Journal: bioRxiv

Article Title: Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

doi: 10.1101/2021.10.01.462712

Figure Lengend Snippet: ( A ) Genetic organization of the pacL1 - ctpC module, and polypeptide composition of DUF1490 protein Rv3269. Red tube indicates DUF1490. Yellow tube and black tube indicate intrinsically disordered region (IDR) and molecular recognition feature (MoRF), respectively, as predicted using D 2 P 2 (ref. ). Protein topology was predicted using TMHMM (TMHMM Server v. 2.0). Extra, extracellular part; TM, transmembrane domain; Intra, intracellular part. ( B ) M. tuberculosis H37Rv (wild-type, WT), M. tuberculosis mutants deleted in pacL1 (Δ pacL1 ) or the pacL1-ctpC operon (Δ pacL1-ctpC ), and their complemented strains (:: gene(s) ), were cultivated in complete 7H9 medium in the absence (Control, black lines) or presence of 100 μM ZnSO 4 (red lines) for 15 days. Bacterial growth was quantified by turbidity measurement (reported in McFarland’s units). Data show mean±s.d. of a biological replicate (n=2) and are representative of 2 independent experiments. ( C ) Killing assay. Strains used in (A) were grown in complete 7H9 medium until an OD 600 of 0.1. Next, bacteria were incubated in complete 7H9 medium containing 500 μM ZnSO 4 and bacterial viability was assessed by CFU scoring over time. Data show mean±s.d of a biological replicate (n=2), and are representative of 2 independent experiments. ( D ) M. smegmatis mc 2 155 (wild-type, WT), or M. smegmatis mutants inactivated in msmeg_0755 ( zitA ), the pacL1-ctpC homologues msmeg_6059-msmeg_6058 , or both modules, were cultivated in complete 7H9 medium containing the indicated concentrations of ZnSO 4 . Data are expressed as % to control (0 ZnSO 4 ), show mean±s.d of a biological replicate (n=3), and are representative of 2 independent experiments. ( E ) M. smegmatis Δ2 was transformed with an integrative vector encoding mCherry under the constitutive promoter P1 , and GFP under the control of the native promoter of the pacL1-ctpC operon ( P pacL1 ). Bacteria were incubated overnight with the indicated concentrations of ZnSO 4 , and GFP and mCherry signals (mean fluorescence intensity, MFI) were measured by flow cytometry. Data (n=1) show GFP MFI/mCherry MFI ratio, and are representative of 3 independent experiments. ( F ) M. smegmatis wild-type (WT), Δ2, or the Δ2 strain complemented with pacL1, ctpC or the pacL1-ctpC module were cultivated in complete 7H9 medium containing the indicated concentrations of ZnSO 4 for 24 hours. Bacterial growth was estimated by turbidity measurement, expressed as % of control (0 ZnSO 4 ), show mean±s.d of a biological replicate (n=4), and are representative of 2 independent experiments. ( G ) The indicated M. smegmatis strains were cultivated in complete 7H9 without ADC containing 100 µM ZnSO 4 , incubated with FluoZin-3 at 1 mM for 1 h, and washed twice in PBS. Bacterial fluorescence was measured by flow cytometry and expressed as mean fluorescence intensity (MFI). Data show mean±s.d of a biological replicate (n=3), and are representative of 2 independent experiments. Data were analyzed using ANOVA. ns, not significant ( P >0.05); ****, P <0.0001.

Article Snippet: M. smegmatis mc 2 155 (ATCC 700084), M. tuberculosis H37Rv (ATCC 27294) and mutants or recombinants derived from these strains were grown at 37°C in 7H9 medium (Difco) supplemented with 10% albumin-dextrose-catalase (ADC, Difco) and 0.05% Tween-80 (Sigma-Aldrich), or on complete 7H11 solid medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, Difco).

Techniques: Control, Bacteria, Incubation, Transformation Assay, Plasmid Preparation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

doi: 10.1101/2021.10.01.462712

Figure Lengend Snippet: ( A ) Western blotting analysis of cytosolic (cyt) and membrane (mb) extracts of M. smegmatis Δ2 expressing recombinant native PacL1 and CtpC (left panel), or FLAG- and HIS 6 -tagged PacL1 and CtpC, respectively (right panel). The top and bottom membranes were detected with anti-HIS 6 and anti-FLAG, respectively. M, molecular size marker. Data are representative of 2 independent experiments. ( B) Western blotting analysis of cytosolic (cyt) and membrane (mb) extracts of M. smegmatis Δ2 expressing recombinant CtpC with FLAG-tagged PacL1 (left panel), or FLAG-tagged PacL1 deleted of its transmembrane domain (ΔTM, right panel). The membrane was treated for FLAG immuno-detection. M, molecular size marker. Data are representative of 2 independent experiments. ( C ) M. tuberculosis H37Rv (wild-type, WT), the Δ pacL1 mutant, or the Δ pacL1 mutant complemented with pacL1 or pacL1 (ΔTM) were cultivated in complete 7H9 medium in the absence (left graph) or presence of 100 μM ZnSO 4 (right panel) for 14 days. Bacterial growth was evaluated by turbidity measurement (reported in McFarland’s units). Data show mean±s.d of a biological replicate (n=3), and are representative of 2 independent experiments.

Article Snippet: M. smegmatis mc 2 155 (ATCC 700084), M. tuberculosis H37Rv (ATCC 27294) and mutants or recombinants derived from these strains were grown at 37°C in 7H9 medium (Difco) supplemented with 10% albumin-dextrose-catalase (ADC, Difco) and 0.05% Tween-80 (Sigma-Aldrich), or on complete 7H11 solid medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, Difco).

Techniques: Western Blot, Membrane, Expressing, Recombinant, Marker, Mutagenesis

Journal: bioRxiv

Article Title: Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

doi: 10.1101/2021.10.01.462712

Figure Lengend Snippet: ( A-C ) M. smegmatis expressing mTurquoise-tagged PacL1 (A,C) or mTurquoise-tagged PacL1ΔTM (B) under the control of the native P pacL1 promoter (A,B) or the “down” P pacL1 promoter, in which T -61 G -60 were mutated into CC to reduce transcription (C), was examined using an Eclipse TI-E/B wide field epifluorescence microscope. The right panel in (C) shows the distribution of spot number/bacterial cell, counted by visual examination in n=173 cells. ( D ) Epifluorescence microscopy image of M. smegmatis expressing mTurquoise- and mVenus-tagged PacL1 and CtpC, respectively. The proteins were expressed under the control of the P pacL1 promoter and bacteria were grown in complete 7H9 medium. ( E ) Formaldehyde-fixed M. tuberculosis expressing mTurquoise- and mVenus-tagged PacL1 and CtpC, respectively, was examined using an Eclipse TI-E/B wide field epifluorescence microscope. Right graph shows co-localization along the white line in the overlay panel. The proteins were expressed under the control of the P pacL1 promoter and bacteria were grown in complete 7H9 medium. ( F ) Co-localization index between blue (PacL1) and yellow (CtpC) pixels in n=413 cells. ( G ) M. smegmatis expressing mTurquoise- and mVenus-tagged PacL1 and Rv1488/FloT, respectively, was examined using an Eclipse TI-E/B wide field epifluorescence microscope. ( H ) Co-localization index between blue (PacL1) and yellow (Rv1488) pixels in n=756 cells. ( I ) M. tuberculosis H37Rv (wild-type, WT, left panel), or a M. tuberculosis mutant deleted in Rv1488 (ΔRv1488) were cultivated in complete 7H9 medium in the absence (Control, black lines) or presence of 100 μM or 250 μM ZnSO 4 for 15 days. Bacterial growth was quantified by turbidity measurement (reported in McFarland’s units). Data show mean±s.d of a biological replicate (n=3), and are representative of 2 independent experiments. ( J ) Formaldehyde-fixed M. tuberculosis mutant ΔRv1488 expressing mTurquoise- and mVenus-tagged PacL1 and CtpC, respectively, was examined using an Eclipse TI-E/B wide field epifluorescence microscope.

Article Snippet: M. smegmatis mc 2 155 (ATCC 700084), M. tuberculosis H37Rv (ATCC 27294) and mutants or recombinants derived from these strains were grown at 37°C in 7H9 medium (Difco) supplemented with 10% albumin-dextrose-catalase (ADC, Difco) and 0.05% Tween-80 (Sigma-Aldrich), or on complete 7H11 solid medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, Difco).

Techniques: Expressing, Control, Microscopy, Epifluorescence Microscopy, Bacteria, Mutagenesis

Journal: bioRxiv

Article Title: Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

doi: 10.1101/2021.10.01.462712

Figure Lengend Snippet: ( A )Measurement of metal binding to solPacL1 and SolPacL1Δ3 (50 µM) by dialysis at equilibrium. Experiment was performed in 50 mM MOPS pH 7, 200 mM NaCl during 21 h at 4°C in the presence of 50 µM of the indicated metal. Data show mean±s.d. of three independent experiments. ( B ) Isothermal titration calorimetry data. Thermograms and corresponding titration curves obtained by successive injections of 2 µL of 1.5 mM Zn[CH 3 COO] 2 into the sample containing 150 µM of purified solPacL1. Molar ratio is defined as the number Zn per solPacL1. The data are representative of two independent experiments. ( C ) Superposition of 2D NMR TOCSY spectra (80 ms mixing time) of solPacL1 (500 µM in 20 mM MES-d 13 pH 6.0, 100 mM NaCl at 7 °C) in the absence (black) and presence (blue) of 2 equivalents of zinc acetate. For clarity, the region of the spectrum containing H N -H β cross-peaks of histidines in the HDHDH zinc-binding motif is shown. The assignment is reported using the sequence numbering and colors represent the strength of perturbation induced by zinc binding; red: strong (broadened beyond detection); orange: medium; green: no effect. ( D ) M. tuberculosis H37Rv (wild-type, WT), the Δ pacL1 mutant, or the Δ pacL1 mutant complemented with pacL1, pacL1 Δ3 (lacking the H 91 DH 93 -encoding sequence) or pacL1 Δ7 (lacking the D 87 LHDHDH 93 -encoding sequence) were cultivated in complete 7H9 medium in the presence of 100 μM (upper panel) or 250 μM (lower graph) ZnSO 4 for 14 days. Bacterial growth was evaluated by turbidity measurement (reported in McFarland’s units). Data show mean±s.d of a biological replicate (n=2), and are representative of 2 independent experiments.

Article Snippet: M. smegmatis mc 2 155 (ATCC 700084), M. tuberculosis H37Rv (ATCC 27294) and mutants or recombinants derived from these strains were grown at 37°C in 7H9 medium (Difco) supplemented with 10% albumin-dextrose-catalase (ADC, Difco) and 0.05% Tween-80 (Sigma-Aldrich), or on complete 7H11 solid medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, Difco).

Techniques: Binding Assay, Isothermal Titration Calorimetry, Titration, Purification, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

doi: 10.1101/2021.10.01.462712

Figure Lengend Snippet: ( A ) Western-blotting analysis of membrane extracts from M. smegmatis Δ2 expressing PacL1 and CtpC, or a combination of HIS 6 -tagged CtpC and FLAG-tagged PacL1, FLAG-tagged PacL1ΔR 37 S 86 , containing the MBM, FLAG-tagged PacL1ΔA 54 S 86 , containing the MBM, or FLAG-tagged PacL1Δ7, lacking the MBM. All genes are expressed under the control of the native P PacL1 promoter and bacteria were grown in 7H9 medium. The upper membrane was treated for HIS 6 immuno-detection and the lower membrane was treated for FLAG immuno-detection. M, molecular size marker. Data are representative of 2 independent experiments. Relative CtpC-HIS 6 protein quantification is displayed in the inset. ( B ) Strains used in (A) were cultivated for 24 h in complete 7H9 medium in the absence (Control, white bars) or presence (orange bars) of 100 μM ZnSO 4 . Bacterial growth was quantified by turbidity measurement (expressed as % of untreated control). Data show mean±s.d of a biological replicate (n=3), and are representative of 2 independent experiments. ( C ) Western-blotting analysis of membrane extracts from M. smegmatis Δ2 expressing Atc-inducible FLAG-tagged PacL1 and constitutive HIS 6 -tagged CtpC (left panel). The membrane was cut into two pieces: the upper part was treated for HIS 6 immuno-detection and the lower part was treated for FLAG immuno-detection. M, molecular size marker. Data are representative of 2 independent experiments. CtpC-HIS 6 protein quantification is displayed in the inset (relative to amount in the non-Atc-induced strain). ( D ) The strain used in (C) was cultivated in complete 7H9 medium in the absence (Control, black circles) or presence (red circles) of 100 μM ZnSO 4 , without (open circles) or with (closed circles) Atc. Bacterial growth was quantified by turbidity measurement (expressed in OD 600 units). Data show mean±s.d of a biological replicate (n=2), and are representative of 2 independent experiments. ( E ) M. smegmatis wild-type strain (WT), Δ2 mutant, or Δ2 expressing recombinant CtpC and native PacL1 or PacL1 E 55 A, E 59 V, E 71 A or 3EA variants were cultivated for 24 h in complete 7H9 medium in the absence (Control, white bars) or presence (orange bars) of 100 μM ZnSO 4 . Bacterial growth was quantified by turbidity measurement (expressed as % of untreated control). Data show mean±s.d of a biological replicate (n=3), and are representative of 2 independent experiments. ( F ) Western-blotting analysis of cytosolic (cyt) and membrane (mb) fractions from M. smegmatis Δ2 expressing recombinant PacL1 and CtpC, or HIS 6 -tagged CtpC and FLAG-tagged PacL1, or the PacL1 E 71 A or 3EA variants. The upper membrane was treated for HIS 6 immuno-detection and the lower membrane was treated for FLAG immune-detection. Data are representative of 2 independent experiments.

Article Snippet: M. smegmatis mc 2 155 (ATCC 700084), M. tuberculosis H37Rv (ATCC 27294) and mutants or recombinants derived from these strains were grown at 37°C in 7H9 medium (Difco) supplemented with 10% albumin-dextrose-catalase (ADC, Difco) and 0.05% Tween-80 (Sigma-Aldrich), or on complete 7H11 solid medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, Difco).

Techniques: Western Blot, Membrane, Expressing, Control, Bacteria, Marker, Mutagenesis, Recombinant

Journal: bioRxiv

Article Title: Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

doi: 10.1101/2021.10.01.462712

Figure Lengend Snippet: ( A ) Epifluorescence microscopy examination of fixed M. smegmatis expressing mTurquoise-tagged PacL1 and mVenus-tagged PacL2 (upper panels) or -PacL3 (lower panels). All taggedproteins were expressed under the control of the P pacL1 promoter, and bacteria were grown in complete 7H9 medium. ( B ) Co-localization index between PacL1 and PacL2, and between PacL1 and PacL3. n, indicates the number of counted cells. All tagged proteins were expressed under the control of the P pacL1 promoter, and bacteria were grown in complete 7H9 medium. ( C ) M. tuberculosis wild-type (WT), a M. tuberculosis triple mutant inactivated in pacL1-3 (Δ pacL1-pacL2-pacL3 ) or the triple mutant complemented with vectors encoding PacL1 and CtpC, PacL3 and CtpC, PacL2 and CtpC or a PacL2:MBM fusion protein and CtpC, were cultivated in complete 7H9 medium containing ZnSO 4 at the indicated concentrations (Control, no zinc). Bacterial growth was quantified by turbidity measurement (expressed in McFarland’s units). Data show mean±s.d of a biological replicate (n=6), and are representative of 2 independent experiments. Data at day 14 were analyzed by Mann-Whitney. NS: Not significant; **, P <0.01 (compared to untreated control). ( D ) M. tuberculosis wild-type (WT) and M. tuberculosis Δ pacL1 or Δ pacL1-ctpC mutants were cultivated in complete 7H9 medium containing 100 μM ZnSO 4 , 5 μM CdCl 2 , 100 μM ZnSO 4 and 5 μM CdCl 2 , or no metal (Control). Bacterial growth was assessed by turbidity measurement as expressed in McFarland’s units. Data show mean±s.d of a biological replicate (n=3), and are representative of 2 independent experiments.

Article Snippet: M. smegmatis mc 2 155 (ATCC 700084), M. tuberculosis H37Rv (ATCC 27294) and mutants or recombinants derived from these strains were grown at 37°C in 7H9 medium (Difco) supplemented with 10% albumin-dextrose-catalase (ADC, Difco) and 0.05% Tween-80 (Sigma-Aldrich), or on complete 7H11 solid medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, Difco).

Techniques: Epifluorescence Microscopy, Expressing, Control, Bacteria, Mutagenesis, MANN-WHITNEY

Journal: Journal of medicinal chemistry

Article Title: Functionalized Dioxonaphthoimidazoliums: A Redox Cycling Chemotype with Potent Bactericidal Activities against Mycobacterium tuberculosis

doi: 10.1021/acs.jmedchem.1c01383

Figure Lengend Snippet: (A) pfurA Promoter activity in a recombinant strain of M. bovis BCG-pfurA-RFP (mid-log phase, inoculum at OD600 = 0.2) treated with test compounds (32, 48, 61, 72, and 73) over a range of concentrations for 24 h. Fluorescent signal (RFP) was normalized against OD600 of treated cultures to account for variations in cell number. DF and INH were employed as drug-free and negative controls. (B) Colony formation by wild-type M. bovis BCG and a katG loss-of-function mutant strain in the presence of test compounds (32, 61) at 1× and 2× MIC90. The control INH was inactive against the mutant strain. (C) Colony formation by M. bovis BCG in the presence of vitamin C (2.5 mM), 32 (0.5×, 1× MIC90), and combinations of vitamin C and 32. For (B) and (C), experiments were carried out in catalase-free 7H9 broth and aliquots withdrawn from the liquid media for CFU counting. *p < 0.05 and **p < 0.01, Student’s t-test (GraphPad Prism, Ver 8.4.3). DF D0 and DF D5 were drug-free controls monitored on day 0 and day 5 of incubation, corresponding to start and end of drug exposure. At least two biological repeats were carried out for each experiment. Figures depicted in (A)–(C) are from representative experiments.

Article Snippet: 53 , 54 , 61 Twofold serial dilutions of 32 and one interested anti-TB drug were made in complete 7H9 broth in a flat-bottom transparent 96-well plate (Costar Corning).

Techniques: Activity Assay, Recombinant, Mutagenesis, Incubation