Journal: bioRxiv

Article Title: Mycolactone causes catastrophic Sec61-dependent loss of the endothelial glycocalyx and basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection

doi: 10.1101/2023.02.21.529382

Figure Lengend Snippet: HDMECs exposed to 10 ng/mL of mycolactone (MYC) or 0.02% DMSO for 24 hours or indicated times were subjected to proteomic analysis ( A-D ), surface immunostaining ( E-F, H ) or immunoblotting ( G ). ( A ) Volcano Plot of differential expression between DMSO and MYC treated samples, plotting mean fold change against false discovery rate adjusted p -values. Pale blue=total detected proteins; dark blue=Type II membrane proteins; yellow=Golgi-localised Type II membrane proteins. ( B ) Violin plot showing fold change in protein levels for Type II membrane proteins grouped according to subcellular location. ns, not significant; **, p < 0.01; ****, p < 0.0001. ( C ) Heat map showing fold change in Golgi-localised O- and N-glycosylation enzymes in mycolactone exposed HDMEC. Dual-colour coding is shown., only one unique peptide detected in asterisks, and significantly downregulated ( p < 0.05) or not ( p ≥ 0.05) in bold or Italic respectively. ( D ) Genes in GAG biosynthesis categorised according to function and side chains of chondroitin sulphate/ dermatan sulphate (CS/DS), heparan sulphate (HS) or keratan sulphate (KS). Heatmap showing Log2 fold change of these genes in response to mycolactone in three independent experiments. Dual-colour coding is shown. Genes undetected are indicated as crossed, only one unique peptide detected in asterisks, and significantly downregulated ( p < 0.05) or not ( p ≥ 0.05) in bold or Italic respectively ( E-F ) Cells were treated with or without chondroitinase ABC (ABC) or heparinase III (HepIII), immunostained with anti-chondroitin sulphate (CS), anti-Δ-heparan sulphate (dHS) antibodies or the isotype controls for flow cytometry analysis. Histogram plot for single cell population of CS (E) and dHS (F) and the respective mean fluorescence intensity (MFI) are shown. Unstained untreated cells filled grey; isotype control of untreated cells, dashed line in black; Untreated cells incubated with chondroitinase ABC prior to CS staining or without HepIII prior to dHS staining, grey line; untreated cells with CS-PE or dHS-PE, black line; cells exposed to DMSO stained with antibodies, blue line; cells exposed to MYC stained with antibodies, red line. MFI is presented as a % of untreated control (mean ± SEM of 3 independent experiments). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ( G ) Cells were lysed, treated with heparinase III and analysed by immunoblotting. HS neoepitopes were visualised with anti-Δ-heparan sulphate (dHS) antibody with the approximate migration of molecular weight markers in kDa. GAPDH as loading control. Images are representative of 3 independent experiments. ( H ) Cells were incubated with HepIII, fixed and immunostained with anti-dHS antibody (green), permeabilised and labelled with TRITC-conjugated phalloidin (magenta). Nuclei were stained with DAPI (blue). Images are representative of 2 independent experiments. Scale bar = 50 μm.

Article Snippet: Antibodies used in this study were: B3Galt6 (H00126792-B01P, Biotechne), Giantin (ab80864, abcam), Laminin α4 (AF7340, Biotechne), Δ-HS (F69-3G10, AMS Biotechnology), perlecan (7B5, ThermoFisher Scientific), TRITC-conjugated phalloidin (FAK100, Merck), Alexa Fluor 594 goat anti-rabbit (A11012), Alexa Fluor 488 donkey anti-mouse (A21202) and Alexa Fluor 488 donkey anti-sheep (A11015) from Invitrogen/ThermoFisher Scientific.

Techniques: Immunostaining, Western Blot, Expressing, Flow Cytometry, Fluorescence, Incubation, Staining, Migration, Molecular Weight

Journal: bioRxiv

Article Title: Mycolactone causes catastrophic Sec61-dependent loss of the endothelial glycocalyx and basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection

doi: 10.1101/2023.02.21.529382

Figure Lengend Snippet: HDMECs exposed to 10 ng/mL mycolactone (MYC), 0.02% DMSO or 20 nM ZIF-80 for 24 hours or indicated times. ( A ) Heatmap showing representative data for genes encoding proteoglycans. Dual-colour coding for log2 fold change in response to MYC is shown. Possible attached glycosaminoglycan chains such as heparan sulphate (HS), chondroitin sulphate (CS) or dermatan sulphate (DS) Candidates with one unique peptide detected indicated with asterisks, significantly downregulated ( p < 0.05) or not ( p ≥ 0.05) in bold or Italic respectively. ( B ) Cells were harvested for flow cytometry analysis. Histogram plots for single cell population of perlecan, glypican-1 and biglycan. Unstained untreated cells, filled grey; isotype control of untreated cells, dashed black line. untreated cells stained with antibodies, black line; cells exposed to DMSO stained with antibodies, blue line; cells exposed to MYC stained with antibodies, red line. MFI is presented as a % of untreated control (mean ± SEM of 3 independent experiments). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ( C ) Cells were fixed and immunostained with anti-perlecan antibody (green), permeabilised and labelled with TRITC-conjugated phalloidin (magenta). Nuclei were stained with DAPI (blue). Images are representative of 3 independent experiments. Scale bar = 50 μm.

Article Snippet: Antibodies used in this study were: B3Galt6 (H00126792-B01P, Biotechne), Giantin (ab80864, abcam), Laminin α4 (AF7340, Biotechne), Δ-HS (F69-3G10, AMS Biotechnology), perlecan (7B5, ThermoFisher Scientific), TRITC-conjugated phalloidin (FAK100, Merck), Alexa Fluor 594 goat anti-rabbit (A11012), Alexa Fluor 488 donkey anti-mouse (A21202) and Alexa Fluor 488 donkey anti-sheep (A11015) from Invitrogen/ThermoFisher Scientific.

Techniques: Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Mycolactone causes catastrophic Sec61-dependent loss of the endothelial glycocalyx and basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection

doi: 10.1101/2023.02.21.529382

Figure Lengend Snippet: HDMECs exposed to 10 ng/mL mycolactone (MYC), 0.02% DMSO or 20 nM ZIF-80 for 24 hours or indicated times. ( A ) Heatmap showing representative data for genes encoding junctional or adhesion molecules, basement membrane components and proteins involved in platelet adhesion. Dual-colour coding for log2 fold change in response to MYC is shown. Candidate with one unique peptide detected is indicated with asterisks, significantly downregulated ( p < 0.05) or not ( p ≥ 0.05) in bold or Italic respectively. ( B ) Cells were fixed and immunostained with anti-laminin α4 antibody (green), permeabilised and labelled with TRITC-conjugated phalloidin (magenta). Nuclei were stained with DAPI (blue). Images are representative of 2 independent experiments. Scale bar = 50 μm. ( C ) Cells were harvested for flow cytometry analysis. Histogram plots for single cell population of integrin β1, integrin β4 and laminin α5. Unstained, untreated cells, filled grey; isotype control of untreated cells, dashed black line. untreated cells stained with antibodies, black line; cells exposed to DMSO stained with antibodies, blue line; cells exposed to MYC stained with antibodies, red line. MFI is presented as a % of untreated control (mean ± SEM of 3 independent experiments). ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ( D ) Alcian blue-periodic acid Schiff stain in the feet of C57BL/6 mice that received vehicle control (PBS) (D1, D4) or intradermal injection of 1*10 5 colony forming units M. ulcerans at 21 (Grade 1; D2, D5) or 28 days (Grade 2/3;I3, D3, D6-7) post-infection. Neutral glycans are indicated by purple staining and acidic glycans by light blue Blood vessel lumens are indicated by an “L”. Scale bars in D1-3: 2 mm; all others: 20 µm.

Article Snippet: Antibodies used in this study were: B3Galt6 (H00126792-B01P, Biotechne), Giantin (ab80864, abcam), Laminin α4 (AF7340, Biotechne), Δ-HS (F69-3G10, AMS Biotechnology), perlecan (7B5, ThermoFisher Scientific), TRITC-conjugated phalloidin (FAK100, Merck), Alexa Fluor 594 goat anti-rabbit (A11012), Alexa Fluor 488 donkey anti-mouse (A21202) and Alexa Fluor 488 donkey anti-sheep (A11015) from Invitrogen/ThermoFisher Scientific.

Techniques: Staining, Flow Cytometry, Injection, Infection