Depletion of M. tuberculosis GlmU from Infected Murine Lungs Effects the Clearance of the Pathogen
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{"title"=>"Depletion of M. tuberculosis GlmU from Infected Murine Lungs Effects the Clearance of the Pathogen", "type"=>"journal", "authors"=>[{"first_name"=>"Vijay", "last_name"=>"Soni", "scopus_author_id"=>"55887616700"}, {"first_name"=>"Sandeep", "last_name"=>"Upadhayay", "scopus_author_id"=>"56941010200"}, {"first_name"=>"Priyanka", "last_name"=>"Suryadevara", "scopus_author_id"=>"55351377300"}, {"first_name"=>"Ganesh", "last_name"=>"Samla", "scopus_author_id"=>"56940787200"}, {"first_name"=>"Archana", "last_name"=>"Singh", "scopus_author_id"=>"56939487300"}, {"first_name"=>"Perumal", "last_name"=>"Yogeeswari", "scopus_author_id"=>"6602479079"}, {"first_name"=>"Dharmarajan", "last_name"=>"Sriram", "scopus_author_id"=>"7005623982"}, {"first_name"=>"Vinay Kumar", "last_name"=>"Nandicoori", "scopus_author_id"=>"23036296300"}], "year"=>2015, "source"=>"PLoS Pathogens", "identifiers"=>{"issn"=>"15537374", "doi"=>"10.1371/journal.ppat.1005235", "sgr"=>"84946059622", "scopus"=>"2-s2.0-84946059622", "pmid"=>"26489015", "pui"=>"606741022"}, "id"=>"c257b9ad-a121-38c5-a009-69863d51140a", "abstract"=>"M. tuberculosis N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmUMtb) is a bi-functional enzyme engaged in the synthesis of two metabolic intermediates N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and UDP-GlcNAc, catalyzed by the C- and N-terminal domains respectively. UDP-GlcNAc is a key metabolite essential for the synthesis of peptidoglycan, disaccharide linker, arabinogalactan and mycothiols. While glmUMtb was predicted to be an essential gene, till date the role of GlmUMtb in modulating the in vitro growth of Mtb or its role in survival of pathogen ex vivo / in vivo have not been deciphered. Here we present the results of a comprehensive study dissecting the role of GlmUMtb in arbitrating the survival of the pathogen both in vitro and in vivo. We find that absence of GlmUMtb leads to extensive perturbation of bacterial morphology and substantial reduction in cell wall thickness under normoxic as well as hypoxic conditions. Complementation studies show that the acetyl- and uridyl- transferase activities of GlmUMtb are independently essential for bacterial survival in vitro, and GlmUMtb is also found to be essential for mycobacterial survival in THP-1 cells as well as in guinea pigs. Depletion of GlmUMtb from infected murine lungs, four weeks post infection, led to significant reduction in the bacillary load. The administration of Oxa33, a novel oxazolidine derivative that specifically inhibits GlmUMtb, to infected mice resulted in significant decrease in the bacillary load. Thus our study establishes GlmUMtb as a strong candidate for intervention measures against established tuberculosis infections.", "link"=>"http://www.mendeley.com/research/depletion-m-tuberculosis-glmu-infected-murine-lungs-effects-clearance-pathogen", "reader_count"=>17, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Librarian"=>2, "Researcher"=>2, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>1, "Student > Master"=>2, "Other"=>1, "Student > Bachelor"=>2, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Librarian"=>2, "Researcher"=>2, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>1, "Student > Master"=>2, "Other"=>1, "Student > Bachelor"=>2, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Engineering"=>2, "Biochemistry, Genetics and Molecular Biology"=>3, "Nursing and Health Professions"=>1, "Agricultural and Biological Sciences"=>5, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Computer Science"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>5}, "Computer Science"=>{"Computer Science"=>2}, "Nursing and Health Professions"=>{"Nursing and Health Professions"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Unspecified"=>{"Unspecified"=>3}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "group_count"=>0}

Scopus | Further Information

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  • {"files"=>["https://ndownloader.figshare.com/files/2368350"], "description"=>"<p><b>(A)</b> Confocal microscopy images of THP1 cells infected with <i>Rv</i> and <i>Rv</i>∆<i>glmU</i> in the presence or absence of ATc as indicated were taken 48 h post infection. Bacteria were stained with FITC (green) and lysosomes were stained with Lyso Tracker red DND 99 (red). Scale bars, 10 μm. <b>(B)</b> Quantification of percentage co-localization of bacterium and lysosomes in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005235#ppat.1005235.g003\" target=\"_blank\">Fig 3A</a>. n = 10 to 15. <b>(C)</b> PMA differentiated THP1 cells were infected with <i>Rv</i> and <i>Rv</i>∆<i>glmU</i> and either ATc (400 ng/ml) or the vehicle were added at 0 h. CFUs were enumerated at different time points post infection. The experiment was performed in triplicates and error bars represent s.e.m. <b>(D)</b> Guinea pigs were infected with 150–200 bacilli CFUs of <i>Rv</i> or <i>Rv</i>∆<i>glmU</i> and Dox was provided in the water as indicated. Guinea pigs (n = 2) were sacrificed on day 1 and homogenates from the lungs were plated in triplicates to determine the implantation. Guinea pigs (6 guinea pigs /group) were sacrificed four weeks post infection and CFUs were determined in the lung homogenates and results were plotted with log<sub>10</sub> /lung on the Y-axis and samples on the X-axis. At four weeks post infection mean CFUs for <i>Rv</i> +Dox or <i>Rv</i>∆<i>glmU</i> +Dox and <i>Rv</i>∆<i>glmU</i>–Dox were 5.2, 0 and 5.11 on log<sub>10</sub> scale.***<i>p</i><0.0001, two tailed non parametric <i>t</i>-test, error bars represent s.e.m. <b>(E)</b> Overall pathology (upper panel) and histopathology (lower panel, HE, 40X) of the infected guinea pig lungs four weeks post infection. Both <i>Rv</i> +Dox and <i>Rv</i>∆<i>glmU</i> -Dox were having prominent granuloma and necrotic lesions in the central and epithelioid cells and lymphocytes around it. Guinea pigs infected with <i>Rv</i>∆<i>glmU</i> +Dox were showing normal lung parenchyma with clear alveolar spaces. G = Granuloma, AS = Alveolar Space. <b>(F)</b> Granuloma scores from histopathology results.</p>", "links"=>[], "tags"=>["survival", "thp", "GlmUMtb", "weeks post infection", "bacillary load", "cell wall thickness", "Complementation studies show", "Infected Murine Lungs Effects"], "article_id"=>1582092, "categories"=>["Biological Sciences"], "users"=>["Vijay Soni", "Sandeep Upadhayay", "Priyanka Suryadevara", "Ganesh Samla", "Archana Singh", "Perumal Yogeeswari", "Dharmarajan Sriram", "Vinay Kumar Nandicoori"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1005235.g005", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Presence_of_GlmU_Mtb_is_obligatory_for_the_survival_of_Mtb_in_the_host_/1582092", "title"=>"Presence of GlmU<sub>Mtb</sub> is obligatory for the survival of <i>Mtb</i> in the host.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-21 03:24:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/2368347"], "description"=>"<p><b> (A)</b> Schematic outline of the experiment. <b>(B)</b><i>Rv</i> and <i>Rv</i>∆<i>glmU</i> cultures were seeded at an initial <i>A</i><sub>600</sub> of 0.1 in 1.5 HPLC tubes or in 500 ml flasks containing penetrable caps. Establishment of hypoxia was monitored with the help of methylene blue color change (blue to colorless). ATc was added to <i>Rv</i> on 20<sup>th</sup> day and to <i>Rv</i>∆<i>glmU</i> on 20<sup>th</sup> and 40<sup>th</sup> day. CFUs were enumerated on day 0, day 20 and day 42. *<i>p</i><0.05 or ***<i>p</i><0.005, two tailed non parametric <i>t</i>-test, Error bars indicate s.e.m. and the experiment was performed in triplicates (n = 3). <b>(C)</b> INH (50 ng/ml) was added to the hypoxic <i>Rv</i> cultures on 20<sup>th</sup> and 40<sup>th</sup> day. CFUs were enumerated on day 42. *<i>p</i><0.05 or NS: non- significant, two tailed non parametric <i>t</i>-test, Error bars indicate s.e.m. and n = 3. <b>(D)</b> Large scale hypoxic cultures were pelleted down on day 42 and processed for scanning electron microscopy (upper panel) or transmission electron microscopy (lower panel) imaging. (SEM: scale bars: 1 μm; TEM scale bar: 20 nm). Cell wall thickness is indicated by red lines. <b>(E)</b> Cell wall thickness was measured in nm for 20 to 54 cells from the TEM images of different samples (representative image shown in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005235#ppat.1005235.g003\" target=\"_blank\">Fig 3C</a>). ***<i>p</i>< 0.0001 and 0.0005, two tailed non parametric <i>t</i>-test, Error bars indicate s.e.m.</p>", "links"=>[], "tags"=>["survival", "thp", "GlmUMtb", "weeks post infection", "bacillary load", "cell wall thickness", "Complementation studies show", "Infected Murine Lungs Effects"], "article_id"=>1582089, "categories"=>["Biological Sciences"], "users"=>["Vijay Soni", "Sandeep Upadhayay", "Priyanka Suryadevara", "Ganesh Samla", "Archana Singh", "Perumal Yogeeswari", "Dharmarajan Sriram", "Vinay Kumar Nandicoori"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1005235.g003", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_GlmU_Mtb_depletion_of_dormant_bacteria_/1582089", "title"=>"Effect of GlmU<sub>Mtb</sub> depletion of dormant bacteria.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-21 03:24:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/2368345"], "description"=>"<p><b>(A)</b><i>Rv</i> and <i>Rv</i>∆<i>glmU</i> cultures grown to an <i>A</i><sub><i>600</i></sub> of 0.8 were used to seed fresh cultures at an initial <i>A</i><sub>600</sub> of 0.1 in the presence or absence of ATc as indicated and the day wise growth was monitored for six days. Inset shows the growth in the culture tubes after six days. The experiment was performed in triplicates (n = 3). Error bars indicate standard error of mean (s.e.m). <b>(B)</b> Day 0 and Day 6 cultures grown in the presence or absence of ATc were serially diluted and spotted on 7H11 agar plates. <b>(C)</b> Large scale cultures were inoculated at A<sub>600</sub> of 0.1 in the presence or absence of ATc and the WCLs were prepared on different days post ATc addition (as indicated). WCLs were resolved and probed with anti-GlmU<sub>Mtb</sub> and anti-GroEL1 antibodies. <b>(D)</b> Scanning electron microscopy of <i>Rv</i> and <i>Rv</i>∆<i>glmU</i> grown for 72 h with or without ATc as indicated. Scale bars in upper panel were 10 nm and for the inset was 200 nm. The experiment was repeated thrice. <b>(E)</b> Transmission electron micrographs results at 50,000X of <i>Rv</i> and <i>Rv</i>∆<i>glmU</i> cultures grown with or without ATc. Red lines depict the cell wall thickness. Scale bar: 20 nm. <b>(F)</b> Cell wall thickness was measured in nm for 15 to 22 cells for each sample. ***<i>p</i><0.0001, two tailed non parametric <i>t</i>-test, mean, s.e.m.</p>", "links"=>[], "tags"=>["survival", "thp", "GlmUMtb", "weeks post infection", "bacillary load", "cell wall thickness", "Complementation studies show", "Infected Murine Lungs Effects"], "article_id"=>1582087, "categories"=>["Biological Sciences"], "users"=>["Vijay Soni", "Sandeep Upadhayay", "Priyanka Suryadevara", "Ganesh Samla", "Archana Singh", "Perumal Yogeeswari", "Dharmarajan Sriram", "Vinay Kumar Nandicoori"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1005235.g002", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_GlmU_Mtb_depletion_affects_the_bacterial_survival_by_perturbing_the_cell_wall_structure_/1582087", "title"=>"GlmU<sub>Mtb</sub> depletion affects the bacterial survival by perturbing the cell wall structure.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-21 03:24:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/2368353"], "description"=>"<p><b>(A)</b> Synthetic scheme for the preparation of <i>4-(4-(benzyloxy)benzylidene)-2-(naphthalen-1-yl)oxazol-5(4H)-one</i> (Oxa33). <b>(B)</b> IC<sub>50</sub> values were determined by varying the concentration of Oxa33 and calculating the percent activity with respect to the uridyltransferase activity of 0.75 pM conc GlmU<sub>Mtb</sub> in the presence of 5% DMSO (vehicle). IC<sub>50</sub> value was calculated by plotting percentage activities against different log<sub>10</sub> values of inhibitor concentrations (μM). Experiment was performed in triplicates and the error bars represent s.e.m. <b>(C)</b> Left panel depicts docking surface representation of the allosteric site on the of the GlmU<sub>Mtb</sub> allosteric site in complex with Oxa33. Right panel represents ribbon cartoon showing the amino acids residues of allosteric site and hinge region interacting with Oxa33 (in ball and stick model). Tyr150, Glu250 and Arg 253 were found to be in hydrogen bonding with carbonyl oxygen over the oxazole ring. While Leu144, Pro147, Phe148, Tyr150, Ala233, Ala236 and Leu247 participate in strong hydrophobic interactions with Oxa33. <b>(D)</b> Based on the data above GlmU<sub>Mtb</sub> allosteric site mutants were created, proteins were purified and the uridyltransferase activity of GlmU<sub>Mtb</sub> mutants was determined. Wild type and mutant GlmU<sub>Mtb</sub> proteins show almost comparable activity. <b>(E)</b> IC<sub>50</sub> values in μM were determined as described above for the wild type and the mutant GlmU<sub>Mtb</sub> proteins. Tables shows the values obtained for wild type and mutant enzymes. Experiments were performed in triplicates and the error bars represent s.e.m. <b>(F)</b> Superimposed view of GlmU<sub>Mtb</sub>-Oxa33 complex before (green) and after (orange) the simulation time period. Showing a partial closure of uridyltransferase site (decreased volume) thereby making it unavailable for its substrates for catalysis.</p>", "links"=>[], "tags"=>["survival", "thp", "GlmUMtb", "weeks post infection", "bacillary load", "cell wall thickness", "Complementation studies show", "Infected Murine Lungs Effects"], "article_id"=>1582095, "categories"=>["Biological Sciences"], "users"=>["Vijay Soni", "Sandeep Upadhayay", "Priyanka Suryadevara", "Ganesh Samla", "Archana Singh", "Perumal Yogeeswari", "Dharmarajan Sriram", "Vinay Kumar Nandicoori"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1005235.g007", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Oxa33_a_novel_inhibitor_against_GlmU_Mtb_binds_at_allosteric_site_of_uridyltransferase_domain_/1582095", "title"=>"Oxa33, a novel inhibitor against GlmU<sub>Mtb</sub> binds at allosteric site of uridyltransferase domain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-21 03:24:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/2368354"], "description"=>"<p><b>(A)</b><i>Rv</i> and <i>Rv</i>::<i>glmU</i><sub><i>tet-on</i></sub> cultures were grown in presence of 1 μg/ml ATc and different concentrations of Oxa33 or INH (as indicated). 0.02% resazurin dye was added on day 7. Pink colour indicates viable bacteria while blue colour indicates dead bacteria. ‘C’ denotes culture media control. The experiment was performed thrice. <b>(B)</b> Differentiated THP1 were infected either with <i>Rv</i> or <i>Rv</i>::<i>glmU</i><sub><i>tet-on</i></sub> and grown in RPMI media containing 400 ng/ml ATc. 24 h post infection different concentrations (as indicated) of Oxa33 or vehicle control was supplemented to the media for the next 72 h followed by CFU enumeration. Experiment was performed in triplicates and the error bars represent s.e.m. <b>(C)</b> BALB/c mice (7 to 11 mice / group) were infected with <i>Rv</i> and the infection was allowed to be established for 28 day. Oxa33 (50 mg/kg, intraperitoneal, in 2.5% Tween 80) or INH (25 mg/kg, in drinking water with 5% sucrose) were administered to the mice for a subsequent period of 56 days. CFU in the lungs of mice were determined on 1, 28 and 84 days post infection. Day 1<sup>st</sup> and day 28<sup>th</sup> CFUs show successful implantation and establishment of infection, respectively. 56 days of Oxa33 or INH treatment partially reduces the mean bacillary load to 2.78 and 1.3 on log<sub>10</sub> scale. ***<i>p</i><0.0005 or 0.0006, two tailed non parametric <i>t</i>-test, error bars represent s.e.m. <b>(D)</b> While the overall pathology of lungs (upper panel) illustrates robust Mtb infection in untreated mice, relatively smaller and fewer lesions and granulomas were observed in Oxa33 or INH treated mice. Lower panel exhibit hematoxylin and eosin stained photomicrograph of lungs from <i>Rv</i>, INH treated and Oxa33 treated mice at 100x magnification. This histopathological analysis shows large or small granuloma with lymphocytes and foamy histiocytes in <i>Rv</i> infected mice or Oxa treated mice respectively G = Granuloma, BL = Bronchial Lumen, AS = Alveolar space. <b>(E)</b> Granuloma scores from lungs of infected and treated mice for 4 weeks and 12 weeks.</p>", "links"=>[], "tags"=>["survival", "thp", "GlmUMtb", "weeks post infection", "bacillary load", "cell wall thickness", "Complementation studies show", "Infected Murine Lungs Effects"], "article_id"=>1582096, "categories"=>["Biological Sciences"], "users"=>["Vijay Soni", "Sandeep Upadhayay", "Priyanka Suryadevara", "Ganesh Samla", "Archana Singh", "Perumal Yogeeswari", "Dharmarajan Sriram", "Vinay Kumar Nandicoori"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1005235.g008", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Specificity_and_efficacy_of_Oxa33_/1582096", "title"=>"Specificity and efficacy of Oxa33.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-21 03:24:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/2368352"], "description"=>"<p><b>(A)</b><i>Rv</i> and <i>Rv</i>∆<i>glmU</i> cultures were inoculated at an initial <i>A</i><sub>600</sub> of 0.1 and the growth was monitored every day for eight days. ATc was added to the <i>Rv</i> culture on day 2 and <i>Rv</i>∆<i>glmU</i> cultures were either grown in the absence of ATc or was supplemented with ATc in the growth media on 2<sup>nd</sup>, 4<sup>th</sup> or 6<sup>th</sup> day. Experiment was performed in triplicates and the error bars represent s.e.m. <b>(B)</b> Differentiated THP-1 cells were infected <i>Rv</i> or <i>Rv</i>∆<i>glmU</i> cultures and the infection was allowed to be established for 24 h. 24 h post infection ATc was supplemented in the media for <i>Rv</i> and <i>Rv</i>∆<i>glmU</i> infected cultures. As a control THP1 cells infected with <i>Rv</i>∆<i>glmU</i> were grown without ATc. CFUs were enumerated at 0, 24 and 96 h post infection. **<i>p</i><0.005, two tailed non parametric <i>t</i>-test, Experiment was performed in triplicates and the error bars represent s.e.m. <b>(C)</b> BALB/c mice (6 to 9 mice / group) were infected with <i>Rv</i> and <i>Rv</i>∆<i>glmU</i> strains and the infection was established for next 28 days. Subsequently, Dox was provided (with 5% dextrose on every alternate day) in the drinking water for <i>Rv</i> infected mice. One group of <i>Rv</i>∆<i>glmU</i> infected mice were administered with Dox and other with vehicle control for the next 56 days. CFUs were enumerated on day 1, day 28 and day 84 post infection. At 28<sup>th</sup> days post infection mean CFUs for <i>Rv</i> and <i>Rv</i>∆<i>glmU</i> infected mice were 4.43 and 4.47 on log<sub>10</sub> scale and 84<sup>th</sup> days post infection mean CFUs for <i>Rv</i> +Dox or <i>Rv</i>∆<i>glmU</i> +Dox and <i>Rv</i>∆<i>glmU</i>–Dox were 5.1, 0.42 and 5.2 on log<sub>10</sub> scale. ***<i>p</i><0.0005, two tailed non parametric <i>t</i>-test, mean, error bars represent s.e.m.. <b>(D)</b> Overall pathology and histopathology of infected BALB/c mice lungs. Infected lungs dissected on 84<sup>th</sup> day after infection from <i>Rv</i> (+Dox) and <i>Rv</i>∆<i>glmU</i> (-Dox) infected mice shows clear lesions (upper panel) and granuloma with lymphocytes and foamy histiocytes (lower panel, HE stain, 100x) while <i>Rv</i>∆<i>glmU</i> (+Dox) was showing normal lung parenchyma and no granuloma. G = Granuloma, BL = Bronchial Lumen, AS = Alveolar Space. <b>(E)</b> Granuloma scores from 4 and 12 weeks of histopathology results.</p>", "links"=>[], "tags"=>["survival", "thp", "GlmUMtb", "weeks post infection", "bacillary load", "cell wall thickness", "Complementation studies show", "Infected Murine Lungs Effects"], "article_id"=>1582094, "categories"=>["Biological Sciences"], "users"=>["Vijay Soni", "Sandeep Upadhayay", "Priyanka Suryadevara", "Ganesh Samla", "Archana Singh", "Perumal Yogeeswari", "Dharmarajan Sriram", "Vinay Kumar Nandicoori"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1005235.g006", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Depletion_of_GlmU_Mtb_from_infected_lungs_leads_to_clearance_of_pathogen_/1582094", "title"=>"Depletion of GlmU<sub>Mtb</sub> from infected lungs leads to clearance of pathogen.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-21 03:24:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/2368348"], "description"=>"<p><b>(A)</b> and <b>(B)</b> Schematic and cartoon representation of GlmU<sub>Mtb</sub> depicting different domains, active site residues and the deletion mutants. <b>(C)</b> GlmU<sub>Mtb</sub> and GlmU<sub>Mtb</sub>-mutants were purified as described earlier [<a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005235#ppat.1005235.ref038\" target=\"_blank\">38</a>] and the 1 μg of the purified proteins were resolved on 10% SDS-PAGE and stained with coomassie. <b>(D)</b> Uridyltransferase (left panel) and acetyltransferase (right panel) activities were carried out as describe in Methods using 0.5 to 20 pmoles of wild type or mutant GlmU<sub>Mtb</sub> proteins. Activity was defined as μM product formed / min / pmole of enzyme. Relative activities of the mutants were calculated with respect to the activity of GlmU<sub>Mtb</sub>, which was normalized to 100%. The experiment was repeated three times and the error bars indicate s.e.m. <b>(E)</b> Wild type and mutated GlmU<sub>Mtb</sub> genes were cloned into pNit vector without any N- or C- terminal tag. pNit-<i>glmU</i><sub>wt</sub> or pNit-<i>glmU</i><sub>mutant</sub> constructs were electroporated into <i>Rv</i>∆<i>glmU</i>, and the WCLs prepared from <i>Rv</i>∆<i>glmU and Rv</i>∆<i>glmU</i>::<i>glmU</i><sub><i>mutant</i></sub> cultures were resolved and probed with anti-GlmU and anti-GroEL1 antibodies. Bands corresponding to FLAG-GlmU<sub>Mtb</sub>, complemented GlmU<sub><i>wt/mutant</i></sub> and the deletion fragments of GlmU are indicated. <b>(F)</b><i>Rv</i>∆<i>glmU and Rv</i>∆<i>glmU</i>::<i>glmU</i><sub><i>mutant</i></sub> cultures were seeded at an initial A<sub>600</sub> of 0.1 and grown for five days in the absence or presence of ATc. The experiment was performed in triplicates and the error bars represent s.e.m.</p>", "links"=>[], "tags"=>["survival", "thp", "GlmUMtb", "weeks post infection", "bacillary load", "cell wall thickness", "Complementation studies show", "Infected Murine Lungs Effects"], "article_id"=>1582090, "categories"=>["Biological Sciences"], "users"=>["Vijay Soni", "Sandeep Upadhayay", "Priyanka Suryadevara", "Ganesh Samla", "Archana Singh", "Perumal Yogeeswari", "Dharmarajan Sriram", "Vinay Kumar Nandicoori"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1005235.g004", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Acetyl_and_uridyltransferase_activities_are_independently_essential_/1582090", "title"=>"Acetyl and uridyltransferase activities are independently essential.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-21 03:24:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/2368361", "https://ndownloader.figshare.com/files/2368362", "https://ndownloader.figshare.com/files/2368363", "https://ndownloader.figshare.com/files/2368364", "https://ndownloader.figshare.com/files/2368365", "https://ndownloader.figshare.com/files/2368366", "https://ndownloader.figshare.com/files/2368367", "https://ndownloader.figshare.com/files/2368368", "https://ndownloader.figshare.com/files/2368369", "https://ndownloader.figshare.com/files/2368370", "https://ndownloader.figshare.com/files/2368371", "https://ndownloader.figshare.com/files/2368372", "https://ndownloader.figshare.com/files/2368373"], "description"=>"<div><p><i>M</i>. <i>tuberculosis</i> N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU<sub>Mtb</sub>) is a bi-functional enzyme engaged in the synthesis of two metabolic intermediates N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and UDP-GlcNAc, catalyzed by the C- and N-terminal domains respectively. UDP-GlcNAc is a key metabolite essential for the synthesis of peptidoglycan, disaccharide linker, arabinogalactan and mycothiols. While <i>glmU</i><sub><i>Mtb</i></sub> was predicted to be an essential gene, till date the role of GlmU<sub>Mtb</sub> in modulating the <i>in vitro</i> growth of <i>Mtb</i> or its role in survival of pathogen <i>ex vivo</i> / <i>in vivo</i> have not been deciphered. Here we present the results of a comprehensive study dissecting the role of GlmU<sub>Mtb</sub> in arbitrating the survival of the pathogen both <i>in vitro</i> and <i>in vivo</i>. We find that absence of GlmU<sub>Mtb</sub> leads to extensive perturbation of bacterial morphology and substantial reduction in cell wall thickness under normoxic as well as hypoxic conditions. Complementation studies show that the acetyl- and uridyl- transferase activities of GlmU<sub>Mtb</sub> are independently essential for bacterial survival <i>in vitro</i>, and GlmU<sub>Mtb</sub> is also found to be essential for mycobacterial survival in THP-1 cells as well as in guinea pigs. Depletion of GlmU<sub>Mtb</sub> from infected murine lungs, four weeks post infection, led to significant reduction in the bacillary load. The administration of Oxa33, a novel oxazolidine derivative that specifically inhibits GlmU<sub>Mtb</sub>, to infected mice resulted in significant decrease in the bacillary load. Thus our study establishes GlmU<sub>Mtb</sub> as a strong candidate for intervention measures against established tuberculosis infections.</p></div>", "links"=>[], "tags"=>["survival", "thp", "GlmUMtb", "weeks post infection", "bacillary load", "cell wall thickness", "Complementation studies show", "Infected Murine Lungs Effects"], "article_id"=>1582103, "categories"=>["Biological Sciences"], "users"=>["Vijay Soni", "Sandeep Upadhayay", "Priyanka Suryadevara", "Ganesh Samla", "Archana Singh", "Perumal Yogeeswari", "Dharmarajan Sriram", "Vinay Kumar Nandicoori"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1005235.s001", "https://dx.doi.org/10.1371/journal.ppat.1005235.s002", "https://dx.doi.org/10.1371/journal.ppat.1005235.s003", "https://dx.doi.org/10.1371/journal.ppat.1005235.s004", "https://dx.doi.org/10.1371/journal.ppat.1005235.s005", "https://dx.doi.org/10.1371/journal.ppat.1005235.s006", "https://dx.doi.org/10.1371/journal.ppat.1005235.s007", "https://dx.doi.org/10.1371/journal.ppat.1005235.s008", "https://dx.doi.org/10.1371/journal.ppat.1005235.s009", "https://dx.doi.org/10.1371/journal.ppat.1005235.s010", "https://dx.doi.org/10.1371/journal.ppat.1005235.s011", "https://dx.doi.org/10.1371/journal.ppat.1005235.s012", "https://dx.doi.org/10.1371/journal.ppat.1005235.s013"], "stats"=>{"downloads"=>13, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Depletion_of_M_tuberculosis_GlmU_from_Infected_Murine_Lungs_Effects_the_Clearance_of_the_Pathogen/1582103", "title"=>"Depletion of <i>M</i>. <i>tuberculosis</i> GlmU from Infected Murine Lungs Effects the Clearance of the Pathogen", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-10-21 03:24:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/2368342"], "description"=>"<p>(<b>A</b>) Schematic depicting the biochemical reaction catalyzed by GlmU<sub>Mtb</sub>. (<b>B)</b> Schematic diagram representing the genomic location of <i>glmU</i><sub><i>Mtb</i></sub> (<i>Rv1018c</i>) and homologous recombination between flanking sequence in the phagemid and the genomic locus. Primers used for the PCR amplification are depicted. Upper right panel shows the immunoblot. WCLs from <i>Rv</i> or <i>Rv</i>::<i>glmU</i> grown in the presence or absence of ATc were resolved and probed with anti-GlmU antibodies. Bands corresponding to the endogenous GlmU and ectopic FLAG-GlmU are indicated. (<b>C)</b> Agarose gel showing the PCR amplification of the <i>Rv</i> & putative <i>Rv</i>∆<i>glmU</i> mutant using specific primers. Primers F1 and R2 are beyond the flanks, R1 and F2 belong to resolvase sites in <i>sacB/hyg</i><sup><i>R</i></sup> cassette and F3 and R3 binds to the native <i>glmU</i>. Amplification with F1-R1 or F2-R2 primers results in 1.23 kb or 1.17 kb size products with <i>Rv</i>∆<i>glmU</i> strain but none with the <i>Rv</i>. Whereas PCRs with F1-R3 or F3-R2 primers amplifies 1.5 kb band with the <i>Rv</i> and none with the <i>Rv</i>∆<i>glmU</i> mutant. <b>(D)</b> Whole cell lysates (WCL) were prepared from the large scale cultures of <i>Rv</i> and <i>Rv</i>∆<i>glmU</i> grown in the absence and presence of ATc for five days. 20 μg of WCLs were resolved and probed with anti-GlmU<sub>Mtb</sub>, anti-PknB and anti-GroEL1 antibodies. Band corresponding to endogenous GlmU<sub>Mtb</sub> and FLAG-GlmU<sub>Mtb</sub> are indicated.</p>", "links"=>[], "tags"=>["survival", "thp", "GlmUMtb", "weeks post infection", "bacillary load", "cell wall thickness", "Complementation studies show", "Infected Murine Lungs Effects"], "article_id"=>1582084, "categories"=>["Biological Sciences"], "users"=>["Vijay Soni", "Sandeep Upadhayay", "Priyanka Suryadevara", "Ganesh Samla", "Archana Singh", "Perumal Yogeeswari", "Dharmarajan Sriram", "Vinay Kumar Nandicoori"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1005235.g001", "stats"=>{"downloads"=>4, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_of_glmU_Mtb_conditional_deletion_mutant_/1582084", "title"=>"Generation of <i>glmU</i><sub><i>Mtb</i></sub> conditional deletion mutant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-21 03:24:43"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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