Journal: BMC Microbiology

Article Title: Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis

doi: 10.1186/1471-2180-11-43

Figure Lengend Snippet: Immunoblot analysis of Obg of M. tuberculosis . A . Immunoblot analysis of Obg from M. tuberculosis strains harboring plasmids. M . tuberculosis strains were grown in 7H9-OADC-TW broth at 37°C to early log phase and lysates prepared using a bead beater and separated (100 μg protein for each lane) on SDS-PAGE. The immunoblots were probed with anti-Obg antiserum (1:500 dilution) followed by alkaline phosphatase labeled anti-rabbit IgG (1:1000 dilution, Zymed). The antibody-incubated blots were then developed with NBT/BCIP substrates. Lane 1, M. tuberculosis carrying the plasmid pMV261(empty vector control); Lane 2, M. tuberculosis carrying the plasmid pMVOBG (plasmid overexpressing Obg). B . Immunoblot analysis of Obg at different growth points in M . tuberculosis culture. Wild type M. tuberculosis was grown in 7H9-OADC-TW broth at 37°C. Lysates were prepared from wild-type M. tuberculosis grown to different ODs at 600 nm, separated (200 μg protein for each lane) on SDS-PAGE, and probed with anti-Obg antiserum (1:500 dilution) followed by peroxidase-labeled anti-rabbit IgG (1:10,000 dilution, Sigma). The blots were developed with an ECL kit (Amersham) and autoradiographed. "Obg" indicates the Obg protein reacting with anti-Obg antiserum. Values below each band indicate the OD value at 600 nm at the time of harvest. The graph above the bands gives the levels of Obg, based on density of the bands using Image J software. C . Immunoblots of Obg in separated soluble vs membrane fractions of M. tuberculosis lysates. The bacteria were grown in 7H9-OADC-TW broth at 37°C to mid-log phase. Lysates were prepared using a bead beater, and the soluble and pellet fractions separated by centrifugation. The protein fractions (200 μg protein for each lane) were separated by SDS-PAGE, blotted and probed with anti-Obg antiserum (1:500 dilution) (marked as Obg) or anti-SigH antiserum (1:1000 dilution) (marked as SigH), followed by peroxidase-labeled anti-rabbit IgG (1:10,000 dilution, Sigma). The blots were developed with an ECL kit (Amersham) and autoradiographed. In the figure, lanes labeled Whole, Supernatant and Pellet represent extracts of whole M. tuberculosis , of the 49,000 g supernatant, and of the 49,000 g pellet, respectively.

Article Snippet: M. tuberculosis H37Rv was grown either in Middlebrook 7H9 broth medium containing Tween (0.05%) and OADC (10%) (7H9-TW-OADC) broth, or in Middlebrook 7H10 agar medium containing Tween (0.05%) and OADC (10%) (7H10-TW-OADC).

Techniques: Western Blot, SDS Page, Labeling, Incubation, Plasmid Preparation, Software, Centrifugation

Journal: BMC Microbiology

Article Title: Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis

doi: 10.1186/1471-2180-11-43

Figure Lengend Snippet: Obg cofractionation with ribosomal subunits . M. tuberculosis was grown in 7H9-OADC-TW broth at 37°C, and lysates prepared using a bead beater. About 500 g protein was separated in 10-40% sucrose gradient. A . The ODs of the separated fractions were measured (manually) at 260 nm. B . The proteins in the fractions were then precipitated with ethanol and separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-Obg antiserum (1:500 dilution), followed by peroxidase-labeled anti-rabbit IgG (1:10,000 dilution, Sigma). The blots were developed with an ECL kit (Amersham) and autoradiographed. Lane C is a whole-cell extract from M. tuberculosis . Lanes 1-15 represent fractions from the top (10% sucrose) to the bottom (40% sucrose) of the sucrose gradient. Fraction 16 was not analyzed in immunoblot.

Article Snippet: M. tuberculosis H37Rv was grown either in Middlebrook 7H9 broth medium containing Tween (0.05%) and OADC (10%) (7H9-TW-OADC) broth, or in Middlebrook 7H10 agar medium containing Tween (0.05%) and OADC (10%) (7H10-TW-OADC).

Techniques: SDS Page, Labeling, Western Blot

Journal: BMC Microbiology

Article Title: Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis

doi: 10.1186/1471-2180-11-43

Figure Lengend Snippet: Growth of M. tuberculosis strains at different time points . M. tuberculosis was grown in 7H9-OADC-TW broth at 37°C. Growth was followed by measuring the OD at 600 nm using 1 ml aliquots. Closed circles: M. tuberculosis carrying the plasmid pMV261 (empty vector control); squares: M. tuberculosis carrying the plasmid pMVOBG (plasmid overexpressing Obg). The data shown are representative findings from three different. experiments.

Article Snippet: M. tuberculosis H37Rv was grown either in Middlebrook 7H9 broth medium containing Tween (0.05%) and OADC (10%) (7H9-TW-OADC) broth, or in Middlebrook 7H10 agar medium containing Tween (0.05%) and OADC (10%) (7H10-TW-OADC).

Techniques: Plasmid Preparation

Journal: Scientific Reports

Article Title: Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria

doi: 10.1038/s41598-019-45164-5

Figure Lengend Snippet: TAG accumulation under the form of ILI is driven by carbon excess and nitrogen starvation in stationary phase. ( A ) Mycobacterial cultures were grown in different media containing increasing glycerol concentrations (1%, 2% and 5%) and were collected at 24 h or 48 h, corresponding to exponential or stationary phase, lyophilized and finally the same amount of dry cell weight was used for apolar lipid extraction. Left panel corresponds to TLC plate analysis of TAG extracted from exponential and stationary-phase cultures with increasing concentrations of Gly, with triolein as standard. Right panel corresponds to TLC densitometric analysis of the relative TAG level in each sample with cultures in classic 7H9 (7H9 Exp ) used as reference. (B) Cultures grown in minimal salt medium containing either 1 g/L or 0.05 g/L of NH 4 Cl and 1% Gly as carbon source were collected after 8 h, 24 h or 48 h incubation periods, lyophilized and equal weights of dry cells used for apolar lipid extraction. TAG levels from each culture were analysed by TLC with triolein as standard. The TLC plate (left panel) is representative of two individual experiments. TLC densitometric analysis of relative TAG levels in each sample with cultures in exponential phase in classic 7H9 (7H9 Exp ) used as reference (right panel). All results are expressed as mean values ± SD of two independent experiments. TAG band intensities were compared using a one-way ANOVA test where * corresponds to a p -value < 0.05. (C) Average Nile-Red fluorescence intensity determined for 6 different 126 µm 2 fields containing between 50 and 150 cells each. Fluorescence intensities were compared using a two-tailed Mann-Whitney test where * corresponds to a p -value < 0.05. (D) Phase contrast, Fluorescence and Merge channels of M. smegmatis cells grown for 24 h in MSM NL Gly 1% medium. Cells harbour distinct morphologies and contain ILI occupying most of the cytoplasm space. Scale bars represent 2 µm. Cells were fixed with glutaraldehyde and processed for EM. (E) Thin section of an in vitro culture of M. smegmatis in classical 7H9 medium. The scale bar represents 1 µm. (F) Thin section of an in vitro culture of M. smegmatis in MSM NL Gly medium. Right panel is a zoomed-in picture providing a better view and resolution of ILI. Scale bars represent 2 µm.

Article Snippet: M. abscessus CIP104536 T , S and R morphotypes, were cultured in 7H9 medium containing 10% BBLTM Middlebrook OADC Enrichment (7H9 OADC ).

Techniques: Extraction, Incubation, Fluorescence, Two Tailed Test, MANN-WHITNEY, In Vitro

Journal: Scientific Reports

Article Title: Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria

doi: 10.1038/s41598-019-45164-5

Figure Lengend Snippet: Drug susceptibility testing of lipid-poor (7H9 Exp ) and lipid-rich (MSM NL Gly 1% medium) M. smegmatis . Evaluation of antibiotic tolerance was performed using the agar plating method.

Article Snippet: M. abscessus CIP104536 T , S and R morphotypes, were cultured in 7H9 medium containing 10% BBLTM Middlebrook OADC Enrichment (7H9 OADC ).

Techniques:

Journal: Scientific Reports

Article Title: Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria

doi: 10.1038/s41598-019-45164-5

Figure Lengend Snippet: Nitrogen/carbon ratio governs TAG accumulation as ILI in Mycobacterium abscessus . ( A ) M. abscessus cells were grown in minimal salt medium containing either 1 g/L or 0.05 g/L NH 4 Cl and 1% Gly as sole carbon source. Cultures were collected after an 8 h, 24 h or 48 h incubation period, lyophilized and equal amounts of dry cells used for apolar lipid extraction. TAG levels from each culture were analysed by TLC with triolein as standard. The TLC plate (left panel) is representative of two independent experiments. Right panel corresponds to TLC densitometric analysis of relative TAG levels in each sample, with cultures in exponential phase in classic 7H9TG OADC used as reference. Results are expressed as mean values ± SD of two individual experiments. TAG band intensities were compared using a one-way ANOVA test where * corresponds to a p -value < 0.05. (B) Thin section of an in vitro culture of M. abscessus in classical 7H9 OADC medium. The scale bar represents 1 µm. (C) Thin section of an in vitro culture of M. abscessus in MSM NL Gly 1% medium. Right panel is a zoomed-in picture providing a better view and resolution of ILI. Scale bars represent 1 µm. (D) TAG levels from M. abscessus grown in MSM NL Gly. Cultures were collected after a 24 h or 48 h incubation period, lyophilized and equal weights of dry cells used for apolar lipid extraction. TAG levels from WT, Δ tgs1 , Δ tgs2 and their respective complemented strains were analysed by TLC with triolein as standard. TLC densitometric analysis of relative TAG levels in each genetic background, with cultures from WT M. abscessus used as a reference. Results are expressed as mean values ± SD of two independent experiments. TAG band intensities of WT and Δ tgs1 were compared using a one-way ANOVA test where * corresponds to a p -value < 0.05.

Article Snippet: M. abscessus CIP104536 T , S and R morphotypes, were cultured in 7H9 medium containing 10% BBLTM Middlebrook OADC Enrichment (7H9 OADC ).

Techniques: Incubation, Extraction, In Vitro

Journal: Scientific Reports

Article Title: Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria

doi: 10.1038/s41598-019-45164-5

Figure Lengend Snippet: Drug susceptibility testing of lipid-poor (7H9 OADC ) and lipid-rich (MSM NL Gly 1%) M. abscessus cells.

Article Snippet: M. abscessus CIP104536 T , S and R morphotypes, were cultured in 7H9 medium containing 10% BBLTM Middlebrook OADC Enrichment (7H9 OADC ).

Techniques:

Journal: Scientific Reports

Article Title: Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria

doi: 10.1038/s41598-019-45164-5

Figure Lengend Snippet: Nitrogen limitation induces hypervirulence in M. abscessus . (A) Simplified diagram of the experimental workflow used to induce lipid-loaded M. abscessus by nitrogen deprivation prior to infection of zebrafish embryos. Zebrafish embryos were infected via caudal vein injection at 30 hpf with approximately 200 CFU of M. abscessus S or R cultured for 48 h in either 7H9 or MSM NL Gly 1%. (B) Nitrogen limitation results in significantly earlier mortality in M. abscessus R-infected zebrafish. Zebrafish embryo survival was monitored daily over a 12-day period following infection. Each group consisted of approximately 20 embryos, with each curve reproduced in triplicate. Statistical analysis was completed using the Mantel-Cox log-rank test. (C) Representative images displaying significantly increased pathology phenotypes at 6 dpi in zebrafish embryos infected with the M. abscessus R morphotype following nitrogen limitation. Transgenic reporter line zebrafish embryos harbouring fluorescent macrophages ( mpeg:mCherry ) (red overlay) were infected with M. abscessus harbouring pTEC15:: mWasabi (green overlay) and the merge was observed in yellow. The open arrow (top) displays an intact abscess, while the closed arrow (bottom) displays a ruptured abscess. Scale bars represent 500 µm. ** p -value ≤ 0.01.

Article Snippet: M. abscessus CIP104536 T , S and R morphotypes, were cultured in 7H9 medium containing 10% BBLTM Middlebrook OADC Enrichment (7H9 OADC ).

Techniques: Infection, Injection, Cell Culture, Transgenic Assay

Journal: Scientific Reports

Article Title: Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria

doi: 10.1038/s41598-019-45164-5

Figure Lengend Snippet: Nitrogen limitation results in increased bacterial burden and granuloma abundance. Transgenic zebrafish embryos harbouring red fluorescent macrophages ( mpeg1:mCherry ) were infected with approximately 200 CFU of M. abscessus S or R (producing mWasabi fluorescent protein) cultured in either 7H9 or in MSM NL Gly 1% and were imaged at 2, 4 and 6 dpi to quantify bacterial burden and granuloma number. (A , B) Bacterial burden was quantified using ImageJ ‘Analyse Particles’ function to determine fluorescent pixel counts. Error bars represent standard deviation, with each data point representing a single embryo. Data shown represent a pool of three individual experiments with approximately 20 embryos per group. Statistical analysis was completed using a Student’s t-test. (C , D) Granuloma number was quantified manually following colocalization (yellow) of bacteria (green) and macrophage aggregates (red) using ImageJ. Error bars represent standard deviation. Data shown is the average of three individual experiments with approximately 20 embryos per group. Statistical analysis was done using a Student’s t-test. (E , F) Representative images of granuloma quantification following bacterial and macrophage colocalization in the cranial and trunk region at 6 dpi. White arrows highlight granuloma presence. Scale bars represent 200 µm. ** p -value ≤ 0.01, *** p -value ≤ 0.001.

Article Snippet: M. abscessus CIP104536 T , S and R morphotypes, were cultured in 7H9 medium containing 10% BBLTM Middlebrook OADC Enrichment (7H9 OADC ).

Techniques: Transgenic Assay, Infection, Cell Culture, Standard Deviation, Bacteria