiko; Mori, Shigetarou

    2014-01-01

    Quinolinic acid phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) is a key enzyme in the de novo pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis and a target for the development of new anti-tuberculosis drugs. QAPRTase catalyzes the synthesis of nicotinic acid mononucleotide from quinolinic acid (QA) and 5-phosphoribosyl-1-pyrophosphate (PRPP) through a phosphoribosyl transfer reaction followed by decarboxylation. The crystal structure of QAPRTase from Mycobacterium tuberculosis H37Rv (MtQAPRTase) has been determined; however, a detailed functional analysis of MtQAPRTase has not been published. Here, we analyzed the enzymatic activities of MtQAPRTase and determined the effect on catalysis of the anti-tuberculosis drug pyrazinamide (PZA). The optimum temperature and pH for MtQAPRTase activity were 60°C and pH 9.2. MtQAPRTase required bivalent metal ions and its activity was highest in the presence of Mg2+. Kinetic analyses revealed that the Km values for QA and PRPP were 0.08 and 0.39 mM, respectively, and the kcat values for QA and PRPP were 0.12 and 0.14 [s-1], respectively. When the amino acid residues of MtQAPRTase, which may interact with QA, were substituted with alanine residues, catalytic activity was undetectable. Further, PZA, which is an anti-tuberculosis drug and a structural analog of QA, markedly inhibited the catalytic activity of MtQAPRTase. The structure of PZA may provide the basis for the design of new inhibitors of MtQAPRTase. These findings provide new insights into the catalytic properties of MtQAPRTase.

  9. Mefloquine and its oxazolidine derivative compound are active against drug-resistant Mycobacterium tuberculosis strains and in a murine model of tuberculosis infection.

    PubMed

    Rodrigues-Junior, Valnês S; Villela, Anne D; Gonçalves, Raoni S B; Abbadi, Bruno Lopes; Trindade, Rogério Valim; López-Gavín, Alexandre; Tudó, Griselda; González-Martín, Julian; Basso, Luiz Augusto; de Souza, Marcus V N; Campos, Maria Martha; Santos, Diógenes Santiago

    2016-08-01

    Repurposing of drugs to treat tuberculosis (TB) has been considered an alternative to overcome the global TB epidemic, especially to combat drug-resistant forms of the disease. Mefloquine has been reported as a potent drug to kill drug-resistant strains of Mycobacterium tuberculosis. In addition, mefloquine-derived molecules have been synthesised and their effectiveness against mycobacteria has been assessed. In this work, we demonstrate for the first time the activities of mefloquine and its oxazolidine derivative compound 1E in a murine model of TB infection following administration of both drugs by the oral route. The effects of associations between mefloquine or 1E with the clinically used antituberculosis drugs isoniazid, rifampicin, ethambutol, moxifloxacin and streptomycin were also investigated. Importantly, combination of mefloquine with isoniazid and of 1E with streptomycin showed a two-fold decrease in their minimum inhibitory concentrations (MICs). Moreover, no tested combinations demonstrated antagonist interactions. Here we describe novel evidence on the activity of mefloquine and 1E against a series of quinolone-resistant M. tuberculosis strains. These data show MICs against quinolone-resistant strains (0.5-8 µg/mL) similar to or lower than those previously reported for multidrug-resistant strains. Taking these results together, we can suggest the use of mefloquine or 1E in combination with clinically available drugs, especially in the case of resistant forms of TB.

  10. Anti-tubercular and antioxidant activities of C-glycosyl carbonic anhydrase inhibitors: towards the development of novel chemotherapeutic agents against Mycobacterium tuberculosis.

    PubMed

    Zaro, María J; Bortolotti, Ana; Riafrecha, Leonardo E; Concellón, Analía; Morbidoni, Héctor R; Colinas, Pedro A

    2016-12-01

    During the treatment of tuberculosis infection, oxidative stress due to anti-tubercular drugs may result in tissue inflammation. It was suggested that treatment with antioxidant drugs could be beneficial as an adjunct to anti-tuberculosis drug therapy. Recently our group has shown that several C-glycosides are inhibitors of Mycobacterium tuberculosis β-carbonic anhydrases (CAs, EC 4.2.1.1). In an effort to develop novel chemotherapeutic agents against tuberculosis, the anti-tubercular and antioxidant activities of a series of C-glycosides containing the phenol or the methoxyaryl moiety were studied. Many compounds showed inhibition of growth of M. tuberculosis H37Rv strain and good antioxidant ability. A glycomimetic incorporating the 3-hydroxyphenyl moiety showed the best activity profile and therefore this functionality represents lead for the development of novel anti-tubercular agents with dual mechanisms of action.

  11. Mycobacterium tuberculosis: Success through dormancy

    PubMed Central

    Gengenbacher, Martin; Kaufmann, Stefan H. E.

    2012-01-01

    Tuberculosis (TB) remains a major health threat, killing near to 2 million individuals around this globe, annually. The sole vaccine developed almost a century ago, provides limited protection only during childhood. After decades without the introduction of new antibiotics, several candidates are currently undergoing clinical investigation. Curing TB requires prolonged combination chemotherapy with several drugs. Moreover, monitoring the success of therapy is questionable due to the lack of reliable biomarkers. To substantially improve the situation, a detailed understanding of the crosstalk between human host and the pathogen Mycobacterium tuberculosis (Mtb) is vital. Principally, Mtb’s enormous success is based on three capacities: First, reprogramming of macrophages after primary infection/phagocytosis in order to prevent its own destruction; second, initiating the formation of well-organized granulomas, comprising different immune cells to create a confined environment for the host–pathogen standoff; third, the capability to shut down its own central metabolism, terminate replication and thereby transit into a stage of dormancy rendering itself extremely resistant to host defense and drug treatment. Here we review the molecular mechanisms underlying these processes, draw conclusions in a working model of mycobacterial dormancy and highlight gaps in our understanding to be addressed in future research. PMID:22320122

  12. Anti-Mycobacterium tuberculosis Activity of Calophyllum brasiliense Extracts Obtained by Supercritical Fluid Extraction and Conventional Techniques.

    PubMed

    Pires, Claudia T A; de L Scodro, Regiane B; Brenzan, Mislaine A; Cortez, Diógenes A G; Siqueira, Vera L D; Cardozo-Filho, Lúcio; Goncalves, Renata M; Caleffi-Ferracioli, Katiany R; Cardoso, Rosilene F

    2016-01-01

    The conventional techniques used to extract natural products have many disadvantages, and alternative methods have been used, such as supercritical fluid extraction (SFE-CO2). We compared the anti-Mycobacterium tuberculosis activity and cytotoxicity of extracts and major pure compounds were obtained from the leaves of Calophyllum brasiliense by SFE-CO2, maceration and Soxhlet. Anti-M tuberculosis activity was evaluated by resazurin microtiter assay plate and cytotoxicity assay was performed using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The (-) mammea A/BB, (-) mammea B/BB, mammea B/BB cyclo D, ponnalide, mammea A/BA cyclo D, and amentoflavone were identified as the majority compounds. SFE-CO2, especially at 313 K and 10.92 MPa showed better yield for (-) mammea A/BB. Anti-M. tuberculosis activity (62.5 μg/mL) and cytotoxicity (Selectivity Index = 0.320-0.576) were similar for the three extracts. Mammea B/BB cyclo D had a minimum inhibitory concentration (MIC) of 125 μg/mL, and ponnalide and mammea A/BA cyclo D had MICs > 250 μg/mL. The pure compounds isolated showed low Selectivity Index (< 0.09). SFE-CO2 may be more promising than conventional methods for the extraction of compound (-) mammea A/BB, which presented the best anti-M. tuberculosis activity in our previous study. This is important for current industrial requirements to obtain extracts from medicinal plants using clean technologies.

  13. The pathology of Mycobacterium tuberculosis infection.

    PubMed

    Sakamoto, K

    2012-05-01

    Mycobacterium tuberculosis is an old enemy of the human race, with evidence of infection observed as early as 5000 years ago. Although more host-restricted than Mycobacterium bovis, which can infect all warm-blooded vertebrates, M. tuberculosis can infect, and cause morbidity and mortality in, several veterinary species as well. As M. tuberculosis is one of the earliest described bacterial pathogens, the literature describing this organism is vast and overwhelming. This review strives to distill what is currently known about this bacterium and the disease it causes for the veterinary pathologist.

  14. Interaction of N-methyl-2-alkenyl-4-quinolones with ATP-dependent MurE ligase of Mycobacterium tuberculosis: antibacterial activity, molecular docking and inhibition kinetics

    PubMed Central

    Guzman, Juan David; Wube, Abraham; Evangelopoulos, Dimitrios; Gupta, Antima; Hüfner, Antje; Basavannacharya, Chandrakala; Rahman, Md. Mukhleshur; Thomaschitz, Christina; Bauer, Rudolf; McHugh, Timothy Daniel; Nobeli, Irene; Prieto, Jose M.; Gibbons, Simon; Bucar, Franz; Bhakta, Sanjib

    2011-01-01

    Objectives The aim of this study was to comprehensively evaluate the antibacterial activity and MurE inhibition of a set of N-methyl-2-alkenyl-4-quinolones found to inhibit the growth of fast-growing mycobacteria. Methods Using the spot culture growth inhibition assay, MICs were determined for Mycobacterium tuberculosis H37Rv, Mycobacterium bovis BCG and Mycobacterium smegmatis mc2155. MICs were determined for Mycobacterium fortuitum, Mycobacterium phlei, methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using microplate dilution assays. Inhibition of M. tuberculosis MurE ligase activity was determined both by colorimetric and HPLC methods. Computational modelling and binding prediction of the quinolones in the MurE structure was performed using Glide. Kinetic experiments were conducted for understanding possible competitive relations of the quinolones with the endogenous substrates of MurE ligase. Results The novel synthetic N-methyl-2-alkenyl-4-quinolones were found to be growth inhibitors of M. tuberculosis and rapid-growing mycobacteria as well as methicillin-resistant S. aureus, while showing no inhibition for E. coli and P. aeruginosa. The quinolones were found to be inhibitory to MurE ligase of M. tuberculosis in the micromolar range (IC50 ∼40–200 μM) when assayed either spectroscopically or by HPLC. Computational docking of the quinolones on the published M. tuberculosis MurE crystal structure suggested that the uracil recognition site is a probable binding site for the quinolones. Conclusions N-methyl-2-alkenyl-4-quinolones are inhibitors of mycobacterial and staphylococcal growth, and show MurE ligase inhibition. Therefore, they are considered as a starting point for the development of increased affinity MurE activity disruptors. PMID:21622974

  15. Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil.

    PubMed

    Coelho, Tatiane; Machado, Diana; Couto, Isabel; Maschmann, Raquel; Ramos, Daniela; von Groll, Andrea; Rossetti, Maria L; Silva, Pedro A; Viveiros, Miguel

    2015-01-01

    Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.

  16. Flourensia cernua: Hexane Extracts a Very Active Mycobactericidal Fraction from an Inactive Leaf Decoction against Pansensitive and Panresistant Mycobacterium tuberculosis

    PubMed Central

    Molina-Salinas, Gloria María; Peña-Rodríguez, Luis Manuel; Mata-Cárdenas, Benito David; Escalante-Erosa, Fabiola; González-Hernández, Silvia; Torres de la Cruz, Víctor Manuel; Martínez-Rodríguez, Herminia Guadalupe; Said-Fernández, Salvador

    2011-01-01

    The efficacy of decoction in extracting mycobactericidal compounds from Flourensia cernua (Hojasé) leaves and fractionation with solvents having ascending polarity was compared with that of (i) ethanol extraction by still maceration, extraction with a Soxhlet device, shake-assisted maceration, or ultrasound-assisted maceration, followed by fractionation with n-hexane, ethyl acetate, and n-butanol; (ii) sequential extraction with n-hexane, ethyl acetate, and n-butanol, by still maceration, using a Soxhlet device, shake-assisted maceration, or ultrasound-assisted maceration. The in vitro mycobactericidal activity of each preparation was measured against drug-sensitive (SMtb) and drug-resistant (RMtb) Mycobacterium tuberculosis strains. The results of which were expressed as absolute mycobactericidal activity (AMA). These data were normalized to the ΣAMA of the decoction fraction set. Although decoction was inactive, the anti-RMtb normalized ΣAMA (NAMA) of its fractions was comparable with the anti-RMtb NAMA of the still maceration extracts and significantly higher than the anti-SMtb and anti-RMtb NAMAs of every other ethanol extract and serial extract and fraction. Hexane extracted, from decoction, material having 55.17% and 92.62% of antituberculosis activity against SMtb and RMtb, respectively. Although the mycobactericidal activity of decoction is undetectable; its efficacy in extracting F. cernua active metabolites against M. tuberculosis is substantially greater than almost all pharmacognostic methods. PMID:21584254

  17. Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG

    PubMed Central

    Mori, Giorgia; Chiarelli, Laurent R.; Esposito, Marta; Makarov, Vadim; Bellinzoni, Marco; Hartkoorn, Ruben C.; Degiacomi, Giulia; Boldrin, Francesca; Ekins, Sean; de Jesus Lopes Ribeiro, Ana Luisa; Marino, Leonardo B.; Centárová, Ivana; Svetlíková, Zuzana; Blaško, Jaroslav; Kazakova, Elena; Lepioshkin, Alexander; Barilone, Nathalie; Zanoni, Giuseppe; Porta, Alessio; Fondi, Marco; Fani, Renato; Baulard, Alain R.; Mikušová, Katarína; Alzari, Pedro M.; Manganelli, Riccardo; de Carvalho, Luiz Pedro S.; Riccardi, Giovanna; Cole, Stewart T.; Pasca, Maria Rosalia

    2015-01-01

    Summary To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target. PMID:26097035

  18. Chemical composition of hexane extract of Citrus aurantifolia and anti-Mycobacterium tuberculosis activity of some of its constituents.

    PubMed

    Sandoval-Montemayor, Nallely E; García, Abraham; Elizondo-Treviño, Elizabeth; Garza-González, Elvira; Alvarez, Laura; del Rayo Camacho-Corona, María

    2012-09-19

    The main aim of this study was to isolate and characterize the active compounds from the hexane extract of the fruit peels of Citrus aurantiifolia, which showed activity against one sensitive and three monoresistant (isoniazid, streptomycin or ethambutol) strains of Mycobacterium tuberculosis H37Rv. The active extract was fractionated by column chromatography, yielding the following major compounds: 5-geranyloxypsoralen (1); 5-geranyloxy-7-methoxycoumarin (2); 5,7-dimethoxycoumarin (3); 5-methoxypsoralen (4); and 5,8-dimethoxypsoralen (5). The structures of these compounds were elucidated by 1D and 2D NMR spectroscopy. In addition, GC-MS analysis of the hexane extract allowed the identification of 44 volatile compounds, being 5,7-dimethoxycoumarin (15.79%), 3-methyl-1,2-cyclopentanedione (8.27%), 1-methoxy-ciclohexene (8.0%), corylone (6.93%), palmitic acid (6.89%), 5,8-dimethoxypsoralen (6.08%), a-terpineol (5.97%), and umbelliferone (4.36%), the major constituents. Four isolated coumarins and 16 commercial compounds identified by GC-MS were tested against M. tuberculosis H37Rv and three multidrug-resistant M. tuberculosis strains using the Microplate Alamar Blue Assay. The constituents that showed activity against all strains were 5 (MICs = 25-50 mg/mL), 1 (MICs = 50-100 mg/mL), palmitic acid (MICs = 25-50 mg/mL), linoleic acid (MICs = 50-100 mg/mL), oleic acid (MICs = 100 mg/mL), 4-hexen-3-one (MICs = 50-100 mg/mL), and citral (MICs = 50-100 mg/mL). Compound 5 and palmitic acid were the most active ones. The antimycobacterial activity of the hexane extract of C. aurantifolia could be attributed to these compounds.

  19. Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

    PubMed Central

    Davidge, Kelly S; Singh, Sandip; Bowman, Lesley AH; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site. PMID:26478812

  20. In vitro susceptibilities of Mycobacterium tuberculosis to 10 antimicrobial agents.

    PubMed Central

    Byrne, S K; Crawford, C E; Geddes, G L; Black, W A

    1988-01-01

    After preliminary in vitro screening of 10 antimicrobial agents against Mycobacterium tuberculosis, the MICs of the 6 most promising agents against 27 clinical isolates were determined by agar dilution. The two quinolone compounds tested (difloxacin and A-56620) were the most active, each inhibiting 50% of the strains at concentrations of 4 micrograms/ml. M. tuberculosis strains previously shown to be resistant to isoniazid, streptomycin, rifampin, or ethambutol were as susceptible to these quinolone compounds as susceptible strains. PMID:3143305

  1. High-content screening technology combined with a human granuloma model as a new approach to evaluate the activities of drugs against Mycobacterium tuberculosis.

    PubMed

    Silva-Miranda, Mayra; Ekaza, Euloge; Breiman, Adrien; Asehnoune, Karim; Barros-Aguirre, David; Pethe, Kevin; Ewann, Fanny; Brodin, Priscille; Ballell-Pages, Lluís; Altare, Frédéric

    2015-01-01

    Tuberculosis remains a major health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis. Some models have provided valuable information about drug resistance and efficacy; however, the translation of these results into effective human treatments has mostly proven unsuccessful. In this study, we adapted high-content screening (HCS) technology to investigate the activities of antitubercular compounds in the context of an in vitro granuloma model. We observed significant shifts in the MIC50s between the activities of the compounds under extracellular and granuloma conditions.

  2. High-Content Screening Technology Combined with a Human Granuloma Model as a New Approach To Evaluate the Activities of Drugs against Mycobacterium tuberculosis

    PubMed Central

    Silva-Miranda, Mayra; Breiman, Adrien; Asehnoune, Karim; Barros-Aguirre, David; Pethe, Kevin; Ewann, Fanny; Brodin, Priscille; Ballell-Pages, Lluís

    2014-01-01

    Tuberculosis remains a major health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis. Some models have provided valuable information about drug resistance and efficacy; however, the translation of these results into effective human treatments has mostly proven unsuccessful. In this study, we adapted high-content screening (HCS) technology to investigate the activities of antitubercular compounds in the context of an in vitro granuloma model. We observed significant shifts in the MIC50s between the activities of the compounds under extracellular and granuloma conditions. PMID:25348525

  3. A new antituberculosis drug that selectively kills nonmultiplying Mycobacterium tuberculosis.

    PubMed

    Mitchison, Denis A

    2008-03-13

    An important report by Bryk et al. in this issue of Cell Host & Microbe describes the properties of a rhodanine prodrug active against nonmultiplying Mycobacterium tuberculosis (Mtb). Considering the tolerance of nonreplicating Mtb to most currently available agents, such a drug could be a major addition to our antituberculosis arsenal and would greatly benefit control of the disease.

  4. Macrophage infection models for Mycobacterium tuberculosis.

    PubMed

    Johnson, Benjamin K; Abramovitch, Robert B

    2015-01-01

    Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging.

  5. Early dynamics of T helper cell cytokines and T regulatory cells in response to treatment of active Mycobacterium tuberculosis infection.

    PubMed

    Feruglio, S L; Tonby, K; Kvale, D; Dyrhol-Riise, A M

    2015-03-01

    Biomarkers that can identify tuberculosis (TB) disease and serve as markers for efficient therapy are requested. We have studied T cell cytokine production [interferon (IFN)-γ, interleukin (IL)-2, tumour necrosis factor (TNF)-α] and degranulation (CD107a) as well as subsets of CD4(+) T regulatory cells (Tregs ) after in-vitro Mycobacterium tuberculosis (Mtb) antigen stimulation [early secretory antigenic target (ESAT)-6, culture filtrate protein (CFP)-10, antigen 85 (Ag85)] in 32 patients with active tuberculosis (TB) disease throughout 24 weeks of effective TB treatment. A significant decline in the fraction of Mtb-specific total IFN-γ and single IFN-γ-producing T cells was already observed after 2 weeks of treatment, whereas the pool of single IL-2(+) cells increased over time for both CD4(+) and CD8(+) T cells. The Treg subsets CD25(high) CD127(low) , CD25(high) CD147(++) and CD25(high) CD127(low) CD161(+) expanded significantly after Mtb antigen stimulation in vitro at all time-points, whereas the CD25(high) CD127(low) CD39(+) Tregs remained unchanged. The fraction of CD25(high) CD127(low) Tregs increased after 8 weeks of treatment. Thus, we revealed an opposing shift of Tregs and intracellular cytokine production during treatment. This may indicate that functional signatures of the CD4(+) and CD8(+) T cells can serve as immunological correlates of early curative host responses. Whether such signatures can be used as biomarkers in monitoring and follow-up of TB treatment needs to be explored further.

  6. Evaluating the anti Mycobacterium tuberculosis activity of Alpinia galanga (L.) Willd. axenically under reducing oxygen conditions and in intracellular assays

    PubMed Central

    2014-01-01

    Background In tuberculosis (TB), the steadily increasing bacterial resistance to existing drugs and latent TB continue to be major concerns. A combination of conventional drugs and plant derived therapeutics can serve to expand the antimicrobial spectrum, prevent the emergence of drug resistant mutants and minimize toxicity. Alpinia galanga, used in various traditional medicines, possesses broad spectrum antibacterial properties. The study was undertaken to assess the antimycobacterial potential of A. galanga in axenic (under aerobic and anaerobic conditions) and intracellular assays. Methods Phytochemical analysis was done using HPTLC. The acetone, aqueous and ethanolic extracts (1, 10, 25, 50 and 100 μg/ml) of A. galanga were tested axenically using Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis (M.tb) H37Rv and three drug sensitive and three multi drug resistant clinical isolates. The activity of the extracts was also evaluated intracellularly in A549 cell line against these strains. The extracts active under intracellular conditions were further tested in an axenic setup under reducing oxygen concentrations using only H37Rv. Results 1´ acetoxychavicol acetate, the reference standard used, was present in all the three extracts. The acetone and ethanolic extracts were active in axenic (aerobic and anaerobic) and intracellular assays. The aqueous extract did not demonstrate activity under the defined assay parameters. Conclusion A. galanga exhibits anti M.tb activity with multiple modes of action. Since the activity of the extracts was observed under reducing oxygen concentrations, it may be effective in treating the dormant and non-replicating bacteria of latent TB. Though the hypothesis needs further testing, A. galanga being a regular dietary component may be utilized in combination with the conventional TB therapy for enhanced efficacy. PMID:24592852

  7. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

    SciTech Connect

    Bai, Lin; Hu, Kuan; Wang, Tong; Jastrab, Jordan B.; Darwin, K. Heran; Li, Huilin

    2016-03-21

    Here, the human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.

  8. Activity of n-propyl pyrazinoate against pyrazinamide-resistant Mycobacterium tuberculosis: investigations into mechanism of action of and mechanism of resistance to pyrazinamide.

    PubMed

    Speirs, R J; Welch, J T; Cynamon, M H

    1995-06-01

    The mechanism of action of pyrazinamide (PZA) is not known. One hypothesis is that PZA functions as a prodrug of pyrazinoic acid. Susceptibility to PZA correlates with amidase activity of the Mycobacterium tuberculosis isolate in question. PZA-resistant isolates retain susceptibility in vitro to pyrazinoic acid and n-propyl pyrazinoate. Esters of pyrazinoic acid appear to circumvent the requirement for activation by mycobacterial amidase. The MICs of n-propyl pyrazinoate for M. tuberculosis isolates are lower than those of pyrazinoic acid. Further studies to assess the effects of modifications of the alcohol and pyrazine moieties of pyrazinoate esters on in vitro and in vivo antituberculosis activity are under way. This may lead to a candidate compound with enhanced activity against both PZA-susceptible and PZA-resistant M. tuberculosis isolates suitable for clinical development.

  9. Mycobacterium tuberculosis wears what it eats.

    PubMed

    Russell, David G; VanderVen, Brian C; Lee, Wonsik; Abramovitch, Robert B; Kim, Mi-jeong; Homolka, Susanne; Niemann, Stefan; Rohde, Kyle H

    2010-07-22

    Mycobacterium tuberculosis remains one of the most pernicious of human pathogens. Current vaccines are ineffective, and drugs, although efficacious, require prolonged treatment with constant medical oversight. Overcoming these problems requires a greater appreciation of M. tuberculosis in the context of its host. Upon infection of either macrophages in culture or animal models, the bacterium realigns its metabolism in response to the new environments it encounters. Understanding these environments, and the stresses that they place on M. tuberculosis, should provide insights invaluable for the development of new chemo- and immunotherapeutic strategies.

  10. Novel Mycobacterium tuberculosis complex pathogen, M. mungi.

    PubMed

    Alexander, Kathleen A; Laver, Pete N; Michel, Anita L; Williams, Mark; van Helden, Paul D; Warren, Robin M; Gey van Pittius, Nicolaas C

    2010-08-01

    Seven outbreaks involving increasing numbers of banded mongoose troops and high death rates have been documented. We identified a Mycobacterium tuberculosis complex pathogen, M. mungi sp. nov., as the causative agent among banded mongooses that live near humans in Chobe District, Botswana. Host spectrum and transmission dynamics remain unknown.

  11. IL-10 down-regulates costimulatory molecules on Mycobacterium tuberculosis-pulsed macrophages and impairs the lytic activity of CD4 and CD8 CTL in tuberculosis patients.

    PubMed

    de la Barrera, S; Aleman, M; Musella, R; Schierloh, P; Pasquinelli, V; Garcia, V; Abbate, E; Sasiain, M del C

    2004-10-01

    Activation of T cells requires both TCR-specific ligation and costimulation through accessory molecules during T cell priming. IFNgamma is a key cytokine responsible for macrophage activation during Mycobacterium tuberculosis (Mtb) infection while IL-10 is associated with suppression of cell mediated immunity in intracellular infection. In this paper we evaluated the role of IFNgamma and IL-10 on the function of cytotoxic T cells (CTL) and on the modulation of costimulatory molecules in healthy controls and patients with active tuberculosis (TB). gamma-irradiated-Mtb (i-Mtb) induced IL-10 production from CD14(+) cells from TB patients. Moreover, CD3(+) T cells of patients with advanced disease also produced IL-10 after i-Mtb stimulation. In healthy donors, IL-10 decreased the lytic activity of CD4(+) and CD8(+) T cells whereas it increased gammadelta-mediated cytotoxicity. Furthermore, we found that the presence of IL-10 induced a loss of the alternative processing pathways of antigen presentation along with a down-regulation of the expression of costimulatory molecule expression on monocytes and macrophages from healthy individuals. Conversely, neutralization of endogenous IL-10 or addition of IFNgamma to either effector or target cells from TB patients induced a strong lytic activity mediated by CD8(+) CTL together with an up-regulation of CD54 and CD86 expression on target cells. Moreover, we observed that macrophages from TB patients could use alternative pathways for i-Mtb presentation. Taken together, our results demonstrate that the presence of IL-10 during Mtb infection might contribute to mycobacteria persistence inside host macrophages through a mechanism that involved inhibition of MHC-restricted cytotoxicity against infected macrophages.

  12. Mycobacterium tuberculosis Differentially Activates cGAS- and Inflammasome-Dependent Intracellular Immune Responses through ESX-1.

    PubMed

    Wassermann, Ruth; Gulen, Muhammet F; Sala, Claudia; Perin, Sonia Garcia; Lou, Ye; Rybniker, Jan; Schmid-Burgk, Jonathan L; Schmidt, Tobias; Hornung, Veit; Cole, Stewart T; Ablasser, Andrea

    2015-06-10

    Cytosolic detection of microbial products is essential for the initiation of an innate immune response against intracellular pathogens such as Mycobacterium tuberculosis (Mtb). During Mtb infection of macrophages, activation of cytosolic surveillance pathways is dependent on the mycobacterial ESX-1 secretion system and leads to type I interferon (IFN) and interleukin-1β (IL-1β) production. Whereas the inflammasome regulates IL-1β secretion, the receptor(s) responsible for the activation of type I IFNs has remained elusive. We demonstrate that the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) is essential for initiating an IFN response to Mtb infection. cGAS associates with Mtb DNA in the cytosol to stimulate cyclic GAMP (cGAMP) synthesis. Notably, activation of cGAS-dependent cytosolic host responses can be uncoupled from inflammasome activation by modulating the secretion of ESX-1 substrates. Our findings identify cGAS as an innate sensor of Mtb and provide insight into how ESX-1 controls the activation of specific intracellular recognition pathways.

  13. Interference of Mycobacterium tuberculosis with macrophage responses.

    PubMed

    Scherr, Nicole; Jayachandran, Rajesh; Mueller, Philipp; Pieters, Jean

    2009-06-01

    Tuberculosis, caused by Mycobacterium tuberculosis, has become an important health and economic burden, with more than four thousand people succumbing to the disease every day. Thus, there is an urgent need to understand the molecular basis of this pathogen's success in causing disease in humans, in order to develop new drugs superior to conventional drugs available at present. One reason why M. tuberculosis is such a dangerous microbe lies within its ability to survive within infected hosts, thereby efficiently circumventing host immune responses. Over the past few years, a number of mechanisms have been unravelled that are utilized by M. tuberculosis to survive within hosts and to avoid immune defence mechanisms. Several of these mechanisms have been described in this communication that may be useful for the development of novel compounds to treat tuberculosis.

  14. Cytokinins beyond plants: synthesis by Mycobacterium tuberculosis

    PubMed Central

    Samanovic, Marie I.; Darwin, K. H.

    2015-01-01

    Mycobacterium tuberculosis (M. tuberculosis) resides mainly inside macrophages, which produce nitric oxide (NO) to combat microbial infections. Earlier studies revealed that proteasome-associated genes are required for M. tuberculosis to resist NO via a previously uncharacterized mechanism. Twelve years later, we elucidated the link between proteasome function and NO resistance in M. tuberculosis in Molecular Cell, 57 (2015), pp. 984-994. In a proteasome degradation-defective mutant, Rv1205, a homologue of the plant enzyme LONELY GUY (LOG) that is involved in the synthesis of phytohormones called cytokinins, accumulates and as a consequence results in the overproduction of cytokinins. Cytokinins break down into aldehydes that kill mycobacteria in the presence of NO. Importantly, this new discovery reveals for the first time that a mammalian bacterial pathogen produces cytokinins and leaves us with the question: why is M. tuberculosis, an exclusively human pathogen, producing cytokinins? PMID:28357289

  15. Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo

    PubMed Central

    Liu, Yancheng; Tan, Shumin; Huang, Lu; Abramovitch, Robert B.; Rohde, Kyle H.; Zimmerman, Matthew D.; Chen, Chao; Dartois, Véronique; VanderVen, Brian C.

    2016-01-01

    Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb’s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs’ primary targets. Mtb’s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb’s response to drug pressure and suggest novel routes for future drug discovery programs. PMID:27114608

  16. Carbamidocyclophanes F and G with Anti-Mycobacterium tuberculosis Activity from the Cultured Freshwater Cyanobacterium Nostoc sp.

    PubMed

    Luo, Shangwen; Kang, Hahk-Soo; Krunic, Aleksej; Chlipala, George E; Cai, Geping; Chen, Wei-Lun; Franzblau, Scott G; Swanson, Steven M; Orjala, Jimmy

    2014-01-15

    Two new (1 and 2) and three known (3-5) carbamidocyclophanes were isolated from a cultured freshwater cyanobacterium Nostoc sp. (UIC 10274) obtained from a sample collected at Des Plaines, Illinois. Their planar structures and stereoconfigurations were determined by extensive spectroscopic analysis including 1D/2D NMR experiments, HRESIMS as well as CD spectroscopy. Carbamidocyclophane F (1) showed potent anti-Mycobacterium tuberculosis activity in the microplate Alamar blue assay and low-oxygen-recovery assay with MIC values of 0.8 and 5.4 µM, respectively. Carbamidocyclophane F (1) also displayed antimicrobial activities against the gram positive bacteria Staphylococcus aureus and Enterococcus faecalis with MIC values of 0.1 and 0.2 µM, respectively. Carbamidocyclophane F (1) and Carbamidocyclophane G (2) both showed antiproliferative activity against MDA-MB-435 and HT-29 human cancer cell lines with IC50 values in the range from 0.5 to 0.7 µM.

  17. Conformational Analysis of an Antibacterial Cyclodepsipeptide Active against Mycobacterium tuberculosis by a Combined ROE and RDC Analysis.

    PubMed

    Fredersdorf, Maic; Kurz, Michael; Bauer, Armin; Ebert, Marc-Olivier; Rigling, Carla; Lannes, Laurie; Thiele, Christina Marie

    2017-01-20

    Griselimycin (GM) and methylgriselimycin (MGM), naturally produced by microorganisms of the genus Streptomyces, are cyclic depsipeptides composed of ten amino acids. They exhibit antibacterial activity against Mycobacterium species by inhibiting the sliding clamp of prokaryotic DNA polymerase III and are therefore considered as potential anti-tuberculosis drugs. The difference between the peptides is the presence of l-(R)-4-methyl-proline in MGM instead of l-proline in GM at position 8 of the amino acid sequence. Methylation increases both metabolic stability and activity of MGM compared to GM. To get deeper insight into the structure-activity relationship, the solution structure of the cyclic part of MGM was determined using rotating-frame nuclear Overhauser effect (ROE) distance restraints and residual dipolar couplings (RDC). The structure of MGM in solution is compared to the structure of GM in a co-crystal with DNA polymerase III subunit beta. As a result, a highly defined structural model of MGM is obtained, which shows related characteristics to the bound GM.

  18. Neurons Are Host Cells for Mycobacterium tuberculosis

    PubMed Central

    Randall, Philippa J.; Hsu, Nai-Jen; Lang, Dirk; Cooper, Susan; Sebesho, Boipelo; Allie, Nasiema; Keeton, Roanne; Francisco, Ngiambudulu M.; Salie, Sumayah; Labuschagné, Antoinette; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston

    2014-01-01

    Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system. PMID:24566619

  19. Neurons are host cells for Mycobacterium tuberculosis.

    PubMed

    Randall, Philippa J; Hsu, Nai-Jen; Lang, Dirk; Cooper, Susan; Sebesho, Boipelo; Allie, Nasiema; Keeton, Roanne; Francisco, Ngiambudulu M; Salie, Sumayah; Labuschagné, Antoinette; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston; Jacobs, Muazzam

    2014-05-01

    Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system.

  20. The Biology of Mycobacterium Tuberculosis Infection

    PubMed Central

    Delogu, Giovanni; Sali, Michela; Fadda, Giovanni

    2013-01-01

    Tuberculosis (TB) still poses a major threat to mankind and during the last thirty years we have seen a recrudescence of the disease even in countries where TB was thought to be conquered. It is common opinion that more effective control tools such as new diagnostics, a new vaccine and new drugs are urgently needed to control the global pandemic, though the so far insufficient understanding of the Mycobacterium tuberculosis (Mtb) mechanism of pathogenesis is a major obstacle for the development of these control tools. In this review, we will summarize the recent advancement in the understanding of Mtb biology and on the pathogenesis of Mtb infection with emphasis on latent infection, with the change in paradigm of the last few years where the dichotomy between latent and active disease has been reconsidered in favor of a dynamic equilibrium between the host and the bacilli, encompassing a continuous spectrum of conditions that has been named TB spectrum. Implications for the diagnosis and control of disease in certain population will also be discussed. PMID:24363885

  1. Crystal Structure and Activity Studies of the Mycobacterium tuberculosis β-Lactamase Reveal Its Critical Role in Resistance to β-Lactam Antibiotics

    PubMed Central

    Wang, Feng; Cassidy, Craig; Sacchettini, James C.

    2006-01-01

    β-Lactam antibiotics are extremely effective in disrupting the synthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. However, they are ineffective against Mycobacterium tuberculosis, due to the production of a β-lactamase enzyme encoded on the chromosome of M. tuberculosis that degrades these antibiotics. Indeed, recent studies have demonstrated that deletion of the blaC gene, the only gene encoding a β-lactamase in M. tuberculosis, or inhibition of the encoded enzyme resulted in significantly increased sensitivity to β-lactam antibiotics. In this paper we present a biochemical and structural characterization of M. tuberculosis BlaC. Recombinant BlaC shows a broad range of specificity with almost equal penicillinase and cepholothinase activity. While clavulanate is a mechanism-based inhibitor to class A β-lactamase with high potency (typically Ki < 0.1 μM), it is a relatively poor inhibitor of the M. tuberculosis BlaC (Ki = 2.4 μM). The crystal structure of the enzyme, determined at a resolution of 1.7 Å, shows that the overall fold of the M. tuberculosis enzyme is similar to other class A β-lactamases. There are, however, several distinct features of the active site, such as the amino acid substitutions N132G, R164A, R244A, and R276E, that explain the broad specificity of the enzyme, relatively low penicillinase activity, and resistance to clavulanate. PMID:16870770

  2. Implications of binding mode and active site flexibility for inhibitor potency against the salicylate synthase from Mycobacterium tuberculosis.

    PubMed

    Chi, Gamma; Manos-Turvey, Alexandra; O'Connor, Patrick D; Johnston, Jodie M; Evans, Genevieve L; Baker, Edward N; Payne, Richard J; Lott, J Shaun; Bulloch, Esther M M

    2012-06-19

    MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family.

  3. Crystal Structures of the Response Regulator DosR From Mycobacterium Tuberculosis Suggest a Helix Rearrangement Mechanism for Phosphorylation Activation

    SciTech Connect

    Wisedchaisri, G.; Wu, M.; Sherman, D.R.; Hol, W.G.J.

    2009-05-26

    The response regulator DosR is essential for promoting long-term survival of Mycobacterium tuberculosis under low oxygen conditions in a dormant state and may be responsible for latent tuberculosis in one-third of the world's population. Here, we report crystal structures of full-length unphosphorylated DosR at 2.2 {angstrom} resolution and its C-terminal DNA-binding domain at 1.7 {angstrom} resolution. The full-length DosR structure reveals several features never seen before in other response regulators. The N-terminal domain of the full-length DosR structure has an unexpected ({beta}{alpha}){sub 4} topology instead of the canonical ({beta}{alpha}){sub 5} fold observed in other response regulators. The linker region adopts a unique conformation that contains two helices forming a four-helix bundle with two helices from another subunit, resulting in dimer formation. The C-terminal domain in the full-length DosR structure displays a novel location of helix {alpha}10, which allows Gln199 to interact with the catalytic Asp54 residue of the N-terminal domain. In contrast, the structure of the DosR C-terminal domain alone displays a remarkable unstructured conformation for helix {alpha}10 residues, different from the well-defined helical conformations in all other known structures, indicating considerable flexibility within the C-terminal domain. Our structures suggest a mode of DosR activation by phosphorylation via a helix rearrangement mechanism.

  4. Validation of a homology model of Mycobacterium tuberculosis DXS: rationalization of observed activities of thiamine derivatives as potent inhibitors of two orthologues of DXS.

    PubMed

    Masini, T; Lacy, B; Monjas, L; Hawksley, D; de Voogd, A R; Illarionov, B; Iqbal, A; Leeper, F J; Fischer, M; Kontoyianni, M; Hirsch, A K H

    2015-12-14

    The enzyme DXS catalyzes the first, rate-limiting step of the 2-C-methyl-d-erythritol-4-phosphate (MEP, 1) pathway using thiamine diphosphate (ThDP) as cofactor; the DXS-catalyzed reaction constitutes also the first step in vitamin B1 and B6 metabolism in bacteria. DXS is the least studied among the enzymes of this pathway in terms of crystallographic information, with only one complete crystal structure deposited in the Protein Data Bank (Deinococcus radiodurans DXS, PDB: ). We synthesized a series of thiamine and ThDP derivatives and tested them for their biochemical activity against two DXS orthologues, namely D. radiodurans DXS and Mycobacterium tuberculosis DXS. These experimental results, combined with advanced docking studies, led to the development and validation of a homology model of M. tuberculosis DXS, which, in turn, will guide medicinal chemists in rationally designing potential inhibitors for M. tuberculosis DXS.

  5. [Strategical use of genotyping of Mycobacterium tuberculosis in tuberculosis control].

    PubMed

    David, Susana

    2008-01-01

    The tuberculosis situation in Portugal justifies the use of a strategy for the genotyping of Mycobacterium tuberculosis, particularly as Portugal is part of the global backdrop of human mobility, something which has a knock-on effect on the pandemic. Several international studies have placed spoligotyping and MIRU- VNTR typing as first line techniques for the molecular epidemiology of Mycobacterium tuberculosis as these techniques rely on simple technologies (PCR) and produce patterns which are easily translated into a direct interpretation numerical code. Spoligotyping has been accordingly proposed for all the isolates, while MIRU-VNTR typing should be applied to isolates with a common spoliotype. Other techniques, including IS6110-RFLP, should be reserved for use ill accordance with selected criteria. Previous studies in Portugal using spoligotyping have underlined the advantages of a strategy based on sampling consecutive patient isolates with no prior selection criteria. This allows characterisation of the M. tuberculosis population structure through monitoring the distribution of the genotypes geographically over time and within the various risk groups. On the other hand, the association of spoligotyping, MIRU-VNTF (typing and, possibly, other techniques, needs evaluating as part of bigger pictures, including identifying recent transmission situations, distinguishing between reinfection and relapse episodes and mapping the size and dynamics of disease transmission. The solution to the tuberculosis problem in Portugal implies structuring genotyping's role in tuberculosis prevention and control and its evaluation through concrete examples and results.

  6. The multistage vaccine H56 boosts the effects of BCG to protect cynomolgus macaques against active tuberculosis and reactivation of latent Mycobacterium tuberculosis infection.

    PubMed

    Lin, Philana Ling; Dietrich, Jes; Tan, Esterlina; Abalos, Rodolfo M; Burgos, Jasmin; Bigbee, Carolyn; Bigbee, Matthew; Milk, Leslie; Gideon, Hannah P; Rodgers, Mark; Cochran, Catherine; Guinn, Kristi M; Sherman, David R; Klein, Edwin; Janssen, Christopher; Flynn, JoAnne L; Andersen, Peter

    2012-01-01

    It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis. Infection typically remains latent, but it can reactivate to cause clinical disease. The only vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is largely ineffective, and ways to enhance its efficacy are being developed. Of note, the candidate booster vaccines currently under clinical development have been designed to improve BCG efficacy but not prevent reactivation of latent infection. Here, we demonstrate that administering a multistage vaccine that we term H56 in the adjuvant IC31 as a boost to vaccination with BCG delays and reduces clinical disease in cynomolgus macaques challenged with M. tuberculosis and prevents reactivation of latent infection. H56 contains Ag85B and ESAT-6, which are two of the M. tuberculosis antigens secreted in the acute phase of infection, and the nutrient stress-induced antigen Rv2660c. Boosting with H56/IC31 resulted in efficient containment of M. tuberculosis infection and reduced rates of clinical disease, as measured by clinical parameters, inflammatory markers, and improved survival of the animals compared with BCG alone. Boosted animals showed reduced pulmonary pathology and extrapulmonary dissemination, and protection correlated with a strong recall response against ESAT-6 and Rv2660c. Importantly, BCG/H56-vaccinated monkeys did not reactivate latent infection after treatment with anti-TNF antibody. Our results indicate that H56/IC31 boosting is able to control late-stage infection with M. tuberculosis and contain latent tuberculosis, providing a rationale for the clinical development of H56.

  7. Esters of Pyrazinoic Acid Are Active against Pyrazinamide-Resistant Strains of Mycobacterium tuberculosis and Other Naturally Resistant Mycobacteria In Vitro and Ex Vivo within Macrophages.

    PubMed

    Pires, David; Valente, Emília; Simões, Marta Filipa; Carmo, Nuno; Testa, Bernard; Constantino, Luís; Anes, Elsa

    2015-12-01

    Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti) but not against M. bovis and M. avium. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257-263, 2009, http://dx.doi.org/10.1016/j.ejps.2009.02.012). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development.

  8. Esters of Pyrazinoic Acid Are Active against Pyrazinamide-Resistant Strains of Mycobacterium tuberculosis and Other Naturally Resistant Mycobacteria In Vitro and Ex Vivo within Macrophages

    PubMed Central

    Valente, Emília; Simões, Marta Filipa; Carmo, Nuno; Testa, Bernard

    2015-01-01

    Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti) but not against M. bovis and M. avium. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257–263, 2009, http://dx.doi.org/10.1016/j.ejps.2009.02.012). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development. PMID:26438493

  9. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis

    PubMed Central

    2016-01-01

    Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb “Hydrolase important for pathogenesis 1” (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections. PMID:27739665

  10. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    PubMed

    Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

    2015-07-01

    Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

  11. Evaluation of the inhibitory activity of (aza)isoindolinone-type compounds: toward in vitro InhA action, Mycobacterium tuberculosis growth and mycolic acid biosynthesis.

    PubMed

    Chollet, Aurélien; Stigliani, Jean-Luc; Pasca, Maria Rosalia; Mori, Giorgia; Lherbet, Christian; Constant, Patricia; Quémard, Annaïk; Bernadou, Jean; Pratviel, Geneviève; Bernardes-Génisson, Vania

    2016-11-01

    Inhibitors of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) are considered as potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that azaisoindolinone-type compounds displayed, in vitro, inhibitory activity toward InhA. Herein, we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15 new compounds and their evaluations toward the in vitro InhA activity. Based on these results, a structure-InhA inhibitory activity relationship analysis and a molecular docking study, using the conformation of InhA found in the 2H7M crystal structure, were carried out to predict a possible mode of interaction of the best (aza)isoindolinone-type inhibitors with InhA in vitro. Then, the work was extended toward evaluations of these compounds against Mycobacterium tuberculosis (Mtb) growth, and finally, some of them were also investigated in respect of their ability to inhibit mycolic acid biosynthesis inside mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.

  12. Structural mechanics of the pH-dependent activity of beta-carbonic anhydrase from Mycobacterium tuberculosis.

    PubMed

    Covarrubias, Adrian Suarez; Bergfors, Terese; Jones, T Alwyn; Högbom, Martin

    2006-02-24

    Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate, a reaction required for many functions, including carbon assimilation and pH homeostasis. Carbonic anhydrases are divided into at least three classes and are believed to share a zinc-hydroxide mechanism for carbon dioxide hydration. beta-carbonic anhydrases are broadly spread among the domains of life, and existing structures from different organisms show two distinct active site setups, one with three protein coordinations to the zinc (accessible) and the other with four (blocked). The latter is believed to be inconsistent with the zinc-hydroxide mechanism. The Mycobacterium tuberculosis Rv3588c gene, shown to be required for in vivo growth of the pathogen, encodes a beta-carbonic anhydrase with a steep pH dependence of its activity, being active at pH 8.4 but not at pH 7.5. We have recently solved the structure of this protein, which was a dimeric protein with a blocked active site. Here we present the structure of the thiocyanate complexed protein in a different crystal form. The protein now forms distinct tetramers and shows large structural changes, including a carboxylate shift yielding the accessible active site. This structure demonstrated for the first time that a beta-carbonic anhydrase can switch between the two states. A pH-dependent dimer to tetramer equilibrium was also demonstrated by dynamic light scattering measurements. The data presented here, therefore, suggest a carboxylate shift on/off switch for the enzyme, which may, in turn, be controlled by a dimer-to-tetramer equilibrium.

  13. Genetic engineering of Mycobacterium tuberculosis: a review.

    PubMed

    Lamrabet, Otmane; Drancourt, Michel

    2012-09-01

    Genetic engineering has been used for decades to mutate and delete genes in the Mycobacterium tuberculosis genome with the translational goal of producing attenuated mutants with conserved susceptibility to antituberculous antibiotics. The development of plasmids and mycobacteriophages that can transfer DNA into the M. tuberculosis chromosome has effectively overcome M. tuberculosis slow growth rate and the capsule and mycolic acid wall, which limit DNA uptake. The use of genetic engineering techniques has shed light on many aspects of pathogenesis mechanisms, including cellular growth, mycolic acid biosynthesis, metabolism, drug resistance and virulence. Moreover, such research gave clues to the development of new vaccines or new drugs for routine clinical practice. The use of genetic engineering tools is mainly based on the underlying concept that altering or reducing the M. tuberculosis genome could decrease its virulence. A contrario, recent post-genomic analyses indicated that reduced bacterial genomes are often associated with increased bacterial virulence and that M. tuberculosis acquired genes by lateral genetic exchange during its evolution. Therefore, ancestors utilizing genetic engineering to add genes to the M. tuberculosis genome may lead to new vaccines and the availability of M. tuberculosis isolates with increased susceptibility to antituberculous antibiotics.

  14. Mycobacterium tuberculosis: Manipulator of Protective Immunity

    PubMed Central

    Korb, Vanessa C.; Chuturgoon, Anil A.; Moodley, Devapregasan

    2016-01-01

    Mycobacterium tuberculosis (MTB) is one of the most successful pathogens in human history and remains a global health challenge. MTB has evolved a plethora of strategies to evade the immune response sufficiently to survive within the macrophage in a bacterial-immunological equilibrium, yet causes sufficient immunopathology to facilitate its transmission. This review highlights MTB as the driver of disease pathogenesis and presents evidence of the mechanisms by which MTB manipulates the protective immune response into a pathological productive infection. PMID:26927066

  15. Comparative Mycobacterium tuberculosis Spoligotype Distribution in Mexico

    PubMed Central

    Ramos-Alvarez, Jessica; Molina-Torres, Carmen A.; Rivera-Morales, Lydia Guadalupe; Rendón, Adrian; Quiñones-Falconi, Francisco; Ocampo-Candiani, Jorge

    2014-01-01

    In the present work, we studied the genetic diversity of Mycobacterium tuberculosis clinical isolates from patients according to their gender, age, and geographic location in Mexico. We did not observe any statistically significant differences in regard to age or gender. We found that spoligo international type 53 (SIT53) is more frequent in the northern states and that SIT119 predominates in central Mexico. PMID:24850349

  16. Pyrosequencing assay for rapid identification of Mycobacterium tuberculosis complex species

    PubMed Central

    2011-01-01

    Background Identification of the Mycobacterium tuberculosis complex organisms to the species level is important for diagnostic, therapeutic and epidemiologic perspectives. Indeed, isolates are routinely identified as belonging to the M. tuberculosis complex without further discrimination in agreement with the high genomic similarity of the M. tuberculosis complex members and the resulting complex available identification tools. Findings We herein develop a pyrosequencing assay analyzing polymorphisms within glpK, pykA and gyrB genes to identify members of the M. tuberculosis complex at the species level. The assay was evaluated with 22 M. tuberculosis, 21 M. bovis, 3 M. caprae, 3 M. microti, 2 M. bovis BCG, 2 M. pinnipedii, 1 M. canettii and 1 M. africanum type I isolates. The resulted pyrograms were consistent with conventional DNA sequencing data and successfully identified all isolates. Additionally, 127 clinical M. tuberculosis complex isolates were analyzed and were unambiguously identified as M. tuberculosis. Conclusion We proposed a pyrosequencing-based scheme for the rapid identification of M. tuberculosis complex isolates at the species level. The assay is robust, specific, rapid and can be easily introduced in the routine activity. PMID:22011383

  17. Verapamil increases the bactericidal activity of bedaquiline against Mycobacterium tuberculosis in a mouse model.

    PubMed

    Gupta, Shashank; Tyagi, Sandeep; Bishai, William R

    2015-01-01

    Bedaquiline is a newly approved drug for the treatment of multidrug-resistant tuberculosis, but there are concerns about its safety in humans. We found that the coadministration of verapamil with subinhibitory doses of bedaquiline gave the same bactericidal effect in mice as did the full human bioequivalent bedaquiline dosing. Adding verapamil to bedaquiline monotherapy also protected against the development of resistant mutants in vivo. The adjunctive use of verapamil may permit use of lower doses of bedaquiline to be used and thereby reduce its dose-related toxicities in tuberculosis patients.

  18. Antimicrobial resistance in Mycobacterium tuberculosis: mechanistic and evolutionary perspectives.

    PubMed

    Gygli, Sebastian M; Borrell, Sonia; Trauner, Andrej; Gagneux, Sebastien

    2017-03-25

    Antibiotic-resistant Mycobacterium tuberculosis strains are threatening progress in containing the global tuberculosis epidemic. Mycobacterium tuberculosis is intrinsically resistant to many antibiotics, limiting the number of compounds available for treatment. This intrinsic resistance is due to a number of mechanisms including a thick, waxy, hydrophobic cell envelope and the presence of drug degrading and modifying enzymes. Resistance to the drugs which are active against M. tuberculosis is, in the absence of horizontally transferred resistance determinants, conferred by chromosomal mutations. These chromosomal mutations may confer drug resistance via modification or overexpression of the drug target, as well as by prevention of prodrug activation. Drug resistance mutations may have pleiotropic effects leading to a reduction in the bacterium's fitness, quantifiable e.g. by a reduction in the in vitro growth rate. Secondary so-called compensatory mutations, not involved in conferring resistance, can ameliorate the fitness cost by interacting epistatically with the resistance mutation. Although the genetic diversity of M. tuberculosis is low compared to other pathogenic bacteria, the strain genetic background has been demonstrated to influence multiple aspects in the evolution of drug resistance. The rate of resistance evolution and the fitness costs of drug resistance mutations may vary as a function of the genetic background.

  19. Early Events in Mycobacterium tuberculosis Infection in Cynomolgus Macaques†

    PubMed Central

    Lin, Philana Ling; Pawar, Santosh; Myers, Amy; Pegu, Amarenda; Fuhrman, Carl; Reinhart, Todd A.; Capuano, Saverio V.; Klein, Edwin; Flynn, JoAnne L.

    2006-01-01

    Little is known regarding the early events of infection of humans with Mycobacterium tuberculosis. The cynomolgus macaque is a useful model of tuberculosis, with strong similarities to human tuberculosis. In this study, eight cynomolgus macaques were infected bronchoscopically with low-dose M. tuberculosis; clinical, immunologic, microbiologic, and pathologic events were assessed 3 to 6 weeks postinfection. Gross pathological abnormalities were observed as early as 3 weeks, including Ghon complex formation by 5 weeks postinfection. Caseous granulomas were observed in the lung as early as 4 weeks postinfection. Only caseous granulomas were observed in the lungs at these early time points, reflecting a rigorous initial response. T-cell activation (CD29 and CD69) and chemokine receptor (CXCR3 and CCR5) expression appeared localized to different anatomic sites. Activation markers were increased on cells from airways and only at modest levels on cells in peripheral blood. The priming of mycobacterium-specific T cells, characterized by the production of gamma interferon occurred slowly, with responses seen only after 4 weeks of infection. These responses were observed from T lymphocytes in blood, airways, and hilar lymph node, with responses predominantly localized to the site of infection. From these studies, we conclude that immune responses to M. tuberculosis are relatively slow in the local and peripheral compartments and that necrosis occurs surprisingly quickly during granuloma formation. PMID:16790751

  20. Active Site Loop Dynamics of a Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

    SciTech Connect

    Pegan, Scott D.; Rukseree, Kamolchanok; Capodagli, Glenn C.; Baker, Erica A.; Krasnykh, Olga; Franzblau, Scott G.; Mesecar, Andrew D.

    2013-01-08

    The class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprises one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation–deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA–PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation–protonation step of the MtFBA reaction mechanism. Furthermore, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.

  1. Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus.

    PubMed

    Kaushik, Amit; Makkar, Nayani; Pandey, Pooja; Parrish, Nicole; Singh, Urvashi; Lamichhane, Gyanu

    2015-10-01

    An effective regimen for treatment of tuberculosis (TB) is comprised of multiple drugs that inhibit a range of essential cellular activities in Mycobacterium tuberculosis. The effectiveness of a regimen is further enhanced if constituent drugs act with synergy. Here, we report that faropenem (a penem) or biapenem, doripenem, or meropenem (carbapenems), which belong to the β-lactam class of antibiotics, and rifampin, one of the drugs that forms the backbone of TB treatment, act with synergy when combined. One of the reasons (carba)penems are seldom used for treatment of TB is the high dosage levels required, often at the therapeutic limits. The synergistic combination of rifampin and these (carba)penems indicates that (carba)penems can be administered at dosages that are therapeutically relevant. The combination of faropenem and rifampin also limits the frequency of resistant mutants, as we were unable to obtain spontaneous mutants in the presence of these two drugs. The combinations of rifampin and (carba)penems were effective not only against drug-sensitive Mycobacterium tuberculosis but also against drug-resistant clinical isolates that are otherwise resistant to rifampin. A combination of doripenem or biapenem and rifampin also exhibited synergistic activity against Mycobacterium abscessus. Although the MICs of these three drugs alone against M. abscessus are too high to be of clinical relevance, their concentrations in combinations are therapeutically relevant; therefore, they warrant further evaluation for clinical utility to treat Mycobacterium abscessus infection, especially in cystic fibrosis patients.

  2. Preclinical testing of the nitroimidazopyran PA-824 for activity against Mycobacterium tuberculosis in a series of in vitro and in vivo models.

    PubMed

    Lenaerts, Anne J; Gruppo, Veronica; Marietta, Karen S; Johnson, Christine M; Driscoll, Diane K; Tompkins, Nicholas M; Rose, Jerry D; Reynolds, Robert C; Orme, Ian M

    2005-06-01

    This study extends earlier reports regarding the in vitro and in vivo efficacies of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis. PA-824 was tested in vitro against a broad panel of multidrug-resistant clinical isolates and was found to be highly active against all isolates (MIC<1 microg/ml). The activity of PA-824 against M. tuberculosis was also assessed grown under conditions of oxygen depletion. PA-824 showed significant activity at 2, 10, and 50 microg/ml, similar to that of metronidazole, in a dose-dependent manner. In a short-course mouse infection model, the efficacy of PA-824 at 50, 100, and 300 mg/kg of body weight formulated in methylcellulose or cyclodextrin/lecithin after nine oral treatments was compared with those of isoniazid, rifampin, and moxifloxacin. PA-824 at 100 mg/kg in cyclodextrin/lecithin was as active as moxifloxacin at 100 mg/kg and isoniazid at 25 mg/kg and was slightly more active than rifampin at 20 mg/kg. Long-term treatment with PA-824 at 100 mg/kg in cyclodextrin/lecithin reduced the bacterial load below 500 CFU in the lungs and spleen. No significant differences in activity between PA-824 and the other single drug treatments tested (isoniazid at 25 mg/kg, rifampin at 10 mg/kg, gatifloxacin at 100 mg/kg, and moxifloxacin at 100 mg/kg) could be observed. In summary, its good activity in in vivo models, as well as its activity against multidrug-resistant M. tuberculosis and against M. tuberculosis isolates in a potentially latent state, makes PA-824 an attractive drug candidate for the therapy of tuberculosis. These data indicate that there is significant potential for effective oral delivery of PA-824 for the treatment of tuberculosis.

  3. Type I IFN Inhibits Alternative Macrophage Activation during Mycobacterium tuberculosis Infection and Leads to Enhanced Protection in the Absence of IFN-γ Signaling

    PubMed Central

    Sousa, Jeremy; McNab, Finlay W.; Torrado, Egídio; Cardoso, Filipa; Machado, Henrique; Castro, Flávia; Cardoso, Vânia; Gaifem, Joana; Wu, Xuemei; Appelberg, Rui; Castro, António Gil; O’Garra, Anne; Saraiva, Margarida

    2016-01-01

    Tuberculosis causes ∼1.5 million deaths every year, thus remaining a leading cause of death from infectious diseases in the world. A growing body of evidence demonstrates that type I IFN plays a detrimental role in tuberculosis pathogenesis, likely by interfering with IFN-γ–dependent immunity. In this article, we reveal a novel mechanism by which type I IFN may confer protection against Mycobacterium tuberculosis infection in the absence of IFN-γ signaling. We show that production of type I IFN by M. tuberculosis–infected macrophages induced NO synthase 2 and inhibited arginase 1 gene expression. In vivo, absence of both type I and type II IFN receptors led to strikingly increased levels of arginase 1 gene expression and protein activity in infected lungs, characteristic of alternatively activated macrophages. This correlated with increased lung bacterial burden and pathology and decreased survival compared with mice deficient in either receptor. Increased expression of other genes associated with alternatively activated macrophages, as well as increased expression of Th2-associated cytokines and decreased TNF expression, were also observed. Thus, in the absence of IFN-γ signaling, type I IFN suppressed the switching of macrophages from a more protective classically activated phenotype to a more permissive alternatively activated phenotype. Together, our data support a model in which suppression of alternative macrophage activation by type I IFN during M. tuberculosis infection, in the absence of IFN-γ signaling, contributes to host protection. PMID:27849167

  4. In Vitro Activity of Copper(II) Complexes, Loaded or Unloaded into a Nanostructured Lipid System, against Mycobacterium tuberculosis

    PubMed Central

    da Silva, Patricia B.; de Souza, Paula C.; Calixto, Giovana Maria Fioramonti; Lopes, Erica de O.; Frem, Regina C. G.; Netto, Adelino V. G.; Mauro, Antonio E.; Pavan, Fernando R.; Chorilli, Marlus

    2016-01-01

    Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis (Mtb), presenting 9.5 million new cases and 1.5 million deaths in 2014. The aim of this study was to evaluate a nanostructured lipid system (NLS) composed of 10% phase oil (cholesterol), 10% surfactant (soy phosphatidylcholine, sodium oleate), and Eumulgin® HRE 40 ([castor oil polyoxyl-40-hydrogenated] in a proportion of 3:6:8), and an 80% aqueous phase (phosphate buffer pH = 7.4) as a tactic to enhance the in vitro anti-Mtb activity of the copper(II) complexes [CuCl2(INH)2]·H2O (1), [Cu(NCS)2(INH)2]·5H2O (2) and [Cu(NCO)2(INH)2]·4H2O (3). The Cu(II) complex-loaded NLS displayed sizes ranging from 169.5 ± 0.7095 to 211.1 ± 0.8963 nm, polydispersity index (PDI) varying from 0.135 ± 0.0130 to 0.236 ± 0.00100, and zeta potential ranging from −0.00690 ± 0.0896 to −8.43 ± 1.63 mV. Rheological analysis showed that the formulations behave as non-Newtonian fluids of the pseudoplastic and viscoelastic type. Antimycobacterial activities of the free complexes and NLS-loaded complexes against Mtb H37Rv ATCC 27294 were evaluated by the REMA methodology, and the selectivity index (SI) was calculated using the cytotoxicity index (IC50) against Vero (ATCC® CCL-81), J774A.1 (ATCC® TIB-67), and MRC-5 (ATCC® CCL-171) cell lines. The data suggest that the incorporation of the complexes into NLS improved the inhibitory action against Mtb by 52-, 27-, and 4.7-fold and the SI values by 173-, 43-, and 7-fold for the compounds 1, 2 and 3, respectively. The incorporation of the complexes 1, 2 and 3 into the NLS also resulted in a significant decrease of toxicity towards an alternative model (Artemia salina L.). These findings suggest that the NLS may be considered as a platform for incorporation of metallic complexes aimed at the treatment of TB. PMID:27196901

  5. In Vitro Activity of Copper(II) Complexes, Loaded or Unloaded into a Nanostructured Lipid System, against Mycobacterium tuberculosis.

    PubMed

    Silva, Patricia B da; Souza, Paula C de; Calixto, Giovana Maria Fioramonti; Lopes, Erica de O; Frem, Regina C G; Netto, Adelino V G; Mauro, Antonio E; Pavan, Fernando R; Chorilli, Marlus

    2016-05-17

    Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis (Mtb), presenting 9.5 million new cases and 1.5 million deaths in 2014. The aim of this study was to evaluate a nanostructured lipid system (NLS) composed of 10% phase oil (cholesterol), 10% surfactant (soy phosphatidylcholine, sodium oleate), and Eumulgin(®) HRE 40 ([castor oil polyoxyl-40-hydrogenated] in a proportion of 3:6:8), and an 80% aqueous phase (phosphate buffer pH = 7.4) as a tactic to enhance the in vitro anti-Mtb activity of the copper(II) complexes [CuCl₂(INH)₂]·H₂O (1), [Cu(NCS)₂(INH)₂]·5H₂O (2) and [Cu(NCO)₂(INH)₂]·4H₂O (3). The Cu(II) complex-loaded NLS displayed sizes ranging from 169.5 ± 0.7095 to 211.1 ± 0.8963 nm, polydispersity index (PDI) varying from 0.135 ± 0.0130 to 0.236 ± 0.00100, and zeta potential ranging from -0.00690 ± 0.0896 to -8.43 ± 1.63 mV. Rheological analysis showed that the formulations behave as non-Newtonian fluids of the pseudoplastic and viscoelastic type. Antimycobacterial activities of the free complexes and NLS-loaded complexes against Mtb H37Rv ATCC 27294 were evaluated by the REMA methodology, and the selectivity index (SI) was calculated using the cytotoxicity index (IC50) against Vero (ATCC(®) CCL-81), J774A.1 (ATCC(®) TIB-67), and MRC-5 (ATCC(®) CCL-171) cell lines. The data suggest that the incorporation of the complexes into NLS improved the inhibitory action against Mtb by 52-, 27-, and 4.7-fold and the SI values by 173-, 43-, and 7-fold for the compounds 1, 2 and 3, respectively. The incorporation of the complexes 1, 2 and 3 into the NLS also resulted in a significant decrease of toxicity towards an alternative model (Artemia salina L.). These findings suggest that the NLS may be considered as a platform for incorporation of metallic complexes aimed at the treatment of TB.

  6. Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants

    PubMed Central

    2004-01-01

    Cell-surface saccharides of Mycobacterium tuberculosis appear to be crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis. In the present study, we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of M. tuberculosis by dodecamer peptides identified by phage-display technology. Using a rabbit antiserum raised against M. tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified. Phage-displayed and chemically synthesized peptides bound to the anticarbohydrate antiserum. Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognize the mannosylated M. tuberculosis cell-wall antigens arabinomannan and lipoarabinomannan, and the glycosylated recombinant protein alanine/proline-rich antigen. Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidylinositol dimannosides or arabinogalactan from mycobacteria. These results suggest that the immunogenic peptide mimics oligomannosidic epitopes. Interestingly, this report provides evidence that, in contrast with previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesized by mycobacteria. The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed. PMID:15560754

  7. 59 FR- Method Development for Airborne Mycobacterium Tuberculosis; Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    1994-03-07

    ... Tuberculosis; Meeting The National Institute for Occupational Safety and Health (NIOSH) of the Centers for... Airborne Mycobacterium Tuberculosis. Time and Date: 1 p.m.-5 p.m., March 29, 1994. Place: Alice Hamilton... peer review of a NIOSH project entitled ``Method Development For Airborne Mycobacterium...

  8. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis

    PubMed Central

    Lerner, Thomas R.; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R.G.; Borel, Sophie; Diedrich, Collin R.; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M.; Wilkinson, Robert J.; Griffiths, Gareth; Gutierrez, Maximiliano G.

    2016-01-01

    In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes. PMID:26901813

  9. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis.

    PubMed

    Lerner, Thomas R; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R G; Borel, Sophie; Diedrich, Collin R; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M; Wilkinson, Robert J; Griffiths, Gareth; Gutierrez, Maximiliano G

    2016-03-01

    In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes.

  10. Efficient heterologous expression and one-step purification of fully active c-terminal histidine-tagged uridine monophosphate kinase from Mycobacterium tuberculosis.

    PubMed

    Penpassakarn, Praweenuch; Chaiyen, Pimchai; Palittapongarnpim, Prasit

    2011-11-01

    Tuberculosis has long been recognized as one of the most significant public health problems. Finding novel antituberculous drugs is always a necessary approach for controlling the disease. Mycobacterium tuberculosis pyrH gene (Rv2883c) encodes for uridine monophosphate kinase (UMK), which is a key enzyme in the uridine nucleotide interconversion pathway. The enzyme is essential for M. tuberculosis to sustain growth and hence is a potential drug target. In this study, we have developed a rapid protocol for production and purification of M. tuberculosis UMK by cloning pyrH (Rv2883c) of M. tuberculosis H37Rv with the addition of 6-histidine residues to the C-terminus of the protein, and expressing in E. coli BL21-CodonPlus (DE3)-RIPL using an auto-induction medium. The enzyme was efficiently purified by a single-step TALON cobalt affinity chromatography with about 8 fold increase in specific activity, which was determined by a coupled assay with the pyruvate kinase and lactate dehydrogenase. The molecular mass of monomeric UMK was 28.2 kDa and that of the native enzyme was 217 kDa. The enzyme uses UMP as a substrate but not CMP and TMP and activity was enhanced by GTP. Measurements of enzyme kinetics revealed the kcat value of 7.6 +/- 0.4 U mg(-1) or 0.127 +/- 0.006 sec(-1).The protocol reported here can be used for expression of M. tuberculosis UMK in large quantity for formulating a high throughput target-based assay for screening anti-tuberculosis UMK compounds.

  11. Structure and Proposed Activity of a Member of the VapBC Family of Toxin-Antitoxin Systems: VapBC-5 from Mycobacterium tuberculosis

    SciTech Connect

    Miallau, L.; Faller, M.; Chiang, J.; Arbing, M.; Guo, F.; Cascio, D.; Eisenberg, D.

    2009-03-02

    In prokaryotes, cognate toxin-antitoxin pairs have long been known, but no three-dimensional structure has been available for any given complex from Mycobacterium tuberculosis. Here we report the crystal structure and activity of a member of the VapBC family of complexes from M. tuberculosis. The toxin VapC-5 is a compact, 150 residues, two domain {alpha}/{beta} protein. Bent around the toxin is the VapB-5 antitoxin, a 33-residue {alpha}-helix. Assays suggest that the toxin is an Mg-enabled endoribonuclease, inhibited by the antitoxin. The lack of DNase activity is consistent with earlier suggestions that the complex represses its own operon. Furthermore, analysis of the interactions in the binding of the antitoxin to the toxin suggest that exquisite control is required to protect the bacteria cell from toxic VapC-5.

  12. EmbA is an essential arabinosyltransferase in Mycobacterium tuberculosis

    PubMed Central

    Amin, Anita G.; Goude, Renan; Shi, Libin; Zhang, Jian; Chatterjee, Delphi; Parish, Tanya

    2008-01-01

    The Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Araβ(1→2)Araα(1→5)] [Araβ(1→2)Araα(1→3)]Araα(1→5)Araα1→(Ara6) in the cell wall polysaccharide arabinogalactan. Studies conducted in Mycobacterium smegmatis revealed that mutants with disruptions in embA or embB are viable, although the growth rate was affected. In contrast, we demonstrate here that embA is an essential gene in Mycobacterium tuberculosis, since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an embA mutant of M. smegmatis by M. tuberculosis embA confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for M. tuberculosis embA located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, embC. Promoter activity from PembA(Mtb) was sevenfold lower when assayed in M. smegmatis compared to M. tuberculosis, indicating that the latter is not a good host for genetic analysis of M. tuberculosis embA expression. PembA(Mtb) activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on PembA(Mtb) activity. These data demonstrate that M. tuberculosis embA encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in M. tuberculosis. PMID:18174142

  13. EmbA is an essential arabinosyltransferase in Mycobacterium tuberculosis.

    PubMed

    Amin, Anita G; Goude, Renan; Shi, Libin; Zhang, Jian; Chatterjee, Delphi; Parish, Tanya

    2008-01-01

    The Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Arabeta(1-->2)Araalpha(1-->5)] [Arabeta(1-->2)Araalpha(1-->3)]Araalpha(1-->5)Araalpha1-->(Ara(6)) in the cell wall polysaccharide arabinogalactan. Studies conducted in Mycobacterium smegmatis revealed that mutants with disruptions in embA or embB are viable, although the growth rate was affected. In contrast, we demonstrate here that embA is an essential gene in Mycobacterium tuberculosis, since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an embA mutant of M. smegmatis by M. tuberculosis embA confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for M. tuberculosis embA located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, embC. Promoter activity from P(embA)((Mtb)) was sevenfold lower when assayed in M. smegmatis compared to M. tuberculosis, indicating that the latter is not a good host for genetic analysis of M. tuberculosis embA expression. P(embA)((Mtb)) activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on P(embA)((Mtb)) activity. These data demonstrate that M. tuberculosis embA encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in M. tuberculosis.

  14. Beta-lactamases of Mycobacterium tuberculosis and Mycobacterium kansasii.

    PubMed

    Segura, C; Salvadó, M

    1997-09-01

    Re-emergence of infectious diseases caused by mycobacteria as well as the emergence of multiresistant strains of Mycobacterium has promoted the research on the use of beta-lactames in the treatment of such diseases. Mycobacteria produce beta-lactamases: M. tuberculosis produces a wide-spectrum beta-lactamase whose behaviour mimicks those of Gram-negative bacteria. M. kansasii produces also beta-lactamase which can be inhibited by clavulanic acid. An overview on beta-lactamases from both species is reported.

  15. Comparative antimycobacterial activities of rifampin, rifapentine, and KRM-1648 against a collection of rifampin-resistant Mycobacterium tuberculosis isolates with known rpoB mutations.

    PubMed Central

    Moghazeh, S L; Pan, X; Arain, T; Stover, C K; Musser, J M; Kreiswirth, B N

    1996-01-01

    A collection of 24 rifampin-resistant clinical isolates of Mycobacterium tuberculosis with characterized RNA polymerase beta-subunit (rpoB) gene mutations was tested against the antimycobacterial agents rifampin, rifapentine, and KRM-1648 to correlate levels of resistance with specific rpoB genotypes. The results indicate that KRM-1648 is more active in vitro than rifampin and rifapentine, and its ability to overcome rifampin resistance in strains with four different genetic alterations may prove to be useful in understanding structure-function relationships. PMID:8913484

  16. mmr, a Mycobacterium tuberculosis Gene Conferring Resistance to Small Cationic Dyes and Inhibitors

    PubMed Central

    De Rossi, Edda; Branzoni, Manuela; Cantoni, Rita; Milano, Anna; Riccardi, Giovanna; Ciferri, Orio

    1998-01-01

    The mmr gene, cloned from Mycobacterium tuberculosis, was shown to confer to Mycobacterium smegmatis resistance to tetraphenylphosphonium (TPP), erythromycin, ethidium bromide, acriflavine, safranin O, and pyronin Y. The gene appears to code for a protein containing four transmembrane domains. Studies of [3H]TPP intracellular accumulation strongly suggest that the resistance mediated by the Mmr protein involves active extrusion of TPP. PMID:9811672

  17. 5-Arylaminouracil Derivatives: New Inhibitors of Mycobacterium tuberculosis.

    PubMed

    Matyugina, Elena; Novikov, Mikhail; Babkov, Denis; Ozerov, Alexander; Chernousova, Larisa; Andreevskaya, Sofia; Smirnova, Tatiana; Karpenko, Inna; Chizhov, Alexander; Murthu, Pravin; Lutz, Stefan; Kochetkov, Sergei; Seley-Radtke, Katherine L; Khandazhinskaya, Anastasia L

    2015-12-01

    Three series of 5-arylaminouracil derivatives, including 5-(phenylamino)uracils, 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-5-(phenylamino)uracils, and 1,3-di-(4'-hydroxy-2'-cyclopenten-1'-yl)-5-(phenylamino)uracils, were synthesized and screened for potential antimicrobial activity. Most of compounds had a negative effect on the growth of the Mycobacterium tuberculosis H37Rv strain, with 100% inhibition observed at concentrations between 5 and 40 μg/mL. Of those, 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-3-(4‴-hydroxy-2‴-cyclopenten-1‴-yl)-5-(4″-butyloxyphenylamino)uracil proved to be the most active among tested compounds against the M. tuberculosis multidrug-resistant strain MS-115 (MIC90 5 μg/mL). In addition, the thymidylate kinase of M. tuberculosis was evaluated as a possible enzymatic target.

  18. Molecular Basis of the Activity and the Regulation of the Eukaryotic-like S/T Protein Kinase PknG from Mycobacterium tuberculosis.

    PubMed

    Lisa, María-Natalia; Gil, Magdalena; André-Leroux, Gwénaëlle; Barilone, Nathalie; Durán, Rosario; Biondi, Ricardo M; Alzari, Pedro M

    2015-06-02

    Tuberculosis remains one of the world's deadliest human diseases, with a high prevalence of antibiotic-resistant Mycobacterium tuberculosis (Mtb) strains. A molecular understanding of processes underlying regulation and adaptation of bacterial physiology may provide novel avenues for the development of antibiotics with unconventional modes of action. Here, we focus on the multidomain S/T protein kinase PknG, a soluble enzyme that controls central metabolism in Actinobacteria and has been linked to Mtb infectivity. Our biochemical and structural studies reveal how different motifs and domains flanking the catalytic core regulate substrate selectivity without significantly affecting the intrinsic kinase activity, whereas a rubredoxin-like domain is shown to downregulate catalysis through specific intramolecular interactions that modulate access to a profound substrate-binding site. Our findings provide the basis for the selective and specific inhibition of PknG, and open new questions about regulation of related bacterial and eukaryotic protein kinases.

  19. Disseminated Mycobacterium tuberculosis Infection in a Dog

    PubMed Central

    Martinho, Anna Paula Vitirito; Franco, Marília Masello Junqueira; Ribeiro, Márcio Garcia; Perrotti, Isabella Belletti Mutt; Mangia, Simone Henriques; Megid, Jane; Vulcano, Luiz Carlos; Lara, Gustavo Henrique Batista; Santos, Adolfo Carlos Barreto; Leite, Clarice Queico Fujimura; de Carvalho Sanches, Osimar; Paes, Antonio Carlos

    2013-01-01

    An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs. PMID:23339199

  20. Disseminated Mycobacterium tuberculosis infection in a dog.

    PubMed

    Martinho, Anna Paula Vitirito; Franco, Marília Masello Junqueira; Ribeiro, Márcio Garcia; Perrotti, Isabella Belletti Mutt; Mangia, Simone Henriques; Megid, Jane; Vulcano, Luiz Carlos; Lara, Gustavo Henrique Batista; Santos, Adolfo Carlos Barreto; Leite, Clarice Queico Fujimura; de Carvalho Sanches, Osimar; Paes, Antonio Carlos

    2013-03-01

    An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs.

  1. Soluble TNFRp75 regulates host protective immunity against Mycobacterium tuberculosis.

    PubMed

    Keeton, Roanne; Allie, Nasiema; Dambuza, Ivy; Abel, Brian; Hsu, Nai-Jen; Sebesho, Boipelo; Randall, Philippa; Burger, Patricia; Fick, Elizabeth; Quesniaux, Valerie F J; Ryffel, Bernhard; Jacobs, Muazzam

    2014-04-01

    Development of host protective immunity against Mycobacterium tuberculosis infection is critically dependent on the inflammatory cytokine TNF. TNF signals through 2 receptors, TNFRp55 and TNFRp75; however, the role of TNFRp75-dependent signaling in immune regulation is poorly defined. Here we found that mice lacking TNFRp75 exhibit greater control of M. tuberculosis infection compared with WT mice. TNFRp75-/- mice developed effective bactericidal granulomas and demonstrated increased pulmonary recruitment of activated DCs. Moreover, IL-12p40-dependent migration of DCs to lung draining LNs of infected TNFRp75-/- mice was substantially higher than that observed in WT M. tuberculosis-infected animals and was associated with enhanced frequencies of activated M. tuberculosis-specific IFN-γ-expressing CD4+ T cells. In WT mice, TNFRp75 shedding correlated with markedly reduced bioactive TNF levels and IL-12p40 expression. Neutralization of TNFRp75 in M. tuberculosis-infected WT BM-derived DCs (BMDCs) increased production of bioactive TNF and IL-12p40 to a level equivalent to that produced by TNFRp75-/- BMDCs. Addition of exogenous TNFRp75 to TNFRp75-/- BMDCs infected with M. tuberculosis decreased IL-12p40 synthesis, demonstrating that TNFRp75 shedding regulates DC activation. These data indicate that TNFRp75 shedding downmodulates protective immune function and reduces host resistance and survival; therefore, targeting TNFRp75 may be beneficial for improving disease outcome.

  2. Prevalence of latent Mycobacterium tuberculosis infection in prisoners

    PubMed Central

    de Navarro, Pedro Daibert; de Almeida, Isabela Neves; Kritski, Afrânio Lineu; Ceccato, Maria das Graças; Maciel, Mônica Maria Delgado; Carvalho, Wânia da Silva; de Miranda, Silvana Spindola

    2016-01-01

    ABSTRACT Objective: To determine the prevalence of and the factors associated with latent Mycobacterium tuberculosis infection (LTBI) in prisoners in the state of Minas Gerais, Brazil. Methods: This was a cross-sectional cohort study conducted in two prisons in Minas Gerais. Tuberculin skin tests were performed in the individuals who agreed to participate in the study. Results: A total of 1,120 individuals were selected for inclusion in this study. The prevalence of LTBI was 25.2%. In the multivariate analysis, LTBI was associated with self-reported contact with active tuberculosis patients within prisons (adjusted OR = 1.51; 95% CI: 1.05-2.18) and use of inhaled drugs (adjusted OR = 1.48; 95% CI: 1.03-2.13). Respiratory symptoms were identified in 131 (11.7%) of the participants. Serological testing for HIV was performed in 940 (83.9%) of the participants, and the result was positive in 5 (0.5%). Two cases of active tuberculosis were identified during the study period. Conclusions: Within the prisons under study, the prevalence of LTBI was high. In addition, LTBI was associated with self-reported contact with active tuberculosis patients and with the use of inhaled drugs. Our findings demonstrate that it is necessary to improve the conditions in prisons, as well as to introduce strategies, such as chest X-ray screening, in order to detect tuberculosis cases and, consequently, reduce M. tuberculosis infection within the prison system. PMID:27812634

  3. Identification of 2-Aminothiazole-4-Carboxylate Derivatives Active against Mycobacterium tuberculosis H37Rv and the β-Ketoacyl-ACP Synthase mtFabH

    PubMed Central

    Al-Balas, Qosay; Anthony, Nahoum G.; Al-Jaidi, Bilal; Alnimr, Amani; Abbott, Grainne; Brown, Alistair K.; Taylor, Rebecca C.; Besra, Gurdyal S.; McHugh, Timothy D.; Gillespie, Stephen H.; Johnston, Blair F.; Mackay, Simon P.; Coxon, Geoffrey D.

    2009-01-01

    Background Tuberculosis (TB) is a disease which kills two million people every year and infects approximately over one-third of the world's population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration. Methodology/Principal Findings Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM) to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM's novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H37Rv and, dissociatively, against the β-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H37Rv with an MIC of 0.06 µg/ml (240 nM), but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido)-5-(3-chlorophenyl)thiazole-4-carboxylate inhibited mtFabH with an IC50 of 0.95±0.05 µg/ml (2.43±0.13 µM) but was not active against the whole cell organism. Conclusions/Significance These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents. PMID:19440303

  4. ATPase activity of Mycobacterium tuberculosis SecA1 and SecA2 proteins and its importance for SecA2 function in macrophages.

    PubMed

    Hou, Jie M; D'Lima, Nadia G; Rigel, Nathan W; Gibbons, Henry S; McCann, Jessica R; Braunstein, Miriam; Teschke, Carolyn M

    2008-07-01

    The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.

  5. The Human Antibody Response to the Surface of Mycobacterium tuberculosis

    PubMed Central

    Perley, Casey C.; Frahm, Marc; Click, Eva M.; Dobos, Karen M.; Ferrari, Guido; Stout, Jason E.; Frothingham, Richard

    2014-01-01

    Background Vaccine-induced human antibodies to surface components of Haemophilus influenzae and Streptococcus pneumonia are correlated with protection. Monoclonal antibodies to surface components of Mycobacterium tuberculosis are also protective in animal models. We have characterized human antibodies that bind to the surface of live M. tuberculosis. Methods Plasma from humans with latent tuberculosis (TB) infection (n = 23), active TB disease (n = 40), and uninfected controls (n = 9) were assayed by ELISA for reactivity to the live M. tuberculosis surface and to inactivated M. tuberculosis fractions (whole cell lysate, lipoarabinomannan, cell wall, and secreted proteins). Results When compared to uninfected controls, patients with active TB disease had higher antibody titers to the surface of live M. tuberculosis (Δ = 0.72 log10), whole cell lysate (Δ = 0.82 log10), and secreted proteins (Δ = 0.62 log10), though there was substantial overlap between the two groups. Individuals with active disease had higher relative IgG avidity (Δ = 1.4 to 2.6) to all inactivated fractions. Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was lower in the active disease group than in uninfected controls (Δ = –1.53, p = 0.004). Patients with active disease had higher IgG than IgM titers for all inactivated fractions (ratios, 2.8 to 10.1), but equal IgG and IgM titers to the live M. tuberculosis surface (ratio, 1.1). Higher antibody titers to the M. tuberculosis surface were observed in active disease patients who were BCG-vaccinated (Δ = 0.55 log10, p = 0.008), foreign-born (Δ = 0.61 log10, p = 0.004), or HIV-seronegative (Δ = 0.60 log10, p = 0.04). Higher relative IgG avidity scores to the M. tuberculosis surface were also observed in active disease patients who were BCG-vaccinated (Δ = 1.12, p<0.001) and foreign-born (Δ = 0.87, p = 0.01). Conclusions/Significance Humans

  6. Real Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection

    DTIC Science & Technology

    2014-09-01

    1 AWARD NUMBER: W81XWH-13-1-0149 TITLE: Real Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection ... Tuberculosis 5a. CONTRACT NUMBER Infection 5b. GRANT NUMBER W81XWH-13-1-0149 5c. PROGRAM ELEMENT NUMBER 6...resistant state, sometimes reactivating to cause tuberculosis (TB) decades after the primary infection , has puzzled scientists for years. This

  7. Mycobacterium tuberculosis Infection among Asian Elephants in Captivity

    PubMed Central

    Simpson, Gary; Zimmerman, Ralph; Shashkina, Elena; Chen, Liang; Richard, Michael; Bradford, Carol M.; Dragoo, Gwen A.; Saiers, Rhonda L.; Peloquin, Charles A.; Daley, Charles L.; Planet, Paul; Narachenia, Apurva; Mathema, Barun

    2017-01-01

    Although awareness of tuberculosis among captive elephants is increasing, antituberculosis therapy for these animals is not standardized. We describe Mycobacterium tuberculosis transmission between captive elephants based on whole genome analysis and report a successful combination treatment. Infection control protocols and careful monitoring of treatment of captive elephants with tuberculosis are warranted. PMID:28221115

  8. Pre-Multidrug-Resistant Mycobacterium tuberculosis Beijing Strain Associated with Disseminated Tuberculosis in a Pet Dog

    PubMed Central

    Perdigão, João; Canto, Ana; Albuquerque, Teresa; Leal, Nuno; Macedo, Rita; Portugal, Isabel; Cunha, Mónica V.

    2014-01-01

    Resistance to isoniazid, ethambutol, and streptomycin was detected in a Mycobacterium tuberculosis strain, belonging to the Beijing family lineage, isolated from two nodule exudates of a Yorkshire terrier with generalized tuberculosis. This report alerts medical practitioners to the risk of dissemination of pre-multidrug-resistant tuberculosis (preMDR-TB) through exposure to M. tuberculosis-shedding pets. PMID:24153119

  9. New Mycobacterium tuberculosis Complex Sublineage, Brazzaville, Congo

    PubMed Central

    Malm, Sven; Linguissi, Laure S. Ghoma; Tekwu, Emmanuel M.; Vouvoungui, Jeannhey C.; Kohl, Thomas A.; Beckert, Patrick; Sidibe, Anissa; Rüsch-Gerdes, Sabine; Madzou-Laboum, Igor K.; Kwedi, Sylvie; Penlap Beng, Véronique; Frank, Matthias; Ntoumi, Francine

    2017-01-01

    Tuberculosis is a leading cause of illness and death in Congo. No data are available about the population structure and transmission dynamics of the Mycobacterium tuberculosis complex strains prevalent in this central Africa country. On the basis of single-nucleotide polymorphisms detected by whole-genome sequencing, we phylogenetically characterized 74 MTBC isolates from Brazzaville, the capital of Congo. The diversity of the study population was high; most strains belonged to the Euro-American lineage, which split into Latin American Mediterranean, Uganda I, Uganda II, Haarlem, X type, and a new dominant sublineage named Congo type (n = 26). Thirty strains were grouped in 5 clusters (each within 12 single-nucleotide polymorphisms), from which 23 belonged to the Congo type. High cluster rates and low genomic diversity indicate recent emergence and transmission of the Congo type, a new Euro-American sublineage of MTBC. PMID:28221129

  10. Structure-based design of diverse inhibitors of Mycobacterium tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase: combined molecular docking, dynamic simulation, and biological activity.

    PubMed

    Soni, Vijay; Suryadevara, Priyanka; Sriram, Dharmarajan; Kumar, Santhosh; Nandicoori, Vinay Kumar; Yogeeswari, Perumal

    2015-07-01

    Persistent nature of Mycobacterium tuberculosis is one of the major factors which make the drug development process monotonous against this organism. The highly lipophilic cell wall, which constituting outer mycolic acid and inner peptidoglycan layers, acts as a barrier for the drugs to enter the bacteria. The rigidity of the cell wall is imparted by the peptidoglycan layer, which is covalently linked to mycolic acid by arabinogalactan. Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) serves as the starting material in the biosynthesis of this peptidoglycan layers. This UDP-GlcNAc is synthesized by N-acetylglucosamine-1-phosphate uridyltransferase (GlmU(Mtb)), a bi-functional enzyme with two functional sites, acetyltransferase site and uridyltransferase site. Here, we report design and screening of nine inhibitors against UTP and NAcGlc-1-P of uridyltransferase active site of glmU(Mtb). Compound 4 was showing good inhibition and was selected for further analysis. The isothermal titration calorimetry (ITC) experiments showed the binding energy pattern of compound 4 to the uridyltransferase active site is similar to that of substrate UTP. In silico molecular dynamics (MD) simulation studies, for compound 4, carried out for 10 ns showed the protein-compound complex to be stable throughout the simulation with relative rmsd in acceptable range. Hence, these compounds can serve as a starting point in the drug discovery processes against Mycobacterium tuberculosis.

  11. Combating highly resistant emerging pathogen Mycobacterium abscessus and Mycobacterium tuberculosis with novel salicylanilide esters and carbamates.

    PubMed

    Baranyai, Zsuzsa; Krátký, Martin; Vinšová, Jarmila; Szabó, Nóra; Senoner, Zsuzsanna; Horváti, Kata; Stolaříková, Jiřina; Dávid, Sándor; Bősze, Szilvia

    2015-08-28

    In the Mycobacterium genus over one hundred species are already described and new ones are periodically reported. Species that form colonies in a week are classified as rapid growers, those requiring longer periods (up to three months) are the mostly pathogenic slow growers. More recently, new emerging species have been identified to lengthen the list, all rapid growers. Of these, Mycobacterium abscessus is also an intracellular pathogen and it is the most chemotherapy-resistant rapid-growing mycobacterium. In addition, the cases of multidrug-resistant Mycobacterium tuberculosis infection are also increasing. Therefore there is an urgent need to find new active molecules against these threatening strains. Based on previous results, a series of salicylanilides, salicylanilide 5-chloropyrazinoates and carbamates was designed, synthesized and characterised. The compounds were evaluated for their in vitro activity on M. abscessus, susceptible M. tuberculosis H37Rv, multidrug-resistant (MDR) M. tuberculosis MDR A8, M. tuberculosis MDR 9449/2006 and on the extremely-resistant Praha 131 (XDR) strains. All derivatives exhibited a significant activity with minimum inhibitory concentrations (MICs) in the low micromolar range. Eight salicylanilide carbamates and two salicylanilide esters exhibited an excellent in vitro activity on M. abscessus with MICs from 0.2 to 2.1 μM, thus being more effective than ciprofloxacin and gentamicin. This finding is potentially promising, particularly, as M. abscessus is a threateningly chemotherapy-resistant species. M. tuberculosis H37Rv was inhibited with MICs from 0.2 μM, and eleven compounds have lower MICs than isoniazid. Salicylanilide esters and carbamates were found that they were effective also on MDR and XDR M. tuberculosis strains with MICs ≥1.0 μM. The in vitro cytotoxicity (IC50) was also determined on human MonoMac-6 cells, and selectivity index (SI) of the compounds was established. In general, salicylanilide

  12. Structural and functional characterization of Mycobacterium tuberculosis triosephosphate isomerase

    SciTech Connect

    Connor, Sean E.; Capodagli, Glenn C.; Deaton, Michelle K.; Pegan, Scott D.

    2012-04-18

    Tuberculosis (TB) is a major infectious disease that accounts for over 1.7 million deaths every year. Mycobacterium tuberculosis, the causative agent of tuberculosis, enters the human host by the inhalation of infectious aerosols. Additionally, one third of the world's population is likely to be infected with latent TB. The incidence of TB is on the rise owing in part to the emergence of multidrug-resistant strains. As a result, there is a growing need to focus on novel M. tuberculosis enzyme targets. M. tuberculosis triosephosphate isomerase (MtTPI) is an essential enzyme for gluconeogenetic pathways, making it a potential target for future therapeutics. In order to determine its structure, the X-ray crystal structure of MtTPI has been determined, as well as that of MtTPI bound with a reaction-intermediate analog. As a result, two forms of the active site were revealed. In conjunction with the kinetic parameters obtained for the MtTPI-facilitated conversion of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate (D-GAP), this provides a greater structural and biochemical understanding of this enzyme. Additionally, isothermal titration calorimetry was used to determine the binding constant for a reaction-intermediate analog bound to the active site of MtTPI.

  13. Mycobacterium tuberculosis: ecology and evolution of a human bacterium.

    PubMed

    Bañuls, Anne-Laure; Sanou, Adama; Anh, Nguyen Thi Van; Godreuil, Sylvain

    2015-11-01

    Some species of the Mycobacterium tuberculosis complex (MTBC), particularly Mycobacterium tuberculosis, which causes human tuberculosis (TB), are the first cause of death linked to a single pathogen worldwide. In the last decades, evolutionary studies have much improved our knowledge on MTBC history and have highlighted its long co-evolution with humans. Its ability to remain latent in humans, the extraordinary proportion of asymptomatic carriers (one-third of the entire human population), the deadly epidemics and the observed increasing level of resistance to antibiotics are proof of its evolutionary success. Many MTBC molecular signatures show not only that these bacteria are a model of adaptation to humans but also that they have influenced human evolution. Owing to the unbalance between the number of asymptomatic carriers and the number of patients with active TB, some authors suggest that infection by MTBC could have a protective role against active TB disease and also against other pathologies. However, it would be inappropriate to consider these infectious pathogens as commensals or symbionts, given the level of morbidity and mortality caused by TB.

  14. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries.

    PubMed Central

    Cosivi, O.; Grange, J. M.; Daborn, C. J.; Raviglione, M. C.; Fujikura, T.; Cousins, D.; Robinson, R. A.; Huchzermeyer, H. F.; de Kantor, I.; Meslin, F. X.

    1998-01-01

    The World Health Organization (WHO) estimates that human tuberculosis (TB) incidence and deaths for 1990 to 1999 will be 88 million and 30 million, respectively, with most cases in developing countries. Zoonotic TB (caused by Mycobacterium bovis) is present in animals in most developing countries where surveillance and control activities are often inadequate or unavailable; therefore, many epidemiologic and public health aspects of infection remain largely unknown. We review available information on zoonotic TB in developing countries, analyze risk factors that may play a role in the disease, review recent WHO activities, and recommend actions to assess the magnitude of the problem and control the disease in humans and animals. PMID:9452399

  15. Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis

    PubMed Central

    Perdigão, João; Couto, Isabel; Portugal, Isabel; Martins, Marta; Amaral, Leonard; Anes, Elsa; Viveiros, Miguel

    2016-01-01

    Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their

  16. Murine Mycobacterium marinum Infection as a Model for Tuberculosis.

    PubMed

    Lienard, Julia; Carlsson, Fredric

    2017-01-01

    Mycobacteria are a major human health problem globally. Regarding tuberculosis the situation is worsened by the poor efficacy of current vaccine regimens and by emergence of drug-resistant strains (Manjelievskaia J et al, Trans R Soc Trop Med Hyg 110: 110, 2016; Pereira et al., Lancet Infect Dis 12:300-306, 2012; http://www.who.int/tb/publications/global_report/en/) undermining both disease-prevention and available treatments. Thus, increased basic understanding of mycobacterial-and particularly Mycobacterium tuberculosis-virulence strategies and pathogenesis is of great importance. To this end several in vivo infection models are available (Guirado and Schlesinger, Front Immunol 4:98, 2013; Leung et al., Eur J Immunol 43:2246-2254, 2013; Patel et al., J Lab Physicians 3:75-79, 2011; van Leeuwen et al., Cold Spring Harb Perspect Med 5:a018580, 2015). While these models all have their merits they also exhibit limitations, and none perfectly mimics all aspects of human tuberculosis. Thus, there is a need for multiple models that may complement each other, ultimately allowing us to gain true insight into the pathogenesis of mycobacterial infections.Here, we describe a recently developed mouse model of Mycobacterium marinum infection that allows kinetic and quantitative studies of disease progression in live animals [8]. Notably, this model exhibits features of human tuberculosis not replicated in M. tuberculosis infected mice, and may provide an important complement to the field. For example, granulomas in the M. marinum model develop central caseating necrosis (Carlsson et al., PLoS Pathog 6:e1000895, 2010), a hallmark of granulomas in human tuberculosis normally not replicated in murine M. tuberculosis infection. Moreover, while tuberculosis is heterogeneous and presents with a continuum of active and latent disease, M. tuberculosis infected mice essentially lack this dynamic range and do not replicate latency (Guirado and Schlesinger, Front Immunol 4:98, 2013

  17. igr Genes and Mycobacterium tuberculosis cholesterol metabolism.

    PubMed

    Chang, Jennifer C; Miner, Maurine D; Pandey, Amit K; Gill, Wendy P; Harik, Nada S; Sassetti, Christopher M; Sherman, David R

    2009-08-01

    Recently, cholesterol was identified as a physiologically important nutrient for Mycobacterium tuberculosis survival in chronically infected mice. However, it remained unclear precisely when cholesterol is available to the bacterium and what additional bacterial functions are required for its metabolism. Here, we show that the igr locus, which we previously found to be essential for intracellular growth and virulence of M. tuberculosis, is required for cholesterol metabolism. While igr-deficient strains grow identically to the wild type in the presence of short- and long-chain fatty acids, the growth of these bacteria is completely inhibited in the presence of cholesterol. Interestingly, this mutant is still able to respire under cholesterol-dependent growth inhibition, suggesting that the bacteria can metabolize other carbon sources during cholesterol toxicity. Consistent with this hypothesis, we found that the growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as mutation of the mce4 sterol uptake system partially suppresses this effect. In addition, the Delta igr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout infection.

  18. Mycobacterium tuberculosis replicates within necrotic human macrophages

    PubMed Central

    Lerner, Thomas R.; Repnik, Urska; Herbst, Susanne; Collinson, Lucy M.; Griffiths, Gareth

    2017-01-01

    Mycobacterium tuberculosis modulation of macrophage cell death is a well-documented phenomenon, but its role during bacterial replication is less characterized. In this study, we investigate the impact of plasma membrane (PM) integrity on bacterial replication in different functional populations of human primary macrophages. We discovered that IFN-γ enhanced bacterial replication in macrophage colony-stimulating factor–differentiated macrophages more than in granulocyte–macrophage colony-stimulating factor–differentiated macrophages. We show that permissiveness in the different populations of macrophages to bacterial growth is the result of a differential ability to preserve PM integrity. By combining live-cell imaging, correlative light electron microscopy, and single-cell analysis, we found that after infection, a population of macrophages became necrotic, providing a niche for M. tuberculosis replication before escaping into the extracellular milieu. Thus, in addition to bacterial dissemination, necrotic cells provide first a niche for bacterial replication. Our results are relevant to understanding the environment of M. tuberculosis replication in the host. PMID:28242744

  19. A species-specific nucleotide sequence of Mycobacterium tuberculosis encodes a protein that exhibits hemolytic activity when expressed in Escherichia coli.

    PubMed Central

    Leão, S C; Rocha, C L; Murillo, L A; Parra, C A; Patarroyo, M E

    1995-01-01

    Species-specific proteins may be implicated in the unique pathogenic mechanisms characteristic of Mycobacterium tuberculosis. In previous studies, a 3.0-kb species-specific DNA fragment of M. tuberculosis was identified (C. A. Parra, L. P. Londoño, P. del Portillo, and M. E. Patarroyo, Immun. 59:3411-3417, 1991). The nucleotide sequence of this 3.0-kb fragment has been obtained. This sequence was shown to contain two open reading frames (ORFs) whose putative gene products share 68.9% identity between each other. The major ORF shows 57.8% similarity with PLC-N and 53.2% similarity with PLC-H, two phospholipase C enzymes from Pseudomonas aeruginosa. The major ORF was amplified by PCR and cloned into the pGEX-5T expression vector. Cell extracts of Escherichia coli overexpressing this glutathione S-transferase fusion protein were shown to produce beta-hemolysis suggestive of phospholipase activity. Since phospholipase C enzymes have been reported as virulence factors of P. aeruginosa and also of the intracellular pathogen Listeria monocytogenes, it is possible that the proteins identified in this study could also play a role in sustaining tuberculosis infection in humans. PMID:7591062

  20. Targeting Mycobacterium tuberculosis topoisomerase I by small-molecule inhibitors.

    PubMed

    Godbole, Adwait Anand; Ahmed, Wareed; Bhat, Rajeshwari Subray; Bradley, Erin K; Ekins, Sean; Nagaraja, Valakunja

    2015-03-01

    We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

  1. Growth hormone activation of human monocytes for superoxide production but not tumor necrosis factor production, cell adherence, or action against Mycobacterium tuberculosis.

    PubMed Central

    Warwick-Davies, J; Lowrie, D B; Cole, P J

    1995-01-01

    We have previously demonstrated that growth hormone (GH) is a human macrophage-activating factor which primes monocytes for enhanced production of H2O2 in vitro. This report extends our observations to other monocyte functions relevant to infection. We find that GH also primes monocytes for O2- production, to a degree similar to the effect of gamma interferon. Neither macrophage-activating factor alone stimulates monocytes to release bioactive tumor necrosis factor. However, GH, unlike gamma interferon, does not synergize with endotoxin for enhanced tumor necrosis factor production. In further contrast, GH does not alter monocyte adherence or morphology, while phagocytosis and killing of Mycobacterium tuberculosis by GH-treated monocytes are also unaffected. Therefore, despite the multiplicity of the effects of GH on the immune system in vivo, its effects on human monocytes in vitro appear to be limited to priming for the release of reactive oxygen intermediates. PMID:7591064

  2. Collagen degrading activity associated with Mycobacterium species

    PubMed Central

    Masso, F; Paez, A; Varela, E; d Diaz; Zenteno, E; Montano, L

    1999-01-01

    BACKGROUND—The mechanism of Mycobacterium tuberculosis penetration into tissues is poorly understood but it is reasonable to assume that there is a contribution from proteases capable of disrupting the extracellular matrix of the pulmonary epithelium and the blood vessels. A study was undertaken to identify and characterise collagen degrading activity of M tuberculosis.
METHODS—Culture filtrate protein extract (CFPE) was obtained from reference mycobacterial strains and mycobacteria isolated from patients with tuberculosis. The collagen degrading activity of CFPE was determined according to the method of Johnson-Wint using 3H-type I collagen. The enzyme was identified by the Birkedal-Hansen and Taylor method and its molecular mass determined by SDS-PAGE and Sephacryl S-300 gel filtration chromatography using an electroelution purified enzyme.
RESULTS—CFPE from Mycobacterium tuberculosis strain H37Rv showed collagenolytic activity that was four times higher than that of the avirulent strain H37Ra. The 75 kDa enzyme responsible was divalent cation dependent. Other mycobacterial species and those isolated from patients with tuberculosis also had collagen degrading activity.
CONCLUSIONS—Mycobacterium species possess a metalloprotease with collagen degrading activity. The highest enzymatic activity was found in the virulent reference strain H37Rv.

 PMID:10212111

  3. Potential Role for Mycobacterium tuberculosis Specific IL-2 and IFN-γ Responses in Discriminating between Latent Infection and Active Disease after Long-Term Stimulation

    PubMed Central

    Sun, Qin; Wei, Wei; Sha, Wei

    2016-01-01

    Interferon gamma release assays (IGRAs) could accurately diagnose Mycobacterium tuberculosis (M.tuberculosis) infection. However, these assays do not discriminate between latent tuberculosis infection (LTBI) and active tuberculosis disease (ATB). Here, a total of 177 subjects, including 65 patients with ATB, 43 subjects with LTBI, and 69 TB-uninfected controls (CON group) were enrolled. The concentration of IFN-γ, IP-10, and IL-2 was determined in peripheral blood mononuclear cells (PBMCs) after short-term (24h) or long-term (72h) stimulation with TB antigens including ESAT-6/CFP-10 (EC) and purified protein derivative (PPD).EC-stimulated IL-2 and gamma interferon-inducible protein 10 (IP-10) release (24h and 72h) showed a good diagnostic performance in distinguishing between TB-infected and TB-uninfected individuals, but failed to discriminate between ATB and LTBI. After 72h of incubation, the release of IL-2 was higher in LTBI patients after stimulation with EC and PPD. The PPD-stimulated IL-2/IFN-γ ratio after 72h incubation had the diagnostic potential to discriminate between ATB and LTBI, with a sensitivity of 90.8% and a specificity of 97.7%. In addition, these new biomarkers, combined with T-SPOT test in a two-step strategy, were validated with high levels of accuracy in a prospective clinical-based cohort. Collectively, the PPD-stimulated IL-2/IFN-γ ratio after long-term incubation may be an alternative diagnostic biomarker in distinguishing between active TB patients and subjects with latent infection. PMID:28033330

  4. Structure-activity relationships for a series of quinoline-based compounds active against replicating and nonreplicating Mycobacterium tuberculosis.

    PubMed

    Lilienkampf, Annamaria; Mao, Jialin; Wan, Baojie; Wang, Yuehong; Franzblau, Scott G; Kozikowski, Alan P

    2009-04-09

    Tuberculosis (TB) remains as a global pandemic that is aggravated by a lack of health care, the spread of HIV, and the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) strains. New anti-TB drugs are urgently required to shorten the long 6-12 month treatment regimen and to battle drug-resistant Mtb strains. We have identified several potent quinoline-based anti-TB compounds, bearing an isoxazole containing side-chain. The most potent compounds, 7g and 13, exhibited submicromolar activity against the replicating bacteria (R-TB), with minimum inhibitory concentrations (MICs) of 0.77 and 0.95 microM, respectively. In general, these compounds also had micromolar activity against the nonreplicating persistent bacteria (NRP-TB) and did not show toxicity on Vero cells up to 128 microM concentration. Compounds 7g and 13 were shown to retain their anti-TB activity against rifampin, isoniazid, and streptomycin resistant Mtb strains. The results suggest that quinoline-isoxazole-based anti-TB compounds are promising leads for new TB drug development.

  5. Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection

    PubMed Central

    Denkinger, Claudia M.; Kik, Sandra V.; Rangaka, Molebogeng X.; Zwerling, Alice; Oxlade, Olivia; Metcalfe, John Z.; Cattamanchi, Adithya; Dowdy, David W.; Dheda, Keertan; Banaei, Niaz

    2014-01-01

    SUMMARY Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-γ) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers. PMID:24396134

  6. Preliminary structure-activity relationships and biological evaluation of novel antitubercular indolecarboxamide derivatives against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains.

    PubMed

    Onajole, Oluseye K; Pieroni, Marco; Tipparaju, Suresh K; Lun, Shichun; Stec, Jozef; Chen, Gang; Gunosewoyo, Hendra; Guo, Haidan; Ammerman, Nicole C; Bishai, William R; Kozikowski, Alan P

    2013-05-23

    Tuberculosis (TB) remains one of the leading causes of mortality and morbidity worldwide, with approximately one-third of the world's population infected with latent TB. This is further aggravated by HIV coinfection and the emergence of multidrug- and extensively drug-resistant (MDR and XDR, respectively) TB; hence the quest for highly effective antitubercular drugs with novel modes of action is imperative. We report herein the discovery of an indole-2-carboxamide analogue, 3, as a highly potent antitubercular agent, and the subsequent chemical modifications aimed at establishing a preliminary body of structure-activity relationships (SARs). These efforts led to the identification of three molecules (12-14) possessing an exceptional activity in the low nanomolar range against actively replicating Mycobacterium tuberculosis , with minimum inhibitory concentration (MIC) values lower than those of the most prominent antitubercular agents currently in use. These compounds were also devoid of apparent toxicity to Vero cells. Importantly, compound 12 was found to be active against the tested XDR-TB strains and orally active in the serum inhibition titration assay.

  7. Haemophagocytic lymphohistiocytosis associated with Mycobacterium tuberculosis infection

    PubMed Central

    Hui, Yee Man Tracy; Pillinger, Toby; Luqmani, Asad; Cooper, Nichola

    2015-01-01

    Haemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal condition that can be primary or secondary. Secondary HLH can occur in association with infections, most commonly viral infections, but has also been reported in association with Mycobacterium tuberculosis (TB). Prompt identification of the underlying cause of HLH is important as it guides treatment decisions. Early initiation of appropriate treatment (eg, anti-TB treatment) reduces morbidity and mortality. We present a case of HLH associated with TB infection. Initial TB investigations were negative and standard combination chemoimmunotherapy for HLH resulted in a limited clinical response. On apparent relapse of HLH, further investigation revealed TB with changes on CT chest, granuloma on bone marrow and eventual positive TB culture on bronchoalveolar lavage. Subsequent treatment with quadruple anti-TB treatment resulted in rapid clinical response and disease remission. We advocate continued monitoring for TB infection in patients with HLH, and prophylaxis or full treatment for those at high risk. PMID:25870214

  8. Immunoinformatics study on highly expressed Mycobacterium tuberculosis genes during infection.

    PubMed

    Nguyen Thi, Le Thuy; Sarmiento, Maria Elena; Calero, Romel; Camacho, Frank; Reyes, Fatima; Hossain, Md Murad; Gonzalez, Gustavo Sierra; Norazmi, Mohd Nor; Acosta, Armando

    2014-09-01

    The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.

  9. Disinfecting endoscopes: how not to transmit Mycobacterium tuberculosis by bronchoscopy.

    PubMed Central

    Leers, W D

    1980-01-01

    Mycobacterium tuberculosis was cultured from the bronchial washings of two patients who underwent bronchoscopy consecutively with the same bronchoscope. Active pulmonary tuberculosis was later confirmed in the first patient, whereas the second patient had clinical and serologic evidence of infection with respiratory syncytial virus. The bronchoscope had been cleaned with an iodophor disinfectant, which had not destroyed the tubercle bacilli. The agent recommended for chemical disinfection of fibreoptic bronchoscopes is 2% glutaraldehyde solution; the instrument should be immersed in it for 10 to 30 minutes. Five hours' exposure to ethylene oxide is recommended for sterilization of instruments. These procedures must be preceded by adequate mechanical cleaning. Then transmission of pathogenic organisms during endoscopy, which can result in nosocomial disease, misdiagnosis or inappropriate treatment, will be avoided. Images FIG. 1 FIG. 2 FIG. 3 PMID:6790150

  10. Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials

    PubMed Central

    Gordon, Sara; Simithy, Johayra; Goodwin, Douglas C; Calderón, Angela I

    2015-01-01

    Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents. PMID:25861218

  11. A Novel in vitro Human Macrophage Model to Study the Persistence of Mycobacterium tuberculosis Using Vitamin D(3) and Retinoic Acid Activated THP-1 Macrophages.

    PubMed

    Estrella, Jaymie L; Kan-Sutton, Celestine; Gong, Xing; Rajagopalan, Malini; Lewis, Dorothy E; Hunter, Robert L; Eissa, N Tony; Jagannath, Chinnaswamy

    2011-01-01

    Mycobacterium tuberculosis (Mtb) replicates within the human macrophages and we investigated the activating effects of retinoic acid (RA) and vitamin D(3) (VD) on macrophages in relation to the viability of intracellular Mtb. A combination of these vitamins (RAVD) enhanced the levels of DC-SIGN and mannose receptors on THP-1 macrophages that increased mycobacterial uptake but inhibited the subsequent intracellular growth of Mtb by inducing reactive oxygen species and autophagy. RAVD also enhanced antigen presenting and chemotactic receptors on THPs suggesting an activated phenotype for RAVD activated THPs. RAVD mediated activation was also associated with a marked phenotypic change in Mtb infected THPs that fused with adjacent THPs to form multinucleated giant cells (MNGCs). Typically, MNGCs occurred over 30 days of in vitro culture and contained non-replicating persisting Mtb for more than 60 days in culture. Latent tuberculosis occurs in over a third of mankind and we propose that RAVD mediated induction of persistent Mtb within human macrophages provides a novel model to develop therapeutic approaches and investigate pathogenesis of latency.

  12. Mycobacterium tuberculosis Infection in Free-Roaming Wild Asian Elephant

    PubMed Central

    Shivashankar, Beechagondahalli Papanna; Umashankar, Kunigal Srinivasa; Nandini, Poojappa; Giridhar, Papanna; Byregowda, Somenahalli Munivenkatappa; Shrinivasa, Basavegowdanadoddi Marinaik

    2017-01-01

    Postmortem examination of a wild Asian elephant at Rajiv Gandhi National Park, India, revealed nodular lesions, granulomas with central caseation, and acid-fast bacilli in the lungs. PCR and nucleotide sequencing confirmed the presence of Mycobacterium tuberculosis. This study indicates that wild elephants can harbor M. tuberculosis that can become fatal. PMID:28221114

  13. Mycobacterium tuberculosis in Wild Asian Elephants, Southern India

    PubMed Central

    Zachariah, Arun; Pandiyan, Jeganathan; Madhavilatha, G.K.; Mundayoor, Sathish; Chandramohan, Bathrachalam; Sajesh, P.K.; Santhosh, Sam

    2017-01-01

    We tested 3 ild Asian elephants (Elephas maximus) in southern India and confirmed infection in 3 animals with Mycobacterium tuberculosis, an obligate human pathogen, by PCR and genetic sequencing. Our results indicate that tuberculosis may be spilling over from humans (reverse zoonosis) and emerging in wild elephants. PMID:28221104

  14. Palaeogenomics of Mycobacterium tuberculosis: epidemic bursts with a degrading genome.

    PubMed

    Djelouadji, Zoheira; Raoult, Didier; Drancourt, Michel

    2011-08-01

    Genome-scale analysis suggests that the last common ancestor of the Mycobacterium tuberculosis complex and Mycobacterium leprae diverged 36 million years ago, and members of the Mycobacterium tuberculosis complex differentiated 40,000 years ago. Analysis of palaeomicrobiological data from a 17,000-year-old sample from a bison and a 9000-year-old sample from a human being suggested that M tuberculosis preceded Mycobacterium bovis and related species. Whole-genome comparisons show that members of the M tuberculosis complex form a unique bacterial species with distinct ecotypes that are transmissible from any infected mammalian species to several others. Genomic deletions identified several M tuberculosis lineages that could be placed on a phylogeographical map, suggesting adaptation to local host populations. The degrees of transmissibility and virulence vary between M tuberculosis clones, with increased virulence mainly linked to gene loss in regulatory pathways. Such data suggest that most M tuberculosis clones have a restricted spreading capacity between the host population, allowing unpredictable bursts of highly transmissible, virulent, and successful clones, such as the east Asian (Beijing) clone. Advances in genomics have helped the development of molecular techniques for accurate identification of species and clones in the M tuberculosis complex, which is essential for tracing the source of infections.

  15. Characterization of Mycobacterium orygis as M. tuberculosis Complex Subspecies

    PubMed Central

    Rahim, Zeaur; Mulder, Arnout; Boeree, Martin J.; Simeone, Roxane; Brosch, Roland; van Soolingen, Dick

    2012-01-01

    The oryx bacilli are Mycobacterium tuberculosis complex organisms for which phylogenetic position and host range are unsettled. We characterized 22 isolates by molecular methods and propose elevation to subspecies status as M. orygis. M. orygis is a causative agent of tuberculosis in animals and humans from Africa and South Asia. PMID:22469053

  16. Transmission of Mycobacterium tuberculosis from Human to Cattle

    PubMed Central

    Ocepek, Matjaž; Pate, Mateja; Žolnir-Dovč, Manca; Poljak, Mario

    2005-01-01

    We describe the first transmission of Mycobacterium tuberculosis from human to cattle confirmed by molecular typing of isolates involved in the transmission. IS6110-based restriction fragment length polymorphism analysis showed that the isolates from the cattle and farm worker who suffered from pulmonary tuberculosis 1 year prior to this case were the same strains. PMID:16000505

  17. Mycobacterium tuberculosis in Wild Asian Elephants, Southern India.

    PubMed

    Zachariah, Arun; Pandiyan, Jeganathan; Madhavilatha, G K; Mundayoor, Sathish; Chandramohan, Bathrachalam; Sajesh, P K; Santhosh, Sam; Mikota, Susan K

    2017-03-01

    We tested 3 ild Asian elephants (Elephas maximus) in southern India and confirmed infection in 3 animals with Mycobacterium tuberculosis, an obligate human pathogen, by PCR and genetic sequencing. Our results indicate that tuberculosis may be spilling over from humans (reverse zoonosis) and emerging in wild elephants.

  18. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    SciTech Connect

    Wubben, T.; Mesecar, A.D.

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  19. Structures of Mycobacterium tuberculosis DosR and DosR-DNA Complex Involved in Gene Activation during Adaptation to Hypoxic Latency

    SciTech Connect

    Wisedchaisri, Goragot; Wu, Meiting; Rice, Adrian E; Roberts, David M; Sherman, David R; Hol, Wim G.J.

    2010-07-20

    On encountering low oxygen conditions, DosR activates the transcription of 47 genes, promoting long-term survival of Mycobacterium tuberculosis in a non-replicating state. Here, we report the crystal structures of the DosR C-terminal domain and its complex with a consensus DNA sequence of the hypoxia-induced gene promoter. The DosR C-terminal domain contains four {alpha}-helices and forms tetramers consisting of two dimers with non-intersecting dyads. In the DNA-bound structure, each DosR C-terminal domain in a dimer places its DNA-binding helix deep into the major groove, causing two bends in the DNA. DosR makes numerous protein-DNA base contacts using only three amino acid residues per subunit: Lys179, Lys182, and Asn183. The DosR tetramer is unique among response regulators with known structures.

  20. Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+

    PubMed Central

    Brown, Alistair K; Meng, Guoyu; Ghadbane, Hemza; Scott, David J; Dover, Lynn G; Nigou, Jérôme; Besra, Gurdyal S; Fütterer, Klaus

    2007-01-01

    Background The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase). Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear. Results We determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own. Conclusion The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity. PMID:17725819

  1. Genotypic characteristics of Mycobacterium tuberculosis isolated from household contacts of tuberculosis patients in the Philippines

    PubMed Central

    2013-01-01

    Background The Philippines has an extremely high rate of tuberculosis but little is known about M. tuberculosis genotypes and transmission dynamics in this country. The aim of this study was to determine the proportion of household contacts who develop active TB due to direct transmission from an index case in that household. Methods Mycobacterium tuberculosis isolates from household contacts of tuberculosis patients in the Philippines were characterized using restriction-fragment-length polymorphism analysis, spoligotyping, and mycobacterial interspersed repetitive units – variable number tandem repeats typing (12-loci) to determine their utility in elucidating transmission in an area of high tuberculosis prevalence. Drug susceptibility patterns for these isolates were also determined. Results Spoligotyping and MIRU-VNTR typing results matched in 10 (62.5%) of 16 index patient-household contact pairs while IS6110 fingerprints matched in only six (37.5%) pairs. Only 3/16 (18.8%) index patient-household contact pairs had identical drug susceptibility results. Conclusions Strain typing of M. tuberculosis isolates from household contacts in the Philippines indicates that transmission of strains does not necessarily occur directly from the index patient living in close proximity in the same household but rather that community-based transmission also frequently occurs. Accurate susceptibility testing of all isolates is necessary to insure optimal care of both the index patients and any culture-positive household contacts. PMID:24308751

  2. Mycobacterium tuberculosis and the macrophage: maintaining a balance.

    PubMed

    Pieters, Jean

    2008-06-12

    Mycobacterium tuberculosis is a highly efficient pathogen, killing millions of infected people annually. The capacity of M. tuberculosis to survive and cause disease is strongly correlated to their ability to escape immune defense mechanisms. In particular, M. tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage. Understanding M. tuberculosis virulence strategies will not only define novel targets for drug development but will also help to uncover previously unknown signaling pathways related to the host's response to M. tuberculosis infection.

  3. The cell envelope glycoconjugates of Mycobacterium tuberculosis

    PubMed Central

    Angala, Shiva Kumar; Belardinelli, Juan Manuel; Huc-Claustre, Emilie; Wheat, William H.; Jackson, Mary

    2015-01-01

    Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative agent of the disease in humans, is a source of unique glycoconjugates and the most distinctive feature of the biology of this organism. It is the basis of much of Mtb pathogenesis and one of the major causes of its intrinsic resistance to chemotherapeutic agents. At the same time, the unique structures of Mtb cell envelope glycoconjugates, their antigenicity and essentiality for mycobacterial growth provide opportunities for drug, vaccine, diagnostic and biomarker development, as clearly illustrated by recent advances in all of these translational aspects. This review focuses on our current understanding of the structure and biogenesis of Mtb glycoconjugates with particular emphasis on one of most intriguing and least understood aspect of the physiology of mycobacteria: the translocation of these complex macromolecules across the different layers of the cell envelope. It further reviews the rather impressive progress made in the last ten years in the discovery and development of novel inhibitors targeting their biogenesis. PMID:24915502

  4. Role of cholesterol in Mycobacterium tuberculosis infection.

    PubMed

    Miner, Maurine D; Chang, Jennifer C; Pandey, Amit K; Sassetti, Christopher M; Sherman, David R

    2009-06-01

    Mycobacterium tuberculosis (MTB) acquisition and utilization of nutrients within the host cell is poorly understood, although it has been hypothesized that host lipids probably play an important role in MTB survival. Cholesterol has recently been identified as an important lipid for mycobacterial infection. The mce4 transport system is required for cholesterol import into bacterial cells, and deletion of mce4 locus resulted in severe attenuation in a chronic mouse model of infection. However, it has remained unclear what additional bacterial functions were required for utilization of this sterol. We have found that the igr locus, which was previously found essential for intracellular growth and virulence of MTB, is required for cholesterol metabolism: igr-deficient bacteria cannot grow using cholesterol as a primary carbon source. The growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as the delta igr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout the course of infection, and that degradation of this sterol is crucial for bacterial persistence.

  5. The Mycobacterium tuberculosis regulatory network and hypoxia

    PubMed Central

    Galagan, James E.; Minch, Kyle; Peterson, Matthew; Lyubetskaya, Anna; Azizi, Elham; Sweet, Linsday; Gomes, Antonio; Rustad, Tige; Dolganov, Gregory; Glotova, Irina; Abeel, Thomas; Mahwinney, Chris; Kennedy, Adam D.; Allard, René; Brabant, William; Krueger, Andrew; Jaini, Suma; Honda, Brent; Yu, Wen-Han; Hickey, Mark J.; Zucker, Jeremy; Garay, Christopher; Weiner, Brian; Sisk, Peter; Stolte, Christian; Winkler, Jessica K.; Van de Peer, Yves; Iazzetti, Paul; Camacho, Diogo; Dreyfuss, Jonathan; Liu, Yang; Dorhoi, Anca; Mollenkopf, Hans-Joachim; Drogaris, Paul; Lamontagne, Julie; Zhou, Yiyong; Piquenot, Julie; Park, Sang Tae; Raman, Sahadevan; Kaufmann, Stefan H. E.; Mohney, Robert P.; Chelsky, Daniel; Moody, D. Branch; Sherman, David R.; Schoolnik, Gary K.

    2014-01-01

    We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub. PMID:23823726

  6. Mycobacterium tuberculosis Spoligotypes in Monterrey, Mexico▿

    PubMed Central

    Molina-Torres, Carmen A.; Moreno-Torres, Elisa; Ocampo-Candiani, Jorge; Rendon, Adrian; Blackwood, Kym; Kremer, Kristin; Rastogi, Nalin; Welsh, Oliverio; Vera-Cabrera, Lucio

    2010-01-01

    Although tuberculosis is still a public health problem in Mexico, there is little information about the genetic characteristics of the isolates. In the present study, we analyzed by spoligotyping 180 Mycobacterium tuberculosis clinical isolates from the urban area of Monterrey, Mexico, including drug-susceptible and drug-resistant isolates. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Four isolates presented spoligotype patterns not found in the database (orphan types); the rest were distributed among 44 spoligo international types (SITs). SIT53 (clade T1) and SIT119 (clade X1) were predominant and included 43 (23.8%) and 28 (15.5%) of the isolates, respectively. In order to determine if there was a dominant spoligotype in the group of multidrug-resistant isolates, 37 of them were analyzed by IS6110-based restriction fragment length polymorphism assays, and scarce clustering of strains with more than five bands was observed. Fourteen isolates of this multidrug-resistant group presented four bands or less and were distributed in four SITs: SIT53 (n = 8), SIT92 (n = 3), SIT70 (n = 2), and SIT3038 (n = 1). When the molecular detection of mutations in the katG and rpoB genes were analyzed in these isolates with low copy numbers of IS6110, only two isolates shared the same IS6110, spoligotyping, and mutations patterns. When the distribution of the spoligotypes was analyzed by age cohort, SIT119 was predominantly found in patients 0 to 20 years old, especially in males, accounting for up to 40% of the isolates. In contrast, SIT53 was more prevalent in older females. This analysis demonstrates the variability of M. tuberculosis isolates in Monterrey and the partial dominance of SIT53 and SIT119 in that area of Mexico. PMID:19940048

  7. Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis.

    PubMed Central

    Hermans, P W; van Soolingen, D; Dale, J W; Schuitema, A R; McAdam, R A; Catty, D; van Embden, J D

    1990-01-01

    IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it has previously been shown to be present in multiple copies in the chromosome of M. tuberculosis. In this study we investigated the value of a IS986-based DNA probe in the diagnosis and epidemiology of tuberculosis. IS986 was found only in species belonging to the M. tuberculosis complex. Independent isolates of M. tuberculosis complex strains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. In contrast, Mycobacterium bovis BCG vaccine strains as well as clinical isolates of M. bovis BCG contained one copy of IS986, which was present at the same location in the chromosome. Different M. tuberculosis isolates from a recent M. tuberculosis outbreak showed an identical banding pattern. We concluded that IS986 is an extremely suitable tool for the diagnosis and epidemiology of tuberculosis. Images PMID:1977765

  8. Mycobacterium tuberculosis PE25/PPE41 protein complex induces activation and maturation of dendritic cells and drives Th2-biased immune responses.

    PubMed

    Chen, Wei; Bao, Yige; Chen, Xuerong; Burton, Jeremy; Gong, Xueli; Gu, Dongqing; Mi, Youjun; Bao, Lang

    2016-04-01

    Mycobacterium tuberculosis evades innate host immune responses by parasitizing macrophages and causes significant morbidity and mortality around the world. A mycobacterial antigen that can activate dendritic cells (DCs) and elicit effective host innate immune responses will be vital to the development of an effective TB vaccine. The M. tuberculosis genes PE25/PPE41 encode proteins which have been associated with evasion of the host immune response. We constructed a PE25/PPE41 complex gene via splicing by overlapping extension and expressed it successfully in E. coli. We investigated whether this protein complex could interact with DCs to induce effective host immune responses. The PE25/PPE41 protein complex induced maturation of isolated mouse DCs in vitro, increasing expression of cell surface markers (CD80, CD86 and MHC-II), thereby promoting Th2 polarization via secretion of pro-inflammatory cytokines IL-4 and IL-10. In addition, PE25/PPE41 protein complex-activated DCs induced proliferation of mouse CD4(+) and CD8(+) T cells, and a strong humoral response in immunized mice. The sera of five TB patients were also highly reactive to this antigen. These findings suggest that interaction of the PE25/PPE41 protein complex with DCs may be of great immunological significance.

  9. The antimicrobial activity of copper and copper alloys against nosocomial pathogens and Mycobacterium tuberculosis isolated from healthcare facilities in the Western Cape: an in-vitro study.

    PubMed

    Mehtar, S; Wiid, I; Todorov, S D

    2008-01-01

    Clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Candida albicans and Mycobacterium tuberculosis (MTB) were tested against copper (Cu) and its alloys. Stainless steel and polyvinylchloride (PVC) were used as controls. The amount of Cu required to inhibit test isolates at room temperature (24 degrees C) and at 4 degrees C was determined. At room temperature, Cu, DZR Brass (Cu 62%, Pb 2.5%, arsenate 0.13% and Zn 22.5%) and Brass 70/30 (Cu 70% and zinc 30%) inhibited C. albicans and K. pneumoniae at 60 min; nickel silver (NiAg) inhibited C. albicans at 60 min and K. pneumoniae at 270 min. P. aeruginosa was inhibited by Brass 70/30 and nickel silver (NiAg) at 180 min and at 270 min by Cu and DZR. Cu and DZR inhibited A. baumannii at 180 min while the other alloys were effective at 360 min. Stainless steel and PVC showed little or no inhibitory activity. Two M. tuberculosis strains, one isoniazid resistant (R267) and the other multidrug resistant (R432), demonstrated growth inhibition with Cu of 98% and 88% respectively compared with PVC; the other alloys were less active. Time to positivity (TTP) for R267 was >15 days with Cu and 11 days for the other alloys; with R432 it was 5 days. Effective inhibition of nosocomial pathogens and MTB by Cu and alloys was best when the Cu content was >55%.

  10. In silico discovery and in vitro activity of inhibitors against Mycobacterium tuberculosis 7,8-diaminopelargonic acid synthase (Mtb BioA)

    PubMed Central

    Billones, Junie B; Carrillo, Maria Constancia O; Organo, Voltaire G; Sy, Jamie Bernadette A; Clavio, Nina Abigail B; Macalino, Stephani Joy Y; Emnacen, Inno A; Lee, Alexandra P; Ko, Paul Kenny L; Concepcion, Gisela P

    2017-01-01

    Computer-aided drug discovery and development approaches such as virtual screening, molecular docking, and in silico drug property calculations have been utilized in this effort to discover new lead compounds against tuberculosis. The enzyme 7,8-diaminopelargonic acid aminotransferase (BioA) in Mycobacterium tuberculosis (Mtb), primarily involved in the lipid biosynthesis pathway, was chosen as the drug target due to the fact that humans are not capable of synthesizing biotin endogenously. The computational screening of 4.5 million compounds from the Enamine REAL database has ultimately yielded 45 high-scoring, high-affinity compounds with desirable in silico absorption, distribution, metabolism, excretion, and toxicity properties. Seventeen of the 45 compounds were subjected to bioactivity validation using the resazurin microtiter assay. Among the 4 actives, compound 7 ((Z)-N-(2-isopropoxyphenyl)-2-oxo-2-((3-(trifluoromethyl)cyclohexyl)amino)acetimidic acid) displayed inhibitory activity up to 83% at 10 μg/mL concentration against the growth of the Mtb H37Ra strain. PMID:28280303

  11. Mycobacterium tuberculosis PstS1 amplifies IFN-γ and induces IL-17/IL-22 responses by unrelated memory CD4+ T cells via dendritic cell activation.

    PubMed

    Palma, Carla; Schiavoni, Giovanna; Abalsamo, Laura; Mattei, Fabrizio; Piccaro, Giovanni; Sanchez, Massimo; Fernandez, Carmen; Singh, Mahavir; Gabriele, Lucia

    2013-09-01

    The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN-γ and, to a lesser extent, of IL-17 by CD4(+) T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag-unrelated CD4(+) T-cell responses. Here we demonstrate that PstS1, a 38 kDa-lipoprotein of Mtb, promotes Ag-independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4(+) and CD8(+) memory T cells, amplifies secretion of IFN-γ and IL-22 and induces IL-17 production by effector memory cells in an Ag-unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α(-) subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL-6, IL-1β and, to a lower extent, IL-23. IL-6 secretion by PstS1-stimulated DCs was required for IFN-γ, and to a lesser extent for IL-22 responses by Ag85B-specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.

  12. Energy Metabolism and Drug Efflux in Mycobacterium tuberculosis

    PubMed Central

    Black, Philippa A.; Warren, Robin M.; Louw, Gail E.; van Helden, Paul D.; Victor, Thomas C.

    2014-01-01

    The inherent drug susceptibility of microorganisms is determined by multiple factors, including growth state, the rate of drug diffusion into and out of the cell, and the intrinsic vulnerability of drug targets with regard to the corresponding antimicrobial agent. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant source of global morbidity and mortality, further exacerbated by its ability to readily evolve drug resistance. It is well accepted that drug resistance in M. tuberculosis is driven by the acquisition of chromosomal mutations in genes encoding drug targets/promoter regions; however, a comprehensive description of the molecular mechanisms that fuel drug resistance in the clinical setting is currently lacking. In this context, there is a growing body of evidence suggesting that active extrusion of drugs from the cell is critical for drug tolerance. M. tuberculosis encodes representatives of a diverse range of multidrug transporters, many of which are dependent on the proton motive force (PMF) or the availability of ATP. This suggests that energy metabolism and ATP production through the PMF, which is established by the electron transport chain (ETC), are critical in determining the drug susceptibility of M. tuberculosis. In this review, we detail advances in the study of the mycobacterial ETC and highlight drugs that target various components of the ETC. We provide an overview of some of the efflux pumps present in M. tuberculosis and their association, if any, with drug transport and concomitant effects on drug resistance. The implications of inhibiting drug extrusion, through the use of efflux pump inhibitors, are also discussed. PMID:24614376

  13. Mycobacterium tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced Fenton reaction

    PubMed Central

    Vilchèze, Catherine; Hartman, Travis; Weinrick, Brian; Jacobs, William R.

    2013-01-01

    Drugs that kill tuberculosis more quickly could shorten chemotherapy significantly. In Escherichia coli, a common mechanism of cell death by bactericidal antibiotics involves the generation of highly reactive hydroxyl radicals via the Fenton reaction. Here we show that vitamin C, a compound known to drive the Fenton reaction, sterilizes cultures of drug-susceptible and drug-resistant Mycobacterium tuberculosis, the causative agent of tuberculosis. While M. tuberculosis is highly susceptible to killing by vitamin C, other Gram-positive and Gram-negative pathogens are not. The bactericidal activity of vitamin C against M. tuberculosis is dependent on high ferrous ion levels and reactive oxygen species production and causes a pleiotropic effect affecting several biological processes. This study enlightens the possible benefits of adding vitamin C to an anti-tuberculosis regimen and suggests that the development of drugs that generate high oxidative burst could be of great use in tuberculosis treatment. PMID:23695675

  14. Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis

    PubMed Central

    Stinear, Timothy P.; Seemann, Torsten; Harrison, Paul F.; Jenkin, Grant A.; Davies, John K.; Johnson, Paul D.R.; Abdellah, Zahra; Arrowsmith, Claire; Chillingworth, Tracey; Churcher, Carol; Clarke, Kay; Cronin, Ann; Davis, Paul; Goodhead, Ian; Holroyd, Nancy; Jagels, Kay; Lord, Angela; Moule, Sharon; Mungall, Karen; Norbertczak, Halina; Quail, Michael A.; Rabbinowitsch, Ester; Walker, Danielle; White, Brian; Whitehead, Sally; Small, Pamela L.C.; Brosch, Roland; Ramakrishnan, Lalita; Fischbach, Michael A.; Parkhill, Julian; Cole, Stewart T.

    2008-01-01

    Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis. PMID:18403782

  15. New nitrofurans amenable by isocyanide multicomponent chemistry are active against multidrug-resistant and poly-resistant Mycobacterium tuberculosis.

    PubMed

    Krasavin, Mikhail; Parchinsky, Vladislav; Kantin, Grigory; Manicheva, Olga; Dogonadze, Marine; Vinogradova, Tatiana; Karge, Bianka; Brönstrup, Mark

    2017-03-15

    A set of structurally diverse N-amino δ-lactams decorated with a 5-nitro-2-furyl moiety was synthesized using isocyanide-based multicomponent chemistry and evaluated for antibacterial activity. Three compounds displayed a selective and potent (MIC 22-33μM) inhibition of M. tuberculosis H37Rv strain growth, while other Gram-positive (MRSA and E. faecium) or Gram-negative (E. coli, P. aeruginosa, A. baumannii, K. pneumoniae) pathogens were not affected. The compounds also displayed moderate-low cytotoxicity, as demonstrated in cell line viability assays. Several multidrug- and poly-resistant patient-derived M. tuberculosis strains were found to be susceptible to treatment with these compounds. The three most potent compounds share a significant structural similarity which provides a basis for further scaffold-hopping analog design.

  16. Transcriptional Profiling of Mycobacterium Tuberculosis During Infection: Lessons Learned

    PubMed Central

    Ward, Sarah K.; Abomoelak, Bassam; Marcus, Sarah A.; Talaat, Adel M.

    2010-01-01

    Infection with Mycobacterium tuberculosis, the causative agent of tuberculosis, is considered one of the biggest infectious disease killers worldwide. A significant amount of attention has been directed toward revealing genes involved in the virulence and pathogenesis of this air-born pathogen. With the advances in technologies for transcriptional profiling, several groups, including ours, took advantage of DNA microarrays to identify transcriptional units differentially regulated by M. tuberculosis within a host. The main idea behind this approach is that pathogens tend to regulate their gene expression levels depending on the host microenvironment, and preferentially express those needed for survival. Identifying this class of genes will improve our understanding of pathogenesis. In our case, we identified an in vivo expressed genomic island that was preferentially active in murine lungs during early infection, as well as groups of genes active during chronic tuberculosis. Other studies have identified additional gene groups that are active during macrophage infection and even in human lungs. Despite all of these findings, one of the lingering questions remaining was whether in vivo expressed transcripts are relevant to the virulence, pathogenesis, and persistence of the organism. The work of our group and others addressed this question by examining the contribution of in vivo expressed genes using a strategy based on gene deletions followed by animal infections. Overall, the analysis of most of the in vivo expressed genes supported a role of these genes in M. tuberculosis pathogenesis. Further, these data suggest that in vivo transcriptional profiling is a valid approach to identify genes required for bacterial pathogenesis. PMID:21738523

  17. Rapid Diagnosis of Pulmonary Tuberculosis with the LCx Mycobacterium tuberculosis Assay and Comparison with Conventional Diagnostic Techniques

    PubMed Central

    Rohner, Peter; Jahn, Esther I. M.; Ninet, Beatrice; Ionati, Concetta; Weber, Rainer; Auckenthaler, Raymond; Pfyffer, Gaby E.

    1998-01-01

    The LCx MTB amplification assay is a nucleic acid amplification test intended for the direct detection of Mycobacterium tuberculosis complex in respiratory specimens. We evaluated its performance on 2,001 consecutive respiratory specimens; 78 were culture positive for M. tuberculosis. Sensitivity, specificity, and positive and negative predictive values of this assay for all specimens compared to culture results were 88.5, 97.7, 60.5, and 99.5%, respectively. When referred to resolved clinical diagnosis of active tuberculosis, these values improved to 90.2, 98.4, 72.8, and 99.5%, respectively. PMID:9738065

  18. Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex.

    PubMed Central

    Roberts, M C; McMillan, C; Coyle, M B

    1987-01-01

    Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified. PMID:3112180

  19. Surviving the macrophage: tools and tricks employed by Mycobacterium tuberculosis.

    PubMed

    Jayachandran, Rajesh; BoseDasgupta, Somdeb; Pieters, Jean

    2013-01-01

    Mycobacterium tuberculosis has evolved to withstand one of the most inhospitable cells within the human body, namely the macrophage, a cell that is normally geared toward the destruction of any invading microbe. How M. tuberculosis achieves this is still incompletely understood; however, a number of mechanisms are now known that provide advantages to M. tuberculosis for its survival and proliferation inside the macrophage. While some of these mechanisms are mediated by factors released by M. tuberculosis, others rely on host components that are being hijacked to benefit survival of M. tuberculosis within the macrophage as well to avoid the generation of an effective immune response. Here, we describe several of these mechanisms, also pointing out the potential usage of this knowledge toward the development of novel strategies to treat tuberculosis. Furthermore, we attempt to put the 'macrophage niche' into context with other intracellular pathogens and discuss some of the generalities as well as specializations that M. tuberculosis employs to survive.

  20. CD8 T cells and Mycobacterium tuberculosis infection.

    PubMed

    Lin, Philana Ling; Flynn, JoAnne L

    2015-05-01

    Tuberculosis is primarily a respiratory disease that is caused by Mycobacterium tuberculosis. M. tuberculosis can persist and replicate in macrophages in vivo, usually in organized cellular structures called granulomas. There is substantial evidence for the importance of CD4 T cells in control of tuberculosis, but the evidence for a requirement for CD8 T cells in this infection has not been proven in humans. However, animal model data support a non-redundant role for CD8 T cells in control of M. tuberculosis infection. In humans, infection with this pathogen leads to generation of specific CD8 T cell responses. These responses include classical (MHC Class I restricted) and non-classical CD8 T cells. Here, we discuss the potential roles of CD8 T cells in defense against tuberculosis, and our current understanding of the wide range of CD8 T cell types seen in M. tuberculosis infection.

  1. Role for Mycobacterium tuberculosis Membrane Vesicles in Iron Acquisition

    PubMed Central

    Prados-Rosales, Rafael; Weinrick, Brian C.; Piqué, Daniel G.; Jacobs, William R.; Casadevall, Arturo

    2014-01-01

    Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host. PMID:24415729

  2. Macrophage polarization drives granuloma outcome during Mycobacterium tuberculosis infection.

    PubMed

    Marino, Simeone; Cilfone, Nicholas A; Mattila, Joshua T; Linderman, Jennifer J; Flynn, JoAnne L; Kirschner, Denise E

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), induces formation of granulomas, structures in which immune cells and bacteria colocalize. Macrophages are among the most abundant cell types in granulomas and have been shown to serve as both critical bactericidal cells and targets for M. tuberculosis infection and proliferation throughout the course of infection. Very little is known about how these processes are regulated, what controls macrophage microenvironment-specific polarization and plasticity, or why some granulomas control bacteria and others permit bacterial dissemination. We take a computational-biology approach to investigate mechanisms that drive macrophage polarization, function, and bacterial control in granulomas. We define a "macrophage polarization ratio" as a metric to understand how cytokine signaling translates into polarization of single macrophages in a granuloma, which in turn modulates cellular functions, including antimicrobial activity and cytokine production. Ultimately, we extend this macrophage ratio to the tissue scale and define a "granuloma polarization ratio" describing mean polarization measures for entire granulomas. Here we coupled experimental data from nonhuman primate TB granulomas to our computational model, and we predict two novel and testable hypotheses regarding macrophage profiles in TB outcomes. First, the temporal dynamics of granuloma polarization ratios are predictive of granuloma outcome. Second, stable necrotic granulomas with low CFU counts and limited inflammation are characterized by short NF-κB signal activation intervals. These results suggest that the dynamics of NF-κB signaling is a viable therapeutic target to promote M1 polarization early during infection and to improve outcome.

  3. Pulmonary disease due to Mycobacterium tuberculosis in a horse: zoonotic concerns and limitations of antemortem testing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A case of pulmonary tuberculosis caused by Mycobacterium tuberculosis was diagnosed in a horse. Clinical evaluation performed prior to euthanasia did not suggest tuberculosis, but postmortem examination provided pathological and bacteriological evidence of disease. In the lungs, multiple tuberculoid...

  4. Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae.

    PubMed

    Wesener, Darryl A; Levengood, Matthew R; Kiessling, Laura L

    2017-02-17

    The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

  5. Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov.

    PubMed

    Cousins, Debby V; Bastida, Ricardo; Cataldi, Angel; Quse, Viviana; Redrobe, Sharon; Dow, Sue; Duignan, Padraig; Murray, Alan; Dupont, Christine; Ahmed, Niyaz; Collins, Des M; Butler, W Ray; Dawson, David; Rodríguez, Diego; Loureiro, Julio; Romano, Maria Isabel; Alito, A; Zumarraga, M; Bernardelli, Amelia

    2003-09-01

    A comparison of Mycobacterium tuberculosis complex isolates from seals (pinnipeds) in Australia, Argentina, Uruguay, Great Britain and New Zealand was undertaken to determine their relationships to each other and their taxonomic position within the complex. Isolates from 30 cases of tuberculosis in six species of pinniped and seven related isolates were compared to representative and standard strains of the M. tuberculosis complex. The seal isolates could be distinguished from other members of the M. tuberculosis complex, including the recently defined 'Mycobacterium canettii' and 'Mycobacterium caprae', on the basis of host preference and phenotypic and genetic tests. Pinnipeds appear to be the natural host for this 'seal bacillus', although the organism is also pathogenic in guinea pigs, rabbits, humans, Brazilian tapir (Tapirus terrestris) and, possibly, cattle. Infection caused by the seal bacillus is predominantly associated with granulomatous lesions in the peripheral lymph nodes, lungs, pleura, spleen and peritoneum. Cases of disseminated disease have been found. As with other members of the M. tuberculosis complex, aerosols are the most likely route of transmission. The name Mycobacterium pinnipedii sp. nov. is proposed for this novel member of the M. tuberculosis complex (the type strain is 6482(T)=ATCC BAA-688(T)=NCTC 13288(T)).

  6. Mycobacterium tuberculosis Isolates from Single Outpatient Clinic in Panama City Exhibit Wide Genetic Diversity

    PubMed Central

    Sambrano, Dilcia; Correa, Ricardo; Almengor, Pedro; Domínguez, Amada; Vega, Silvio; Goodridge, Amador

    2014-01-01

    Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities. PMID:24865686

  7. Mycobacterium tuberculosis isolates from single outpatient clinic in Panama City exhibit wide genetic diversity.

    PubMed

    Sambrano, Dilcia; Correa, Ricardo; Almengor, Pedro; Domínguez, Amada; Vega, Silvio; Goodridge, Amador

    2014-08-01

    Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities.

  8. Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae

    PubMed Central

    Youm, Jiwon; Saier, Milton H.

    2012-01-01

    The co-emergence of multidrug resistant pathogenic bacterial strains and the HIV pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyses of transport proteins encoded within the genomes of these two organisms. A minimal set of genes required for intracellular and extracellular life were identified. Drug efflux systems utilizing primary active transport mechanisms have been preferentially retained in Mle and still others preferentially lost. Transporters associated with environmental adaptation found in Mtu were mostly lost in Mle. These findings provide starting points for experimental studies that may elucidate the dependencies of pathogenesis on transport for these two pathogenic mycobacteria. They also lead to suggestions regarding transporters that function in intra- versus extra-cellular growth. PMID:22179038

  9. Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae.

    PubMed

    Youm, Jiwon; Saier, Milton H

    2012-03-01

    The co-emergence of multidrug resistant pathogenic bacterial strains and the Human Immunodeficiency Virus pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyses of transport proteins encoded within the genomes of these two organisms. A minimal set of genes required for intracellular and extracellular life was identified. Drug efflux systems utilizing primary active transport mechanisms have been preferentially retained in Mle and still others preferentially lost. Transporters associated with environmental adaptation found in Mtu were mostly lost in Mle. These findings provide starting points for experimental studies that may elucidate the dependencies of pathogenesis on transport for these two pathogenic mycobacteria. They also lead to suggestions regarding transporters that function in intra- versus extra-cellular growth.

  10. Global Efforts in the Development of Vaccines for Tuberculosis: Requirements for Improved Vaccines Against Mycobacterium tuberculosis.

    PubMed

    Méndez-Samperio, P

    2016-10-01

    Currently, more than 9.0 million people develop acute pulmonary tuberculosis (TB) each year and about 1.5 million people worldwide die from this infection. Thus, developing vaccines to prevent active TB disease remains a priority. This article discusses recent progress in the development of new vaccines against TB and focusses on the main requirements for development of improved vaccines against Mycobacterium tuberculosis (M. tb). Over the last two decades, significant progress has been made in TB vaccine development, and some TB vaccine candidates have currently completed a phase III clinical trial. The potential public health benefits of these vaccines are possible, but it will need much more effort, including new global governance investment on this research. This investment would certainly be less than the annual global financial toll of TB treatment.

  11. MTBreg: The Database of Conditionally Regulated Proteins in Mycobacterium Tuberculosis

    DOE Data Explorer

    Kaufman, Markus; Pal, Debnath; Eisenberg, David

    Proteins up- and down- regulated in Mycobacterium tuberculosis grown under conditions mimicking infection are included in this database. It also includes information on proteins that are regulated by selected transcription factors or other regulatory proteins. The literature data provided here is complimentary to the databases provided by Michael Strong that include recent TB computational functional linkages and the Prolinks Database by Peter Bowers. The experimental condition, the experimental dataset and a literature reference will be displayed, including links to the computationally linked proteins in the Prolinks Database and the entry in the Mycobacterium tuberculosis Structural Genomics Database.[Copied from information at http://www.doe-mbi.ucla.edu/Services/MTBreg/

  12. A Case of False-Positive Mycobacterium tuberculosis Caused by Mycobacterium celatum

    PubMed Central

    Abdel-Rahman, Zaid; Sengupta, Ruchira; Johnson, Laura

    2016-01-01

    Mycobacterium celatum is a nontuberculous mycobacterium shown to cause symptoms similar to pulmonary M. tuberculosis. Certain strains have been shown to cross-react with the probes used to detect M. tuberculosis, making this a diagnostic challenge. We present a 56-year-old gentleman who developed signs and symptoms of lung infection with computed tomography scan of the chest showing right lung apex cavitation. Serial sputum samples were positive for acid-fast bacilli and nucleic acid amplification testing identified M. tuberculosis ribosomal RNA, resulting in treatment initiation. Further testing with high performance liquid chromatography showed a pattern consistent with M. celatum. This case illustrates the potential for M. celatum to mimic M. tuberculosis in both its clinical history and laboratory testing due to the identical oligonucleotide sequence contained in both. An increasing number of case reports suggest that early reliable differentiation could reduce unnecessary treatment and public health intervention associated with misdiagnosed tuberculosis. PMID:27895946

  13. Radiometric selective inhibition tests for differentiation of Mycobacterium tuberculosis, Mycobacterium bovis, and other mycobacteria.

    PubMed Central

    Gross, W M; Hawkins, J E

    1985-01-01

    In the context of a busy reference laboratory, radiometric selective inhibition tests were evaluated for rapid differentiation of Mycobacterium tuberculosis and Mycobacterium bovis and of the M. tuberculosis complex from other mycobacteria. p-Nitro-alpha-acetylamino-beta-hydroxypropiophenone at 5 micrograms and hydroxylamine hydrochloride at 62.5 and 125 micrograms per ml of 7H12 medium were used to separate the M. tuberculosis complex from other mycobacteria (MOTT bacilli). Since it is important epidemiologically to distinguish M. tuberculosis from M. bovis, susceptibility to 1 microgram of thiophene-2-carboxylic acid per ml was also determined radiometrically. By using these three agents as selective inhibitors, M. tuberculosis, M. bovis, and MOTT bacilli were differentiated with a high degree of specificity by a BACTEC radiometric procedure. Results of tests performed on clinical isolates submitted on solid medium to our reference laboratory were available within 5 days. PMID:3921561

  14. Sub-speciation of Mycobacterium tuberculosis complex from tuberculosis patients in Japan.

    PubMed

    Ueyama, Masako; Chikamatsu, Kinuyo; Aono, Akio; Murase, Yoshiro; Kuse, Naoyuki; Morimoto, Kozo; Okumura, Masao; Yoshiyama, Takashi; Ogata, Hideo; Yoshimori, Kozo; Kudoh, Shoji; Azuma, Arata; Gemma, Akihiko; Mitarai, Satoshi

    2014-01-01

    Mycobacterium tuberculosis is the major causative agent of tuberculosis in humans. It is well known that Mycobacterium bovis and other species in the M. tuberculosis complex (MTC) can cause respiratory diseases as zoonosis. We analyzed the MTC isolates collected from tuberculosis patients from Japan in 2002 using a multiplex PCR system that detected cfp32, RD9 and RD12. A total of 970 MTC isolates that were representative of the tuberculosis cases throughout Japan, were examined using this method. As a result, 966 (99.6%) M. tuberculosis, two Mycobacterium africanum and two Mycobacterium canettii were identified using a multiplex PCR system, while no M. bovis was detected. Two isolates that lacked RD9 were initially considered to be M. canettii, but further analysis of the hsp65 sequence revealed them to be M. tuberculosis. Also two M. africanum were identified as M. tuberculosis using the -215 narG nucleotide polymorphism. Though PCR-linked methods have been used for a rapid differentiation of MTC and NTM, from our cases we suggest careful interpretation of RD based identification.

  15. A subunit vaccine based on rH-NS induces protection against Mycobacterium tuberculosis infection by inducing the Th1 immune response and activating macrophages.

    PubMed

    Liu, Yuan; Chen, Suting; Pan, Bowen; Guan, Zhu; Yang, Zhenjun; Duan, Linfei; Cai, Hong

    2016-10-01

    Mycobacterium tuberculosis (Mtb) is a Gram-positive pathogen which causes tuberculosis in both animals and humans. All tested rH-NS formulations induced a specific Th1 response, as indicated by increased production of interferon γ (IFN-γ) and interleukin 2 (IL-2) by lymphocytes in the spleen of mice which were immunized with rH-NS alone or with rH-NS and the adjuvant cyclic GMP-AMP (cGAMP). Serum from mice immunized with rH-NS with or without adjuvant also had higher levels of IL-12p40 and TNF-α, compared with those from control mice immunized with phosphate-buffered saline. Both vaccines increased protective efficacy in mice which were challenged with Mtb H37Rv, as measured by reduced relative CFU counts in the lungs. We found that rH-NS induced the production of TNF-α, IL-6, and IL-12p40, which relied on the activation of mitogen-activated protein kinases by stimulating the rapid phosphorylation of ERK1/2, p38, and JNK, and on the activation of transcription factor NF-κB in macrophages. Additionally, we also found that rH-NS could interact with TLR2 but not TLR4 in pull-down assays. The rH-NS-induced cytokine production from TLR2-silenced RAW264.7 cells was lower than that from BALB/c macrophages. Prolonged exposure (>24 h) of RAW264.7 cells to rH-NS resulted in a significant enhancement in IFN-γ-induced MHC II expression, which was not found in shTLR2-treated RAW264.7 cells. These results suggest that rH-NS is a TLR2 agonist which induces the production of cytokines by macrophages and up-regulates macrophage function.

  16. Early Secreted Antigenic Target of 6-kDa of Mycobacterium tuberculosis Stimulates IL-6 Production by Macrophages through Activation of STAT3

    PubMed Central

    Jung, Bock-Gie; Wang, Xisheng; Yi, Na; Ma, Justin; Turner, Joanne; Samten, Buka

    2017-01-01

    As early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis (Mtb) is an essential virulence factor and macrophages are critical for tuberculosis infection and immunity, we studied ESAT-6 stimulated IL-6 production by macrophages. ESAT-6 stimulated significantly higher IL-6 secretion by murine bone marrow derived macrophages (BMDM) compared to culture filtrate protein 10 kDa (CFP10) and antigen 85A. Polymyxin B, an LPS blocker, did not affect ESAT-6 stimulated macrophage IL-6 production. ESAT-6 but not Pam3CSK4 induced IL-6 by TLR2 knockout BMDM. ESAT-6 induced phosphorylation and DNA binding of STAT3 and this was blocked by STAT3 inhibitors but not by rapamycin. STAT3 inhibitors suppressed ESAT-6-induced IL-6 transcription and secretion without affecting cell viability. This was confirmed by silencing STAT3 in macrophages. Blocking neither IL-6Rα/IL-6 nor IL-10 affected ESAT-6-induced STAT3 activation and IL-6 production. Infection of BMDM and human macrophages with Mtb with esat-6 deletion induced diminished STAT3 activation and reduced IL-6 production compared to wild type and esat-6 complemented Mtb strains. Administration of ESAT-6 but not CFP10 induced STAT3 phosphorylation and IL-6 expression in the mouse lungs, consistent with expression of ESAT-6, IL-6 and phosphorylated-STAT3 in Mtb-infected mouse lungs. We conclude that ESAT-6 stimulates macrophage IL-6 production through STAT3 activation. PMID:28106119

  17. Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

    PubMed

    Cho, Ha Yeon; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Eungbin; Kang, Beom Sik

    2013-05-03

    The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.

  18. The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis

    SciTech Connect

    Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

    2013-04-22

    Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constants for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5–α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5–α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.

  19. Molecular dynamics simulation of the last step of a catalytic cycle: product release from the active site of the enzyme chorismate mutase from Mycobacterium tuberculosis.

    PubMed

    Choutko, Alexandra; van Gunsteren, Wilfred F

    2012-11-01

    The protein chorismate mutase MtCM from Mycobacterium tuberculosis catalyzes one of the few pericyclic reactions known in biology: the transformation of chorismate to prephenate. Chorismate mutases have been widely studied experimentally and computationally to elucidate the transition state of the enzyme catalyzed reaction and the origin of the high catalytic rate. However, studies about substrate entry and product exit to and from the highly occluded active site of the enzyme have to our knowledge not been performed on this enzyme. Crystallographic data suggest a possible substrate entry gate, that involves a slight opening of the enzyme for the substrate to access the active site. Using multiple molecular dynamics simulations, we investigate the natural dynamic process of the product exiting from the binding pocket of MtCM. We identify a dominant exit pathway, which is in agreement with the gate proposed from the available crystallographic data. Helices H2 and H4 move apart from each other which enables the product to exit from the active site. Interestingly, in almost all exit trajectories, two residues arginine 72 and arginine 134, which participate in the burying of the active site, are accompanying the product on its exit journey from the catalytic site.

  20. Activation of apoptosis, but not necrosis, during Mycobacterium tuberculosis infection correlated with decreased bacterial growth: role of TNF-alpha, IL-10, caspases and phospholipase A2.

    PubMed

    Arcila, Mary Luz; Sánchez, María Dulfary; Ortiz, Blair; Barrera, Luis Fernando; García, Luis F; Rojas, Mauricio

    2007-10-01

    Monocyte/macrophage cell death is an important event during mycobacterial infection. To get insights about the influence of mononuclear phagocyte maturation in this event we compared the response to Mycobacterium tuberculosis (Mtb) infection of fresh isolated monocytes and monocyte-derived macrophages (MDM) from healthy tuberculin positive individuals. Both monocytes and MDM underwent apoptosis, however, there was a higher numbers of apoptotic macrophages with active Caspases 8 and 9. We also compared Mtb-induced cell death in U937 pro-monocytes and PMA-differentiated cells (U937D). In response to Mtb infection, U937D cells underwent apoptosis and promonocytes both apoptosis and necrosis. There were high number of U937D cells producing TNF-alpha and high number of IL-10+ promonocytes. These evidences suggest that U937 could be a valid model to study the mechanisms that rule Mtb-induced cell death. Experiments with the cell line and fresh isolated mononuclear cells with pharmacological inhibitors showed that induction of necrosis involved calcium and cAMP signals resulting in IL-10 production. Necrosis also correlated with Caspase 3, PLA2 activity and bacterial growth. In U937D cells and monocytes from healthy donors there was activation of calcium, TNF-alpha and Caspase 8 activation and decreased bacterial load. Understanding the mechanisms that control the dichotomy events between apoptosis and necrosis/oncosis associated with cell maturity might open new strategies to better control the course of mycobacterial infections.

  1. Dramatic reduction of culture time of Mycobacterium tuberculosis

    NASA Astrophysics Data System (ADS)

    Ghodbane, Ramzi; Raoult, Didier; Drancourt, Michel

    2014-02-01

    Mycobacterium tuberculosis culture, a critical technique for routine diagnosis of tuberculosis, takes more than two weeks. Here, step-by-step improvements in the protocol including a new medium, microaerophlic atmosphere or ascorbic-acid supplement and autofluorescence detection dramatically shortened this delay. In the best case, primary culture and rifampicin susceptibility testing were achieved in 72 hours when specimens were inoculated directly on the medium supplemented by antibiotic at the beginning of the culture.

  2. Efflux inhibition with verapamil potentiates bedaquiline in Mycobacterium tuberculosis.

    PubMed

    Gupta, Shashank; Cohen, Keira A; Winglee, Kathryn; Maiga, Mamoudou; Diarra, Bassirou; Bishai, William R

    2014-01-01

    Drug efflux is an important resistance mechanism in Mycobacterium tuberculosis. We found that verapamil, an efflux inhibitor, profoundly decreases the MIC of bedaquiline and clofazimine to M. tuberculosis by 8- to 16-fold. This exquisite susceptibility was noted among drug-susceptible and drug-resistant clinical isolates. Thus, efflux inhibition is an important sensitizer of bedaquiline and clofazimine, and efflux may emerge as a resistance mechanism to these drugs.

  3. Rifampin induces hydroxyl radical formation in Mycobacterium tuberculosis.

    PubMed

    Piccaro, Giovanni; Pietraforte, Donatella; Giannoni, Federico; Mustazzolu, Alessandro; Fattorini, Lanfranco

    2014-12-01

    The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·(-) and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·(-), with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·(-)-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.

  4. Mycobacterium tuberculosis epidemiology in South Dakota.

    PubMed

    Iverson, D A; Hurley, B; Pueringer, R

    1994-03-01

    Tuberculosis incidence in the United States has recently increased from its rate of decline resulting in an excess of cases nationwide. The increase has been attributed largely to the HIV epidemic. Although tuberculosis incidence in South Dakota has increased similar to the national trend, South Dakota has not reported a single HIV-associated case of tuberculosis. Tuberculosis incidence in South Dakota has decreased in younger individuals. As a result, the percentage of tuberculosis cases in the elderly has increased. Though the reported cases of pulmonary tuberculosis have decreased, the reported cases of extrapulmonary tuberculosis have not changed. Furthermore, the percentage of extrapulmonary tuberculosis occurring in the elderly has increased. Tuberculosis incidence in South Dakota is, in part, increasing because of the persistence of extrapulmonary tuberculosis in the elderly.

  5. Purified recombinant hypothetical protein coded by open reading frame Rv1885c of Mycobacterium tuberculosis exhibits a monofunctional AroQ class of periplasmic chorismate mutase activity.

    PubMed

    Prakash, Prachee; Aruna, Bandi; Sardesai, Abhijit A; Hasnain, Seyed E

    2005-05-20

    Naturally occurring variants of the enzyme chorismate mutase are known to exist that exhibit diversity in enzyme structure, regulatory properties, and association with other proteins. Chorismate mutase was not annotated in the initial genome sequence of Mycobacterium tuberculosis (Mtb) because of low sequence similarity between known chorismate mutases. Recombinant protein coded by open reading frame Rv1885c of Mtb exhibited chorismate mutase activity in vitro. Biochemical and biophysical characterization of the recombinant protein suggests its resemblance to the AroQ class of chorismate mutases, prototype examples of which include the Escherichia coli and yeast chorismate mutases. We also demonstrate that unlike the corresponding proteins of E. coli, Mtb chorismate mutase does not have any associated prephenate dehydratase or dehydrogenase activity, indicating its monofunctional nature. The Rv1885c-encoded chorismate mutase showed allosteric regulation by pathway-specific as well as cross-pathway-specific ligands, as evident from proteolytic cleavage protection and enzyme assays. The predicted N-terminal signal sequence of Mtb chorismate mutase was capable of functioning as one in E. coli, suggesting that Mtb chorismate mutase belongs to the AroQ class of chorismate mutases. It was evident that Rv1885c may not be the only enzyme with chorismate mutase enzyme function within Mtb, based on our observation of the presence of chorismate mutase activity displayed by another hypothetical protein coded by open reading frame Rv0948c, a novel instance of the existence of two monofunctional chorismate mutases ever reported in any pathogenic bacterium.

  6. Turning the respiratory flexibility of Mycobacterium tuberculosis against itself

    PubMed Central

    Lamprecht, Dirk A.; Finin, Peter M.; Rahman, Md. Aejazur; Cumming, Bridgette M.; Russell, Shannon L.; Jonnala, Surendranadha R.; Adamson, John H.; Steyn, Adrie J. C.

    2016-01-01

    The Mycobacterium tuberculosis (Mtb) electron transport chain (ETC) has received significant attention as a drug target, however its vulnerability may be affected by its flexibility in response to disruption. Here we determine the effect of the ETC inhibitors bedaquiline, Q203 and clofazimine on the Mtb ETC, and the value of the ETC as a drug target, by measuring Mtb's respiration using extracellular flux technology. We find that Mtb's ETC rapidly reroutes around inhibition by these drugs and increases total respiration to maintain ATP levels. Rerouting is possible because Mtb rapidly switches between terminal oxidases, and, unlike eukaryotes, is not susceptible to back pressure. Increased ETC activity potentiates clofazimine's production of reactive oxygen species, causing rapid killing in vitro and in a macrophage model. Our results indicate that combination therapy targeting the ETC can be exploited to enhance killing of Mtb. PMID:27506290

  7. Genomic signal analysis of Mycobacterium tuberculosis

    NASA Astrophysics Data System (ADS)

    Cristea, Paul Dan; Banica, Dorina; Tuduce, Rodica

    2007-02-01

    As previously shown the conversion of nucleotide sequences into digital signals offers the possibility to apply signal processing methods for the analysis of genomic data. Genomic Signal Analysis (GSA) has been used to analyze large scale features of DNA sequences, at the scale of whole chromosomes, including both coding and non-coding regions. The striking regularities of genomic signals reveal restrictions in the way nucleotides and pairs of nucleotides are distributed along nucleotide sequences. Structurally, a chromosome appears to be less of a "plain text", corresponding to certain semantic and grammar rules, but more of a "poem", satisfying additional symmetry restrictions that evoke the "rhythm" and "rhyme". Recurrent patterns in nucleotide sequences are reflected in simple mathematical regularities observed in genomic signals. GSA has also been used to track pathogen variability, especially concerning their resistance to drugs. Previous work has been dedicated to the study of HIV-1, Clade F and Avian Flu. The present paper applies GSA methodology to study Mycobacterium tuberculosis (MT) rpoB gene variability, relevant to its resistance to antibiotics. Isolates from 50 Romanian patients have been studied both by rapid LightCycler PCR and by sequencing of a segment of 190-250 nucleotides covering the region of interest. The variability is caused by SNPs occurring at specific sites along the gene strand, as well as by inclusions. Because of the mentioned symmetry restrictions, the GS variations tend to compensate. An important result is that MT can act as a vector for HIV virus, which is able to retrotranscribe its specific genes both into human and MT genomes.

  8. Keratinocyte growth factor administration attenuates murine pulmonary mycobacterium tuberculosis infection through granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent macrophage activation and phagolysosome fusion.

    PubMed

    Pasula, Rajamouli; Azad, Abul K; Gardner, Jason C; Schlesinger, Larry S; McCormack, Francis X

    2015-03-13

    Augmentation of innate immune defenses is an appealing adjunctive strategy for treatment of pulmonary Mycobacterium tuberculosis infections, especially those caused by drug-resistant strains. The effect of intranasal administration of keratinocyte growth factor (KGF), an epithelial mitogen and differentiation factor, on M. tuberculosis infection in mice was tested in prophylaxis, treatment, and rescue scenarios. Infection of C57BL6 mice with M. tuberculosis resulted in inoculum size-dependent weight loss and mortality. A single dose of KGF given 1 day prior to infection with 10(5) M. tuberculosis bacilli prevented weight loss and enhanced pulmonary mycobacterial clearance (compared with saline-pretreated mice) for up to 28 days. Similar effects were seen when KGF was delivered intranasally every third day for 15 days, but weight loss and bacillary growth resumed when KGF was withdrawn. For mice with a well established M. tuberculosis infection, KGF given every 3 days beginning on day 15 postinoculation was associated with reversal of weight loss and an increase in M. tuberculosis clearance. In in vitro co-culture experiments, M. tuberculosis-infected macrophages exposed to conditioned medium from KGF-treated alveolar type II cell (MLE-15) monolayers exhibited enhanced GM-CSF-dependent killing through mechanisms that included promotion of phagolysosome fusion and induction of nitric oxide. Alveolar macrophages from KGF-treated mice also exhibited enhanced GM-CSF-dependent phagolysosomal fusion. These results provide evidence that administration of KGF promotes M. tuberculosis clearance through GM-CSF-dependent mechanisms and enhances host defense against M. tuberculosis infection.

  9. The β2 clamp in the Mycobacterium tuberculosis DNA polymerase III αβ2ε replicase promotes polymerization and reduces exonuclease activity

    PubMed Central

    Gu, Shoujin; Li, Wenjuan; Zhang, Hongtai; Fleming, Joy; Yang, Weiqiang; Wang, Shihua; Wei, Wenjing; Zhou, Jie; Zhu, Guofeng; Deng, Jiaoyu; Hou, Jian; Zhou, Ying; Lin, Shiqiang; Zhang, Xian-En; Bi, Lijun

    2016-01-01

    DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an αβ2ε polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its α subunit, Mtb DNA pol III has two potential proofreading subunits; the α and ε subunits. During DNA replication, the presence of the β2 clamp strongly promotes the polymerization of the αβ2ε replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb. PMID:26822057

  10. Co-evolution of Mycobacterium tuberculosis and Homo sapiens

    PubMed Central

    Brites, Daniela; Gagneux, Sebastien

    2015-01-01

    The causative agent of human tuberculosis (TB), Mycobacterium tuberculosis, is an obligate pathogen that evolved to exclusively persist in human populations. For M. tuberculosis to transmit from person to person, it has to cause pulmonary disease. Therefore, M. tuberculosis virulence has likely been a significant determinant of the association between M. tuberculosis and humans. Indeed, the evolutionary success of some M. tuberculosis genotypes seems at least partially attributable to their increased virulence. The latter possibly evolved as a consequence of human demographic expansions. If co-evolution occurred, humans would have counteracted to minimize the deleterious effects of M. tuberculosis virulence. The fact that human resistance to infection has a strong genetic basis is a likely consequence of such a counter-response. The genetic architecture underlying human resistance to M. tuberculosis remains largely elusive. However, interactions between human genetic polymorphisms and M. tuberculosis genotypes have been reported. Such interactions are consistent with local adaptation and allow for a better understanding of protective immunity in TB. Future ‘genome-to-genome’ studies, in which locally associated human and M. tuberculosis genotypes are interrogated in conjunction, will help identify new protective antigens for the development of better TB vaccines. PMID:25703549

  11. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  12. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  13. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  14. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  15. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  16. A strip array for spoligotyping of Mycobacterium tuberculosis complex isolates.

    PubMed

    Tu, Yiling; Zeng, Xiaohong; Li, Hui; Zheng, Rongrong; Xu, Ye; Li, Qingge

    2016-03-01

    A novel strip array was developed for a nine-spacer spoligotyping scheme of Mycobacterium tuberculosis complex (MTBC). The new method was evaluated using 211 MTBC isolates and the results were fully concordant with the traditional spoligotyping approach. The strip array proved to be rapid and convenient for spoligotyping of MTBC.

  17. Tuberculosis in Alpacas (Lama pacos) Caused by Mycobacterium bovis▿

    PubMed Central

    García-Bocanegra, I.; Barranco, I.; Rodríguez-Gómez, I. M.; Pérez, B.; Gómez-Laguna, J.; Rodríguez, S.; Ruiz-Villamayor, E.; Perea, A.

    2010-01-01

    We report three cases of tuberculosis in alpacas from Spain caused by Mycobacterium bovis. The animals revealed two different lesional patterns. Mycobacterial culture and PCR assay yielded positive results for M. bovis. Molecular typing of the isolates identified spoligotype SB0295 and identical variable-number tandem repeat (VNTR) allele sizes. PMID:20237097

  18. [A case of pulmonary multiresistant Mycobacterium bovis tuberculosis in Madagascar].

    PubMed

    Ramarokoto, H; Andrianasolo, D; Rasolonavalona, T; Ramaroson, F; Razafitsiarovana, I; Vincent, V; Ratsimba, L; Rasolofo Razanamparany, V

    2003-01-01

    We report a chronic case of pulmonary tuberculosis in a Malagasy citizen from Antsohihy (West of Madagascar), who was infected with a multi-drug resistant Mycobacterium bovis strain. This is the first case reported of the isolation of such a strain in Madagascar.

  19. Lack of protection afforded by ribonucleic acid preparations from Mycobacterium tuberculosis against Mycobacterium leprae infections in mice.

    PubMed Central

    Shepard, C C; Youmans, A Y; Youmans, G P

    1977-01-01

    Mycobacterial ribonucleic acid preparations from H37Ra, an attenuated strain of Mycobacterium tuberculosis, provide their usual marked protection against M. tuberculosis challenge; however, they provided no protection against Mycobacterium leprae challenge. Suspensions of intact H37Ra were not effective against M. leprae. Suspensions of BCG gave their usual distinct protection against M. leprae challenge. PMID:404242

  20. The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae.

    PubMed

    Phelan, Jody; Maitra, Arundhati; McNerney, Ruth; Nair, Mridul; Gupta, Antima; Coll, Francesc; Pain, Arnab; Bhakta, Sanjib; Clark, Taane G

    2015-09-01

    Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

  1. Mycobacterium tuberculosis Bacteremia Among Acutely Febrile Children in Western Kenya.

    PubMed

    Pavlinac, Patricia B; Naulikha, Jaqueline M; John-Stewart, Grace C; Onchiri, Frankline M; Okumu, Albert O; Sitati, Ruth R; Cranmer, Lisa M; Lokken, Erica M; Singa, Benson O; Walson, Judd L

    2015-11-01

    In children, Mycobacterium tuberculosis (M. tuberculosis) frequently disseminates systemically, presenting with nonspecific signs including fever. We determined prevalence of M. tuberculosis bacteremia among febrile children presenting to hospitals in Nyanza, Kenya (a region with high human immunodeficiency virus (HIV) and M. tuberculosis prevalence). Between March 2013 and February 2014, we enrolled children aged 6 months to 5 years presenting with fever (axillary temperature ≥ 37.5°C) and no recent antibiotic use. Blood samples were collected for bacterial and mycobacterial culture using standard methods. Among 148 children enrolled, median age was 3.1 years (interquartile range: 1.8-4.1 years); 10.3% of children were living with a household member diagnosed with M. tuberculosis in the last year. Seventeen percent of children were stunted (height-for-age z-score < -2), 18.6% wasted (weight-for-height z-score < -2), 2.7% were HIV-infected, and 14.2% were HIV-exposed uninfected. Seventeen children (11.5%) had one or more signs of tuberculosis (TB). All children had a Bacille Calmette-Guerin vaccination scar. Among 134 viable blood cultures, none (95% confidence interval: 0-2.7%) had Mycobacterium isolated. Despite exposure to household TB contacts, HIV exposure, and malnutrition, M. tuberculosis bacteremia was not detected in this pediatric febrile cohort, a finding consistent with other pediatric studies.

  2. Mycobacterium tuberculosis Bacteremia among Acutely Febrile Children in Western Kenya

    PubMed Central

    Pavlinac, Patricia B.; Naulikha, Jaqueline M.; John-Stewart, Grace C.; Onchiri, Frankline M.; Okumu, Albert O.; Sitati, Ruth R.; Cranmer, Lisa M.; Lokken, Erica M.; Singa, Benson O.; Walson, Judd L.

    2015-01-01

    In children, Mycobacterium tuberculosis (M. tuberculosis) frequently disseminates systemically, presenting with nonspecific signs including fever. We determined prevalence of M. tuberculosis bacteremia among febrile children presenting to hospitals in Nyanza, Kenya (a region with high human immunodeficiency virus (HIV) and M. tuberculosis prevalence). Between March 2013 and February 2014, we enrolled children aged 6 months to 5 years presenting with fever (axillary temperature ≥ 37.5°C) and no recent antibiotic use. Blood samples were collected for bacterial and mycobacterial culture using standard methods. Among 148 children enrolled, median age was 3.1 years (interquartile range: 1.8–4.1 years); 10.3% of children were living with a household member diagnosed with M. tuberculosis in the last year. Seventeen percent of children were stunted (height-for-age z-score < −2), 18.6% wasted (weight-for-height z-score < −2), 2.7% were HIV-infected, and 14.2% were HIV-exposed uninfected. Seventeen children (11.5%) had one or more signs of tuberculosis (TB). All children had a Bacille Calmette-Guerin vaccination scar. Among 134 viable blood cultures, none (95% confidence interval: 0–2.7%) had Mycobacterium isolated. Despite exposure to household TB contacts, HIV exposure, and malnutrition, M. tuberculosis bacteremia was not detected in this pediatric febrile cohort, a finding consistent with other pediatric studies. PMID:26324730

  3. Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids

    SciTech Connect

    Touchette, Megan H.; Bommineni, Gopal R.; Delle Bovi, Richard J.; Gadbery, John; Nicora, Carrie D.; Shukla, Anil K.; Kyle, Jennifer E.; Metz, Thomas O.; Martin, Dwight W.; Sampson, Nicole S.; Miller, W. T.; Tonge, Peter J.; Seeliger, Jessica C.

    2015-09-08

    Although classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl beta-diol, phthiocerol, with branched-chain fatty acids know as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. We here show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl beta-diol substrate analogues. Applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinase PknB modifies PapA5 on three Thr residues, including two (T196, T198) located on an unresolved loop. These results clarify the DIM biosynthetic pathway and suggest possible mechanisms by which DIM biosynthesis may be regulated by the post-translational modification of PapA5.

  4. Comparative genomics of archived pyrazinamide resistant Mycobacterium tuberculosis complex isolates from Uganda

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine tuberculosis is a ‘neglected zoonosis’ and its contribution to the proportion of Mycobacterium tuberculosis complex infections in humans is unknown. A retrospective study on archived Mycobacterium tuberculosis complex (MTC) isolates from a reference laboratory in Uganda was undertaken to iden...

  5. First case of Mycobacterium tuberculosis transmission by heart transplantation from donor to recipient.

    PubMed

    Weile, Jan; Eickmeyer, Holm; Dreier, Jens; Liebke, Michael; Fuchs, Uwe; Wittke, Johann-Wolfgang; Richter, Elvira; Gummert, Jan; Knabbe, Cornelius; Schulz, Uwe

    2013-12-01

    We report the first documented case of a Mycobacterium tuberculosis transmission by an orthotopic heart transplantation from the donor to the recipient. Mycobacterium tuberculosis positive blood culture showed systemic prevalence of the Mycobacteria, however, prophylactic therapy was able to prevent a clinical manifestation of tuberculosis in the recipient.

  6. Structural and functional studies of phosphoenolpyruvate carboxykinase from Mycobacterium tuberculosis.

    PubMed

    Machová, Iva; Snášel, Jan; Dostál, Jiří; Brynda, Jiří; Fanfrlík, Jindřich; Singh, Mahavir; Tarábek, Ján; Vaněk, Ondřej; Bednárová, Lucie; Pichová, Iva

    2015-01-01

    Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.

  7. Preparation and biological evaluation of ethionamide-mesoporous silicon nanoparticles against Mycobacterium tuberculosis.

    PubMed

    Vale, Nuno; Correia, Alexandra; Silva, Sara; Figueiredo, Patrícia; Mäkilä, Ermei; Salonen, Jarno; Hirvonen, Jouni; Pedrosa, Jorge; Santos, Hélder A; Fraga, Alexandra

    2017-02-01

    Ethionamide (ETH) is an important second-line antituberculosis drug used for the treatment of patients infected with multidrug-resistant Mycobacterium tuberculosis. Recently, we reported that the loading of ETH into thermally carbonized-porous silicon (TCPSi) nanoparticles enhanced the solubility and permeability of ETH at different pH-values and also increased its metabolization process. Based on these results, we synthesized carboxylic acid functionalized thermally hydrocarbonized porous silicon nanoparticles (UnTHCPSi NPs) conjugated with ETH and its antimicrobial effect was evaluated against Mycobacterium tuberculosis strain H37Rv. The activity of the conjugate was increased when compared to free-ETH, which suggests that the nature of the synergy between the NPs and ETH is likely due to the weakening of the bacterial cell wall that improves conjugate-penetration. These ETH-conjugated NPs have great potential in reducing dosing frequency of ETH in the treatment of multidrug-resistant tuberculosis (MDR-TB).

  8. Mycobacterium tuberculosis resistance to antituberculosis drugs in Mozambique*, **

    PubMed Central

    Pires, Germano Manuel; Folgosa, Elena; Nquobile, Ndlovu; Gitta, Sheba; Cadir, Nureisha

    2014-01-01

    OBJECTIVE: To determine the drug resistance profile of Mycobacterium tuberculosis in Mozambique. METHODS: We analyzed secondary data from the National Tuberculosis Referral Laboratory, in the city of Maputo, Mozambique, and from the Beira Regional Tuberculosis Referral Laboratory, in the city of Beira, Mozambique. The data were based on culture-positive samples submitted to first-line drug susceptibility testing (DST) between January and December of 2011. We attempted to determine whether the frequency of DST positivity was associated with patient type or provenance. RESULTS: During the study period, 641 strains were isolated in culture and submitted to DST. We found that 374 (58.3%) were resistant to at least one antituberculosis drug and 280 (43.7%) were resistant to multiple antituberculosis drugs. Of the 280 multidrug-resistant tuberculosis cases, 184 (65.7%) were in previously treated patients, most of whom were from southern Mozambique. Two (0.71%) of the cases of multidrug-resistant tuberculosis were confirmed to be cases of extensively drug-resistant tuberculosis. Multidrug-resistant tuberculosis was most common in males, particularly those in the 21-40 year age bracket. CONCLUSIONS: M. tuberculosis resistance to antituberculosis drugs is high in Mozambique, especially in previously treated patients. The frequency of M. tuberculosis strains that were resistant to isoniazid, rifampin, and streptomycin in combination was found to be high, particularly in samples from previously treated patients. PMID:24831398

  9. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR.

    PubMed Central

    Folgueira, L; Delgado, R; Palenque, E; Aguado, J M; Noriega, A R

    1996-01-01

    A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population. PMID:8904404

  10. Management of latent Mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries.

    PubMed

    Getahun, Haileyesus; Matteelli, Alberto; Abubakar, Ibrahim; Aziz, Mohamed Abdel; Baddeley, Annabel; Barreira, Draurio; Den Boon, Saskia; Borroto Gutierrez, Susana Marta; Bruchfeld, Judith; Burhan, Erlina; Cavalcante, Solange; Cedillos, Rolando; Chaisson, Richard; Chee, Cynthia Bin-Eng; Chesire, Lucy; Corbett, Elizabeth; Dara, Masoud; Denholm, Justin; de Vries, Gerard; Falzon, Dennis; Ford, Nathan; Gale-Rowe, Margaret; Gilpin, Chris; Girardi, Enrico; Go, Un-Yeong; Govindasamy, Darshini; D Grant, Alison; Grzemska, Malgorzata; Harris, Ross; Horsburgh, C Robert; Ismayilov, Asker; Jaramillo, Ernesto; Kik, Sandra; Kranzer, Katharina; Lienhardt, Christian; LoBue, Philip; Lönnroth, Knut; Marks, Guy; Menzies, Dick; Migliori, Giovanni Battista; Mosca, Davide; Mukadi, Ya Diul; Mwinga, Alwyn; Nelson, Lisa; Nishikiori, Nobuyuki; Oordt-Speets, Anouk; Rangaka, Molebogeng Xheedha; Reis, Andreas; Rotz, Lisa; Sandgren, Andreas; Sañé Schepisi, Monica; Schünemann, Holger J; Sharma, Surender Kumar; Sotgiu, Giovanni; Stagg, Helen R; Sterling, Timothy R; Tayeb, Tamara; Uplekar, Mukund; van der Werf, Marieke J; Vandevelde, Wim; van Kessel, Femke; van't Hoog, Anna; Varma, Jay K; Vezhnina, Natalia; Voniatis, Constantia; Vonk Noordegraaf-Schouten, Marije; Weil, Diana; Weyer, Karin; Wilkinson, Robert John; Yoshiyama, Takashi; Zellweger, Jean Pierre; Raviglione, Mario

    2015-12-01

    Latent tuberculosis infection (LTBI) is characterised by the presence of immune responses to previously acquired Mycobacterium tuberculosis infection without clinical evidence of active tuberculosis (TB). Here we report evidence-based guidelines from the World Health Organization for a public health approach to the management of LTBI in high risk individuals in countries with high or middle upper income and TB incidence of <100 per 100 000 per year. The guidelines strongly recommend systematic testing and treatment of LTBI in people living with HIV, adult and child contacts of pulmonary TB cases, patients initiating anti-tumour necrosis factor treatment, patients receiving dialysis, patients preparing for organ or haematological transplantation, and patients with silicosis. In prisoners, healthcare workers, immigrants from high TB burden countries, homeless persons and illicit drug users, systematic testing and treatment of LTBI is conditionally recommended, according to TB epidemiology and resource availability. Either commercial interferon-gamma release assays or Mantoux tuberculin skin testing could be used to test for LTBI. Chest radiography should be performed before LTBI treatment to rule out active TB disease. Recommended treatment regimens for LTBI include: 6 or 9 month isoniazid; 12 week rifapentine plus isoniazid; 3-4 month isoniazid plus rifampicin; or 3-4 month rifampicin alone.

  11. Management of latent Mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries

    PubMed Central

    Matteelli, Alberto; Abubakar, Ibrahim; Aziz, Mohamed Abdel; Baddeley, Annabel; Barreira, Draurio; Den Boon, Saskia; Borroto Gutierrez, Susana Marta; Bruchfeld, Judith; Burhan, Erlina; Cavalcante, Solange; Cedillos, Rolando; Chaisson, Richard; Chee, Cynthia Bin-Eng; Chesire, Lucy; Corbett, Elizabeth; Dara, Masoud; Denholm, Justin; de Vries, Gerard; Falzon, Dennis; Ford, Nathan; Gale-Rowe, Margaret; Gilpin, Chris; Girardi, Enrico; Go, Un-Yeong; Govindasamy, Darshini; D. Grant, Alison; Grzemska, Malgorzata; Harris, Ross; Horsburgh Jr, C. Robert; Ismayilov, Asker; Jaramillo, Ernesto; Kik, Sandra; Kranzer, Katharina; Lienhardt, Christian; LoBue, Philip; Lönnroth, Knut; Marks, Guy; Menzies, Dick; Migliori, Giovanni Battista; Mosca, Davide; Mukadi, Ya Diul; Mwinga, Alwyn; Nelson, Lisa; Nishikiori, Nobuyuki; Oordt-Speets, Anouk; Rangaka, Molebogeng Xheedha; Reis, Andreas; Rotz, Lisa; Sandgren, Andreas; Sañé Schepisi, Monica; Schünemann, Holger J.; Sharma, Surender Kumar; Sotgiu, Giovanni; Stagg, Helen R.; Sterling, Timothy R.; Tayeb, Tamara; Uplekar, Mukund; van der Werf, Marieke J.; Vandevelde, Wim; van Kessel, Femke; van't Hoog, Anna; Varma, Jay K.; Vezhnina, Natalia; Voniatis, Constantia; Vonk Noordegraaf-Schouten, Marije; Weil, Diana; Weyer, Karin; Wilkinson, Robert John; Yoshiyama, Takashi; Zellweger, Jean Pierre; Raviglione, Mario

    2015-01-01

    Latent tuberculosis infection (LTBI) is characterised by the presence of immune responses to previously acquired Mycobacterium tuberculosis infection without clinical evidence of active tuberculosis (TB). Here we report evidence-based guidelines from the World Health Organization for a public health approach to the management of LTBI in high risk individuals in countries with high or middle upper income and TB incidence of <100 per 100 000 per year. The guidelines strongly recommend systematic testing and treatment of LTBI in people living with HIV, adult and child contacts of pulmonary TB cases, patients initiating anti-tumour necrosis factor treatment, patients receiving dialysis, patients preparing for organ or haematological transplantation, and patients with silicosis. In prisoners, healthcare workers, immigrants from high TB burden countries, homeless persons and illicit drug users, systematic testing and treatment of LTBI is conditionally recommended, according to TB epidemiology and resource availability. Either commercial interferon-gamma release assays or Mantoux tuberculin skin testing could be used to test for LTBI. Chest radiography should be performed before LTBI treatment to rule out active TB disease. Recommended treatment regimens for LTBI include: 6 or 9 month isoniazid; 12 week rifapentine plus isoniazid; 3–4 month isoniazid plus rifampicin; or 3–4 month rifampicin alone. PMID:26405286

  12. Mycobacterium tuberculosis Infection Interferes with HIV Vaccination in Mice

    PubMed Central

    Ignatowicz, Lech; Mazurek, Jolanta; Leepiyasakulchai, Chaniya; Sköld, Markus; Hinkula, Jorma; Källenius, Gunilla; Pawlowski, Andrzej

    2012-01-01

    Tuberculosis (TB) has emerged as the most prominent bacterial disease found in human immunodeficiency virus (HIV)-positive individuals worldwide. Due to high prevalence of asymptomatic Mycobacterium tuberculosis (Mtb) infections, the future HIV vaccine in areas highly endemic for TB will often be administrated to individuals with an ongoing Mtb infection. The impact of concurrent Mtb infection on the immunogenicity of a HIV vaccine candidate, MultiHIV DNA/protein, was investigated in mice. We found that, depending on the vaccination route, mice infected with Mtb before the administration of the HIV vaccine showed impairment in both the magnitude and the quality of antibody and T cell responses to the vaccine components p24Gag and gp160Env. Mice infected with Mtb prior to intranasal HIV vaccination exhibited reduced p24Gag-specific serum IgG and IgA, and suppressed gp160Env-specific serum IgG as compared to respective titers in uninfected HIV-vaccinated controls. Importantly, in Mtb-infected mice that were HIV-vaccinated by the intramuscular route the virus neutralizing activity in serum was significantly decreased, relative to uninfected counterparts. In addition mice concurrently infected with Mtb had fewer p24Gag-specific IFN-γ-expressing T cells and multifunctional T cells in their spleens. These results suggest that Mtb infection might interfere with the outcome of prospective HIV vaccination in humans. PMID:22848444

  13. Macrophage polarization: convergence point targeted by mycobacterium tuberculosis and HIV.

    PubMed

    Lugo-Villarino, Geanncarlo; Vérollet, Christel; Maridonneau-Parini, Isabelle; Neyrolles, Olivier

    2011-01-01

    In the arms race of host-microbe co-evolution, macrophages (Mɸs) have been endowed with strategies to neutralize pathogenic challenge while preserving host integrity. During steady-states conditions, Mɸs perform multiple house-keeping functions governed by their differentiation state, tissue distribution, and signals from the microenvironment. In response to pathogenic challenge and host mediators, however, Mɸs undergo different programs of activation rendering them either pro-inflammatory and microbicidal (M1), or immunosuppressants and tissue repairers (M2). An excessive or prolonged polarization of either program may be detrimental to the host due to potential tissue injury or contribution to pathogenesis. Conversely, intracellular microbes that cause chronic diseases such as tuberculosis and acquired immunodeficiency syndrome exemplify strategies for survival in the host. Indeed, both Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV-1) are successful intracellular microbes that thrive in Mɸs. Given these microbes not only co-circulate throughout the developing world but each has contributed to prevalence and mortality caused by the other, substantial insights into microbe physiology and host defenses then rest in the attempt to fully understand their influence on Mɸ polarization. This review addresses the role of Mɸ polarization in the immune response to, and pathogenesis of, Mtb and HIV.

  14. Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence.

    PubMed

    Zheng, Huiqing; Colvin, Christopher J; Johnson, Benjamin K; Kirchhoff, Paul D; Wilson, Michael; Jorgensen-Muga, Katriana; Larsen, Scott D; Abramovitch, Robert B

    2017-02-01

    The Mycobacterium tuberculosis (Mtb) DosRST two-component regulatory system promotes the survival of Mtb during non-replicating persistence (NRP). NRP bacteria help drive the long course of tuberculosis therapy; therefore, chemical inhibition of DosRST may inhibit the ability of Mtb to establish persistence and thus shorten treatment. Using a DosRST-dependent fluorescent Mtb reporter strain, a whole-cell phenotypic high-throughput screen of a ∼540,000 compound small-molecule library was conducted. The screen discovered novel inhibitors of the DosRST regulon, including three compounds that were subject to follow-up studies: artemisinin, HC102A and HC103A. Under hypoxia, all three compounds inhibit Mtb-persistence-associated physiological processes, including triacylglycerol synthesis, survival and antibiotic tolerance. Artemisinin functions by disabling the heme-based DosS and DosT sensor kinases by oxidizing ferrous heme and generating heme-artemisinin adducts. In contrast, HC103A inhibits DosS and DosT autophosphorylation activity without targeting the sensor kinase heme.

  15. Macrophage Migration Inhibition Studies with Cells from Mice Vaccinated with Cell Walls of Mycobacterium bovis BCG: Relationship Between Inhibitory Activity of Lung Cells and Resistance to Airborne Challenge with Mycobacterium tuberculosis H37Rv.

    PubMed

    Yamamoto, K; Anacker, R L; Ribi, E

    1970-06-01

    In an effort to evaluate the role of delayed hypersensitivity in acquired resistance of mice to airborne infection with Mycobacterium tuberculosis H37Rv, the ability of lung and peritoneal cells from mice vaccinated in various ways with mycobacterial fractions or with M. bovis BCG to inhibit, in the presence of purified protein derivative, in vitro migration of normal peritoneal cells was determined. The degree of inhibition induced by lung cells was correlated with immunity, but that induced by peritoneal cells could not be associated with enhanced resistance. Live BCG given intravenously to mice stimulated greater resistance to infection and inhibitory activity of lung cells than did live BCG given subcutaneously. Vaccines with a protective index greater than 1 also induced a significant increase in lung weight. Although a correlation between ability of lung cells to inhibit cell migration and acquired resistance of the host to airborne infection with H37Rv was demonstrated, the data do not exclude the possibility that the two phenomena are independent responses to the immunologically complex mycobacterial antigens.

  16. Whole genome analysis of Mycobacterium tuberculosis isolates from recurrent episodes of tuberculosis, Finland, 1995-2013.

    PubMed

    Korhonen, V; Smit, P W; Haanperä, M; Casali, N; Ruutu, P; Vasankari, T; Soini, H

    2016-06-01

    Recurrent tuberculosis (TB) is caused by an endogenous re-activation of the same strain of Mycobacterium tuberculosis (relapse) or exogenous infection with a new strain (re-infection). Recurrence of TB in Finland was analysed in a population-based, 19-year study, and genotyping was used to define relapse and re-infection. The M. tuberculosis isolates from patients with suspected relapse were further analysed by whole genome sequencing (WGS) to determine the number and type of mutations occurring in the bacterial genome between the first and second disease episodes. In addition, publicly available tools (PhyResSE and SpolPred) were used to predict drug resistance and spoligotype profile from the WGS data. Of the 8299 notified TB cases, 48 (0.6%) patients had episodes classified as recurrent. Forty-two patients had more than one culture-confirmed TB episode, and isolates from two episodes in 21 patients were available for genotyping. In 18 patients, the M. tuberculosis isolates obtained from the first and second TB episodes had identical spoligotypes. The WGS analysis of the 36 M. tuberculosis isolates from the 18 suspected relapse patients (average time between isolates 2.8 years) revealed 0 to 38 single nucleotide polymorphisms (median 1, mean 3.78) between the first and second isolate. There seemed to be no direct relation between the number of years between the two isolates, or treatment outcome, and the number of single nucleotide polymorphisms. The results suggest that the mutation rate may depend on multiple host-, strain- and treatment-related factors.

  17. Mean Platelet Volume in Mycobacterium tuberculosis Infection

    PubMed Central

    Lee, Min Young; Kim, Young Jin; Lee, Hee Joo; Park, Tae Sung

    2016-01-01

    Introduction. Mean platelet volume (MPV) has been thought as a useful index of platelet activation. It is supposed that MPV is also associated with several inflammatory and infectious diseases. Korea still has a high incidence of tuberculosis (TB). The aim of this study was to investigate MPV as an inflammatory marker in TB patients. Materials and Methods. MPV were determined in 221 patients with TB and 143 individuals for control group. MPV was estimated by an Advia 2120 (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Results. In the TB patient group, a positive correlation was found between CRP and MPV. Age and MPV had a positive correlation in TB patient group. Conclusions. We conclude that there is a significant relation between MPV and inflammatory conditions. MPV can be an inflammatory marker to determine the disease activity in TB patients. PMID:27419136

  18. Interaction of Pattern Recognition Receptors with Mycobacterium Tuberculosis.

    PubMed

    Mortaz, Esmaeil; Adcock, Ian M; Tabarsi, Payam; Masjedi, Mohammad Reza; Mansouri, Davood; Velayati, Ali Akbar; Casanova, Jean-Laurent; Barnes, Peter J

    2015-01-01

    Tuberculosis (TB) is considered a major worldwide health problem with 10 million new cases diagnosed each year. Our understanding of TB immunology has become greater and more refined since the identification of Mycobacterium tuberculosis (MTB) as an etiologic agent and the recognition of new signaling pathways modulating infection. Understanding the mechanisms through which the cells of the immune system recognize MTB can be an important step in designing novel therapeutic approaches, as well as improving the limited success of current vaccination strategies. A great challenge in chronic disease is to understand the complexities, mechanisms, and consequences of host interactions with pathogens. Innate immune responses along with the involvement of distinct inflammatory mediators and cells play an important role in the host defense against the MTB. Several classes of pattern recognition receptors (PRRs) are involved in the recognition of MTB including Toll-Like Receptors (TLRs), C-type lectin receptors (CLRs) and Nod-like receptors (NLRs) linked to inflammasome activation. Among the TLR family, TLR1, TLR2, TLR4, and TLR9 and their down-stream signaling proteins play critical roles in the initiation of the immune response in the pathogenesis of TB. The inflammasome pathway is associated with the coordinated release of cytokines such as IL-1β and IL-18 which also play a role in the pathogenesis of TB. Understanding the cross-talk between these signaling pathways will impact on the design of novel therapeutic strategies and in the development of vaccines and immunotherapy regimes. Abnormalities in PRR signaling pathways regulated by TB will affect disease pathogenesis and need to be elucidated. In this review we provide an update on PRR signaling during M. tuberculosis infection and indicate how greater knowledge of these pathways may lead to new therapeutic opportunities.

  19. Mode of action of pyrazinamide: disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid.

    PubMed

    Zhang, Ying; Wade, Mary Margaret; Scorpio, Angelo; Zhang, Hao; Sun, Zhonghe

    2003-11-01

    Pyrazinamide is an important sterilizing drug that shortens tuberculosis (TB) therapy. However, the mechanism of action of pyrazinamide is poorly understood because of its unusual properties. Here we show that pyrazinoic acid, the active moiety of pyrazinamide, disrupted membrane energetics and inhibited membrane transport function in Mycobacterium tuberculosis. The preferential activity of pyrazinamide against old non-replicating bacilli correlated with their low membrane potential and the disruption of membrane potential by pyrazinoic acid and acid pH. Inhibitors of membrane energetics increased the antituberculous activity of pyrazinamide. These findings shed new light on the mode of action of pyrazinamide and may help in the design of new drugs that shorten therapy.

  20. Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme; Insights of N-Terminal [beta]-Sandwich in Sustrate Specifity and Enzymatic Activity

    SciTech Connect

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H. Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam

    2010-07-13

    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an {alpha}-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1 {yields} 4 bond and making a new 1 {yields} 6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-{angstrom} resolution. MtbGlgBWT contains four domains: N1 {beta}-sandwich, N2 {beta}-sandwich, a central ({beta}/{alpha}){sub 8} domain that houses the catalytic site, and a C-terminal {beta}-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb{Delta}108GlgB protein. The N1 {beta}-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 {beta}-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb{Delta}108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1 {yields} 4 bond breakage) and isomerization (1 {yields} 6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb{Delta}108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC{Delta}112GlgB).

  1. Asymmetric cell division in Mycobacterium tuberculosis and its unique features.

    PubMed

    Vijay, Srinivasan; Nagaraja, Mukkayyan; Sebastian, Jees; Ajitkumar, Parthasarathi

    2014-03-01

    Recently, several reports showed that about 80 % of mid-log phase Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG cells divide symmetrically with 5-10 % deviation in the septum position from the median. However, the mode of cell division of the pathogenic mycobacterial species, Mycobacterium tuberculosis, remained unclear. Therefore, in the present study, using electron microscopy, fluorescence microscopy of septum- and nucleoid-stained live and fixed cells, and live cell time-lapse imaging, we show the occurrence of asymmetric cell division with unusually deviated septum/constriction in 20 % of the 15 % septating M. tuberculosis cells in the mid-log phase population. The remaining 80 % of the 15 % septating cells divided symmetrically but with 2-5 % deviation in the septum/constriction position, as reported for M. smegmatis, M. marinum, and M. bovis BCG cells. Both the long and the short portions of the asymmetrically dividing M. tuberculosis cells with unusually deviated septum contained nucleoids, thereby generating viable short and long cells from each asymmetric division. M. tuberculosis short cells were acid fast positive and, like the long cells, further readily underwent growth and division to generate micro-colony, thereby showing that they were neither mini cells, spores nor dormant forms of mycobacteria. The freshly diagnosed pulmonary tuberculosis patients' sputum samples, which are known for the prevalence of oxidative stress conditions, also contained short cells at the same proportion as that in the mid-log phase population. The probable physiological significance of the generation of the short cells through unusually deviated asymmetric cell division is discussed.

  2. Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

    PubMed Central

    Silva, Marcio Roberto; Rocha, Adalgiza da Silva; da Costa, Ronaldo Rodrigues; de Alencar, Andrea Padilha; de Oliveira, Vania Maria; Fonseca, Antônio Augusto; Sales, Mariana Lázaro; Issa, Marina de Azevedo; Soares, Paulo Martins; Pereira, Omara Tereza Vianello; dos Santos, Eduardo Calazans; Mendes, Rejane Silva; Ferreira, Ângela Maria de Jesus; Mota, Pedro Moacyr Pinto Coelho; Suffys, Philip Noel; Guimarães, Mark Drew Crosland

    2013-01-01

    In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials. PMID:23778657

  3. Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil.

    PubMed

    Silva, Marcio Roberto; Rocha, Adalgiza da Silva; da Costa, Ronaldo Rodrigues; de Alencar, Andrea Padilha; de Oliveira, Vania Maria; Fonseca Júnior, Antônio Augusto; Sales, Mariana Lázaro; Issa, Marina de Azevedo; Filho, Paulo Martins Soares; Pereira, Omara Tereza Vianello; dos Santos, Eduardo Calazans; Mendes, Rejane Silva; Ferreira, Angela Maria de Jesus; Mota, Pedro Moacyr Pinto Coelho; Suffys, Philip Noel; Guimarães, Mark Drew Crosland

    2013-05-01

    In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

  4. Cyclic GMP-AMP Synthase Is an Innate Immune DNA Sensor for Mycobacterium tuberculosis.

    PubMed

    Collins, Angela C; Cai, Haocheng; Li, Tuo; Franco, Luis H; Li, Xiao-Dong; Nair, Vidhya R; Scharn, Caitlyn R; Stamm, Chelsea E; Levine, Beth; Chen, Zhijian J; Shiloh, Michael U

    2015-06-10

    Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection.

  5. Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis.

    PubMed

    Zimhony, O; Cox, J S; Welch, J T; Vilchèze, C; Jacobs, W R

    2000-09-01

    Tuberculosis treatment is shortened to six months by the indispensable addition of pyrazinamide (PZA) to the drug regimen that includes isoniazid and rifampin. PZA is a pro-drug of pyrazinoic acid (POA) (ref. 3), whose target of action has never been identified. Although PZA is active only against Mycobacterium tuberculosis, the PZA analog 5-chloro-pyrazinamide (5-Cl-PZA) displays a broader range of anti-mycobacterial activity. We have found that the eukaryotic-like fas1 gene (encoding fatty acid synthetase I, FASI) from M. avium, M. bovis BCG or M. tuberculosis confers resistance to 5-Cl-PZA when present on multi-copy vectors in M. smegmatis. 5-Cl-PZA and PZA markedly inhibited the activity of M. tuberculosis FASI, the biosynthesis of C16 to C24/C26 fatty acids from acetyl-CoA (ref. 6). Importantly, PZA inhibited FASI in M. tuberculosis in correlation with PZA susceptibility. These results indicate that FASI is a primary target of action for PZA in M. tuberculosis. Further characterization of FASI as a drug target for PZA may allow the development of new drugs to shorten the therapy against M. tuberculosis and may provide more options for treatment against M. bovis, M. avium and drug resistant M. tuberculosis.

  6. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA.

    PubMed Central

    Young, R A; Bloom, B R; Grosskinsky, C M; Ivanyi, J; Thomas, D; Davis, R W

    1985-01-01

    A recombinant DNA strategy has been used systematically to survey the Mycobacterium tuberculosis genome for sequences that encode specific antigens detected by monoclonal antibodies. M. tuberculosis genomic DNA fragments with randomly generated endpoints were used to construct a large lambda gt11 recombinant DNA expression library. Sufficient numbers of recombinants were produced to contain inserts whose endpoints occur at nearly every base pair in the pathogen genome. Protein antigens specified by linear segments of pathogen DNA and produced by the recombinant phage of Escherichia coli were screened with monoclonal antibody probes. This approach was coupled with an improved detection method for gene isolation using antibodies to clonally isolate DNA sequences that specify polypeptide components of M. tuberculosis. The methodology described here, which is applicable to other pathogens, offers possibilities for the development of more sensitive and specific immunodiagnostic and seroepidemiological tests for tuberculosis and, ultimately, for the development of more effective vaccines. Images PMID:2581251

  7. Prevalence of Latent and Active Tuberculosis among Dairy Farm Workers Exposed to Cattle Infected by Mycobacterium bovis

    PubMed Central

    Torres-Gonzalez, Pedro; Soberanis-Ramos, Orbelin; Martinez-Gamboa, Areli; Chavez-Mazari, Barbara; Barrios-Herrera, Ma Teresa; Torres-Rojas, Martha; Cruz-Hervert, Luis Pablo; Garcia-Garcia, Lourdes; Singh, Mahavir; Gonzalez-Aguirre, Adrian; Ponce de Leon-Garduño, Alfredo; Sifuentes-Osornio, José; Bobadilla-del-Valle, Miriam

    2013-01-01

    Background Human tuberculosis caused by M. bovis is a zoonosis presently considered sporadic in developed countries, but remains a poorly studied problem in low and middle resource countries. The disease in humans is mainly attributed to unpasteurized dairy products consumption. However, transmission due to exposure of humans to infected animals has been also recognized. The prevalence of tuberculosis infection and associated risk factors have been insufficiently characterized among dairy farm workers (DFW) exposed in settings with poor control of bovine tuberculosis. Methodology/Principal Findings Tuberculin skin test (TST) and Interferon-gamma release assay (IGRA) were administered to 311 dairy farm and abattoir workers and their household contacts linked to a dairy production and livestock facility in Mexico. Sputa of individuals with respiratory symptoms and samples from routine cattle necropsies were cultured for M. bovis and resulting spoligotypes were compared. The overall prevalence of latent tuberculosis infection (LTBI) was 76.2% (95% CI, 71.4–80.9%) by TST and 58.5% (95% CI, 53.0–64.0%) by IGRA. Occupational exposure was associated to TST (OR 2.72; 95% CI, 1.31–5.64) and IGRA (OR 2.38; 95% CI, 1.31–4.30) adjusting for relevant variables. Two subjects were diagnosed with pulmonary tuberculosis, both caused by M. bovis. In one case, the spoligotype was identical to a strain isolated from bovines. Conclusions We documented a high prevalence of latent and pulmonary TB among workers exposed to cattle infected with M. bovis, and increased risk among those occupationally exposed in non-ventilated spaces. Interspecies transmission is frequent and represents an occupational hazard in this setting. PMID:23638198

  8. Evaluation of the semiautomated Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis in respiratory specimens.

    PubMed Central

    Ausina, V; Gamboa, F; Gazapo, E; Manterola, J M; Lonca, J; Matas, L; Manzano, J R; Rodrigo, C; Cardona, P J; Padilla, E

    1997-01-01

    Five hundred twenty processed respiratory specimens from 326 patients received for the diagnosis of tuberculosis or other mycobacterial infections were tested by means of the LCx Mycobacterium tuberculosis Assay from Abbott Laboratories, which uses ligase chain reaction technology for the direct detection of M. tuberculosis complex in respiratory specimens. The results of the LCx M. tuberculosis Assay were compared with the results of culture and staining techniques. After a combination of culture results and the patient's clinical data, a total of 195 specimens were collected from 110 patients who were positively diagnosed as having pulmonary tuberculosis. Twenty-three of these 195 specimens which corresponded to 10 patients with a history of pulmonary tuberculosis (TB) and anti-TB treatment ranging from 1 to 6 months were culture negative. The other 172 specimens were culture positive for M. tuberculosis. With an overall positivity rate of 37.5% (195 of 520 specimens), the sensitivity, specificity, and positive and negative predictive values were 90.8, 100, 100, and 94.7%, respectively, for the LCx M. tuberculosis Assay; 88.2, 100, 100, and 93.4%, respectively, for culture; and 82.6, 92, 72.9, and 97.6%, respectively, for acid-fast staining. For 161 specimens (82.6%) from patients smear positive for the disease and 34 specimens (17.4%) from patients smear negative for the disease, the sensitivity values for the LCx M. tuberculosis Assay were 98.8 and 53%, respectively. There were no statistically significant differences in the sensitivities and specificities between the LCx M. tuberculosis Assay and culture (P > 0.05). Conclusively, the LCx M. tuberculosis Assay has proved to have an acceptable sensitivity and a high specificity in detecting M. tuberculosis and has the potential of reducing the diagnosis time to an 8-h working day. PMID:9230369

  9. Dispersal of Mycobacterium tuberculosis via the Canadian fur trade.

    PubMed

    Pepperell, Caitlin S; Granka, Julie M; Alexander, David C; Behr, Marcel A; Chui, Linda; Gordon, Janet; Guthrie, Jennifer L; Jamieson, Frances B; Langlois-Klassen, Deanne; Long, Richard; Nguyen, Dao; Wobeser, Wendy; Feldman, Marcus W

    2011-04-19

    Patterns of gene flow can have marked effects on the evolution of populations. To better understand the migration dynamics of Mycobacterium tuberculosis, we studied genetic data from European M. tuberculosis lineages currently circulating in Aboriginal and French Canadian communities. A single M. tuberculosis lineage, characterized by the DS6(Quebec) genomic deletion, is at highest frequency among Aboriginal populations in Ontario, Saskatchewan, and Alberta; this bacterial lineage is also dominant among tuberculosis (TB) cases in French Canadians resident in Quebec. Substantial contact between these human populations is limited to a specific historical era (1710-1870), during which individuals from these populations met to barter furs. Statistical analyses of extant M. tuberculosis minisatellite data are consistent with Quebec as a source population for M. tuberculosis gene flow into Aboriginal populations during the fur trade era. Historical and genetic analyses suggest that tiny M. tuberculosis populations persisted for ∼100 y among indigenous populations and subsequently expanded in the late 19th century after environmental changes favoring the pathogen. Our study suggests that spread of TB can occur by two asynchronous processes: (i) dispersal of M. tuberculosis by minimal numbers of human migrants, during which small pathogen populations are sustained by ongoing migration and slow disease dynamics, and (ii) expansion of the M. tuberculosis population facilitated by shifts in host ecology. If generalizable, these migration dynamics can help explain the low DNA sequence diversity observed among isolates of M. tuberculosis and the difficulties in global elimination of tuberculosis, as small, widely dispersed pathogen populations are difficult both to detect and to eradicate.

  10. Dispersal of Mycobacterium tuberculosis via the Canadian fur trade

    PubMed Central

    Pepperell, Caitlin S.; Granka, Julie M.; Alexander, David C.; Behr, Marcel A.; Chui, Linda; Gordon, Janet; Guthrie, Jennifer L.; Jamieson, Frances B.; Langlois-Klassen, Deanne; Long, Richard; Nguyen, Dao; Wobeser, Wendy; Feldman, Marcus W.

    2011-01-01

    Patterns of gene flow can have marked effects on the evolution of populations. To better understand the migration dynamics of Mycobacterium tuberculosis, we studied genetic data from European M. tuberculosis lineages currently circulating in Aboriginal and French Canadian communities. A single M. tuberculosis lineage, characterized by the DS6Quebec genomic deletion, is at highest frequency among Aboriginal populations in Ontario, Saskatchewan, and Alberta; this bacterial lineage is also dominant among tuberculosis (TB) cases in French Canadians resident in Quebec. Substantial contact between these human populations is limited to a specific historical era (1710–1870), during which individuals from these populations met to barter furs. Statistical analyses of extant M. tuberculosis minisatellite data are consistent with Quebec as a source population for M. tuberculosis gene flow into Aboriginal populations during the fur trade era. Historical and genetic analyses suggest that tiny M. tuberculosis populations persisted for ∼100 y among indigenous populations and subsequently expanded in the late 19th century after environmental changes favoring the pathogen. Our study suggests that spread of TB can occur by two asynchronous processes: (i) dispersal of M. tuberculosis by minimal numbers of human migrants, during which small pathogen populations are sustained by ongoing migration and slow disease dynamics, and (ii) expansion of the M. tuberculosis population facilitated by shifts in host ecology. If generalizable, these migration dynamics can help explain the low DNA sequence diversity observed among isolates of M. tuberculosis and the difficulties in global elimination of tuberculosis, as small, widely dispersed pathogen populations are difficult both to detect and to eradicate. PMID:21464295

  11. The Endothelin System Has a Significant Role in the Pathogenesis and Progression of Mycobacterium tuberculosis Infection

    PubMed Central

    Correa, Andre F.; Bailão, Alexandre M.; Bastos, Izabela M. D.; Orme, Ian M.; Soares, Célia M. A.; Kipnis, Andre

    2014-01-01

    Tuberculosis (TB) remains a major global health problem, and although multiple studies have addressed the relationship between Mycobacterium tuberculosis and the host on an immunological level, few studies have addressed the impact of host physiological responses. Proteases produced by bacteria have been associated with important alterations in the host tissues, and a limited number of these enzymes have been characterized in mycobacterial species. M. tuberculosis produces a protease called Zmp1, which appears to be associated with virulence and has a putative action as an endothelin-converting enzyme. Endothelins are a family of vasoactive peptides, of which 3 distinct isoforms exist, and endothelin 1 (ET-1) is the most abundant and the best-characterized isoform. The aim of this work was to characterize the Zmp1 protease and evaluate its role in pathogenicity. Here, we have shown that M. tuberculosis produces and secretes an enzyme with ET-1 cleavage activity. These data demonstrate a possible role of Zmp1 for mycobacterium-host interactions and highlights its potential as a drug target. Moreover, the results suggest that endothelin pathways have a role in the pathogenesis of M. tuberculosis infections, and ETA or ETB receptor signaling can modulate the host response to the infection. We hypothesize that a balance between Zmp1 control of ET-1 levels and ETA/ETB signaling can allow M. tuberculosis adaptation and survival in the lung tissues. PMID:25267836

  12. Zirconia based nucleic acid sensor for Mycobacterium tuberculosis detection

    NASA Astrophysics Data System (ADS)

    Das, Maumita; Sumana, Gajjala; Nagarajan, R.; Malhotra, B. D.

    2010-03-01

    Nanostructured zirconium oxide (ZrO2) film (particle size˜35 nm), electrochemically deposited onto gold(Au) surface, has been used to immobilize 21-mer oligonucleotide probe (ssDNA) specific to Mycobacterium tuberculosis by utilizing affinity between oxygen atom of phosphoric group and zirconium to fabricate DNA biosensor. This DNA-ZrO2/Au bioelectrode, characterized using x-ray diffraction, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy techniques, can be used for early and rapid diagnosis of M. tuberculosis with detection limit of 0.065 ng/μL within 60s.

  13. Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis

    PubMed Central

    Ouellet, Hugues; Johnston, Jonathan B.; Ortiz de Montellano, Paul R.

    2011-01-01

    Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects 10 million worldwide and kills 2 million people every year. The uptake and utilization of nutrients by Mtb within the host cell is still poorly understood, although lipids play an important role in Mtb persistence. The recent identification of a large regulon of cholesterol catabolic genes suggests that Mtb can use host sterol for infection and persistence. In this review, we report on recent progress in elucidation of the Mtb cholesterol catabolic reactions and their potential utility as targets for tuberculosis therapeutic agents. PMID:21924910

  14. Mycobacterium leprae RecA is structurally analogous but functionally distinct from Mycobacterium tuberculosis RecA protein.

    PubMed

    Patil, K Neelakanteshwar; Singh, Pawan; Harsha, Sri; Muniyappa, K

    2011-12-01

    Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein.

  15. Genotypic and Spatial Analysis of Mycobacterium tuberculosis Transmission in a High-Incidence Urban Setting

    PubMed Central

    Ribeiro, Fabíola Karla Correa; Pan, William; Bertolde, Adelmo; Vinhas, Solange Alves; Peres, Renata Lyrio; Riley, Lee; Palaci, Moisés; Maciel, Ethel Leonor

    2015-01-01

    Background. Genotyping Mycobacterium tuberculosis isolates allows study of dynamics of tuberculosis transmission, while geoprocessing allows spatial analysis of clinical and epidemiological data. Here, genotyping data and spatial analysis were combined to characterize tuberculosis transmission in Vitória, Brazil, to identify distinct neighborhoods and risk factors associated with recent tuberculosis transmission. Methods. From 2003 to 2007, 503 isolates were genotyped by IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The analysis included kernel density estimation, K-function analysis, and a t test distance analysis. Mycobacterium tuberculosis isolates belonging to identical RFLP patterns (clusters) were considered to represent recent tuberculosis infection (cases). Results. Of 503 genotyped isolates, 242 (48%) were categorized into 70 distinct clusters belonging to 12 RFLP families. The proportion of recent transmission was 34.2%. Kernel density maps indicated 3 areas of intense concentration of cases. K-function analysis of the largest RFLP clusters and families showed they co-localized in space. The distance analysis confirmed these results and demonstrated that unique strain patterns (controls) randomly distributed in space. A logit model identified young age, positive smear test, and lower Index of Quality of Urban Municipality as risk factors for recent transmission. The predicted probabilities for each neighborhood were mapped and identified neighborhoods with high risk for recent transmission. Conclusions. Spatial and genotypic clustering of M. tuberculosis isolates revealed ongoing active transmission of tuberculosis caused by a small subset of strains in specific neighborhoods of the city. Such information provides an opportunity to target tuberculosis transmission control, such as through rigorous and more focused contact investigation programs. PMID:25948063

  16. Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis*

    PubMed Central

    Li, Mi; Kandror, Olga; Akopian, Tatos; Dharkar, Poorva; Wlodawer, Alexander; Maurizi, Michael R.; Goldberg, Alfred L.

    2016-01-01

    The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, in Mycobacterium tuberculosis (Mtb) are essential and, therefore, promising drug targets. The Mtb ClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3–4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity of Mtb ClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2–3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates. PMID:26858247

  17. Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl Beta-Diol Lipids

    PubMed Central

    Touchette, Megan H.; Bommineni, Gopal R.; Delle Bovi, Richard J.; Gadbery, John E.; Nicora, Carrie D.; Shukla, Anil K.; Kyle, Jennifer E.; Metz, Thomas O.; Martin, Dwight W.; Sampson, Nicole S.; Miller, W. Todd; Tonge, Peter J.; Seeliger, Jessica C.

    2015-01-01

    Although classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl beta-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. We here show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl beta-diol substrate analogues. Applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in regulation DIM biosynthesis. PMID:26271001

  18. Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids.

    PubMed

    Touchette, Megan H; Bommineni, Gopal R; Delle Bovi, Richard J; Gadbery, John E; Nicora, Carrie D; Shukla, Anil K; Kyle, Jennifer E; Metz, Thomas O; Martin, Dwight W; Sampson, Nicole S; Miller, W Todd; Tonge, Peter J; Seeliger, Jessica C

    2015-09-08

    Although they are classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl β-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. Here, we show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl β-diol substrate analogues. By applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and that a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in the regulation of DIM biosynthesis.

  19. Inhibition of the in-vitro growth of Mycobacterium tuberculosis by a phytosiderophore.

    PubMed

    Rajiv, J; Dam, T; Kumar, S; Bose, M; Aggarwal, K K; Babu, C R

    2001-10-01

    Non-compliance by patients and poor clinical management due to the use of incorrect regimens are the main reasons for the development of drug resistance by mycobacterial strains. New strategies for the control of multi-drug-resistant mycobacterial strains have become a necessity for proper management of tuberculosis, which, according to the WHO report (1997), is estimated to remain among the top 10 mortality-causing diseases of the twenty-first century. One of the strategies is the use of iron-sequestering agents like siderophores as active therapeutic agents in the treatment of tuberculosis. This report describes for the first time the inhibition of the growth of Mycobacterium tuberculosis H37Ra in vitro by a phytosiderophore isolated from the root washings of Tephrosia purpurea. This finding may help in the establishment of a new drug regimen which will be more effective in the treatment of tuberculosis.

  20. Mycobacterium tuberculosis Hip1 Dampens Macrophage Proinflammatory Responses by Limiting Toll-Like Receptor 2 Activation▿

    PubMed Central

    Madan-Lala, Ranjna; Peixoto, Katia Vitorello; Re, Fabio; Rengarajan, Jyothi

    2011-01-01

    Mycobacterium tuberculosis is a highly successful human pathogen that evades host innate immunity by interfering with macrophage functions. In addition to avoiding macrophage microbicidal activities, M. tuberculosis triggers secretion of proinflammatory cytokines and chemokines in macrophages. The levels of proinflammatory cytokines induced by clinical M. tuberculosis isolates are thought to play an important role in determining tuberculosis disease progression and severity, but the mechanisms by which M. tuberculosis modulates the magnitude of inflammatory responses remain unclear. Here we show that M. tuberculosis restricts robust macrophage activation and dampens proinflammatory responses through the cell envelope-associated serine hydrolase Hip1 (hydrolase important for pathogenesis 1). By transcriptionally profiling macrophages infected with either wild-type or hip1 mutant bacteria, we found that the hip1 mutant induced earlier and significantly higher levels of several proinflammatory cytokines and chemokines. We show that increased activation of Toll-like receptor 2 (TLR2)- and MyD88-dependent signaling pathways mediates the enhanced cytokine secretion induced by the hip1 mutant. Thus, Hip1 restricts the onset and magnitude of proinflammatory cytokines by limiting TLR2-dependent activation. We also show that Hip1 dampens TLR2-independent activation of the inflammasome and limits secretion of interleukin-18 (IL-18). Dampening of TLR2 signaling does not require viable M. tuberculosis or phagocytosis but does require Hip1 catalytic activity. We propose that M. tuberculosis restricts proinflammatory responses by masking cell surface interactions between TLR2 agonists on M. tuberculosis and TLR2 on macrophages. This strategy may allow M. tuberculosis to evade early detection by host immunity, delay the onset of adaptive immune responses, and accelerate disease progression. PMID:21947769

  1. Suboptimal Antigen Presentation Contributes to Virulence of Mycobacterium tuberculosis In Vivo.

    PubMed

    Grace, Patricia S; Ernst, Joel D

    2016-01-01

    Mycobacterium tuberculosis commonly causes persistent or chronic infection, despite the development of Ag-specific CD4 T cell responses. We hypothesized that M. tuberculosis evades elimination by CD4 T cell responses by manipulating MHC class II Ag presentation and CD4 T cell activation and tested this hypothesis by comparing activation of Ag85B-specific CD4 T cell responses to M. tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) Pasteur in vivo and in vitro. We found that, although M. tuberculosis persists in lungs of immunocompetent mice, M. bovis BCG is cleared, and clearance is T cell dependent. We further discovered that M. tuberculosis-infected macrophages and dendritic cells activate Ag85B-specific CD4 T cells less efficiently and less effectively than do BCG-infected cells, in vivo and in vitro, despite higher production and secretion of Ag85B by M. tuberculosis. During BCG infection, activation of Ag85B-specific CD4 T cells requires fewer infected dendritic cells and fewer Ag-producing bacteria than during M. tuberculosis infection. When dendritic cells containing equivalent numbers of M. tuberculosis or BCG were transferred to mice, BCG-infected cells activated proliferation of more Ag85B-specific CD4 T cells than did M. tuberculosis-infected cells. Differences in Ag85B-specific CD4 T cell activation were attributable to differential Ag presentation rather than differential expression of costimulatory or inhibitory molecules. These data indicate that suboptimal Ag presentation contributes to persistent infection and that limiting Ag presentation is a virulence property of M. tuberculosis.

  2. NOD2 and Toll-Like Receptors Are Nonredundant Recognition Systems of Mycobacterium tuberculosis

    PubMed Central

    2005-01-01

    Infection with Mycobacterium tuberculosis is one of the leading causes of death worldwide. Recognition of M. tuberculosis by pattern recognition receptors is crucial for activation of both innate and adaptive immune responses. In the present study, we demonstrate that nucleotide-binding oligomerization domain 2 (NOD2) and Toll-like receptors (TLRs) are two nonredundant recognition mechanisms of M. tuberculosis. CHO cell lines transfected with human TLR2 or TLR4 were responsive to M. tuberculosis. TLR2 knock-out mice displayed more than 50% defective cytokine production after stimulation with mycobacteria, whereas TLR4-defective mice also released 30% less cytokines compared to controls. Similarly, HEK293T cells transfected with NOD2 responded to stimulation with M. tuberculosis. The important role of NOD2 for the recognition of M. tuberculosis was demonstrated in mononuclear cells of individuals homozygous for the 3020insC NOD2 mutation, who showed an 80% defective cytokine response after stimulation with M. tuberculosis. Finally, the mycobacterial TLR2 ligand 19-kDa lipoprotein and the NOD2 ligand muramyl dipeptide synergized for the induction of cytokines, and this synergism was lost in cells defective in either TLR2 or NOD2. Together, these results demonstrate that NOD2 and TLR pathways are nonredundant recognition mechanisms of M. tuberculosis that synergize for the induction of proinflammatory cytokines. PMID:16322770

  3. Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro.

    PubMed

    Castaño, Diana; Barrera, Luis F; Rojas, Mauricio

    2011-01-01

    This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.

  4. Mycobacterium tuberculosis DevR/DosR Dormancy Regulator Activation Mechanism: Dispensability of Phosphorylation, Cooperativity and Essentiality of α10 Helix.

    PubMed

    Sharma, Saurabh; Tyagi, Jaya Sivaswami

    2016-01-01

    DevR/DosR is a well-characterized regulator in Mycobacterium tuberculosis which is implicated in various processes ranging from dormancy/persistence to drug tolerance. DevR induces the expression of an ~48-gene dormancy regulon in response to gaseous stresses, including hypoxia. Strains of the Beijing lineage constitutively express this regulon, which may confer upon them a significant advantage, since they would be 'pre-adapted' to the environmental stresses that predominate during infection. Aerobic DevR regulon expression in laboratory-manipulated overexpression strains is also reported. In both instances, the need for an inducing signal is bypassed. While a phosphorylation-mediated conformational change in DevR was proposed as the activation mechanism under hypoxia, the mechanism underlying constitutive expression is not understood. Because DevR is implicated in bacterial dormancy/persistence and is a promising drug target, it is relevant to resolve the mechanistic puzzle of hypoxic activation on one hand and constitutive expression under 'non-inducing' conditions on the other. Here, an overexpression strategy was employed to elucidate the DevR activation mechanism. Using a panel of kinase and transcription factor mutants, we establish that DevR, upon overexpression, circumvents DevS/DosT sensor kinase-mediated or small molecule phosphodonor-dependent activation, and also cooperativity-mediated effects, which are key aspects of hypoxic activation mechanism. However, overexpression failed to rescue the defect of C-terminal-truncated DevR lacking the α10 helix, establishing the α10 helix as an indispensable component of DevR activation mechanism. We propose that aerobic overexpression of DevR likely increases the concentration of α10 helix-mediated active dimer species to above the threshold level, as during hypoxia, and enables regulon expression. This advance in the understanding of DevR activation mechanism clarifies a long standing question as to the

  5. Mycobacterium tuberculosis DevR/DosR Dormancy Regulator Activation Mechanism: Dispensability of Phosphorylation, Cooperativity and Essentiality of α10 Helix

    PubMed Central

    Sharma, Saurabh; Tyagi, Jaya Sivaswami

    2016-01-01

    DevR/DosR is a well-characterized regulator in Mycobacterium tuberculosis which is implicated in various processes ranging from dormancy/persistence to drug tolerance. DevR induces the expression of an ~48-gene dormancy regulon in response to gaseous stresses, including hypoxia. Strains of the Beijing lineage constitutively express this regulon, which may confer upon them a significant advantage, since they would be ‘pre-adapted’ to the environmental stresses that predominate during infection. Aerobic DevR regulon expression in laboratory-manipulated overexpression strains is also reported. In both instances, the need for an inducing signal is bypassed. While a phosphorylation-mediated conformational change in DevR was proposed as the activation mechanism under hypoxia, the mechanism underlying constitutive expression is not understood. Because DevR is implicated in bacterial dormancy/persistence and is a promising drug target, it is relevant to resolve the mechanistic puzzle of hypoxic activation on one hand and constitutive expression under ‘non-inducing’ conditions on the other. Here, an overexpression strategy was employed to elucidate the DevR activation mechanism. Using a panel of kinase and transcription factor mutants, we establish that DevR, upon overexpression, circumvents DevS/DosT sensor kinase-mediated or small molecule phosphodonor-dependent activation, and also cooperativity-mediated effects, which are key aspects of hypoxic activation mechanism. However, overexpression failed to rescue the defect of C-terminal-truncated DevR lacking the α10 helix, establishing the α10 helix as an indispensable component of DevR activation mechanism. We propose that aerobic overexpression of DevR likely increases the concentration of α10 helix-mediated active dimer species to above the threshold level, as during hypoxia, and enables regulon expression. This advance in the understanding of DevR activation mechanism clarifies a long standing question as to

  6. The rpoB gene of Mycobacterium tuberculosis.

    PubMed Central

    Miller, L P; Crawford, J T; Shinnick, T M

    1994-01-01

    A portion of the Mycobacterium tuberculosis gene encoding the beta subunit of RNA polymerase (rpoB) was amplified by PCR using degenerate oligonucleotides and used as a hybridization probe to isolate plasmid clones carrying the entire rpoB gene of M. tuberculosis H37Rv, a virulent, rifampin-susceptible strain. Sequence analysis of a 5,084-bp SacI genomic DNA fragment revealed a 3,534-bp open reading frame encoding an 1,178-amino-acid protein with 57% identity with the Escherichia coli beta subunit. This SacI fragment also carried a portion of the rpoC gene located 43 bp downstream from the 3' end of the rpoB open reading frame; this organization is similar to that of the rpoBC operon of E. coli. The M. tuberculosis rpoB gene was cloned into the shuttle plasmid pMV261 and electroporated into the LR223 strain of Mycobacterium smegmatis, which is highly resistant to rifampin (MIC > 200 micrograms/ml). The resulting transformants were relatively rifampin susceptible (MIC = 50 micrograms/ml). Using PCR mutagenesis techniques, we introduced a specific rpoB point mutation (associated with clinical strains of rifampin-resistant M. tuberculosis) into the cloned M. tuberculosis rpoB gene and expressed this altered gene in the LR222 strain of M. smegmatis, which is susceptible to rifampin (MIC = 25 micrograms/ml). The resulting transformants were rifampin resistant (MIC = 200 micrograms/ml). The mutagenesis and expression strategy of the cloned M. tuberculosis rpoB gene that we have employed in this study will allow us to determine the rpoB mutations that are responsible for rifampin resistance in M. tuberculosis. PMID:8031050

  7. Mixed-Strain Mycobacterium tuberculosis Infections and the Implications for Tuberculosis Treatment and Control

    PubMed Central

    van Helden, Paul D.; Wilson, Douglas; Colijn, Caroline; McLaughlin, Megan M.; Abubakar, Ibrahim; Warren, Robin M.

    2012-01-01

    Summary: Numerous studies have reported that individuals can simultaneously harbor multiple distinct strains of Mycobacterium tuberculosis. To date, there has been limited discussion of the consequences for the individual or the epidemiological importance of mixed infections. Here, we review studies that documented mixed infections, highlight challenges associated with the detection of mixed infections, and discuss possible implications of mixed infections for the diagnosis and treatment of patients and for the community impact of tuberculosis control strategies. We conclude by highlighting questions that should be resolved in order to improve our understanding of the importance of mixed-strain M. tuberculosis infections. PMID:23034327

  8. Mycobacterium tuberculosis pili (MTP), a putative biomarker for a tuberculosis diagnostic test.

    PubMed

    Naidoo, Natasha; Ramsugit, Saiyur; Pillay, Manormoney

    2014-05-01

    Novel biomarkers are urgently needed for point of care TB diagnostics. In this study, we investigated the potential of the pilin subunit protein encoded by the mtp gene as a diagnostic biomarker. BLAST analysis of the mtp gene on published genome databases, and amplicon sequencing were performed in Mycobacterium tuberculosis Complex (MTBC) strains and other organisms. The protein secondary structure of the amino acid sequences of non-tuberculous Mycobacteria that partially aligned with the mtp sequence was analysed with PredictProtein software. The mtp gene and corresponding amino acid sequence of MTBC were 100% homologous with H37Rv, in contrast to the partial alignment of the non-tuberculous Mycobacteria. The mtp gene was present in all 91 clinical isolates of MTBC. Except for 2 strains with point mutations, the sequence was 100% conserved among the clinical strains. The mtp gene could not be amplified in all non-tuberculous Mycobacteria and respiratory organisms. The predicted MTP protein structure of Mycobacterium avium, Mycobacterium ulcerans and Mycobacterium abscessus differed significantly from that of the M. tuberculosis, which was similar to Mycobacterium marinum. The absence of the mtp gene in non-tuberculous Mycobacteria and other respiratory bacteria suggests that its encoded product, the pilin subunit protein of M. tuberculosis may be a suitable marker for a point of care TB test.

  9. Phagosomal processing of Mycobacterium tuberculosis antigen 85B is modulated independently of mycobacterial viability and phagosome maturation.

    PubMed

    Ramachandra, Lakshmi; Smialek, Jamie L; Shank, Sam S; Convery, Marilyn; Boom, W Henry; Harding, Clifford V

    2005-02-01

    Control of Mycobacterium tuberculosis infection requires CD4 T-cell responses and major histocompatibility complex class II (MHC-II) processing of M. tuberculosis antigens (Ags). We have previously demonstrated that macrophages process heat-killed (HK) M. tuberculosis more efficiently than live M. tuberculosis. These observations suggested that live M. tuberculosis may inhibit Ag processing by inhibiting phagosome maturation or that HK M. tuberculosis may be less resistant to Ag processing. In the present study we examined the correlation between M. tuberculosis viability and phagosome maturation and efficiency of Ag processing. Since heat treatment could render M. tuberculosis Ags more accessible to proteolysis, M. tuberculosis was additionally killed by antibiotic treatment and radiation. Processing of HK, live, radiation-killed (RadK), or rifampin-killed (RifK) M. tuberculosis in activated murine bone marrow macrophages was examined by using an I-A(b)-restricted T-cell hybridoma cell line (BB7) that recognizes an epitope derived from Ag 85B. Macrophages processed HK M. tuberculosis more rapidly and efficiently than they processed live, RadK, or RifK M. tuberculosis. Live, RadK, and RifK M. tuberculosis cells were processed with similar efficiencies for presentation to BB7 T hybridoma cells. Furthermore, phagosomes containing live or RadK M. tuberculosis expressed fewer M. tuberculosis peptide-MHC-II complexes than phagosomes containing HK M. tuberculosis expressed. Since only live M. tuberculosis was able to prevent acidification of the phagosome, our results suggest that regulation of phagosome maturation does not explain the differences in processing of different forms of M. tuberculosis. These findings suggest that the mechanisms used by M. tuberculosis to inhibit phagosomal maturation differ from the mechanisms involved in modulating phagosome Ag processing.

  10. Differentiation of antigen-specific T cells with limited functional capacity during Mycobacterium tuberculosis infection.

    PubMed

    Jeong, Yun Hee; Jeon, Bo-Young; Gu, Sun-Hwa; Cho, Sang-Nae; Shin, Sung Jae; Chang, Jun; Ha, Sang-Jun

    2014-01-01

    Despite the generation of Mycobacterium tuberculosis-specific T cell immune responses during the course of infection, only 5 to 10% of exposed individuals develop active disease, while others develop a latent infection. This phenomenon suggests defective M. tuberculosis-specific immunity, which necessitates more careful characterization of M. tuberculosis-specific T cell responses. Here, we longitudinally analyzed the phenotypes and functions of M. tuberculosis-specific T cells. In contrast to the functional exhaustion of T cells observed after chronic infection, M. tuberculosis-specific CD8(+) T cells differentiated into either effector (CD127(lo) CD62L(lo)) or effector memory (CD127(hi) CD62L(lo)) cells, but not central memory cells (CD127(hi) CD62L(hi)), with low programmed death 1 (PD-1) expression, even in the presence of high levels of bacteria. Additionally, M. tuberculosis-specific CD8(+) and CD4(+) T cells produced substantial levels of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), but not interleukin 2 (IL-2), upon in vitro restimulation. Among M. tuberculosis-specific CD8(+) T cells, CD127(hi) effector memory cells displayed slower ongoing turnover but greater survival potential. In addition, these cells produced more IFN-γ and TNF-α and displayed lytic activity upon antigen stimulation. However, the effector function of M. tuberculosis-specific CD8(+) CD127(hi) effector memory T cells was inferior to that of canonical CD8(+) CD127(hi) memory T cells generated after acute lymphocytic choriomeningitis virus infection. Collectively, our data demonstrate that M. tuberculosis-specific T cells can differentiate into memory T cells during the course of M. tuberculosis infection independent of the bacterial burden but with limited functionality. These results provide a framework for further understanding the mechanisms of M. tuberculosis infection that can be used to develop more effective vaccines.

  11. The MTCY428.08 Gene of Mycobacterium tuberculosis Codes for NAD+ Synthetase

    PubMed Central

    Cantoni, Rita; Branzoni, Manuela; Labò, Monica; Rizzi, Menico; Riccardi, Giovanna

    1998-01-01

    The product of the MTCY428.08 gene of Mycobacterium tuberculosis shows sequence homology with several NAD+ synthetases. The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the MTCY428.08 gene of M. tuberculosis is the structural gene for NAD+ synthetase. PMID:9620974

  12. Machine learning and docking models for Mycobacterium tuberculosis topoisomerase I.

    PubMed

    Ekins, Sean; Godbole, Adwait Anand; Kéri, György; Orfi, Lászlo; Pato, János; Bhat, Rajeshwari Subray; Verma, Rinkee; Bradley, Erin K; Nagaraja, Valakunja

    2017-03-01

    There is a shortage of compounds that are directed towards new targets apart from those targeted by the FDA approved drugs used against Mycobacterium tuberculosis. Topoisomerase I (Mttopo I) is an essential mycobacterial enzyme and a promising target in this regard. However, it suffers from a shortage of known inhibitors. We have previously used computational approaches such as homology modeling and docking to propose 38 FDA approved drugs for testing and identified several active molecules. To follow on from this, we now describe the in vitro testing of a library of 639 compounds. These data were used to create machine learning models for Mttopo I which were further validated. The combined Mttopo I Bayesian model had a 5 fold cross validation receiver operator characteristic of 0.74 and sensitivity, specificity and concordance values above 0.76 and was used to select commercially available compounds for testing in vitro. The recently described crystal structure of Mttopo I was also compared with the previously described homology model and then used to dock the Mttopo I actives norclomipramine and imipramine. In summary, we describe our efforts to identify small molecule inhibitors of Mttopo I using a combination of machine learning modeling and docking studies in conjunction with screening of the selected molecules for enzyme inhibition. We demonstrate the experimental inhibition of Mttopo I by small molecule inhibitors and show that the enzyme can be readily targeted for lead molecule development.

  13. Role of Cathepsins in Mycobacterium tuberculosis Survival in Human Macrophages

    PubMed Central

    Pires, David; Marques, Joana; Pombo, João Palma; Carmo, Nuno; Bettencourt, Paulo; Neyrolles, Olivier; Lugo-Villarino, Geanncarlo; Anes, Elsa

    2016-01-01

    Cathepsins are proteolytic enzymes that function in the endocytic pathway, especially in lysosomes, where they contribute directly to pathogen killing or indirectly, by their involvement in the antigen presentation pathways. Mycobacterium tuberculosis (MTB) is a facultative intracellular pathogen that survives inside the macrophage phagosomes by inhibiting their maturation to phagolysosomes and thus avoiding a low pH and protease-rich environment. We previously showed that mycobacterial inhibition of the proinflammatory transcription factor NF-κB results in impaired delivery of lysosomal enzymes to phagosomes and reduced pathogen killing. Here, we elucidate how MTB also controls cathepsins and their inhibitors, cystatins, at the level of gene expression and proteolytic activity. MTB induced a general down-regulation of cathepsin expression in infected cells, and inhibited IFNγ-mediated increase of cathepsin mRNA. We further show that a decrease in cathepsins B, S and L favours bacterial survival within human primary macrophages. A siRNA knockdown screen of a large set of cathepsins revealed that almost half of these enzymes have a role in pathogen killing, while only cathepsin F coincided with MTB resilience. Overall, we show that cathepsins are important for the control of MTB infection, and as a response, it manipulates their expression and activity to favour its intracellular survival. PMID:27572605

  14. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    SciTech Connect

    Ansong, Charles; Ortega, Corrie; Payne, Samuel H.; Haft, Daniel H.; Chauvigne-Hines, Lacie M.; Lewis, Michael P.; Ollodart, Anja R.; Purvine, Samuel O.; Shukla, Anil K.; Fortuin, Suereta; Smith, Richard D.; Adkins, Joshua N.; Grundner, Christoph; Wright, Aaron T.

    2013-01-24

    The annotation of protein function is almost completely performed by in silico approaches. However, computational prediction of protein function is frequently incomplete and error prone. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins. This lack of functional information severely limits our understanding of Mtb pathogenicity. Current tools for experimental functional annotation are limited and often do not scale to entire protein families. Here, we report a generally applicable chemical biology platform to functionally annotate bacterial proteins by combining activity-based protein profiling (ABPP) and quantitative LC-MS-based proteomics. As an example of this approach for high-throughput protein functional validation and discovery, we experimentally annotate the families of ATP-binding proteins in Mtb. Our data experimentally validate prior in silico predictions of >250 ATPases and adenosine nucleotide-binding proteins, and reveal 73 hypothetical proteins as novel ATP-binding proteins. We identify adenosine cofactor interactions with many hypothetical proteins containing a diversity of unrelated sequences, providing a new and expanded view of adenosine nucleotide binding in Mtb. Furthermore, many of these hypothetical proteins are both unique to Mycobacteria and essential for infection, suggesting specialized functions in mycobacterial physiology and pathogenicity. Thus, we provide a generally applicable approach for high throughput protein function discovery and validation, and highlight several ways in which application of activity-based proteomics data can improve the quality of functional annotations to facilitate novel biological insights.

  15. Biosynthesis and translocation of unsulfated acyltrehaloses in Mycobacterium tuberculosis.

    PubMed

    Belardinelli, Juan Manuel; Larrouy-Maumus, Gérald; Jones, Victoria; Sorio de Carvalho, Luiz Pedro; McNeil, Michael R; Jackson, Mary

    2014-10-03

    A number of species-specific polymethyl-branched fatty acid-containing trehalose esters populate the outer membrane of Mycobacterium tuberculosis. Among them, 2,3-diacyltrehaloses (DAT) and penta-acyltrehaloses (PAT) not only play a structural role in the cell envelope but also contribute to the ability of M. tuberculosis to multiply and persist in the infected host, promoting the intracellular survival of the bacterium and modulating host immune responses. The nature of the machinery, topology, and sequential order of the reactions leading to the biosynthesis, assembly, and export of these complex glycolipids to the cell surface are the object of the present study. Our genetic and biochemical evidence corroborates a model wherein the biosynthesis and translocation of DAT and PAT to the periplasmic space are coupled and topologically split across the plasma membrane. The formation of DAT occurs on the cytosolic face of the plasma membrane through the action of PapA3, FadD21, and Pks3/4; that of PAT occurs on the periplasmic face via transesterification reactions between DAT substrates catalyzed by the acyltransferase Chp2 (Rv1184c). The integral membrane transporter MmpL10 is essential for DAT to reach the cell surface, and its presence in the membrane is required for Chp2 to be active. Disruption of mmpL10 or chp2 leads to an important build-up of DAT inside the cells and to the formation of a novel form of unsulfated acyltrehalose esterified with polymethyl-branched fatty acids normally found in sulfolipids that is translocated to the cell surface.

  16. Mycolic Acid Index Susceptibility Method for Mycobacterium tuberculosis

    PubMed Central

    Viader-Salvadó, José M.; Garza-González, Elvira; Valdez-Leal, Ramón; de los Angeles del Bosque-Moncayo, M.; Tijerina-Menchaca, Rolando; Guerrero-Olazarán, Martha

    2001-01-01

    A rapid drug susceptibility test to measure the susceptibility of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RIF) using clinical isolates and a newly defined mycolic acid index (MAI) was evaluated. A total of 200 clinical isolates of M. tuberculosis were tested for susceptibility or resistance to INH and RIF by the MAI susceptibility and indirect-proportion methods. Overall, there was agreement between the two methods for 398 (99.5%) of the 400 total tests. Specifically, the sensitivity of the MAI susceptibility method for INH and RIF was 97.6 and 100%, respectively. The specificity and positive predictive value were 100% for both drugs, and the negative predictive value for INH and RIF was 98.3 and 100%, respectively. In conclusion, the MAI susceptibility method described here can be used for rapid drug susceptibility testing of M. tuberculosis clinical isolates within 5 days after clinical isolates are incubated in the presence or absence of an antituberculosis drug. PMID:11427584

  17. Development of Rifapentine Susceptibility Tests for Mycobacterium tuberculosis

    PubMed Central

    Heifets, L.; Sanchez, T.; Vanderkolk, J.; Pham, V.

    1999-01-01

    Two methods for testing the susceptibility of Mycobacterium tuberculosis to rifapentine have been developed: the agar proportion method and the radiometric BACTEC technique. A critical concentration of 0.5 μg of rifapentine per ml is proposed for both methods since it provides a reliable means of distinguishing between susceptible and resistant M. tuberculosis isolates. It is recommended that two quality control M. tuberculosis strains be used at the introduction of these tests in a clinical laboratory: one that is pansusceptible (H37Rv) and one that is resistant to rifapentine. The resistant strain can be obtained from the American Type Culture Collection, where it is deposited under the number ATCC 700457. PMID:9869560

  18. The efficacy of the heat killing of Mycobacterium tuberculosis

    PubMed Central

    Doig, C; Seagar, A L; Watt, B; Forbes, K J

    2002-01-01

    There is concern that current procedures for the heat inactivation of Mycobacterium tuberculosis may not be adequate. This raises serious safety issues for laboratory staff performing molecular investigations such as IS6110 restriction fragment length polymorphism typing. This paper confirms that the protocol of van Embden et al, as performed routinely in this laboratory, is safe and effective for the heat inactivation of M tuberculosis. This procedure involves complete immersion of a tube containing a suspension of one loopfull of growth in a water bath at 80°C for 20 minutes. Seventy four isolates were included in this investigation. Despite prolonged incubation for 20 weeks, none of the heat killed M tuberculosis suspensions produced visible colonies or gave a positive growth signal from liquid culture. This method did not affect the integrity of the DNA for subsequent molecular investigations. PMID:12354807

  19. Novel Mycobacterium tuberculosis complex isolate from a wild chimpanzee.

    PubMed

    Coscolla, Mireia; Lewin, Astrid; Metzger, Sonja; Maetz-Rennsing, Kerstin; Calvignac-Spencer, Sébastien; Nitsche, Andreas; Dabrowski, Pjotr Wojtek; Radonic, Aleksandar; Niemann, Stefan; Parkhill, Julian; Couacy-Hymann, Emmanuel; Feldman, Julia; Comas, Iñaki; Boesch, Christophe; Gagneux, Sebastien; Leendertz, Fabian H

    2013-06-01

    Tuberculosis (TB) is caused by gram-positive bacteria known as the Mycobacterium tuberculosis complex (MTBC). MTBC include several human-associated lineages and several variants adapted to domestic and, more rarely, wild animal species. We report an M. tuberculosis strain isolated from a wild chimpanzee in Côte d'Ivoire that was shown by comparative genomic and phylogenomic analyses to belong to a new lineage of MTBC, closer to the human-associated lineage 6 (also known as M. africanum West Africa 2) than to the other classical animal-associated MTBC strains. These results show that the general view of the genetic diversity of MTBC is limited and support the possibility that other MTBC variants exist, particularly in wild mammals in Africa. Exploring this diversity is crucial to the understanding of the biology and evolutionary history of this widespread infectious disease.

  20. Interaction of antimicrobial peptide with mycolyl transferase in Mycobacterium tuberculosis.

    PubMed

    Banerjee, Devjani I; Gohil, Tejas P

    2016-03-01

    It is estimated that about 40% of the Indian population are infected with tuberculosis (TB) and that ∼3,000,000 people die as a result of TB annually. TB is caused by Mycobacterium tuberculosis. In 2011, the World Health Organization declared India as having the highest TB burden worldwide. An important criteria for pathogenicity is the presence of mycolic acid linked to the protective outer membrane of bacteria. Mycolyl transferase catalyzes the transfer of mycolic acid and promotes cell wall synthesis. This is also considered as a novel target for drug-mediated intervention strategies. Here, we have attempted to understand the interaction between the antimicrobial peptide (AMP), dermcidin, and mycolyl transferase in M. tuberculosis using a computational approach. The present study was undertaken in order to elucidate the capability of AMPs to treat this bacteria, which is less sensitive to available antibiotics, and to design a novel method for new therapies.

  1. Manipulation of the Mononuclear Phagocyte System by Mycobacterium tuberculosis

    PubMed Central

    Lugo-Villarino, Geanncarlo; Neyrolles, Olivier

    2014-01-01

    Over the past 20 years, there has been an emerging appreciation about the role of the mononuclear phagocyte system (MPS) to control and eradicate pathogens. Likewise, there have been significant advances in dissecting the mechanisms involved in the microbial subversion of MPS cells, mainly affecting their differentiation and effector functions. Mycobacterium tuberculosis is a chronic bacterial pathogen that represents an enigma to the field because of its remarkable ability to thrive in humans. One reason is that M. tuberculosis renders a defective MPS compartment, which is perhaps the most ingenious strategy for survival in the host given the prominence of these cells to modulate microenvironments, their function as sentinels and orchestrators of the immune response, and their pathogenic role as reservoirs for microbial persistence. In this article, the principal strategies used by M. tuberculosis to subvert the MPS compartment are presented along with emerging concepts. PMID:25147188

  2. Advances in Mycobacterium tuberculosis therapeutics discovery utlizing structural biology

    PubMed Central

    Chim, Nicholas; Owens, Cedric P.; Contreras, Heidi; Goulding, Celia W.

    2013-01-01

    In 2012, tuberculosis (TB) remains a global health threat and is exacerbated both by the emergence of drug resistant Mycobacterium tuberculosis strains and its synergy with HIV infection. The waning effectiveness of current treatment regimens necessitates the development of new or repurposed anti-TB therapeutics for improved combination therapies against the disease. Exploiting atomic resolution structural information of proteins in complex with their substrates and/or inhibitors can facilitate structure-based rational drug design. Since our last review in 2009, there has been a wealth of new M. tuberculosis protein structural information. Once again, we have compiled the most promising structures with regards to potential anti-TB drug development and present them in this updated review. PMID:23167715

  3. A vitamin B12 transporter in Mycobacterium tuberculosis

    PubMed Central

    Gopinath, Krishnamoorthy; Venclovas, Česlovas; Ioerger, Thomas R.; Sacchettini, James C.; McKinney, John D.; Mizrahi, Valerie; Warner, Digby F.

    2013-01-01

    Vitamin B12-dependent enzymes function in core biochemical pathways in Mycobacterium tuberculosis, an obligate pathogen whose metabolism in vivo is poorly understood. Although M. tuberculosis can access vitamin B12 in vitro, it is uncertain whether the organism is able to scavenge B12 during host infection. This question is crucial to predictions of metabolic function, but its resolution is complicated by the absence in the M. tuberculosis genome of a direct homologue of BtuFCD, the only bacterial B12 transport system described to date. We applied genome-wide transposon mutagenesis to identify M. tuberculosis mutants defective in their ability to use exogenous B12. A small proportion of these mapped to Rv1314c, identifying the putative PduO-type ATP : co(I)rrinoid adenosyltransferase as essential for B12 assimilation. Most notably, however, insertions in Rv1819c dominated the mutant pool, revealing an unexpected function in B12 acquisition for an ATP-binding cassette (ABC)-type protein previously investigated as the mycobacterial BacA homologue. Moreover, targeted deletion of Rv1819c eliminated the ability of M. tuberculosis to transport B12 and related corrinoids in vitro. Our results establish an alternative to the canonical BtuCD-type system for B12 uptake in M. tuberculosis, and elucidate a role in B12 metabolism for an ABC protein implicated in chronic mycobacterial infection. PMID:23407640

  4. Differentiation of human mononuclear phagocytes increases their innate response to Mycobacterium tuberculosis infection.

    PubMed

    Castaño, Diana; García, Luis F; Rojas, Mauricio

    2014-05-01

    The heterogeneity of mononuclear phagocytes, partially explained by cell differentiation, influences the activation of innate responses. It has been reported that Mycobacterium tuberculosis inhibits monocyte differentiation into either dendritic cells or macrophages. To evaluate whether the activation of effector mechanisms against M. tuberculosis differ between less and more differentiated mononuclear phagocytes, we compared monocytes differentiated in vitro for 24 h (MON24) and 120 h (MDM120) infected with M. tuberculosis H37Rv, H37Ra and the clinical isolate UT127 at different multiplicity of infection. MDM120 phagocytosed more M. tuberculosis, inhibited mycobacterial growth and did not die in response to the infection, compared with MON24. In contrast, MON24 become Annexin V and Propidium iodide positive after 36 h of M. tuberculosis infection. Although, there were striking differences between MON24 and MDM120, there were also some differences in the response to the mycobacterial strains used. Finally, in MDM120 infected with M. tuberculosis H37Rv, a lower percentage of mycobacterial phagosomes accumulated transferrin and a higher percentage co-localized with cathelicidin than in MON24. These results demonstrate that innate responses induced by M. tuberculosis depends upon the stage of differentiation of mononuclear phagocytes and support that terminally differentiated cells are more efficient anti-mycobacterial effectors than the less differentiated ones.

  5. Structure and conformational variability of the mycobacterium tuberculosis fatty acid synthase multienzyme complex.

    PubMed

    Ciccarelli, Luciano; Connell, Sean R; Enderle, Mathias; Mills, Deryck J; Vonck, Janet; Grininger, Martin

    2013-07-02

    Antibiotic therapy in response to Mycobacterium tuberculosis infections targets de novo fatty acid biosynthesis, which is orchestrated by a 1.9 MDa type I fatty acid synthase (FAS). Here, we characterize M. tuberculosis FAS by single-particle cryo-electron microscopy and interpret the data by docking the molecular models of yeast and Mycobacterium smegmatis FAS. Our analysis reveals a porous barrel-like structure of considerable conformational variability that is illustrated by the identification of several conformational states with altered topology in the multienzymatic assembly. This demonstrates that the barrel-like structure of M. tuberculosis FAS is not just a static scaffold for the catalytic domains, but may play an active role in coordinating fatty acid synthesis. The conception of M. tuberculosis FAS as a highly dynamic assembly of domains revises the view on bacterial type I fatty acid synthesis and might inspire new strategies for inhibition of de novo fatty acid synthesis in M. tuberculosis.

  6. A Molecular Biological and Biochemical Investigation on Mycobacterium tuberculosis MutT Protein

    PubMed Central

    Huang, Hsiu-Lin; Su, Ho-Ting; Wu, Chung-Hsiun Herbert; Tsai-Wu, Jyy-Jih

    2014-01-01

    Background: Mycobacterium tuberculosis is a vicious microbe co-existing with the infected host. This pathogen exploited opportunities to spread during periods of urbanization and social upheaval, and got retreated with improved hygiene. Objectives: This investigation was designed to clone and characterize M. tuberculosis mutT gene, a homologue of a DNA repair protein in Escherichia coli. The aim was to depict the possible role of this homologue in the virulent microbe. Materials and Methods: A DNA fragment of the mutT gene was amplified with PCR from the genomic DNA of strain H37Rv M. tuberculosis. The expression vector was transformed into E. coli strains BL21 (DE3) and MK602 (DE3) (mutT-). The protein activity assay was performed by biochemical methods. Results: M. tuberculosis MutT shares 23% identity with the E. coli MutT protein. The mutT gene DNA fragment was subcloned into the expression vector pET28a(+) and the recombinant plasmid was overexpressed in E. coli. Purified and refolded M. tuberculosis MutT possesses a dGTPase activity, which is one of the most well-known preference nucleotidase activities of MutT in E. coli. This study also showed that the dGTPase activity of M. tuberculosis MutT was enhanced by magnesium and inhibited by Ni2+ or EDTA. Endogenous MutT protein in M. tuberculosis lysate displayed a smear pattern in the Western blot, suggesting instability of this protein in the bacteria similar to the important proteins, such as P53 protein, tightly regulated by protein degradation. Conclusions: The cloned M. tuberculosis mutT gene and MutT protein were characterized. M. tuberculosis MutT has a dGTPase activity, which is one of the most well-known preference nucleotidase activities of MutT in E. coli. These findings provide further understanding about the vicious bacterium. PMID:25147690

  7. Mixed Infection of Mycobacterium abscessus subsp. abscessus and Mycobacterium tuberculosis in the Lung

    PubMed Central

    Sohn, Sungmin; Wang, Sungho; Shi, Hyejin; Park, Sungrock; Lee, Sangki; Park, Kyoung Taek

    2017-01-01

    A mixed infection of Mycobacterium abscessus subsp. abscessus (Mab) and Mycobacterium tuberculosis (MTB) in the lung is an unusual clinical manifestation and has not yet been reported. A 61-year-old woman had been treated for Mab lung disease and concomitant pneumonia, and was diagnosed with pulmonary tuberculosis (PTB). Despite both anti-PTB and anti-Mab therapy, her entire left lung was destroyed and collapsed. She underwent left pneumonectomy and received medical therapy. We were able to successfully treat her mixed infection by pneumonectomy followed by inhaled amikacin therapy. To the best of our knowledge, thus far, this is the first description of a mixed Mab and MTB lung infection. PMID:28180105

  8. Determination of in vitro susceptibility of Mycobacterium tuberculosis to cephalosporins by radiometric and conventional methods.

    PubMed Central

    Heifets, L B; Iseman, M D; Cook, J L; Lindholm-Levy, P J; Drupa, I

    1985-01-01

    Among eight cephalosporins and cephamycins tested in preliminary in vitro screening against Mycobacterium tuberculosis, the most promising for further study was found to be ceforanide, followed by ceftizoxime, cephapirin, and cefotaxime. Moxalactam, cefoxitin, cefamandole, and cephalothin were found to be not active enough against M. tuberculosis to be considered for further in vitro studies. The antibacterial activity of various ceforanide concentrations was investigated by three methods: (i) the dynamics of radiometric readings (growth index) in 7H12 broth; (ii) the number of CFU in the same medium; and (iii) the proportion method on 7H11 agar plates. There was a good correlation among the results obtained with these methods. The MIC for most strains ranged from 6.0 to 25.0 micrograms/ml. The BACTEC radiometric method is a reliable, rapid, and convenient method for preliminary screening and determination of the level of antibacterial activity of drugs not commonly used against M. tuberculosis. PMID:3920957

  9. Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study

    PubMed Central

    Cabibbe, Andrea M.; Feuerriegel, Silke; Casali, Nicola; Drobniewski, Francis; Rodionova, Yulia; Bakonyte, Daiva; Stakenas, Petras; Pimkina, Edita; Augustynowicz-Kopeć, Ewa; Degano, Massimo; Ambrosi, Alessandro; Hoffner, Sven; Mansjö, Mikael; Werngren, Jim; Rüsch-Gerdes, Sabine; Niemann, Stefan; Cirillo, Daniela M.

    2014-01-01

    ABSTRACT Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZAr). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZAr strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZAr strains, (iii) mutations with an unclear role found in less than 70% of PZAr strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZAr; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. PMID:25336456

  10. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    SciTech Connect

    Wang, T.; Li, H; Lin, G; Tang, C; Li, D; Nathan, C; Heran Darwin, K

    2009-01-01

    Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double {beta} barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

  11. Different responses of human mononuclear phagocyte populations to Mycobacterium tuberculosis.

    PubMed

    Duque, Camilo; Arroyo, Leonar; Ortega, Héctor; Montúfar, Franco; Ortíz, Blanca; Rojas, Mauricio; Barrera, Luis F

    2014-03-01

    Mycobacterium tuberculosis (Mtb) infects different populations of macrophages. Alveolar macrophages (AMs) are initially infected, and their response may contribute to controlling Mtb infection and dissemination. However, Mtb infection may disseminate to other tissues, infecting a wide variety of macrophages. Given the difficulty in obtaining AMs, monocyte-derived macrophages (MDMs) are used to model macrophage-mycobacteria interactions in humans. However, the response of other tissue macrophages to Mtb infection has been poorly explored. We have compared MDMs, AMs and splenic human macrophages (SMs) for their in vitro capacity to control Mtb growth, cytokine production, and induction of cell death in response to Mtb H37Rv, and the Colombian isolate UT205, and to the virulence factor ESAT-6. Significant differences in the magnitude of cell death and cytokine production depending mainly on the Mtb strain were observed; however, no major differences in the mycobacteriostatic/mycobacteriocidal activity were detected among the macrophage populations. Infection with the clinical isolate UT205 was associated with an increased cell death with membrane damage, particularly in IFNγ-treated SMs and H37Rv induced a higher production of cytokines compared to UT205. These results are concordant with the interpretation of a differential response to Mtb infection mainly depending upon the strain of Mtb.

  12. Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex.

    PubMed

    Spositto, F L E; Campanerut, P A Z; Ghiraldi, L D; Leite, C Q F; Hirata, M H; Hirata, R D C; Siqueira, V L D; Cardoso, R Fressatti

    2014-01-01

    We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169 C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

  13. Synthesis and Biological Evaluation of New Hydrazone Derivatives of Quinoline and Their Cu(II) and Zn(II) Complexes against Mycobacterium tuberculosis

    PubMed Central

    Mandewale, Mustapha C.; Thorat, Bapu; Shelke, Dnyaneshwar; Yamgar, Ramesh

    2015-01-01

    A new series of quinoline hydrazone derivatives and their metal complexes have been synthesized and their biological properties have been evaluated against Mycobacterium tuberculosis (H37 RV strain). Most of the newly synthesized compounds displayed 100% inhibitory activity at a concentration of 6.25–25 μg/mL, against Mycobacterium tuberculosis. Fluorescence properties of all the synthesized compounds have been studied. PMID:26759537

  14. CsoR Is Essential for Maintaining Copper Homeostasis in Mycobacterium tuberculosis.

    PubMed

    Marcus, Sarah A; Sidiropoulos, Sarah W; Steinberg, Howard; Talaat, Adel M

    2016-01-01

    Mycobacterium tuberculosis, a pathogen infecting one third of the world population, faces numerous challenges within the host, including high levels of copper. We have previously shown that M. tuberculosis CsoR is a copper inducible transcriptional regulator. Here we examined the hypothesis that csoR is necessary for maintaining copper homeostasis and surviving under various stress conditions. With an unmarked csoR knockout strain, we were able to characterize the role of csoR in M. tuberculosis as it faced copper and host stress. Growth under high levels of copper demonstrated that M. tuberculosis survives copper stress significantly better in the absence of csoR. Yet under minimal levels of copper, differential expression analysis revealed that the loss of csoR results in a cell wide hypoxia-type stress response with the induction of the DosR regulon. Despite the stress placed on M. tuberculosis by the loss of csoR, survival of the knockout strain was increased compared to wild type during the early chronic stages of mouse infection, suggesting that csoR could play an active role in modulating M. tuberculosis fitness within the host. Overall, analysis of CsoR provided an increased understanding of the M. tuberculosis copper response with implications for other intracellular pathogens harboring CsoR.

  15. M2 macrophages or IL-33 treatment attenuate ongoing Mycobacterium tuberculosis infection

    PubMed Central

    Piñeros, A. R.; Campos, L. W.; Fonseca, D. M.; Bertolini, T. B.; Gembre, A. F.; Prado, R. Q.; Alves-Filho, J. C.; Ramos, S. G.; Russo, M.; Bonato, V. L. D.

    2017-01-01

    The protective effects of mycobacterial infections on lung allergy are well documented. However, the inverse relationship between tuberculosis and type 2 immunity is still elusive. Although type 1 immunity is essential to protection against Mycobacterium tuberculosis it might be also detrimental to the host due to the induction of extensive tissue damage. Here, we determined whether lung type 2 immunity induced by allergen sensitization and challenge could affect the outcome of M. tuberculosis infection. We used two different protocols in which sensitization and allergen challenge were performed before or after M. tuberculosis infection. We found an increased resistance to M. tuberculosis only when allergen exposure was given after, but not before infection. Infected mice exposed to allergen exhibited lower bacterial load and cellular infiltrates in the lungs. Enhanced resistance to infection after allergen challenge was associated with increased gene expression of alternatively activated macrophages (M2 macrophages) and IL-33 levels. Accordingly, either adoptive transfer of M2 macrophages or systemic IL-33 treatment was effective in attenuating M. tuberculosis infection. Notably, the enhanced resistance induced by allergen exposure was dependent on IL-33 receptor ST2. Our work indicates that IL-33 might be an alternative therapeutic treatment for severe tuberculosis. PMID:28128217

  16. CsoR Is Essential for Maintaining Copper Homeostasis in Mycobacterium tuberculosis

    PubMed Central

    Marcus, Sarah A.; Sidiropoulos, Sarah W.; Steinberg, Howard; Talaat, Adel M.

    2016-01-01

    Mycobacterium tuberculosis, a pathogen infecting one third of the world population, faces numerous challenges within the host, including high levels of copper. We have previously shown that M. tuberculosis CsoR is a copper inducible transcriptional regulator. Here we examined the hypothesis that csoR is necessary for maintaining copper homeostasis and surviving under various stress conditions. With an unmarked csoR knockout strain, we were able to characterize the role of csoR in M. tuberculosis as it faced copper and host stress. Growth under high levels of copper demonstrated that M. tuberculosis survives copper stress significantly better in the absence of csoR. Yet under minimal levels of copper, differential expression analysis revealed that the loss of csoR results in a cell wide hypoxia-type stress response with the induction of the DosR regulon. Despite the stress placed on M. tuberculosis by the loss of csoR, survival of the knockout strain was increased compared to wild type during the early chronic stages of mouse infection, suggesting that csoR could play an active role in modulating M. tuberculosis fitness within the host. Overall, analysis of CsoR provided an increased understanding of the M. tuberculosis copper response with implications for other intracellular pathogens harboring CsoR. PMID:26999439

  17. Culture and molecular method for detection of Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis in milk and dairy products.

    PubMed

    Messelhäusser, U; Kämpf, P; Hörmansdorfer, S; Wagner, B; Schalch, B; Busch, U; Höller, C; Wallner, P; Barth, G; Rampp, A

    2012-01-01

    A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.

  18. Evidences for anti-mycobacterium activities of lipids and surfactants.

    PubMed

    Hussain, Afzal; Singh, Sandeep Kumar

    2016-01-01

    Tuberculosis is the most widespread and deadly airborne disease caused by Mycobacterium tuberculosis. The two-pronged lethal effect on the bacteria using lipids/surfactants and anti-tubercular drugs may render the miniaturization of dose owing to synergistic and tandem effect of both. The current research has been focused on screening and evaluating various lipids/surfactants possessing inherent anti-mycobacterium activity that can ferry the anti-tubercular drugs. In vitro anti-mycobacterium activity was evaluated using agar well diffusion method. Furthermore, time-concentration dependent killing and DNA/RNA content release studies were performed to correlate the findings. The exact mechanism of bacterial killing was further elucidated by electron/atomic force microscopy studies. Finally, to negate any toxicity, in vitro hemolysis and toxicity studies were performed. The study revealed that capmul MCM C-8, labrasol and acconon C-80 possessed highest in vitro anti-mycobacterium activity. Electron/atomic force microscopy results confirmed in vitro studies and verified the killing of Mycobacterium owing to the release of cytoplasmic content after cell wall fragmentation and disruption. Moreover, the least hemolysis and hundred percent survivals rate of mice using the excipients demonstrated the safety aspects of explored excipients that can ferry the anti-tubercular drugs. The present study concluded the safe, efficient and synergistic activity of the explored excipients and anti-tubercular drugs in controlling the menace of tuberculosis.

  19. Discovery and structure optimization of a series of isatin derivatives as Mycobacterium tuberculosis chorismate mutase inhibitors.

    PubMed

    Jeankumar, Variam U; Alokam, Reshma; Sridevi, Jonnalagadda P; Suryadevara, Priyanka; Matikonda, Siddharth S; Peddi, Santosh; Sahithi, Seedarala; Alvala, Mallika; Yogeeswari, Perumal; Sriram, Dharmarajan

    2014-04-01

    In this study, the crystal structure of the Mycobacterium tuberculosis (MTB) enzyme chorismate mutase (CM) bound to transition state analogue (PDB: 2FP2) was used as a framework for virtual screening of the BITS-Pilani in-house database (2500 compounds) to identify new scaffold. We identified isatin as novel small molecule MTB CM inhibitors; further twenty-four isatin derivatives were synthesized and evaluated in vitro for their ability to inhibit MTB CM, and activity against M. tuberculosis as steps towards the derivation of structure-activity relationships (SAR) and lead optimization. Compound 3-(4-nitrobenzylidene)indolin-2-one, 24 emerged as the most promising lead with an IC50 of 1.01 ± 0.22 μm for purified CM and MIC of 23.5 μm for M. tuberculosis, with little or no cytotoxicity.

  20. Real-Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection

    DTIC Science & Technology

    2015-05-01

    AWARD NUMBER: W81XWH-13-1-0149 TITLE: “Real-Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection ...Mycobacteria Meeting. Birmingham, Alabama. January 24-26, 2014. Energy and redox homeostasis during Mycobacterium tuberculosis infection . Adrie JC Steyn. 4... Infections . June 26- 29, 2014. Saltsjöbaden, Sweden. Metabolomic discovery of a redox and bioenergetic hierarchy in M. tuberculosis and in human TB. Adrie

  1. How B cells shape the immune response against Mycobacterium tuberculosis.

    PubMed

    Maglione, Paul J; Chan, John

    2009-03-01

    Extensive work illustrating the importance of cellular immune mechanisms for protection against Mycobacterium tuberculosis has largely relegated B-cell biology to an afterthought within the tuberculosis (TB) field. However, recent studies have illustrated that B lymphocytes, through a variety of interactions with the cellular immune response, play previously underappreciated roles in shaping host defense against non-viral intracellular pathogens, including M. tuberculosis. Work in our laboratory has recently shown that, by considering these lymphocytes more broadly within their variety of interactions with cellular immunity, B cells have a significant impact on the outcome of airborne challenge with M. tuberculosis as well as the resultant inflammatory response. In this review, we advocate for a revised view of TB immunology in which roles of cellular and humoral immunity are not mutually exclusive. In the context of our current understanding of host defense against non-viral intracellular infections, we review recent data supporting a more significant role of B cells during M. tuberculosis infection than previously thought.

  2. Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment

    PubMed Central

    JACOBS, WILLIAM R.

    2016-01-01

    Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids—chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages—was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research. “I had to know my enemy in order to prevail against him.”Nelson Mandela PMID:26105819

  3. Bystander Macrophage Apoptosis after Mycobacterium tuberculosis H37Ra Infection▿

    PubMed Central

    Kelly, Deirdre M.; ten Bokum, Annemieke M. C.; O'Leary, Seonadh M.; O'Sullivan, Mary P.; Keane, Joseph

    2008-01-01

    Human macrophages infected with Mycobacterium tuberculosis may undergo apoptosis. Macrophage apoptosis contributes to the innate immune response against M. tuberculosis by containing and limiting the growth of mycobacteria and also by depriving the bacillus of its niche cell. Apoptosis of infected macrophages is well documented; however, bystander apoptosis of uninfected macrophages has not been described in the setting of M. tuberculosis. We observed that uninfected human macrophages underwent significant bystander apoptosis 48 and 96 h after they came into contact with macrophages infected with avirulent M. tuberculosis. The bystander apoptosis was significantly greater than the background apoptosis observed in uninfected control cells cultured for the same length of time. There was no evidence of the involvement of tumor necrosis factor alpha, Fas, tumor necrosis factor-related apoptosis-inducing ligand, transforming growth factor β, Toll-like receptor 2, or MyD88 in contact-mediated bystander apoptosis. This newly described phenomenon may further limit the spread of M. tuberculosis by eliminating the niche cells on which the bacillus relies. PMID:17954721

  4. TIM3 Mediates T Cell Exhaustion during Mycobacterium tuberculosis Infection.

    PubMed

    Jayaraman, Pushpa; Jacques, Miye K; Zhu, Chen; Steblenko, Katherine M; Stowell, Britni L; Madi, Asaf; Anderson, Ana C; Kuchroo, Vijay K; Behar, Samuel M

    2016-03-01

    While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain-containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis.

  5. The progress made in determining the Mycobacterium tuberculosis structural proteome

    PubMed Central

    Hecker, Michael

    2011-01-01

    Mycobacterium tuberculosis is a highly infectious pathogen that is still responsible for millions of deaths annually. Effectively treating this disease typically requires a course of antibiotics, most of which were developed decades ago. These drugs are, however, not effective against persistent tubercle bacilli and the emergence of drug-resistant stains threatens to make many of them obsolete. The identification of new drug targets, allowing the development of new potential drugs, is therefore imperative. Both proteomics and structural biology have important roles to play in this process, the former as a means of identifying promising drug targets and the latter allowing understanding of protein function and protein–drug interactions at atomic resolution. The determination of M. tuberculosis protein structures has been a goal of the scientific community for the last decade, who have aimed to supply a large amount of structural data that can be used in structure-based approaches for drug discovery and design. Only since the genome sequence of M. tuberculosis has been available has the determination of large numbers of tuberculosis protein structures been possible. Currently, the molecular structures of 8.5% of all the pathogen's protein-encoding ORFs have been determined. In this review, we look at the progress made in determining the M. tuberculosis structural proteome and the impact this has had on the development of potential new drugs, as well as the discovery of the function of crucial mycobaterial proteins. PMID:21674801

  6. Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence

    PubMed Central

    Smith, Issar

    2003-01-01

    Tuberculosis (TB), one of the oldest known human diseases. is still is one of the major causes of mortality, since two million people die each year from this malady. TB has many manifestations, affecting bone, the central nervous system, and many other organ systems, but it is primarily a pulmonary disease that is initiated by the deposition of Mycobacterium tuberculosis, contained in aerosol droplets, onto lung alveolar surfaces. From this point, the progression of the disease can have several outcomes, determined largely by the response of the host immune system. The efficacy of this response is affected by intrinsic factors such as the genetics of the immune system as well as extrinsic factors, e.g., insults to the immune system and the nutritional and physiological state of the host. In addition, the pathogen may play a role in disease progression since some M. tuberculosis strains are reportedly more virulent than others, as defined by increased transmissibility as well as being associated with higher morbidity and mortality in infected individuals. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is more TB than ever before, requiring new vaccines and drugs and more specific and rapid diagnostics. Researchers are utilizing information obtained from the complete sequence of the M. tuberculosis genome and from new genetic and physiological methods to identify targets in M. tuberculosis that will aid in the development of these sorely needed antitubercular agents. PMID:12857778

  7. Evasion of Innate and Adaptive Immunity by Mycobacterium tuberculosis.

    PubMed

    Goldberg, Michael F; Saini, Neeraj K; Porcelli, Steven A

    2014-10-01

    Through thousands of years of reciprocal coevolution, Mycobacterium tuberculosis has become one of humanity's most successful pathogens, acquiring the ability to establish latent or progressive infection and persist even in the presence of a fully functioning immune system. The ability of M. tuberculosis to avoid immune-mediated clearance is likely to reflect a highly evolved and coordinated program of immune evasion strategies that interfere with both innate and adaptive immunity. These include the manipulation of their phagosomal environment within host macrophages, the selective avoidance or engagement of pattern recognition receptors, modulation of host cytokine production, and the manipulation of antigen presentation to prevent or alter the quality of T-cell responses. In this article we review an extensive array of published studies that have begun to unravel the sophisticated program of specific mechanisms that enable M. tuberculosis and other pathogenic mycobacteria to persist and replicate in the face of considerable immunological pressure from their hosts. Unraveling the mechanisms by which M. tuberculosis evades or modulates host immune function is likely to be of major importance for the development of more effective new vaccines and targeted immunotherapy against tuberculosis.

  8. Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates.

    PubMed Central

    Balasubramanian, V; Pavelka, M S; Bardarov, S S; Martin, J; Weisbrod, T R; McAdam, R A; Bloom, B R; Jacobs, W R

    1996-01-01

    Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines. PMID:8550428

  9. Building a better bacillus: the emergence of Mycobacterium tuberculosis

    PubMed Central

    Wang, Joyce; Behr, Marcel A.

    2014-01-01

    The genus Mycobacterium is comprised of more than 150 species that reside in a wide variety of habitats. Most mycobacteria are environmental organisms that are either not associated with disease or are opportunistic pathogens that cause non-transmissible disease in immunocompromised individuals. In contrast, a small number of species, such as the tubercle bacillus, Mycobacterium tuberculosis, are host-adapted pathogens for which there is no known environmental reservoir. In recent years, gene disruption studies using the host-adapted pathogen have uncovered a number of “virulence factors,” yet genomic data indicate that many of these elements are present in non-pathogenic mycobacteria. This suggests that much of the genetic make-up that enables virulence in the host-adapted pathogen is already present in environmental members of the genus. In addition to these generic factors, we hypothesize that molecules elaborated exclusively by professional pathogens may be particularly implicated in the ability of M. tuberculosis to infect, persist, and cause transmissible pathology in its host species, Homo sapiens. One approach to identify these molecules is to employ comparative analysis of mycobacterial genomes, to define evolutionary events such as horizontal gene transfer (HGT) that contributed M. tuberculosis-specific genetic elements. Independent studies have now revealed the presence of HGT genes in the M. tuberculosis genome and their role in the pathogenesis of disease is the subject of ongoing investigations. Here we review these studies, focusing on the hypothesized role played by HGT loci in the emergence of M. tuberculosis from a related environmental species into a highly specialized human-adapted pathogen. PMID:24765091

  10. Dielectrophoretic characterization of antibiotic-treated Mycobacterium tuberculosis complex cells.

    PubMed

    Inoue, Shinnosuke; Lee, Hyun-Boo; Becker, Annie L; Weigel, Kris M; Kim, Jong-Hoon; Lee, Kyong-Hoon; Cangelosi, Gerard A; Chung, Jae-Hyun

    2015-10-01

    Multi-drug resistant tuberculosis (MDR-TB) has become a serious concern for proper treatment of patients. As a phenotypic method, dielectrophoresis can be useful but is yet to be attempted to evaluate Mycobacterium tuberculosis complex cells. This paper investigates the dielectrophoretic behavior of Mycobacterium bovis (Bacillus Calmette-Guérin, BCG) cells that are treated with heat or antibiotics rifampin (RIF) or isoniazid (INH). The experimental parameters are designed on the basis of our sensitivity analysis. The medium conductivity (σ(m)) and the frequency (f) for a crossover frequency (f(xo1)) test are decided to detect the change of σ(m)-f(xo1) in conjunction with the drug mechanism. Statistical modeling is conducted to estimate the distributions of viable and nonviable cells from the discrete measurement of f (xo1). Finally, the parameters of the electrophysiology of BCG cells, C(envelope) and σ(cyto), are extracted through a sampling algorithm. This is the first evaluation of the dielectrophoresis (DEP) approach as a means to assess the effects of antimicrobial drugs on M. tuberculosis complex cells.

  11. Mycobacterium tuberculosis infection in women with unexplained infertility

    PubMed Central

    Eftekhar, Maryam; Pourmasumi, Soheila; Sabeti, Parvin; Aflatoonian, Abbas; Sheikhha, Mohammad Hasan

    2015-01-01

    Background: Genital tuberculosis (GTB) is an important cause of female infertility, especially in developing countries. The positive results of polymerase chain reaction (PCR) in endometrial GTB in the absence of tubal damage raise the possibility of the detection of sub-clinical or latent disease, with doubtful benefits of treatment. Objective: To evaluate the mycobacterium tuberculosis infection in endometrial biopsy samples collected from unexplained infertile women attending Yazd Research and Clinical Center for Infertility by using PCR techniques. Materials and Methods: In this cross sectional study, 144 infertile women with unexplained infertility aged 20-35 years old and normal Histro-saplango graphy findings were enrolled. Endometrial biopsy samples from each participant were tested for mycobacterium tuberculosis detecting by PCR. In 93 patients, peritoneal fluid was also taken for culture and PCR. Results: The PCR results of endometrial specimens were negative in all cases, demonstrating that there was no GTB infection among our patients. Conclusion: Our results showed that GTB could not be considered as a major problem in women with unexplained infertility. Although, studies have indicated that PCR is a useful method in diagnosing early GTB disease in infertile women with no demonstrable evidence of tubal or endometrial involvement. PMID:27141534

  12. The Copper-Responsive RicR Regulon Contributes to Mycobacterium tuberculosis Virulence

    PubMed Central

    Shi, Xiaoshan; Festa, Richard A.; Ioerger, Thomas R.; Butler-Wu, Susan; Sacchettini, James C.; Darwin, K. Heran; Samanovic, Marie I.

    2014-01-01

    ABSTRACT As with most life on Earth, the transition metal copper (Cu) is essential for the viability of the human pathogen Mycobacterium tuberculosis. However, infected hosts can also use Cu to control microbial growth. Several Cu-responsive pathways are present in M. tuberculosis, including the regulated in copper repressor (RicR) regulon, which is unique to pathogenic mycobacteria. In this work, we describe the contribution of each RicR-regulated gene to Cu resistance in vitro and to virulence in animals. We found that the deletion or disruption of individual RicR-regulated genes had no impact on virulence in mice, although several mutants had Cu hypersensitivity. In contrast, a mutant unable to activate the RicR regulon was not only highly susceptible to Cu but also attenuated in mice. Thus, these data suggest that several genes of the RicR regulon are required simultaneously to combat Cu toxicity in vivo or that this regulon is also important for resistance against Cu-independent mechanisms of host defense. IMPORTANCE Mycobacterium tuberculosis is the causative agent of tuberculosis, killing millions of people every year. Therefore, understanding the biology of M. tuberculosis is crucial for the development of new therapies to treat this devastating disease. Our studies reveal that although host-supplied Cu can suppress bacterial growth, M. tuberculosis has a unique pathway, the RicR regulon, to defend against Cu toxicity. These findings suggest that Cu homeostasis pathways in both the host and the pathogen could be exploited for the treatment of tuberculosis. PMID:24549843

  13. Mycobacterium tuberculosis adhesins: potential biomarkers as anti-tuberculosis therapeutic and diagnostic targets.

    PubMed

    Govender, Viveshree S; Ramsugit, Saiyur; Pillay, Manormoney

    2014-09-01

    Adhesion to host cells is a precursor to host colonization and evasion of the host immune response. Conversely, it triggers the induction of the immune response, a process vital to the host's defence against infection. Adhesins are microbial cell surface molecules or structures that mediate the attachment of the microbe to host cells and thus the host-pathogen interaction. They also play a crucial role in bacterial aggregation and biofilm formation. In this review, we discuss the role of adhesins in the pathogenesis of the aetiological agent of tuberculosis, Mycobacterium tuberculosis. We also provide insight into the structure and characteristics of some of the characterized and putative M. tuberculosis adhesins. Finally, we examine the potential of adhesins as targets for the development of tuberculosis control strategies.

  14. Genetic Lineages of Mycobacterium tuberculosis Isolates in Isfahan, Iran.

    PubMed

    Riyahi Zaniani, Fatemeh; Moghim, Sharareh; Mirhendi, Hossein; Ghasemian Safaei, Hajieh; Fazeli, Hossein; Salehi, Mahshid; Nasr Esfahani, Bahram

    2017-01-01

    In this study, we aimed to identify the genetic lineages of Mycobacterium tuberculosis isolates in Isfahan via the mycobacterial interspersed repetitive-unit-variable number tandem repeat typing method based on 15 loci. Forty-nine M. tuberculosis isolates were collected between 2013 and 2015 from Tuberculosis patients in Mollahadi Sabzevari Tuberculosis Center in Isfahan. All isolates were typed by 15-locus MIRU-VNTR typing. The highest percentage of isolates, 44.89 % (22/49), belonged to the Euro-American lineage, while the frequencies of the East-African-Indian, East-Asian, and Indo-Oceanic lineages were 28.57 % (14/49), 24.4 % (12/49), and 2.04 % (1/49), respectively. Among the 22 isolates of the Euro-American lineage, those belonging to the NEW-1 sub-lineage were most prevalent (24.4 %). Approximately, the same proportion of isolates belonging to the Delhi/CAS, Beijing, and NEW-1 sub-lineages were identified in Iranian and Afghan immigrant patients. The Delhi/CAS and Beijing sub-lineage isolates were prevalent among patients who had been previously treated for TB. Results showed that all of the 49 MIRU-VNTR patterns were unique and the clustering rate of the 15-locus MIRU-VNTR was 0.0 (minimum recent transmission). The results of this study show that the lineages of M. tuberculosis isolates in Isfahan are similar to those reported in the Eastern Mediterranean region (indicative of the epidemiological relationship between the countries in the region). The low clustering rate in our results reveals that transmission of tuberculosis in Isfahan is, in most cases, a reactivation of previous tuberculosis infection and the role of recently transmitted disease is minor.

  15. Catalysis and Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidase

    SciTech Connect

    Lu, Jing-Ping; Chai, Sergio C.; Ye, Qi-Zhuang

    2010-09-07

    Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II), and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity toward the Fe(II)-form, the Mn(II)-form, or the Co(II) and Ni(II) forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues.

  16. Catalysis and inhibition of Mycobacterium tuberculosis methionine aminopeptidase

    PubMed Central

    Lu, Jing-Ping; Chai, Sergio C.; Ye, Qi-Zhuang

    2010-01-01

    Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II) and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells, because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity towards the Fe(II)-form, the Mn(II)-form, or the Co(II)- and Ni(II)-forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues. PMID:20038112

  17. Strain specific transcriptional response in Mycobacterium tuberculosis infected macrophages

    PubMed Central

    2012-01-01

    Background Tuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis (Mtb) remains a significant health problem worldwide with a third of the world population infected and nearly nine million new cases claiming 1.1 million deaths every year. The outcome following infection by Mtb is determined by a complex and dynamic host-pathogen interaction in which the phenotype of the pathogen and the immune status of the host play a role. However, the molecular mechanism by which Mtb strains induce different responses during intracellular infection of the host macrophage is not fully understood. To explore the early molecular events triggered upon Mtb infection of macrophages, we studied the transcriptional responses of murine bone marrow-derived macrophages (BMM) to infection with two clinical Mtb strains, CDC1551 and HN878. These strains have previously been shown to differ in their virulence/immunogenicity in the mouse and rabbit models of pulmonary TB. Results In spite of similar intracellular growth rates, we observed that compared to HN878, infection by CDC1551 of BMM was associated with an increased global transcriptome, up-regulation of a specific early (6 hours) immune response network and significantly elevated nitric oxide production. In contrast, at 24 hours post-infection of BMM by HN878, more host genes involved in lipid metabolism, including cholesterol metabolism and prostaglandin synthesis were up-regulated, compared to infection with CDC1551. In association with the differences in the macrophage responses to infection with the 2 Mtb strains, intracellular CDC1551 expressed higher levels of stress response genes than did HN878. Conclusions In association with the early and more robust macrophage activation, intracellular CDC1551 cells were exposed to a higher level of stress leading to increased up-regulation of the bacterial stress response genes. In contrast, sub-optimal activation of macrophages and induction of a dysregulated host cell

  18. Salicylanilide pyrazinoates inhibit in vitro multidrug-resistant Mycobacterium tuberculosis strains, atypical mycobacteria and isocitrate lyase.

    PubMed

    Krátký, Martin; Vinšová, Jarmila; Novotná, Eva; Stolaříková, Jiřina

    2014-03-12

    The development of antimicrobial agents represents an up-to-date topic. This study investigated in vitro antimycobacterial activity, mycobacterial isocitrate lyase inhibition and cytotoxicity of salicylanilide pyrazinoates. They may be considered being mutual prodrugs of both antimycobacterial active salicylanilides and pyrazinoic acid (POA), an active metabolite of pyrazinamide, in which these esters are likely hydrolysed without presence of pyrazinamidase/nicotinamidase. Minimum inhibitory concentrations (MICs) of the esters were within the range 0.5-8 μmol/l for Mycobacterium tuberculosis and 1-32 μmol/l for nontuberculous mycobacteria (Mycobacterium avium, Mycobacterium kansasii). All esters showed a weak inhibition (8-17%) of isocitrate lyase at the concentration of 10 μmol/l. The most active pyrazinoates showed MICs for multidrug-resistant tuberculosis strains in the range of 0.125-2 μmol/l and no cross-resistance with clinically used drugs, thus being the most in vitro efficacious salicylanilide esters with 4-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl pyrazine-2-carboxylate superiority (MICs⩽0.25 μmol/l). This promising activity is likely due to an additive or synergistic effect of released POA and salicylanilides. Selectivity indexes for the most active salicylanilide pyrazinoates ranged up to 64, making some derivatives being attractive candidates for the next research; 4-bromo-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl pyrazine-2-carboxylate showed the most convenient toxicity profile.

  19. Mycobacterium tuberculosis DosR is Required for Activity of the PmbtB and PmbtI Promoters under Hypoxia

    PubMed Central

    Schreuder, Lise J.; Parish, Tanya

    2014-01-01

    Mycobacterium tuberculosis has the ability to survive for extended periods of time under conditions of low oxygen, low pH, low iron and low nutrients. The mycobactins (M. tuberculosis siderophores) play a key role in scavenging iron from the environment and are induced in response to low iron in an IdeR-regulated manner. We demonstrate that the promoters of two mycobactin gene (mbt) operons are also expressed during adaptation to low oxygen, and that this expression is dependent on the DosR regulator. Up-regulation of mbt operons induced by low iron was not DosR-dependent. DosR is a member of a two component regulatory system which responds to oxygen availability. Deletion of the DosR regulator led to increased expression of bacterioferritin and increased capacity to grow under iron depletion. These data provide a link between the mycobacterial response to two conditions likely to be encountered in vivo, low iron and low oxygen. PMID:25211224

  20. Insights on the Emergence of Mycobacterium tuberculosis from the Analysis of Mycobacterium kansasii

    PubMed Central

    Wang, Joyce; McIntosh, Fiona; Radomski, Nicolas; Dewar, Ken; Simeone, Roxane; Enninga, Jost; Brosch, Roland; Rocha, Eduardo P.; Veyrier, Frédéric J.; Behr, Marcel A.

    2015-01-01

    By phylogenetic analysis, Mycobacterium kansasii is closely related to Mycobacterium tuberculosis. Yet, although both organisms cause pulmonary disease, M. tuberculosis is a global health menace, whereas M. kansasii is an opportunistic pathogen. To illuminate the differences between these organisms, we have sequenced the genome of M. kansasii ATCC 12478 and its plasmid (pMK12478) and conducted side-by-side in vitro and in vivo investigations of these two organisms. The M. kansasii genome is 6,432,277 bp, more than 2 Mb longer than that of M. tuberculosis H37Rv, and the plasmid contains 144,951 bp. Pairwise comparisons reveal conserved and discordant genes and genomic regions. A notable example of genomic conservation is the virulence locus ESX-1, which is intact and functional in the low-virulence M. kansasii, potentially mediating phagosomal disruption. Differences between these organisms include a decreased predicted metabolic capacity, an increased proportion of toxin–antitoxin genes, and the acquisition of M. tuberculosis-specific genes in the pathogen since their common ancestor. Consistent with their distinct epidemiologic profiles, following infection of C57BL/6 mice, M. kansasii counts increased by less than 10-fold over 6 weeks, whereas M. tuberculosis counts increased by over 10,000-fold in just 3 weeks. Together, these data suggest that M. kansasii can serve as an image of the environmental ancestor of M. tuberculosis before its emergence as a professional pathogen, and can be used as a model organism to study the switch from an environmental opportunistic pathogen to a professional host-restricted pathogen. PMID:25716827

  1. Tuberculosis Caused by Mycobacterium bovis in a Capybara (Hydrochoerus hydrochaeris).

    PubMed

    Mol, J P S; Carvalho, T F; Fonseca, A A; Sales, E B; Issa, M A; Rezende, L C; Hodon, M A; Tinoco, H P; Malta, M C C; Pessanha, A T; Pierezan, F; Mota, P M P C; Paixão, T A; Santos, R L

    2016-01-01

    Tuberculosis, associated with Mycobacterium bovis, was diagnosed post mortem in an adult female capybara (Hydrochoerus hydrochaeris), kept at the Pampulha Ecological Park, Belo Horizonte, Brazil, in a large metropolitan area. On post-mortem examination, there were numerous firm white nodules scattered throughout all lobes of both lungs. Tissue samples were collected for histological and microbiological examination. Microscopically, the pulmonary nodules were multifocal to coalescing granulomas and intralesional acid-fast bacilli were evident in Ziehl-Neelsen-stained sections of the lung and spleen. Colonies with morphological features of Mycobacterium spp. were isolated from lung samples and conventional polymerase chain reaction (PCR) with genomic DNA from the isolates was positive for M. bovis; sequencing indicated 100% identity with the region of difference 4 (RD4) of M. bovis. In addition, M. bovis DNA was detected in the lung by quantitative PCR. The finding of M. bovis in a capybara indicates a potential public health risk in a zoological collection.

  2. Detection of Mycobacterium tuberculosis in latently infected lungs by immunohistochemistry and confocal microscopy

    PubMed Central

    Eugenin, Eliseo; Kaplan, Gilla

    2014-01-01

    Detection of latent Mycobacterium tuberculosis is a challenge in the diagnosis of asymptomatic, subclinical tuberculosis. We report the development of an immunofluorescence technique to visualize and enumerate M. tuberculosis in latently infected rabbit lungs where no acid-fast–stained organisms were seen and no cultivable bacilli were obtained by the agar-plating method. PMID:25161200

  3. Mechanisms of cell recruitment in the immune response to Mycobacterium tuberculosis.

    PubMed

    Peters, Wendy; Ernst, Joel D

    2003-02-01

    Recent advances in understanding cell traffic, especially the roles of adhesion proteins, chemokines, and chemokine receptors, provide the opportunity for understanding mechanisms involved in the immune response to tuberculosis. This review concentrates on the roles of these molecules and the immune response in tuberculosis, based on studies of humans and mice infected with Mycobacterium tuberculosis.

  4. Extracellular adenosine triphosphate affects the response of human macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Dubois-Colas, Nicolas; Petit-Jentreau, Laetitia; Barreiro, Luis B; Durand, Sylvère; Soubigou, Guillaume; Lecointe, Cécile; Klibi, Jihène; Rezaï, Keyvan; Lokiec, François; Coppée, Jean-Yves; Gicquel, Brigitte; Tailleux, Ludovic

    2014-09-01

    Granulomas are the hallmark of Mycobacterium tuberculosis infection. As the host fails to control the bacteria, the center of the granuloma exhibits necrosis resulting from the dying of infected macrophages. The release of the intracellular pool of nucleotides into the surrounding medium may modulate the response of newly infected macrophages, although this has never been investigated. Here, we show that extracellular adenosine triphosphate (ATP) indirectly modulates the expression of 272 genes in human macrophages infected with M. tuberculosis and that it induces their alternative activation. ATP is rapidly hydrolyzed by the ecto-ATPase CD39 into adenosine monophosphate (AMP), and it is AMP that regulates the macrophage response through the adenosine A2A receptor. Our findings reveal a previously unrecognized role for the purinergic pathway in the host response to M. tuberculosis. Dampening inflammation through signaling via the adenosine A2A receptor may limit tissue damage but may also favor bacterial immune escape.

  5. Reconstruction and topological characterization of the sigma factor regulatory network of Mycobacterium tuberculosis

    PubMed Central

    Chauhan, Rinki; Ravi, Janani; Datta, Pratik; Chen, Tianlong; Schnappinger, Dirk; Bassler, Kevin E.; Balázsi, Gábor; Gennaro, Maria Laura

    2016-01-01

    Accessory sigma factors, which reprogram RNA polymerase to transcribe specific gene sets, activate bacterial adaptive responses to noxious environments. Here we reconstruct the complete sigma factor regulatory network of the human pathogen Mycobacterium tuberculosis by an integrated approach. The approach combines identification of direct regulatory interactions between M. tuberculosis sigma factors in an E. coli model system, validation of selected links in M. tuberculosis, and extensive literature review. The resulting network comprises 41 direct interactions among all 13 sigma factors. Analysis of network topology reveals (i) a three-tiered hierarchy initiating at master regulators, (ii) high connectivity and (iii) distinct communities containing multiple sigma factors. These topological features are likely associated with multi-layer signal processing and specialized stress responses involving multiple sigma factors. Moreover, the identification of overrepresented network motifs, such as autoregulation and coregulation of sigma and anti-sigma factor pairs, provides structural information that is relevant for studies of network dynamics. PMID:27029515

  6. Reconstruction and topological characterization of the sigma factor regulatory network of Mycobacterium tuberculosis.

    PubMed

    Chauhan, Rinki; Ravi, Janani; Datta, Pratik; Chen, Tianlong; Schnappinger, Dirk; Bassler, Kevin E; Balázsi, Gábor; Gennaro, Maria Laura

    2016-03-31

    Accessory sigma factors, which reprogram RNA polymerase to transcribe specific gene sets, activate bacterial adaptive responses to noxious environments. Here we reconstruct the complete sigma factor regulatory network of the human pathogen Mycobacterium tuberculosis by an integrated approach. The approach combines identification of direct regulatory interactions between M. tuberculosis sigma factors in an E. coli model system, validation of selected links in M. tuberculosis, and extensive literature review. The resulting network comprises 41 direct interactions among all 13 sigma factors. Analysis of network topology reveals (i) a three-tiered hierarchy initiating at master regulators, (ii) high connectivity and (iii) distinct communities containing multiple sigma factors. These topological features are likely associated with multi-layer signal processing and specialized stress responses involving multiple sigma factors. Moreover, the identification of overrepresented network motifs, such as autoregulation and coregulation of sigma and anti-sigma factor pairs, provides structural information that is relevant for studies of network dynamics.

  7. Molecular modeling of Mycobacterium tuberculosis dUTpase: docking and catalytic mechanism studies.

    PubMed

    Ramalho, Teodorico C; Caetano, Melissa S; Josa, Daniela; Luz, Gustavo P; Freitas, Elisangela A; da Cunha, Elaine F F

    2011-06-01

    Mycobacterium tuberculosis is a leading cause of infectious disease in the world today. This outlook is aggravated by a growing number of M. tuberculosis infections in individuals who are immunocompromised as a result of HIV infections. Thus, new and more potent anti-TB agents are necessary. Therefore, dUTpase was selected as a target enzyme to combat M. tuberculosis. In this work, molecular modeling methods involving docking and QM/MM calculations were carried out to investigate the binding orientation and predict binding affinities of some potential dUTpase inhibitors. Our results suggest that the best potential inhibitor investigated, among the compounds studied in this work, is the compound dUPNPP. Regarding the reaction mechanism, we concluded that the decisive stage for the reaction is the stage 1. Furthermore, it was also observed that the compounds with a -1 electrostatic charge presented lower activation energy in relation to the compounds with a -2 charge.

  8. Mycobacterium tuberculosis infection in an orangutan (Pongo pygmaeus).

    PubMed

    Shin, N S; Kwon, S W; Han, D H; Bai, G H; Yoon, J; Cheon, D S; Son, Y S; Ahn, K; Chae, C; Lee, Y S

    1995-10-01

    A respiratory disorder was noted in a 5-year-old female orangutan kept in the Yongin Farmland. Radiographically, multiple radiodense foci ranging from 2 to 6 mm diameter were seen throughout the lung lobes. Grossly, the thoracic cavity revealed a firm texture and grayish-pink discoloration of the left apical lung lobe. Histopathologically, multifocal areas of granulomatous pneumonia present the right and left apical lung lobes. Both primers from IS1081 and IS6110 targeting 196 bp and 245 bp respectively were used in polymerase chain reaction, Mycobacterium tuberculosis was isolated from liver and confirmed by polymerase chain reaction.

  9. Development of a murine mycobacterial growth inhibition assay for evaluating vaccines against Mycobacterium tuberculosis.

    PubMed

    Parra, Marcela; Yang, Amy L; Lim, JaeHyun; Kolibab, Kristopher; Derrick, Steven; Cadieux, Nathalie; Perera, Liyanage P; Jacobs, William R; Brennan, Michael; Morris, Sheldon L

    2009-07-01

    The development and characterization of new tuberculosis (TB) vaccines has been impeded by the lack of reproducible and reliable in vitro assays for measuring vaccine activity. In this study, we developed a murine in vitro mycobacterial growth inhibition assay for evaluating TB vaccines that directly assesses the capacity of immune splenocytes to control the growth of Mycobacterium tuberculosis within infected macrophages. Using this in vitro assay, protective immune responses induced by immunization with five different types of TB vaccine preparations (Mycobacterium bovis BCG, an attenuated M. tuberculosis mutant strain, a DNA vaccine, a modified vaccinia virus strain Ankara [MVA] construct expressing four TB antigens, and a TB fusion protein formulated in adjuvant) can be detected. Importantly, the levels of vaccine-induced mycobacterial growth-inhibitory responses seen in vitro after 1 week of coculture correlated with the protective immune responses detected in vivo at 28 days postchallenge in a mouse model of pulmonary tuberculosis. In addition, similar patterns of cytokine expression were evoked at day 7 of the in vitro culture by immune splenocytes taken from animals immunized with the different TB vaccines. Among the consistently upregulated cytokines detected in the immune cocultures are gamma interferon, growth differentiation factor 15, interleukin-21 (IL-21), IL-27, and tumor necrosis factor alpha. Overall, we have developed an in vitro functional assay that may be useful for screening and comparing new TB vaccine preparations, investigating vaccine-induced protective mechanisms, and assessing manufacturing issues, including product potency and stability.

  10. Streptomyces as host for recombinant production of Mycobacterium tuberculosis proteins.

    PubMed

    Vallin, Carlos; Ramos, Astrid; Pimienta, Elsa; Rodríguez, Caridad; Hernández, Tairí; Hernández, Ivones; Del Sol, Ricardo; Rosabal, Grisel; Van Mellaert, Lieve; Anné, Jozef

    2006-01-01

    The 45/47 kDa APA protein (Rv1860) of Mycobacterium tuberculosis was produced by Streptomyces lividans. The recombinant protein could be recovered from the culture medium of an S. lividans clone containing the apa gene under control of the promoter and signal sequence of the Streptomyces coelicolor agarase gene. The recombinant protein production was further scaled-up using fermentation conditions. The APA protein was subsequently purified from the culture supernatant by means of immunochromatography. About 80 mg of recombinant protein were obtained per liter of culture media. In vivo tests with the APA protein purified from S. lividans TK24/pRGAPA1 revealed that the recombinant protein was antigenic and could induce high titers of specific antibodies in the mouse biological model. Results obtained concerning heterologous production of APA, its immunogenic and antigenic capacity, demonstrated the potential of S. lividans as a valuable host for the production of recombinant proteins from M. tuberculosis.

  11. Structural enzymology of sulphur metabolism in Mycobacterium tuberculosis.

    PubMed

    Schnell, Robert; Schneider, Gunter

    2010-05-21

    The emergence of multidrug-resistant strains of Mycobacterium tuberculosis poses a serious threat to human health and has led to world-wide efforts focusing on the development of novel vaccines and antibiotics against this pathogen. Sulphur metabolism in this organism has been linked to essential processes such as virulence and redox defence. The cysteine biosynthetic pathway is up-regulated in models of persistent M. tuberculosis infections and provides potential targets for novel anti-mycobacterial agents, directed specifically toward the pathogen in its persistent phase. Functional and structural characterization of enzymes from sulfur metabolism establishes a necessary framework for the design of strong binding inhibitors that might be developed into new drugs. This review summarizes recent progress in the elucidation of the structural enzymology of the sulphate reduction and cysteine biosynthesis pathways.

  12. Clonal complexity in Mycobacterium tuberculosis can hamper diagnostic procedures.

    PubMed

    Pérez-Lago, Laura; Herranz, Marta; Navarro, Yurena; Ruiz Serrano, María Jesús; Miralles, Pilar; Bouza, Emilio; García-de-Viedma, Darío

    2017-02-15

    Clonal complexity is increasingly accepted in Mycobacterium tuberculosis infection, including mixed infections by ≥2 strains, which usually occur in settings with a high burden of tuberculosis and/or a high risk of overexposure to infected patients. Mixed infections can hamper diagnostic procedures: obtaining an accurate antibiogram is difficult when the susceptibility patterns of the strains differ. Here, we show how mixed infections can also prove challenging for other diagnostic procedures, even outside settings where mixed infections are traditionally expected. We show how an unnoticed mixed infection in an HIV-positive patient diagnosed in Madrid, Spain, with differences in the representativeness of the coinfecting strains in different sputum samples, markedly complicated the resolution of a laboratory cross-contamination false-positivity alert.

  13. Eosinophilic cystitis and cholangitis - systemic disease triggered by mycobacterium tuberculosis?

    PubMed

    Buda, Piotr; Grenda, Ryszard; Wieteska-Klimczak, Anna; Gietka, Piotr; Skobejko-Włodarska, Lidia; Felberg, Karina; Książyk, Janusz

    Eosinophilic cystitis (EC) is a rare inflammatory disorder of the urinary tract characterized by infiltration of bladder with eosinophils. The cause remains unclear, immunological mechanisms have been implicated in pathogenesis. Potential etiological factors include: tumors, allergy, parasitic infections, trauma. The disease may have a variable course, from a mild self-limiting, through common symptoms like: dysuria, hematuria, abdominal pain, tumor, to severe renal failure, with eosinophilic infiltration of the other organs and systemic complications. Treatment depending on disease severity and etiology is pharmacological and/or surgical. Here we report a case of a previously healthy 16-year old girl with inflammatory tumor in the liver hilum infiltrating extrahepatic biliary tract who developed three months later haematuria with acute dysuric signs and renal failure. Based on histopathological findings diagnosis of eosinophilic cystitis was established. Tests for Mycobacterium tuberculosis were positive. To our knowledge, EC association with cholangitis and tuberculosis have never been reported before.

  14. The transmission of Mycobacterium tuberculosis in high burden settings.

    PubMed

    Yates, Tom A; Khan, Palwasha Y; Knight, Gwenan M; Taylor, Jonathon G; McHugh, Timothy D; Lipman, Marc; White, Richard G; Cohen, Ted; Cobelens, Frank G; Wood, Robin; Moore, David A J; Abubakar, Ibrahim

    2016-02-01

    Unacceptable levels of Mycobacterium tuberculosis transmission are noted in high burden settings and a renewed focus on reducing person-to-person transmission in these communities is needed. We review recent developments in the understanding of airborne transmission. We outline approaches to measure transmission in populations and trials and describe the Wells-Riley equation, which is used to estimate transmission risk in indoor spaces. Present research priorities include the identification of effective strategies for tuberculosis infection control, improved understanding of where transmission occurs and the transmissibility of drug-resistant strains, and estimates of the effect of HIV and antiretroviral therapy on transmission dynamics. When research is planned and interventions are designed to interrupt transmission, resource constraints that are common in high burden settings-including shortages of health-care workers-must be considered.

  15. Mycobacterium tuberculosis Infection following Kidney Transplantation

    PubMed Central

    Boubaker, Karima; Gargah, Tahar; Abderrahim, Ezzedine; Ben Abdallah, Taieb; Kheder, Adel

    2013-01-01

    Introduction and Aims. Post-transplant tuberculosis (TB) is a problem in successful long-term outcome of renal transplantation recipients. Our objective was to describe the pattern and risk factors of TB infection and the prognosis in our transplant recipients. Patients and Methods. This study was a retrospective review of the records of 491 renal transplant recipients in our hospital during the period from January 1986 to December 2009. The demographic data, transplant characteristics, clinical manifestations, diagnostic criteria, treatment protocol, and long-term outcome of this cohort of patients were analyzed. Results. 16 patients (3,2%) developed post-transplant TB with a mean age of 32,5 ± 12,7 (range: 13–60) years and a mean post-transplant period of 36,6months (range: 12,3 months–15,9 years). The forms of the diseases were pulmonary in 10/16 (62,6%), disseminated in 3/16 (18,7%), and extrapulmonary in 3/16 (18,7%). Graft dysfunction was observed in 7 cases (43,7%) with tissue-proof acute rejection in 3 cases and loss of the graft in 4 cases. Hepatotoxicity developed in 3 patients (18,7%) during treatment. Recurrences were observed in 4 cases after early stop of treatment. Two patients (12.5%) died. Conclusion. Extra pulmonary and disseminated tuberculosis were observed in third of our patients. More than 9months of treatment may be necessary to prevent recurrence. PMID:24222903

  16. Relationship Between HIV Coinfection, Interleukin 10 Production, and Mycobacterium tuberculosis in Human Lymph Node Granulomas

    PubMed Central

    Diedrich, Collin R.; O'Hern, Jennifer; Gutierrez, Maximiliano G.; Allie, Nafiesa; Papier, Patricia; Meintjes, Graeme; Coussens, Anna K.; Wainwright, Helen; Wilkinson, Robert J.

    2016-01-01

    Background. Human immunodeficiency virus type 1 (HIV)–infected persons are more susceptible to tuberculosis than HIV–uninfected persons. Low peripheral CD4+ T-cell count is not the sole cause of higher susceptibility, because HIV–infected persons with a high peripheral CD4+ T-cell count and those prescribed successful antiretroviral therapy (ART) remain more prone to active tuberculosis than HIV–uninfected persons. We hypothesized that the increase in susceptibility is caused by the ability of HIV to manipulate Mycobacterium tuberculosis–associated granulomas. Methods. We examined 71 excised cervical lymph nodes (LNs) from persons with HIV and M. tuberculosis coinfection, those with HIV monoinfection, and those with M. tuberculosis monoinfection with a spectrum of peripheral CD4+ T-cell counts and ART statuses. We quantified differences in M. tuberculosis levels, HIV p24 levels, cellular response, and cytokine presence within granulomas. Results. HIV increased M. tuberculosis numbers and reduced CD4+ T-cell counts within granulomas. Peripheral CD4+ T-cell depletion correlated with granulomas that contained fewer CD4+ and CD8+ T cells, less interferon γ, more neutrophils, more interleukin 10 (IL-10), and increased M. tuberculosis numbers. M. tuberculosis numbers correlated positively with IL-10 and interferon α levels and fewer CD4+ and CD8+ T cells. ART reduced IL-10 production. Conclusions. Peripheral CD4+ T-cell depletion correlated with increased M. tuberculosis presence, increased IL-10 production, and other phenotypic changes within granulomas, demonstrating the HIV infection progressively changes these granulomas. PMID:27462092

  17. Mannosylated lipoarabinomannan antagonizes Mycobacterium tuberculosis-induced macrophage apoptosis by altering Ca+2-dependent cell signaling.

    PubMed

    Rojas, M; García, L F; Nigou, J; Puzo, G; Olivier, M

    2000-07-01

    Mycobacterium tuberculosis-induced macrophage apoptosis can be inhibited by mannosylated lipoarabinomannan (ManLAM), although it induces tumor necrosis factor (TNF)-alpha and NO production, which participate in apoptosis induction. ManLAM also modulates Ca(+2)-dependent intracellular events, and Ca(+2) participates in apoptosis in different systems. Ca(+2) was assessed for involvement in M. tuberculosis-induced macrophage apoptosis and for modulation by ManLAM. The role of Ca(+2) was supported by the blockade of apoptosis by cAMP inhibitors and the Ca(+2) chelator, BAPTA/AM. These agents also inhibited caspase-1 activation and cAMP-responsive element-binding protein translocation without affecting TNF-alpha production. Infection of macrophages with M. tuberculosis induced an influx of Ca(+2) that was prevented by ManLAM. Similarly, M. tuberculosis infection-altered mitochondrial permeability transition was prevented by ManLAM and BAPTA/AM. Finally, ManLAM and BAPTA/AM reversed the effects of M. tuberculosis on p53 and Bcl-2 expression. ManLAM counteracts the alterations of calcium-dependent intracellular events that occur during M. tuberculosis-induced macrophage apoptosis.

  18. Genetic diversity and dynamic distribution of Mycobacterium tuberculosis isolates causing pulmonary and extrapulmonary tuberculosis in Thailand.

    PubMed

    Srilohasin, Prapaporn; Chaiprasert, Angkana; Tokunaga, Katsushi; Nishida, Nao; Prammananan, Therdsak; Smittipat, Nat; Mahasirimongkol, Surakameth; Chaiyasirinroje, Boonchai; Yanai, Hideki; Palittapongarnpim, Prasit

    2014-12-01

    This study examined the genetic diversity and dynamicity of circulating Mycobacterium tuberculosis strains in Thailand using nearly neutral molecular markers. The single nucleotide polymorphism (SNP)-based genotypes of 1,414 culture-positive M. tuberculosis isolates from 1,282 pulmonary tuberculosis (PTB) and 132 extrapulmonary TB (EPTB) patients collected from 1995 to 2011 were characterized. Among the eight SNP cluster groups (SCG), SCG2 (44.1%), which included the Beijing (BJ) genotype, and SCG1 (39.4%), an East African Indian genotype, were dominant. Comparisons between the genotypes of M. tuberculosis isolates causing PTB and EPTB in HIV-negative cases revealed similar prevalence trends although genetic diversity was higher in the PTB patients. The identification of 10 reported sequence types (STs) and three novel STs was hypothesized to indicate preferential expansion of the SCG2 genotype, especially the modern BJ ST10 (15.6%) and ancestral BJ ST19 (13.1%). An association between SCG2 and SCG1 genotypes and particular patient age groups implies the existence of different genetic advantages among the bacterial populations. The results revealed that increasing numbers of young patients were infected with M. tuberculosis SCGs 2 and 5, which contrasts with the reduction of the SCG1 genotype. Our results indicate the selection and dissemination of potent M. tuberculosis genotypes in this population. The determination of heterogeneity and dynamic population changes of circulating M. tuberculosis strains in countries using the Mycobacterium bovis BCG (bacillus Calmette-Guérin) vaccine are beneficial for vaccine development and control strategies.

  19. Enhanced Specialized Transduction Using Recombineering in Mycobacterium tuberculosis

    PubMed Central

    Tufariello, JoAnn M.; Malek, Adel A.; Vilchèze, Catherine; Cole, Laura E.; Ratner, Hannah K.; González, Pablo A.; Jain, Paras; Hatfull, Graham F.; Larsen, Michelle H.

    2014-01-01

    ABSTRACT Genetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100-fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the recovery of deletion mutants and are expected to facilitate construction of a comprehensive library of mutants with every nonessential gene of M. tuberculosis deleted. The findings also open up the potential for sophisticated genetic screens, such as synthetic lethal analyses, which have so far not been feasible for the slow-growing mycobacteria. PMID:24865558

  20. Molecular Biology of Drug Resistance in Mycobacterium tuberculosis

    PubMed Central

    Smith, Tasha; Wolff, Kerstin A.; Nguyen, Liem

    2014-01-01

    Tuberculosis (TB) has become a curable disease thanks to the discovery of antibiotics. However, it has remained one of the most difficult infections to treat. Most current TB regimens consist of six to nine months of daily doses of four drugs that are highly toxic to patients. The purpose of these lengthy treatments is to completely eradicate Mycobacterium tuberculosis, notorious for its ability to resist most antibacterial agents, thereby preventing the formation of drug resistant mutants. On the contrary, the prolonged therapies have led to poor patient adherence. This, together with a severe limit of drug choices, has resulted in the emergence of strains that are increasingly resistant to the few available antibiotics. Here we review our current understanding of molecular mechanisms underlying the profound drug resistance of M. tuberculosis. This knowledge is essential for the development of more effective antibiotics that not only are potent against drug resistant M. tuberculosis strains but also help shorten the current treatment courses required for drug susceptible TB. PMID:23179675

  1. Inhibition studies of Mycobacterium tuberculosis salicylate synthase (MbtI).

    PubMed

    Manos-Turvey, Alexandra; Bulloch, Esther M M; Rutledge, Peter J; Baker, Edward N; Lott, J Shaun; Payne, Richard J

    2010-07-05

    Mycobacterium tuberculosis salicylate synthase (MbtI), a member of the chorismate-utilizing enzyme family, catalyses the first committed step in the biosynthesis of the siderophore mycobactin T. This complex secondary metabolite is essential for both virulence and survival of M. tuberculosis, the etiological agent of tuberculosis (TB). It is therefore anticipated that inhibitors of this enzyme may serve as TB therapies with a novel mode of action. Herein we describe the first inhibition study of M. tuberculosis MbtI using a library of functionalized benzoate-based inhibitors designed to mimic the substrate (chorismate) and intermediate (isochorismate) of the MbtI-catalyzed reaction. The most potent inhibitors prepared were those designed to mimic the enzyme intermediate, isochorismate. These compounds, based on a 2,3-dihydroxybenzoate scaffold, proved to be low-micromolar inhibitors of MbtI. The most potent inhibitors in this series possessed hydrophobic enol ether side chains at C3 in place of the enol-pyruvyl side chain found in chorismate and isochorismate.

  2. Prospective genotyping of Mycobacterium tuberculosis from fresh clinical samples.

    PubMed

    Bidovec-Stojkovič, Urška; Seme, Katja; Žolnir-Dovč, Manca; Supply, Philip

    2014-01-01

    Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of Mycobacterium tuberculosis directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (p = 0.0003) and for 53 follow-up samples (p = 0.002). For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of M. tuberculosis can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample.

  3. The T cell response to secreted antigens of Mycobacterium tuberculosis.

    PubMed

    Andersen, P

    1994-10-01

    Recent information from several laboratories points to proteins secreted from live Mycobacterium tuberculosis as being involved in protective immunity. We have studied protein release from M. tuberculosis during growth and have defined 3 different groups of proteins: excreted proteins, secreted proteins of the outer cell wall and cytoplasmic proteins released at late culture timepoints. These findings have lead to the definition of a short-term culture filtrate (ST-CF) enriched in excreted/secreted proteins and with a minimal content of autolytic products. ST-CF was tested as antigen in experimental vaccines against tuberculosis. A vaccine based on the adjuvant dimethyldioctadecylammonium chloride (DDA) was constructed and demonstrated to induce a potent cell mediated immune response of the Th-1 type. The vaccine was tested in parallel with a BCG standard vaccine and both vaccines induced a highly significant protection of the same magnitude. Molecules within the Ag85 complex and a 6-kDA secreted protein were mapped as the major antigenic targets for long-lived T cells involved in protective immunity against M. tuberculosis.

  4. Molecular Epidemiology of Mycobacterium tuberculosis among South African Gold Miners

    PubMed Central

    Lewis, James J.; Connors, Jeremy; Chihota, Violet N.; Shashkina, Elena; van der Meulen, Minty; Graviss, Edward A.; Ha, Ngan P.; Kreiswirth, Barry N.; Grant, Alison D.; Fielding, Katherine L.; Dorman, Susan E.; Churchyard, Gavin J.

    2015-01-01

    Rationale: HIV-associated tuberculosis remains a major health problem among the gold-mining workforce in South Africa. We postulate that high levels of recent transmission, indicated by strain clustering, are fueling the tuberculosis epidemic among gold miners. Objectives: To combine molecular and epidemiologic data to describe Mycobacterium tuberculosis genetic diversity, estimate levels of transmission, and examine risk factors for clustering. Methods: We conducted a cross-sectional study of culture-positive M. tuberculosis isolates in 15 gold mine shafts across three provinces in South Africa. All isolates were subject IS6110-based restriction fragment length polymorphisms, and we performed spoligotyping analysis and combined it with basic demographic and clinical information. Measurements and Main Results: Of the 1,602 M. tuberculosis patient isolates, 1,240 (78%) had genotyping data available for analysis. A highly diverse bacillary population was identified, comprising a total of 730 discrete genotypes. Four genotypic families (Latin American Mediterranean spoligotype family; W-Beijing; AH or X; and T1–T4) accounted for over 50% of all strains. Overall, 45% (560/1,240) of strains were genotypically clustered. The minimum estimate for recent transmission (n − 1 method) was 32% (range, 27–34%). There were no individual-level risk factors for clustering, apart from borderline evidence for being non–South African and having self-reported HIV infection. Conclusions: The high M. tuberculosis genetic diversity and lack of risk factors for clustering are indicative of a universal risk for disease among gold miners and likely mixing with nonmining populations. Our results underscore the urgent need to intensify interventions to interrupt transmission across the entire gold-mining workforce in South Africa. PMID:25419914

  5. Essential Metabolites of Mycobacterium tuberculosis and Their Mimics

    PubMed Central

    Lamichhane, Gyanu; Freundlich, Joel S.; Ekins, Sean; Wickramaratne, Niluka; Nolan, Scott T.; Bishai, William R.

    2011-01-01

    An organism requires a range of biomolecules for its growth. By definition, these are essential molecules which constitute the basic metabolic requirements of an organism. A small organic molecule with chemical similarity to that of an essential metabolite may bind to the enzyme that catalyzes its production and inhibit it, likely resulting in the stasis or death of the organism. Here, we report a high-throughput approach for identifying essential metabolites of an organism using genetic and biochemical approaches and then implement computational approaches to identify metabolite mimics. We generated and genotyped 5,126 Mycobacterium tuberculosis mutants and performed a statistical analysis to determine putative essential genes. The essential molecules of M. tuberculosis were classified as products of enzymes that are encoded by genes in this list. Although incomplete, as many enzymes of M. tuberculosis have yet to be identified and characterized, this is the first report of a large number of essential molecules of the organism. We identified essential metabolites of three distinct metabolic pathways in M. tuberculosis and selected molecules with chemical similarity using cheminformatics strategies that illustrate a variety of different pharmacophores. Our approach is aimed at systematic identification of essential molecules and their mimics as a blueprint for development of effective chemical probes of M. tuberculosis metabolism, with the ultimate goal of seeking drugs that can kill this pathogen. As an illustration of this approach, we report that compounds JFD01307SC and l-methionine-S-sulfoximine, which share chemical similarity with an essential molecule of M. tuberculosis, inhibited the growth of this organism at micromolar concentrations. PMID:21285434

  6. Response of Mycobacterium tuberculosis to reactive oxygen and nitrogen intermediates.

    PubMed Central

    Garbe, T. R.; Hibler, N. S.; Deretic, V.

    1996-01-01

    BACKGROUND: Mycobacterium tuberculosis is a significant human pathogen capable of replicating in mononuclear phagocytic cells. Exposure to reactive oxygen and nitrogen intermediates is likely to represent an important aspect of the life cycle of this organism. The response of M. tuberculosis to these agents may be of significance for its survival in the host. MATERIALS AND METHODS: Patterns of de novo proteins synthesized in M. tuberculosis H37Rv exposed to compounds that generate reactive oxygen and nitrogen intermediates were studied by metabolic labeling and two-dimensional electrophoresis. RESULTS: Menadione, a redox cycling compound which increases intracellular superoxide levels, caused enhanced synthesis of seven polypeptides, six of which appeared to be heat shock proteins. Chemical release of nitric oxide induced eight polypeptides of which only one could be identified as a heat shock protein. Nitric oxide also exhibited a mild inhibitory action on general protein synthesis in the concentration range tested. Hydrogen peroxide did not cause differential gene expression and exerted a generalized inhibition in a dose-dependent manner. Cumene hydroperoxide caused mostly inhibition but induction of two heat shock proteins was detectable. CONCLUSIONS: The presented findings indicate major differences between M. tuberculosis and the paradigms of oxidative stress response in enteric bacteria, and are consistent with the multiple lesions found in oxyR of this organism. The effect of hydrogen peroxide, which in Escherichia coli induces eight polypeptides known to be controlled by the central regulator oxyR, appears to be absent in M. tuberculosis. Superoxide and nitric oxide responses, which in E. coli overlap and are controlled by the same regulatory system soxRS, represent discrete and independent phenomena in M. tuberculosis. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 PMID:8900541

  7. Essential metabolites of Mycobacterium tuberculosis and their mimics.

    PubMed

    Lamichhane, Gyanu; Freundlich, Joel S; Ekins, Sean; Wickramaratne, Niluka; Nolan, Scott T; Bishai, William R

    2011-02-01

    An organism requires a range of biomolecules for its growth. By definition, these are essential molecules which constitute the basic metabolic requirements of an organism. A small organic molecule with chemical similarity to that of an essential metabolite may bind to the enzyme that catalyzes its production and inhibit it, likely resulting in the stasis or death of the organism. Here, we report a high-throughput approach for identifying essential metabolites of an organism using genetic and biochemical approaches and then implement computational approaches to identify metabolite mimics. We generated and genotyped 5,126 Mycobacterium tuberculosis mutants and performed a statistical analysis to determine putative essential genes. The essential molecules of M. tuberculosis were classified as products of enzymes that are encoded by genes in this list. Although incomplete, as many enzymes of M. tuberculosis have yet to be identified and characterized, this is the first report of a large number of essential molecules of the organism. We identified essential metabolites of three distinct metabolic pathways in M. tuberculosis and selected molecules with chemical similarity using cheminformatics strategies that illustrate a variety of different pharmacophores. Our approach is aimed at systematic identification of essential molecules and their mimics as a blueprint for development of effective chemical probes of M. tuberculosis metabolism, with the ultimate goal of seeking drugs that can kill this pathogen. As an illustration of this approach, we report that compounds JFD01307SC and l-methionine-S-sulfoximine, which share chemical similarity with an essential molecule of M. tuberculosis, inhibited the growth of this organism at micromolar concentrations.

  8. Recurrence due to Relapse or Reinfection With Mycobacterium tuberculosis: A Whole-Genome Sequencing Approach in a Large, Population-Based Cohort With a High HIV Infection Prevalence and Active Follow-up

    PubMed Central

    Guerra-Assunção, José Afonso; Houben, Rein M. G. J.; Crampin, Amelia C.; Mzembe, Themba; Mallard, Kim; Coll, Francesc; Khan, Palwasha; Banda, Louis; Chiwaya, Arthur; Pereira, Rui P. A.; McNerney, Ruth; Harris, David; Parkhill, Julian; Clark, Taane G.; Glynn, Judith R.

    2015-01-01

    Background. Recurrent tuberculosis is a major health burden and may be due to relapse with the original strain or reinfection with a new strain. Methods. In a population-based study in northern Malawi, patients with tuberculosis diagnosed from 1996 to 2010 were actively followed after the end of treatment. Whole-genome sequencing with approximately 100-fold coverage was performed on all available cultures. Results of IS6110 restriction fragment-length polymorphism analyses were available for cultures performed up to 2008. Results. Based on our data, a difference of ≤10 single-nucleotide polymorphisms (SNPs) was used to define relapse, and a difference of >100 SNPs was used to define reinfection. There was no evidence of mixed infections among those classified as reinfections. Of 1471 patients, 139 had laboratory-confirmed recurrences: 55 had relapse, and 20 had reinfection; for 64 type of recurrence was unclassified. Almost all relapses occurred in the first 2 years. Human immunodeficiency virus infection was associated with reinfection but not relapse. Relapses were associated with isoniazid resistance, treatment before 2007, and lineage-3 strains. We identified several gene variants associated with relapse. Lineage-2 (Beijing) was overrepresented and lineage-1 underrepresented among the reinfecting strains (P = .004). Conclusions. While some of the factors determining recurrence depend on the patient and their treatment, differences in the Mycobacterium tuberculosis genome appear to have a role in both relapse and reinfection. PMID:25336729

  9. Acquired resistance of Mycobacterium tuberculosis to bedaquiline.

    PubMed

    Andries, Koen; Villellas, Cristina; Coeck, Nele; Thys, Kim; Gevers, Tom; Vranckx, Luc; Lounis, Nacer; de Jong, Bouke C; Koul, Anil

    2014-01-01

    Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multi-drug resistant tuberculosis in decades. In vitro resistance to BDQ was previously shown to be due to target-based mutations. Here we report that non-target based resistance to BDQ, and cross-resistance to clofazimine (CFZ), is due to mutations in Rv0678, a transcriptional repressor of the genes encoding the MmpS5-MmpL5 efflux pump. Efflux-based resistance was identified in paired isolates from patients treated with BDQ, as well as in mice, in which it was confirmed to decrease bactericidal efficacy. The efflux inhibitors verapamil and reserpine decreased the minimum inhibitory concentrations of BDQ and CFZ in vitro, but verapamil failed to increase the bactericidal effect of BDQ in mice and was unable to reverse efflux-based resistance in vivo. Cross-resistance between BDQ and CFZ may have important clinical implications.

  10. High Throughput Phenotypic Analysis of Mycobacterium tuberculosis and Mycobacterium bovis Strains' Metabolism Using Biolog Phenotype Microarrays

    PubMed Central

    Khatri, Bhagwati; Fielder, Mark; Jones, Gareth; Newell, William; Abu-Oun, Manal; Wheeler, Paul R.

    2013-01-01

    Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has

  11. Rv2744c Is a PspA Ortholog That Regulates Lipid Droplet Homeostasis and Nonreplicating Persistence in Mycobacterium tuberculosis

    PubMed Central

    Armstrong, Richard M.; Adams, Katherine L.; Zilisch, Joseph E.; Bretl, Daniel J.; Sato, Hiromi; Anderson, David M.

    2016-01-01

    . IMPORTANCE Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease associated with significant morbidity and mortality worldwide. M. tuberculosis is capable of establishing lifelong asymptomatic infections in susceptible individuals and reactivating during periods of immune suppression to cause active disease. The determinants that are important for persistent infection of M. tuberculosis or for reactivation of this organism from latency are poorly understood. In this study, we describe our initial characterizations of Rv2744c, an ortholog of phage shock protein A (PspA) that regulates the homeostasis of lipid bodies and nonreplicating persistence in M. tuberculosis. This function of PspA in M. tuberculosis is novel and suggests that PspA may represent a unique bacterial target upon which to base therapeutic interventions against this organism. PMID:27002134

  12. Disseminated Mycobacterium tuberculosis Infection Masquerading as Metastasis after Heavy Ion Radiotherapy for Prostate Cancer

    PubMed Central

    Ando, Masaru; Mukai, Yutaka; Ushijima, Ryo-ichi; Shioyama, Yoshiyuki; Umeki, Kenji; Okada, Fumito; Nureki, Shin-ichi; Mimata, Hiromitsu; Kadota, Jun-ichi

    2016-01-01

    Fluorodeoxyglucose (FDG)-positron emission tomography with computed tomography (FDG-PET/CT) is useful in disease monitoring of malignancies after therapy, while an FDG uptake may also be present in benign diseases. We herein demonstrate a case of disseminated Mycobacterium tuberculosis mimicking systemic metastasis of prostate cancer. This case highlights that clinicians should consider Mycobacterium tuberculosis in patients with prostate cancer who demonstrate multifocal FDG uptakes masquerading as metastasis, even when the chest photographs reveal a normal appearance and a sputum examination demonstrates negative results. An invasive surgical biopsy may be required and a pathological analysis would be critical in the diagnosis of Mycobacterium tuberculosis. PMID:27853089

  13. Mycobacterium tuberculosis triggers apoptosis in peripheral neutrophils involving toll-like receptor 2 and p38 mitogen protein kinase in tuberculosis patients.

    PubMed

    Alemán, Mercedes; Schierloh, Pablo; de la Barrera, Silvia S; Musella, Rosa M; Saab, María A; Baldini, Matías; Abbate, Eduardo; Sasiain, María C

    2004-09-01

    Polymorphonuclear neutrophils (PMN) exposed to Mycobacterium tuberculosis display bactericidal responses and produce inflammatory proteins. This PMN-mediated inflammatory response is regulated by an activation of the apoptotic program, which collaborates to avoid tissue injury. In vitro, circulating PMN from patients with tuberculosis (TB) show an increased spontaneous apoptosis, and M. tuberculosis-induced activation accelerates the PMN apoptosis. In this study, we evaluated the mechanisms involved in spontaneous and M. tuberculosis-induced apoptosis. We demonstrate that apoptosis of PMN is not induced by lipoarabinomannan or by a whole-cell lysate of M. tuberculosis and that neither tumor necrosis factor alpha nor CD11b, CD14, and Fcgamma receptors are involved. Apoptosis of PMN from patients with active TB (TB-PMN) is induced by the interaction with the whole M. tuberculosis via Toll-like receptor 2 (TLR2), and, in contrast to spontaneous apoptosis, it involves the p38 mitogen-activated protein kinase (MAPK) pathway. These results correlate with a high expression of phosphorylated p38 (p-p38) in circulating TB-PMN and with the ability of M. tuberculosis to induce in vitro the expression of p-p38 in PMN. Therefore, when the bacterial burden is low, TB-PMN could be detecting nonopsonized M. tuberculosis via TLR2, leading to the activation of the p38 MAPK pathway, which in turn would induce PMN activation and apoptosis. This mechanism needs further confirmation at the site of infection.

  14. Identification of Mannich Base as a Novel Inhibitor of Mycobacterium Tuberculosis Isocitrate by High-Throughput Screening

    PubMed Central

    Ji, Lei; Long, Quanxin; Yang, Dacheng; Xie, Jianping

    2011-01-01

    Mycobacterium tuberculosis (MTB) remains one of the most significant human pathogens since its discovery in 1882. An estimated 1.5 million people died from tubercle bacillus (TB) in 2006, and globally, there were an estimated 9.27 million incident cases of TB in 2007. Glyoxylate bypass pathway occurs in a wide range of pathogens and plays a key role in the pathogenesis of Mycobacterium tuberculosis. Isocitrate lyase (ICL) can catalyses the first step of this pathway, and reversibly cleaves isocitrate into succinate and glyoxylate. So, ICL may represent a good drug target for the treatment of tuberculosis. ICL was cloned, expressed, and purified, and a high-throughput screen (HTS) developed to screen active molecule from a mannich base compounds library for inhibition of ICL. This assay had signal to noise (S/N) of 650.6990 and Z' factor of 0.8141, indicating that the assay was suitable for HTS. Screening of a collection of 124 mannich base compounds resulted in the identification of one mannich base compound, which has a significant inhibitory activity. So, a new family of compound was first reported to inhibit the activity of Mycobacterium tuberculosis ICL. This family of compound might offer new avenue to explore better anti-tuberculosis and fungi drugs. PMID:21494431

  15. Impaired Cytokine but Enhanced Cytotoxic Marker Expression in Mycobacterium tuberculosis-Induced CD8+ T Cells in Individuals With Type 2 Diabetes and Latent Mycobacterium tuberculosis Infection.

    PubMed

    Kumar, Nathella Pavan; Moideen, Kadar; George, Parakkal Jovvian; Dolla, Chandrakumar; Kumaran, Paul; Babu, Subash

    2016-03-01

    Type 2 diabetes mellitus (DM) is a risk factor for tuberculosis among individuals with latent Mycobacterium tuberculosis infection. To explore the influence of DM on CD8(+) T-cell responses during latent M. tuberculosis infection, we estimated the cytokine and cytotoxic marker expression pattern in individuals with latent M. tuberculosis infection with DM and those with latent M. tuberculosis infection without DM. Among individuals with latent M. tuberculosis infection, those with DM had diminished frequencies of CD8(+) T-helper type 1 (Th1), Th2, and Th17 cells following stimulation by M. tuberculosis antigen and enhanced frequencies of CD8(+) T cells expressing cytotoxic markers, compared with those without DM. Thus, our results suggest that coincident DM modulates CD8(+) T-cell function during latent M. tuberculosis infection.

  16. Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

    PubMed Central

    Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

    2013-01-01

    Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

  17. Synthesis and biological evaluation against Mycobacterium tuberculosis and Leishmania amazonensis of a series of diaminated terpenoids.

    PubMed

    Dos Reis, Dijovani Batista; Souza, Thalita Cristina Alves; Lourenço, Maria Cristina S; de Almeida, Mauro Vieira; Barbosa, Adriano; Eger, Iriane; Saraiva, Mauricio Frota

    2016-12-01

    We report the synthesis of a series of diaminated terpenoids containing, as side-chain of the diamine core, the "head-to-tail" prenyl derivatives, with amino amino spacers of variable length. In vitro biological activity of these compounds was evaluated against Mycobacterium tuberculosis and Leishmania amazonensis, and the structure-activity relationships are discussed. Different biological results were observed depending on the terpenic side-chain length. The best results were obtained for trans,trans-farnesol derivatives. Moreover, these results demonstrated that the stereochemistry of the double bond could play an important role in determining antitubercular and antileishmanial activities of these compounds.

  18. Identification of Rv2041c, a novel immunogenic antigen from Mycobacterium tuberculosis with serodiagnostic potential.

    PubMed

    Shin, S J; Kim, S-Y; Shin, A-R; Kim, H-J; Cho, S-N; Park, J-K

    2009-11-01

    Novel immunogenic antigens are continually required for the improvement of diagnostic techniques for Mycobacterium tuberculosis infection. Some proteins with serodiagnostic value are not expressed under normal culture conditions, but may be induced under specific conditions such as gradual oxygen depletion and low pH, and from inside macrophages. Using a customized amplification library, we previously found that Rv2041c from M. tuberculosis H37Rv was highly expressed in vitro under conditions of low pH and hypoxia. In this study, recombinant (r)Rv2041c was produced in Escherichia coli to examine its role in immune responses. Increased Rv2041c expression in vitro during dormancy and during infection in human macrophages was confirmed by Western blotting and reverse transcription polymerase chain reaction, respectively. Interestingly, positive antibody responses to rRv2041c were detected only in those patients with active tuberculosis (TB) and in mice infected with M. tuberculosis H37Rv. Finally, Rv2041c was used successfully in the serodiagnosis of active M. tuberculosis infection in Korean patients in conjunction with other M. tuberculosis proteins, including Ag85 complex, 38 kDa, rESAT-6, rHSP-X and rCFP-10. Our Rv2041c-ELISA had comparable diagnostic sensitivity and equivalent specificity to the use of an M. tuberculosis H37Rv cellular extract. In addition, seven of 46 serum samples collected from TB patients (15.28%) showed positive antibody responses to Rv2041c, but not to the other proteins. These results suggest that Rv2041c can be used to increase assay sensitivity alongside well-known antigens for the serodiagnosis of M. tuberculosis infection.

  19. Mechanisms of β-lactam killing and resistance in the context of Mycobacterium tuberculosis.

    PubMed

    Wivagg, Carl N; Bhattacharyya, Roby P; Hung, Deborah T

    2014-09-01

    β-Lactams are one of the most useful classes of antibiotics against many common bacterial pathogens. One exception is Mycobacterium tuberculosis. However, with increasing incidence of multidrug-resistant tuberculosis and a need for new agents to treat it, the use of β-lactams, specifically the combination of carbapenem and clavulanate, is now being revisited. With this attention, comes the need to better understand both the mechanisms of action of β-lactams against M. tuberculosis as well as possible mechanisms of resistance, within the context of what is known about the β-lactam action in other bacteria. M. tuberculosis has two major mechanisms of intrinsic resistance: a highly active β-lactamase and a poorly permeable outer membrane. Within the cell wall, β-lactams bind several enzymes with differing peptidoglycan-synthetic and -lytic functions. The inhibition of these enzymes may lead to cell death through several mechanisms, involving disruption of the balance of synthetic and lethal activities. Currently, all known means of resistance to the β-lactams rely on diminishing the proportion of peptidoglycan-synthetic proteins bound and inhibited by β-lactams, through either exclusion or destruction of the antibiotic, or through replacement or supplementation of target enzymes. In this review, we discuss possible mechanisms for β-lactam activity in M. tuberculosis and the means by which it may acquire resistance, within the context of what is known in other bacterial species.

  20. The Mycobacterium tuberculosis Ag85A is a novel diacylglycerol acyltransferase involved in lipid body formation.

    PubMed

    Elamin, Ayssar A; Stehr, Matthias; Spallek, Ralf; Rohde, Manfred; Singh, Mahavir

    2011-09-01

    Mycobacterium tuberculosis accumulates large amounts of triacylglycerol (TAG) which acts as storage compounds for energy and carbon. The mycobacterial triacylglycerols stored in the form of intracellular lipid droplets are essential for long-term survival of M. tuberculosis during a dormant state. We report here that when the M. tuberculosis mycolytransferase Ag85A is overexpressed in Mycobacterium smegmatis mc(2)155, cell morphology was changed and the cells became grossly enlarged. A massive formation of lipid bodies and a change in lipid pattern was observed simultaneously. We suspected a possible role of Ag85A in the acyl lipid metabolism and discovered that the enzyme possesses acyl-CoA:diacylglycerol acyltransferase (DGAT) activity in addition to its well-known function as mycolyltransferase. Ag85A mediates the transesterification of diacylglycerol using long-chain acyl-CoA as acyl donors. The K(m) and K(cat) values for palmitoleoyl-coenzyme A were 390 µM and 55.54 min(-1) respectively. A docking model suggests that palmitoleoyl-coenzyme A and 1,2-dipalmitin occupy the same active site as trehalose 6,6'-dimycolate and trehalose 6'-monomycolate. The site-directed Ser126Ala mutation of the active site proved that this residue is involved in the catalytic activity of this enzyme. Although not proven conclusively for dormant stage of M. tuberculosis, our novel finding about the synthesis of TAGs by Ag85A strongly suggests that Ag85A may play a significant role in the formation of lipid storage bodies and thus also in the establishment and maintenance of a persistent tuberculosis infection.

  1. [Advances in the research of an animal model of wound due to Mycobacterium tuberculosis infection].

    PubMed

    Chen, Ling; Jia, Chiyu

    2015-12-01

    Tuberculosis ranks as the second deadly infectious disease worldwide. The incidence of tuberculosis is high in China. Refractory wound caused by Mycobacterium tuberculosis infection ranks high in misdiagnosis, and it is accompanied by a protracted course, and its pathogenic mechanism is still not so clear. In order to study its pathogenic mechanism, it is necessary to reproduce an appropriate animal model. Up to now the study of the refractory wound caused by Mycobacterium tuberculosis infection is just beginning, and there is still no unimpeachable model for study. This review describes two models which may reproduce a wound similar to the wound caused by Mycobacterium tuberculosis infection, so that they could be used to study the pathogenesis and characteristics of a tuberculosis wound in an animal.

  2. Structures of the Michaelis Complex (1.2A) and the Covalent Acyl Intermediate (2.0A ) of Cefamandole Bound in the Active Sites of the Mycobacterium tuberculosis beta-Lactamase K72A and E166A Mutants

    SciTech Connect

    L Tremblay; h Xu; J Blanchard

    2011-12-31

    The genome of Mycobacterium tuberculosis (TB) contains a gene that encodes a highly active {beta}-lactamase, BlaC, that imparts TB with resistance to {beta}-lactam chemotherapy. The structure of covalent BlaC-{beta}-lactam complexes suggests that active site residues K73 and E166 are essential for acylation and deacylation, respectively. We have prepared the K73A and E166A mutant forms of BlaC and have determined the structures of the Michaelis complex of cefamandole and the covalently bound acyl intermediate of cefamandole at resolutions of 1.2 and 2.0 {angstrom}, respectively. These structures provide insight into the details of the catalytic mechanism.

  3. A High-Throughput Cidality Screen for Mycobacterium Tuberculosis

    PubMed Central

    Kaur, Parvinder; Ghosh, Anirban; Krishnamurthy, Ramya Vadageri; Bhattacharjee, Deepa Gagwani; Achar, Vijayashree; Datta, Santanu; Narayanan, Shridhar; Anbarasu, Anand; Ramaiah, Sudha

    2015-01-01

    Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well ‘spot-assay’ for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process. PMID:25693161

  4. Irreversible Inhibition of the Mycobacterium tuberculosis β-lactamase by Clavulanate

    PubMed Central

    Hugonnet, Jean-Emmanuel; Blanchard, John S.

    2008-01-01

    The β-lactam class of antibiotics, which inhibit the bacterial d,d transpeptidases involved in cell wall biosynthesis, have never been used systematically in the treatment of Mycobacterium tuberculosis infections because of this organism’s resistance to β-lactams. The critical resistance factor is the constitutive production of a chromosomally encoded, Ambler class A β-lactamase, BlaC in M. tuberculosis. We show that BlaC is an extended spectrum β-lactamase (ESBL) with high levels of penicillinase and cephalosporinase activity as well as measurable activity with carbapenems, including imipenem and meropenem. We have characterized the enzyme’s inhibition by three FDA-approved β-lactamase inhibitors: sulbactam, tazobactam and clavulanate. Sulbactam inhibits the enzyme competitively and reversibly with respect to nitrocefin. Tazobactam inhibits the enzyme in a time-dependent manner, but the activity of the enzyme reappears due to the slow hydrolysis of the covalently acylated enzyme. In contrast, clavulanate reacts with the enzyme quickly to form hydrolytically stable, inactive forms of the enzyme that have been characterized by mass spectrometry. Clavulanate has potential to be used in combination with approved β-lactam antibiotics to treat multi-drug resistant (MDR) and extremely-drug resistant (XDR) strains of Mycobacterium tuberculosis. PMID:17915954

  5. Effect of common and experimental anti-tuberculosis treatments on Mycobacterium tuberculosis growing as biofilms

    PubMed Central

    Dalton, James P.; Uy, Benedict; Phummarin, Narisa; Copp, Brent R.; Denny, William A.; Swift, Simon

    2016-01-01

    Much is known regarding the antibiotic susceptibility of planktonic cultures of Mycobacterium tuberculosis, the bacterium responsible for the lung disease tuberculosis (TB). As planktonically-grown M. tuberculosis are unlikely to be entirely representative of the bacterium during infection, we set out to determine how effective a range of anti-mycobacterial treatments were against M. tuberculosis growing as a biofilm, a bacterial phenotype known to be more resistant to antibiotic treatment. Light levels from bioluminescently-labelled M. tuberculosis H37Rv (strain BSG001) were used as a surrogate for bacterial viability, and were monitored before and after one week of treatment. After treatment, biofilms were disrupted, washed and inoculated into fresh broth and plated onto solid media to rescue any surviving bacteria. We found that in this phenotypic state M. tuberculosis was resistant to the majority of the compounds tested. Minimum inhibitory concentrations (MICs) increased by 20-fold to greater than 1,000-fold, underlying the potential of this phenotype to cause significant problems during treatment. PMID:27904808

  6. The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis

    PubMed Central

    Flint, Jessica L.; Kowalski, Joseph C.; Karnati, Pavan K.; Derbyshire, Keith M.

    2004-01-01

    Conjugal DNA transfer occurs by an atypical mechanism in Mycobacterium smegmatis. The transfer system is chromosomally encoded and requires recipient recombination functions for both chromosome and plasmid transfer. Cis-acting sequences have been identified that confer mobility on nontransferable plasmids, but these are larger and have different properties to canonical oriT sites found in bacterial plasmids. To identify trans-acting factors required for mediating DNA transfer, a library of transposon insertion mutants was generated in the donor strain, and individual mutants were screened for their effect on transfer. From this screen, a collection of insertion mutants was isolated that increased conjugation frequencies relative to wild type. Remarkably, the mutations map to a 25-kb region of the M. smegmatis chromosome that is syntenous with the RD1 region of Mycobacterium tuberculosis, which is considered to be the primary attenuating deletion in the related vaccine strain Mycobacterium bovis bacillus Calmette–Guérin. The genes of the RD1 region encode a secretory apparatus responsible for exporting Cfp10- and Esat-6, both potent antigens and virulence factors. In crosses using two M. smegmatis donors, we show that wild-type cells can suppress the elevated transfer phenotype of mutant donors, which is consistent with the secretion of a factor that suppresses conjugation. Most importantly, the RD1 region of M. tuberculosis complements the conjugation phenotype of the RD1 mutants in M. smegmatis. Our results indicate that the M. tuberculosis and M. smegmatis RD1 regions are functionally equivalent and provide a unique perspective on the role of this critical secretion apparatus. PMID:15314236

  7. Mycobacterium tuberculosis upregulates microglial matrix metalloproteinase-1 and -3 expression and secretion via NF-kappaB- and Activator Protein-1-dependent monocyte networks.

    PubMed

    Green, Justin A; Elkington, Paul T; Pennington, Caroline J; Roncaroli, Federico; Dholakia, Shruti; Moores, Rachel C; Bullen, Anwen; Porter, Joanna C; Agranoff, Dan; Edwards, Dylan R; Friedland, Jon S

    2010-06-01

    Inflammatory tissue destruction is central to pathology in CNS tuberculosis (TB). We hypothesized that microglial-derived matrix metalloproteinases (MMPs) have a key role in driving such damage. Analysis of all of the MMPs demonstrated that conditioned medium from Mycobacterium tuberculosis-infected human monocytes (CoMTb) stimulated greater MMP-1, -3, and -9 gene expression in human microglial cells than direct infection. In patients with CNS TB, MMP-1/-3 immunoreactivity was demonstrated in the center of brain granulomas. Concurrently, CoMTb decreased expression of the inhibitors, tissue inhibitor of metalloproteinase-2, -3, and -4. MMP-1/-3 secretion was significantly inhibited by dexamethasone, which reduces mortality in CNS TB. Surface-enhanced laser desorption ionization time-of-flight analysis of CoMTb showed that TNF-alpha and IL-1beta are necessary but not sufficient for upregulating MMP-1 secretion and act synergistically to drive MMP-3 secretion. Chemical inhibition and promoter-reporter analyses showed that NF-kappaB and AP-1 c-Jun/FosB heterodimers regulate CoMTb-induced MMP-1/-3 secretion. Furthermore, NF-kappaB p65 and AP-1 c-Jun subunits were upregulated in biopsy granulomas from patients with cerebral TB. In summary, functionally unopposed, network-dependent microglial MMP-1/-3 gene expression and secretion regulated by NF-kappaB and AP-1 subunits were demonstrated in vitro and, for the first time, in CNS TB patients. Dexamethasone suppression of MMP-1/-3 gene expression provides a novel mechanism explaining the benefit of steroid therapy in these patients.

  8. Uncoupling Environmental pH and Intrabacterial Acidification from Pyrazinamide Susceptibility in Mycobacterium tuberculosis.

    PubMed

    Peterson, Nicholas D; Rosen, Brandon C; Dillon, Nicholas A; Baughn, Anthony D

    2015-12-01

    Pyrazinamide (PZA) is a first-line antitubercular drug for which the mode of action remains unresolved. Mycobacterium tuberculosis lacks measurable susceptibility to PZA under standard laboratory growth conditions. However, susceptibility to this drug can be induced by cultivation of the bacilli in an acidified growth medium. Previous reports suggested that the active form of PZA, pyrazinoic acid (POA), operates as a proton ionophore that confers cytoplasmic acidification when M. tuberculosis is exposed to an acidic environment. In this study, we demonstrate that overexpression of the PZA-activating enzyme PncA can confer PZA susceptibility to M. tuberculosis under neutral and even alkaline growth conditions. Furthermore, we find that wild-type M. tuberculosis displays increased susceptibility to POA relative to PZA in neutral and alkaline media. Utilizing a strain of M. tuberculosis that expresses a pH-sensitive green fluorescent protein (GFP), we find that unlike the bona fide ionophores monensin and carbonyl cyanide 3-chlorophenylhydrazone, PZA and POA do not induce rapid uncoupling or cytoplasmic acidification under conditions that promote susceptibility. Thus, based on these observations, we conclude that the antitubercular action of POA is independent of environmental pH and intrabacterial acidification.

  9. PapA3 is an acyltransferase required for polyacyltrehalose biosynthesis in Mycobacterium tuberculosis.

    PubMed

    Hatzios, Stavroula K; Schelle, Michael W; Holsclaw, Cynthia M; Behrens, Christopher R; Botyanszki, Zsofia; Lin, Fiona L; Carlson, Brian L; Kumar, Pawan; Leary, Julie A; Bertozzi, Carolyn R

    2009-05-08

    Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.

  10. Suppression of Mcl-1 induces apoptosis in mouse peritoneal macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Wang, Fei-Yu; Wang, Xin-Min; Wang, Chan; Wang, Xiao-Fang; Zhang, Yu-Qing; Wu, Jiang-Dong; Wu, Fang; Zhang, Wan-Jiang; Zhang, Le

    2016-04-01

    The effect of myeloid cell leukemia-1 (Mcl-1) inhibition on apoptosis of peritoneal macrophages in mice infected with Mycobacterium tuberculosis was investigated and the primary signaling pathway associated with the transcriptional regulation of Mcl-1 was identified. Real-time PCR and western blotting indicated that Mcl-1 transcript and protein expression are upregulated during infection with virulent M. tuberculosis H37Rv and Xinjiang strains but not with attenuated M. tuberculosis strain H37Ra or Bacillus Calmette-Guérin. Mcl-1 transcript and protein expression were downregulated by specific inhibitors of the Janus kinase/signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways (AG490, PD98059 and LY294002, respectively). The strongest inhibitor of Mcl-1 expression was PD98059, the MAPK inhibitor. Flow cytometry demonstrated that the rate of apoptosis in peritoneal macrophages is significantly higher in mice infected with M. tuberculosis and the rate of apoptosis is correlated with the virulence of the strain of M. tuberculosis. Apoptosis was found to be upregulated by AG490, PD98059 and LY294002, whereas inhibition of the MAPK pathway sensitized the infected macrophages to apoptosis. Taken together, these results suggest that specific downregulation of Mcl-1 significantly increases apoptosis of peritoneal macrophages and that the MAPK signaling pathway is the primary mediator of Mcl-1 expression.

  11. [A case of pulmonary Mycobacterium kansasii infection with pleural effusion, distinguished from pulmonary tuberculosis].

    PubMed

    Kimura, Yosuke; Kurosawa, Takayuki; Hosaka, Kiminori

    2014-09-01

    A case of pulmonary Mycobacterium kansasii infection with pleural effusion is very rare. We report a case of pulmonary Mycobacterium kansasii infection with pleural effusion, distinguished from pulmonary tuberculosis. A 44-year-old man presented to a clinic with a productive cough, sputum, and loss of appetite for several months. Chest X-ray and chest computed tomography (CT) showed right pleural effusion, centrilobular nodules and infiltrative shadows with cavities in the bilateral lung fields. The direct smear examination showed positive acid-fast bacilli (Gaffky 5). He was referred to our hospital for suspected recurrent pulmonary tuberculosis. We started anti-tuberculosis drugs because pulmonary tuberculosis complicated with pleurisy was first suspected from the findings of high ADA level (78.6 IU/l) of the effusion and positive result of interferon-gamma release assay (QuantiFERON TB-2G). But Mycobacterium tuberculosis and M. avium complex was not identified by the polymerase chain reaction method and the culture of the sputum was negative. At a later date, Mycobacterium kansasii was detected by sputum culture. The patient was diagnosed as pulmonary Mycobacterium kansasii infection and treatment with anti-tuberculosis drugs including RFP resulted in a good clinical response. This case was a rare case of pulmonary Mycobacterium kansasii infection with pleural effusion, distinguished from pulmonary tuberculosis.

  12. Mycobacterium tuberculosis Malate Synthase- and MPT51-Based Serodiagnostic Assay as an Adjunct to Rapid Identification of Pulmonary Tuberculosis

    PubMed Central

    Achkar, Jacqueline M.; Dong, Yuxin; Holzman, Robert S.; Belisle, John; Kourbeti, Irene S.; Sherpa, Tsering; Condos, Rany; Rom, William N.; Laal, Suman

    2006-01-01

    The 81-kDa malate synthase (MS; Rv 1837c) and the 27-kDa MPT51 (Rv 3803c) of Mycobacterium tuberculosis are immunodominant antigens recognized by serum antibodies from ∼80% of human immunodeficiency virus-negative smear-positive tuberculosis patients from India. We now provide evidence that the use of the MS/MPT51-based serodiagnostic assay can serve as an adjunct to sputum microscopy in the rapid diagnosis of pulmonary tuberculosis. PMID:17090645

  13. Constitutive expression of SMAR1 confers susceptibility to Mycobacterium tuberculosis infection in a transgenic mouse model

    PubMed Central

    Yadav, Bhawna; Malonia, Sunil K.; Majumdar, Subeer S.; Gupta, Pushpa; Wadhwa, Neerja; Badhwar, Archana; Gupta, Umesh D.; Katoch, Vishwa M.; Chattopadhyay, Samit

    2015-01-01

    Background & objectives: Studies involving animal models of experimental tuberculosis have elucidated the predominant role of cytokines secreted by T cells and macrophages to be an essential component of the immune response against Mycobacterium tuberculosis infection. The immune activities of CD4+ T cells are mediated in part by Th1 cytokine interferon gamma (IFN-γ) which is produced primarily by T cells and natural killer (NK) cells and critical for initiating the immune response against intracellular pathogen such as M. tuberculosis. Nuclear matrix protein SMAR1 plays an important role in V(D)J recombination, T helper cell differentiation and inflammatory diseases. In this study a transgenic mouse model was used to study the role of SMAR1 in M. tuberculosis infection. Methods: Wild type BALB/c, C57BL/6, BALB/c-EGFP-SMAR1 and C57BL/6-SMAR1 transgenic mice were infected with M. tuberculosis (H37Rv). A dose of 100 bacilli was used for infection via respiratory route. Bacterial load in lung and spleen of infected mice was determined at 2, 4, 6 and 8 wk post-infection. Gene expression analysis for Th1 cytokines and inducible nitric oxide synthase (iNOS) was performed in infected lung tissues by quantitative reverse transcription (RT)-PCR. Results: SMAR1 transgenic mice from both BALB/c and C57BL/6 genetic background displayed higher bacillary load and susceptibility to M. tuberculosis infection compared to wild type mice. This susceptibility was attributed due to compromised of Th1 response exhibited by transgenic mice. Interpretation & conclusions: SMAR1 transgenic mice exhibited susceptibility to M. tuberculosis infection in vivo irrespective of genetic background. This susceptibility was attributed to downregulation of Th1 response and its hallmark cytokine IFN-γ. Hence, SMAR1 plays an important role in modulating host immune response after M. tuberculosis infection. PMID:26831422

  14. Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

    SciTech Connect

    Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C.; Jacobs, Jr, William R.; Kremer, Laurent

    2011-08-24

    The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA{_}T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.

  15. Rapid susceptibility testing of Mycobacterium avium complex and Mycobacterium tuberculosis isolated from AIDS patients

    NASA Technical Reports Server (NTRS)

    Dhople, Arvind M.

    1994-01-01

    In ominous projections issued by both U.S. Public Health Service and the World Health Organization, the epidemic of HIV infection will continue to rise more rapidly worldwide than predicted earlier. The AIDS patients are susceptible to diseases called opportunistic infections of which tuberculosis and Mycobacterium avium complex (MAC) infection are most common. This has created an urgent need to uncover new drugs for the treatment of these infections. In the seventies, NASA scientists at Goddard Space Flight Center, Greenbelt, MD, had adopted a biochemical indicator, adenosine triphosphate (ATP), to detect presence of life in extraterrestrial space. We proposed to develop ATP assay technique to determine sensitivity of antibacterial compounds against MAC and M. tuberculosis.

  16. MetaMerge: scaling up genome-scale metabolic reconstructions with application to Mycobacterium tuberculosis

    PubMed Central

    2012-01-01

    Reconstructed models of metabolic networks are widely used for studying metabolism in various organisms. Many different reconstructions of the same organism often exist concurrently, forcing researchers to choose one of them at the exclusion of the others. We describe MetaMerge, an algorithm for semi-automatically reconciling a pair of existing metabolic network reconstructions into a single metabolic network model. We use MetaMerge to combine two published metabolic networks for Mycobacterium tuberculosis into a single network, which allows many reactions that could not be active in the individual models to become active, and predicts essential genes with a higher positive predictive value. PMID:22292986

  17. Mycobacterium tuberculosis Is Selectively Killed by Rifampin and Rifapentine in Hypoxia at Neutral pH.

    PubMed

    Iacobino, Angelo; Piccaro, Giovanni; Giannoni, Federico; Mustazzolu, Alessandro; Fattorini, Lanfranco

    2017-03-01

    The activities of rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide, isoniazid, amikacin, moxifloxacin, niclosamide, thioridazine, and pyrazinamide were tested against nonreplicating (dormant) Mycobacterium tuberculosis H37Rv under conditions of hypoxia at pHs 5.8 and 7.3, mimicking environments of cellular granulomas and caseous granulomas, respectively. At pH 5.8, several drugs killed dormant bacilli, with the best being rifampin and rifapentine. At pH 7.3, only rifampin and rifapentine efficiently killed dormant bacilli, while all other drugs showed little activity.

  18. Syntheses of Mycobactin Analogs as Potent and Selective Inhibitors of Mycobacterium tuberculosis

    PubMed Central

    Juárez-Hernández, Raúl E.; Franzblau, Scott G.

    2012-01-01

    Three analogs of mycobactin T, the siderophore secreted by Mycobacterium tuberculosis (Mtb) were synthesized and screened for their antibiotic activity against Mtb H37Rv and a broad panel of Gram-positive and Gram-negative bacteria. The synthetic mycobactins were potent (MIC90 0.02–0.88 μM in 7H12 media) and selective Mtb inhibitors, with no inhibitory activity observed against any other of the microorganisms tested. The maleimide-containing analog 40 represents a versatile platform for the development of mycobactin-drug conjugates, as well as other applications. PMID:22895786

  19. [Pyrazinamide monoresistant Mycobacterium tuberculosis in Manisa region, Turkey].

    PubMed

    Ozkütük, Nuri; Ecemiş, Talat; Sürücüoğlu, Süheyla

    2008-10-01

    Pyrazinamide (PZA) is a primary antituberculous drug. BACTEC 460TB is the recommended reference method for the detection of PZA resistance in Mycobacterium tuberculosis. This method is more expensive than the conventional susceptibility methods and therefore, it is recommended that each laboratory should establish their own protocol for the inclusion of PZA in the panel of primary drugs tested. One of the most important factors that help this decision is the prevalence of PZA resistance, particularly PZA monoresistance in the related community. The aim of the present study was to determine the extent of PZA monoresistance in M. tuberculosis complex (MTBC) isolates in our region. In this study, PZA susceptibility testing of 109 MTBC strains (susceptible to isoniazid, rifampicin, ethambutol and streptomycin) isolated from Manisa province in the Aegean region of Turkey was performed by using the BACTEC 460TB radiometric system (Becton Dickinson, MD). Two (1.8%) of the 109 isolates which were susceptible to all primary drugs revealed monoresistance against PZA. One of the PZA-monoresistant isolates has been identified as M. bovis and the other as M. tuberculosis by molecular method (Genotype MTBC, Hain Lifescience, Germany). The results of our study indicated that since the rate of PZA monoresistance was low, susceptibility testing of a panel of primary drugs without PZA may be an economical alternative in our region.

  20. Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis.

    PubMed

    Shi, Wanliang; Zhang, Xuelian; Jiang, Xin; Yuan, Haiming; Lee, Jong Seok; Barry, Clifton E; Wang, Honghai; Zhang, Wenhong; Zhang, Ying

    2011-09-16

    Pyrazinamide (PZA) is a first-line tuberculosis drug that plays a unique role in shortening the duration of tuberculosis chemotherapy. PZA is hydrolyzed intracellularly to pyrazinoic acid (POA) by pyrazinamidase (PZase, encoded by pncA), an enzyme frequently lost in PZA-resistant strains, but the target of POA in Mycobacterium tuberculosis has remained elusive. Here, we identify a previously unknown target of POA as the ribosomal protein S1 (RpsA), a vital protein involved in protein translation and the ribosome-sparing process of trans-translation. Three PZA-resistant clinical isolates without pncA mutation harbored RpsA mutations. RpsA overexpression conferred increased PZA resistance, and we confirmed that POA bound to RpsA (but not a clinically identified ΔAla mutant) and subsequently inhibited trans-translation rather than canonical translation. Trans-translation is essential for freeing scarce ribosomes in nonreplicating organisms, and its inhibition may explain the ability of PZA to eradicate persisting organisms.

  1. Immunological consequences of strain variation within the Mycobacterium tuberculosis complex

    PubMed Central

    Tientcheu, Leopold D.; Ndengane, Mthawelenga; Andoseh, Genevieve; Kampmann, Beate; Wilkinson, Robert J

    2017-01-01

    In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype‐dependent host–pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies. PMID:28150302

  2. Genotyping of ancient Mycobacterium tuberculosis strains reveals historic genetic diversity

    PubMed Central

    Müller, Romy; Roberts, Charlotte A.; Brown, Terence A.

    2014-01-01

    The evolutionary history of the Mycobacterium tuberculosis complex (MTBC) has previously been studied by analysis of sequence diversity in extant strains, but not addressed by direct examination of strain genotypes in archaeological remains. Here, we use ancient DNA sequencing to type 11 single nucleotide polymorphisms and two large sequence polymorphisms in the MTBC strains present in 10 archaeological samples from skeletons from Britain and Europe dating to the second–nineteenth centuries AD. The results enable us to assign the strains to groupings and lineages recognized in the extant MTBC. We show that at least during the eighteenth–nineteenth centuries AD, strains of M. tuberculosis belonging to different genetic groups were present in Britain at the same time, possibly even at a single location, and we present evidence for a mixed infection in at least one individual. Our study shows that ancient DNA typing applied to multiple samples can provide sufficiently detailed information to contribute to both archaeological and evolutionary knowledge of the history of tuberculosis. PMID:24573854

  3. A New Approach for Pyrazinamide Susceptibility Testing in Mycobacterium tuberculosis

    PubMed Central

    Loli, Sebastian; Gilman, Robert H.; Gutierrez, Andrés; Fuentes, Patricia; Cotrina, Milagros; Kirwan, Daniela; Sheen, Patricia

    2012-01-01

    Background: Pyrazinamide (PZA) is an important drug in the treatment of tuberculosis. Microbiological methods of PZA susceptibility testing are controversial and have low reproducibility. After conversion of PZA into pyrazinoic acid (POA) by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. Objective: To evaluate the rate of POA extrusion from Mycobacterium tuberculosis as a parameter to detect PZA resistance. Methods: The rate of POA extrusion and PZA susceptibility determined by BACTEC 460 were measured for 34 strains in a previous study. PZA resistance was modeled in a logistic regression with the pyrazinoic efflux rate. Result: POA efflux rate predicted PZA resistance with 70.83%–92.85% sensitivity and 100% specificity compared with BACTEC 460. Conclusion: POA efflux rate could be a useful tool for predicting PZA resistance in M. tuberculosis. Further exploration of this approach may lead to the development of new tools for diagnosing PZA resistance, which may be of public health importance. PMID:22372927

  4. Mycobacterium tuberculosis Transcription Machinery: Ready To Respond to Host Attacks

    PubMed Central

    Flentie, Kelly; Garner, Ashley L.

    2016-01-01

    Regulating responses to stress is critical for all bacteria, whether they are environmental, commensal, or pathogenic species. For pathogenic bacteria, successful colonization and survival in the host are dependent on adaptation to diverse conditions imposed by the host tissue architecture and the immune response. Once the bacterium senses a hostile environment, it must enact a change in physiology that contributes to the organism's survival strategy. Inappropriate responses have consequences; hence, the execution of the appropriate response is essential for survival of the bacterium in its niche. Stress responses are most often regulated at the level of gene expression and, more specifically, transcription. This minireview focuses on mechanisms of regulating transcription initiation that are required by Mycobacterium tuberculosis to respond to the arsenal of defenses imposed by the host during infection. In particular, we highlight how certain features of M. tuberculosis physiology allow this pathogen to respond swiftly and effectively to host defenses. By enacting highly integrated and coordinated gene expression changes in response to stress, M. tuberculosis is prepared for battle against the host defense and able to persist within the human population. PMID:26883824

  5. Isolation of Mycobacterium tuberculosis from captive Ateles paniscus.

    PubMed

    Rocha, Vivianne Cambuí Mesquita; Ikuta, Cássia Yumi; Gomes, Marcelo S; Quaglia, Fausto; Matushima, Eliana R; Ferreira Neto, José Soares

    2011-05-01

    An adult female red-faced black spider monkey (Ateles paniscus), housed for 2 years in the Parque Estoril Zoo in São Paulo, Brazil, showed apathy. Clinical examination revealed discrete emaciation, swelling and induration of lymph nodes, and presence of a mass in the abdominal cavity. Therapies with enrofloxacin, azithromycin, and ceftiofur were ineffective. The animal died after 6 months. Necropsy and histopathology confirmed granulommas in lymph nodes, parietal and visceral pleura, lungs, liver, spleen, and kidneys. Acid-fast bacilli were isolated and identified as Mycobacterium tuberculosis by polymerase chain reaction restriction analysis and Spoligotyping techniques. The zoo personnel and other animals that had had contact with the infected primate were negative to tuberculosis diagnostic procedures, such as sputum exam (baciloscopy) and thorax radiography. It was impossible to determine whether the infection occurred before or after the arrival of the animal to the Parque Estoril Zoo. This is the first report of M. tuberculosis infection in Ateles paniscus, a neotropical primate.

  6. Modulation of cAMP metabolism in Mycobacterium tuberculosis and its effect on host infection.

    PubMed

    Barba, Jeannette; Alvarez, Angel H; Flores-Valdez, Mario Alberto

    2010-05-01

    Mycobacterium tuberculosis remains the single most relevant bacterial infectious agent as Tuberculosis is estimated to affect one-third of the world population. Like other microorganisms, M. tuberculosis needs to sense and adapt to changes in the several niches where it is found, ranging from the environment to a number of host-adapted programs, including infection of cell types such as macrophages, dendritic cells, epithelial cells and adipocytes. A strategy commonly used by cells to respond to such changes consists of producing small molecules known as second messengers. 3',5'-cyclic adenosine monophosphate (cAMP) is one of the best-studied second messengers in many organisms, and in recent years its participation during the M. tuberculosis infection cycle has just begun to be thoroughly considered. In this work, we aimed to provide a perspective of how cAMP metabolism proceeds in M. tuberculosis, which genes are activated in response to cAMP signaling in this organism, and discuss the evidence for bacterially produced cAMP use during infection. Furthermore, key issues needing to be addressed for better understanding cAMP physiology in slow-growing pathogenic mycobacteria are presented.

  7. Adaptation to environmental stimuli within the host: two-component signal transduction systems of Mycobacterium tuberculosis.

    PubMed

    Bretl, Daniel J; Demetriadou, Chrystalla; Zahrt, Thomas C

    2011-12-01

    Pathogenic microorganisms encounter a variety of environmental stresses following infection of their respective hosts. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is an unusual bacterial pathogen in that it is able to establish lifelong infections in individuals within granulomatous lesions that are formed following a productive immune response. Adaptation to this highly dynamic environment is thought to be mediated primarily through transcriptional reprogramming initiated in response to recognition of stimuli, including low-oxygen tension, nutrient depletion, reactive oxygen and nitrogen species, altered pH, toxic lipid moieties, cell wall/cell membrane-perturbing agents, and other environmental cues. To survive continued exposure to these potentially adverse factors, M. tuberculosis encodes a variety of regulatory factors, including 11 complete two-component signal transduction systems (TCSSs) and several orphaned response regulators (RRs) and sensor kinases (SKs). This report reviews our current knowledge of the TCSSs present in M. tuberculosis. In particular, we discuss the biochemical and functional characteristics of individual RRs and SKs, the environmental stimuli regulating their activation, the regulons controlled by the various TCSSs, and the known or postulated role(s) of individual TCSSs in the context of M. tuberculosis physiology and/or pathogenesis.

  8. Adaptation to Environmental Stimuli within the Host: Two-Component Signal Transduction Systems of Mycobacterium tuberculosis

    PubMed Central

    Bretl, Daniel J.; Demetriadou, Chrystalla; Zahrt, Thomas C.

    2011-01-01

    Summary: Pathogenic microorganisms encounter a variety of environmental stresses following infection of their respective hosts. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is an unusual bacterial pathogen in that it is able to establish lifelong infections in individuals within granulomatous lesions that are formed following a productive immune response. Adaptation to this highly dynamic environment is thought to be mediated primarily through transcriptional reprogramming initiated in response to recognition of stimuli, including low-oxygen tension, nutrient depletion, reactive oxygen and nitrogen species, altered pH, toxic lipid moieties, cell wall/cell membrane-perturbing agents, and other environmental cues. To survive continued exposure to these potentially adverse factors, M. tuberculosis encodes a variety of regulatory factors, including 11 complete two-component signal transduction systems (TCSSs) and several orphaned response regulators (RRs) and sensor kinases (SKs). This report reviews our current knowledge of the TCSSs present in M. tuberculosis. In particular, we discuss the biochemical and functional characteristics of individual RRs and SKs, the environmental stimuli regulating their activation, the regulons controlled by the various TCSSs, and the known or postulated role(s) of individual TCSSs in the context of M. tuberculosis physiology and/or pathogenesis. PMID:22126994

  9. Bioactive Potential of Actinomycetes from Less Explored Ecosystems against Mycobacterium tuberculosis and Other Nonmycobacterial Pathogens

    PubMed Central

    Venugopal, Gopikrishnan; Subramaniam, Balaji; Ramasamy, Balagurunathan

    2014-01-01

    Bioactive potential of actinomycetes isolated from certain less explored Indian ecosystems against Mycobacterium tuberculosis and other nonmycobacterial pathogens was investigated. Actinomycetes were isolated from the soil samples collected from desert, coffee plantation, rubber forest, and hill area and their cultural and micromorphological characteristics were studied. Crude extracts were prepared by agar surface fermentation and tested against M. tuberculosis isolates by luciferase reporter phage (LRP) assay at 100 µg/mL. Activity against nonmycobacterial pathogens was studied by agar plug method. Totally 54 purified cultures of actinomycetes including 43 Streptomyces and 11 non-Streptomyces were isolated. While screening for antitubercular activity, extracts of 39 actinomycetes showed activity against one or more M. tuberculosis isolates whereas 27 isolates exhibited antagonistic activity against nonmycobacterial pathogens. In particular crude extracts from sixteen actinomycete isolates inhibited all the three M. tuberculosis isolates tested. Findings of the present study concluded that less explored ecosystems investigated in this study are the potential resource for bioactive actinomycetes. Further purification and characterization of active molecule from the potential extracts will pave the way for determination of MIC, toxicity, and specificity studies. PMID:27437460

  10. Combined Bioinformatic and Rational Design Approach To Develop Antimicrobial Peptides against Mycobacterium tuberculosis

    PubMed Central

    Pearson, C. Seth; Kloos, Zachary; Murray, Brian; Tabe, Ebot; Gupta, Monica; Kwak, Jun Ha; Karande, Pankaj

    2016-01-01

    Drug-resistant pathogens are a growing problem, and novel strategies are needed to combat this threat. Among the most significant of these resistant pathogens is Mycobacterium tuberculosis, which is an unusually difficult microbial target due to its complex membrane. Here, we design peptides for specific activity against M. tuberculosis using a combination of “database filtering” bioinformatics, protein engineering, and de novo design. Several variants of these peptides are structurally characterized to validate the design process. The designed peptides exhibit potent activity (MIC values as low as 4 μM) against M. tuberculosis and also exhibit broad activity against a host of other clinically relevant pathogenic bacteria such as Gram-positive bacteria (Streptococcus) and Gram-negative bacteria (Escherichia coli). They also display excellent selectivity, with low cytotoxicity against cultured macrophages and lung epithelial cells. These first-generation antimicrobial peptides serve as a platform for the design of antibiotics and for investigating structure-activity relationships in the context of the M. tuberculosis membrane. The antimicrobial peptide design strategy is expected to be generalizable for any pathogen for which an activity database can be created. PMID:26902758

  11. Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide.

    PubMed

    Zhang, Y; Scorpio, A; Nikaido, H; Sun, Z

    1999-04-01

    Pyrazinamide (PZA) is an important antituberculosis drug. Unlike most antibacterial agents, PZA, despite its remarkable in vivo activity, has no activity against Mycobacterium tuberculosis in vitro except at an acidic pH. M. tuberculosis is uniquely susceptible to PZA, but other mycobacteria as well as nonmycobacteria are intrinsically resistant. The role of acidic pH in PZA action and the basis for the unique PZA susceptibility of M. tuberculosis are unknown. We found that in M. tuberculosis, acidic pH enhanced the intracellular accumulation of pyrazinoic acid (POA), the active derivative of PZA, after conversion of PZA by pyrazinamidase. In contrast, at neutral or alkaline pH, POA was mainly found outside M. tuberculosis cells. PZA-resistant M. tuberculosis complex organisms did not convert PZA into POA. Unlike M. tuberculosis, intrinsically PZA-resistant M. smegmatis converted PZA into POA, but it did not accumulate POA even at an acidic pH, due to a very active POA efflux mechanism. We propose that a deficient POA efflux mechanism underlies the unique susceptibility of M. tuberculosis to PZA and that the natural PZA resistance of M. smegmatis is due to a highly active efflux pump. These findings may have implications with regard to the design of new antimycobacterial drugs.

  12. Characterization of a Mycobacterium tuberculosis Nanocompartment and Its Potential Cargo Proteins*

    PubMed Central

    Contreras, Heidi; Joens, Matthew S.; McMath, Lisa M.; Le, Vincent P.; Tullius, Michael V.; Kimmey, Jaqueline M.; Bionghi, Neda; Horwitz, Marcus A.; Fitzpatrick, James A. J.; Goulding, Celia W.

    2014-01-01

    Mycobacterium tuberculosis has evolved various mechanisms by which the bacterium can maintain homeostasis under numerous environmental assaults generated by the host immune response. M. tuberculosis harbors enzymes involved in the oxidative stress response that aid in survival during the production of reactive oxygen species in activated macrophages. Previous studies have shown that a dye-decolorizing peroxidase (DyP) is encapsulated by a bacterial nanocompartment, encapsulin (Enc), whereby packaged DyP interacts with Enc via a unique C-terminal extension. M. tuberculosis also harbors an encapsulin homolog (CFP-29, Mt-Enc), within an operon with M. tuberculosis DyP (Mt-DyP), which contains a C-terminal extension. Together these observations suggest that Mt-DyP interacts with Mt-Enc. Furthermore, it has been suggested that DyPs may function as either a heme-dependent peroxidase or a deferrochelatase. Like Mt-DyP, M. tuberculosis iron storage ferritin protein, Mt-BfrB, and an M. tuberculosis protein involved in folate biosynthesis, 7,8-dihydroneopterin aldolase (Mt-FolB), have C-terminal tails that could also interact with Mt-Enc. For the first time, we show by co-purification and electron microscopy that mycobacteria via Mt-Enc can encapsulate Mt-DyP, Mt-BfrB, and Mt-FolB. Functional studies of free or encapsulated proteins demonstrate that they retain their enzymatic activity within the Mt-Enc nanocompartment. Mt-DyP, Mt-FolB, and Mt-BfrB all have antioxidant properties, suggesting that if these proteins are encapsulated by Mt-Enc, then this nanocage may play a role in the M. tuberculosis oxidative stress response. This report provides initial structural and biochemical clues regarding the molecular mechanisms that utilize compartmentalization by which the mycobacterial cell may aid in detoxification of the local environment to ensure long term survival. PMID:24855650

  13. Immune Subversion by Mycobacterium tuberculosis through CCR5 Mediated Signaling: Involvement of IL-10

    PubMed Central

    Das, Shibali; Banerjee, Sayantan; Majumder, Saikat; Paul Chowdhury, Bidisha; Goswami, Avranil; Halder, Kuntal; Chakraborty, Urmita; Pal, Nishith K.; Majumdar, Subrata

    2014-01-01

    Tuberculosis is characterized by severe immunosuppression of the host macrophages, resulting in the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates C-C Chemokine Receptor 5 (CCR5) to enhance IL-10 production, indicating the possible involvement of CCR5 in regulation of the host immune response. Here, we found that Mycobacterium infection significantly increased CCR5 expression in macrophages there by facilitating the activation of its downstream signaling. These events culminated in up-regulation of the immunosuppressive cytokine IL-10 production, which was further associated with the down-regulation of macrophage MHC-II expression along with the up-regulation of CCR5 expression via engagement of STAT-3 in a positive feedback loop. Treatment of macrophages with CCR5 specific siRNA abrogated the IL-10 production and restored MHCII expression. While, in vivo CCR5 silencing was also effective for the restoration of host immune responses against tuberculosis. This study demonstrated that CCR5 played a very critical role for the immune subversion mechanism employed by the pathogen. PMID:24695099

  14. Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420.

    PubMed

    Blondiaux, Nicolas; Moune, Martin; Desroses, Matthieu; Frita, Rosangela; Flipo, Marion; Mathys, Vanessa; Soetaert, Karine; Kiass, Mehdi; Delorme, Vincent; Djaout, Kamel; Trebosc, Vincent; Kemmer, Christian; Wintjens, René; Wohlkönig, Alexandre; Antoine, Rudy; Huot, Ludovic; Hot, David; Coscolla, Mireia; Feldmann, Julia; Gagneux, Sebastien; Locht, Camille; Brodin, Priscille; Gitzinger, Marc; Déprez, Benoit; Willand, Nicolas; Baulard, Alain R

    2017-03-17

    Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in M. tuberculosis, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.

  15. Structure of the Covalent Adduct Formed Between Mycobacterium tuberculosis beta-Lactamase and Clavulanate

    SciTech Connect

    Tremblay,L.; Hugonnet, J.; Blanchard, J.

    2008-01-01

    The intrinsic resistance of Mycobacterium tuberculosis to the {beta}-lactam class of antibiotics arises from a chromosomally encoded, extended spectrum, class A {beta}-lactamase, BlaC. Herein, we report the X-ray crystallographic structure of BlaC inhibited with clavulanate at a resolution of 1.7 Angstroms with an R-factor value of 0.180 and R-free value of 0.212 for the m/z +154 clavulanate-derived fragment observed in the active site. Structural evidence reveals the presence of hydrogen bonds to the C1 carbonyl along with a coplanar arrangement of C1, C2, C3, and N4, which favors enolization to generate a trans-a, {beta}-eneamine, stabilizing the +154 adduct from hydrolysis. The irreversible inhibition of BlaC suggests that treatment of M. tuberculosis with a combination of a {beta}-lactam antibiotic and clavulanate may lead to rapid bactericidal activity.

  16. Implication of the RD(Rio) Mycobacterium tuberculosis sublineage in multidrug resistant tuberculosis in Portugal.

    PubMed

    David, Susana; Duarte, Elsa L; Leite, Clarice Queico Fugimura; Ribeiro, João-Nuno; Maio, José-Nuno; Paixão, Eleonora; Portugal, Clara; Sancho, Luísa; Germano de Sousa, José

    2012-10-01

    Multidrug and extensively drug resistant Mycobacterium tuberculosis are a threat to tuberculosis contro