Mycolactone inhibits co-translational translocation of proteins into the ER via a mechanism that does not disrupt the structural integrity of the ER.

<p>A–E. <i>In vitro</i> translation (IVT) reactions of different capped transcripts were performed as described +/− mycolactone (MYC; 200 ng/ml unless indicated otherwise), Eeyarestatin 1 (ES1; 250 µM) or DMSO (-; 0.02%). Data representative of 3 independent experiments. A. IVT of luciferase and TNF mRNAs detected by ³⁵S incorporation and Western blot (∼26 kDa) respectively. B. IVT of TNF mRNA was performed in the presence of semi-permeabilised RAW264.7 cells then incubated with no addition (-), or Proteinase K (PK) +/−0.1% Triton-x-100 (PKT) for 1 hr at 4°C before stopping the reaction. C. Dose dependence of loss of the PK protected band. Signal intensity was quantified by ImageJ analysis of non-saturated blots and the protected band (PK) was normalised to total pro-TNF (-). D. IVT of prepro-α Factor (PPAF) and β-lactamase (LACTB) mRNAs in the absence or presence of canine pancreatic microsomal membranes (CPMM). Black arrowhead, glycosylated forms of α Factor; *, signal peptide-cleaved LACTB. E. IVT of PPAF, Sec61β and cytochrome B5 (Cyt-B5) mRNAs in the presence CPMM. After labelling with ³⁵S as described, membranes were isolated by centrifugation through a sucrose cushion <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004061#ppat.1004061-Cross1" target="_blank">[31]</a>. Endoglycosidase H (EndoH) is used to confirm glycosylation of proteins (black arrowhead), the thin line represents where an empty lane was removed from the image for ease of interpretation. F. RAW264.7 cells were incubated +/−125 ng/ml mycolactone (MYC), washed and incubated without mycolactone for various periods (recovery time) before stimulating with LPS for 4 hrs. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays).</p

Mycolactone specifically targets membrane and secreted proteins.

<p>Cells in Met/Cys free medium were incubated +/−125 ng/ml mycolactone (MYC), 10 µg/ml cycloheximide (CHX) or 0.0125% DMSO for 1 hr and stimulated with LPS for 1 hr before replacement with fresh medium containing Tran35slabel for 2 hrs. Data representative of 3 independent experiments (A–D). A. Cytosolic and digitonin-resistant membrane fractions from treated RAW264.7 cells (10⁵ cell equivalents/lane). B. Quantification of ³⁵S incorporation in (A) by scintillation counting (mean±SEM, n = 3). *, P<0.05; **, P<0.01; ***, P<0.001. C. Total cell lysates, Concanavalin A (ConA) agarose precipitated proteins (Glycosylated) and supernatants (Media) from RAW264.7 cells. D. Dose dependence of the suppression of supernatant protein production in RAW264.7 cells. E. ConA precipitated (Glycos) and supernatant proteins (Media) from ³⁵S labelled Human microvascular dermal epithelial cells (HDMVEC), L929 fibroblasts and Hela cells. In each case total cell labelling was comparable between samples (not shown).</p

Proinflammatory mRNAs are actively translating in the presence of mycolactone.

<p>RAW264.7 cells were incubated for 1+/−125 ng/ml mycolactone (MYC), then stimulated with LPS. A and B. After 4 hr 100 µg/ml puromycin (PURO) or 5 µM homoharringtonine (HH) were added for 3 min, then CHX was added before lysis and separation of polysomes on a 10–20% sucrose gradient. LPS (solid black line), LPS+PURO (solid green line) and LPS+HH (dotted blue line). A. RNA profiles measured by absorbance at 254 nm. B. Quantitation of specific mRNAs purified from each fraction and analysed by Northern blotting. Signal intensity was quantified by ImageJ analysis of non-saturated phosphorscreen images. Values are presented as percentage of total signal. C and D. Cytosolic and digitonin-resistant membrane fractions were prepared from treated cells as described. C. Western blot of cell fractions (0.5×10⁵ cell equivalents/lane). GCS1; glucosidase I (∼92 kDa), GAPDH (∼40 kDa). D. total RNA was used as a template in qRT-PCR absolute quantitation assays and is presented as % total RNA for each gene (mean±SEM). All data representative of 3 independent experiments.</p

Mycolactone causes degradation of TNF and Cox-2 by the 26S proteasome in the cytosol.

<p>RAW264.7 cells were incubated +/−125 ng/ml mycolactone (MYC) or 5 µg/ml tunicamycin (TUN) for 1 hr and stimulated with LPS for 4 hrs. In certain samples 5 µM PSI was added 2 hrs after the LPS stimulation. This allowed time for the proteasome-dependent activation of NFκB required for transcriptional activation to occur before proteasome activity was inhibited (see text). A. Supernatant TNF levels from cells treated as above, measured by ELISA (mean±SEM). B. Western blot of cell lysates (0.5×10⁶ cell equivalents/lane), the dotted line separates samples with and without PSI treatment for ease of interpretation. C. Signal intensity was quantified by ImageJ analysis of non-saturated blots and normalised to LPS+TUN (not shown). Values are mean intensity for each lane±SEM of 3 independent experiments. For Cox-2 this is the unglycosylated mol wt band only (UG Cox-2). *, P<0.05; ***, P<0.001. D. Western blot of cytosolic and digitonin-resistant membrane fractions from cells treated with PSI as described above. All data representative of (or pooled from, C) 3 independent experiments. G; glycosylated Cox-2 (∼80 kDa), UG; unglycosylated Cox-2 (∼69 kDa), pro-TNF (∼26 kDa), GAPDH (∼40 KDa), GCS1; glucosidase I (∼92 KDa). Open arrowheads indicate cells treated with mycolactone but not PSI. Closed arrowheads indicate cells treated with mycolactone and PSI.</p

Mycolactone does not change the polysomal association of proinflammatory mRNAs.

<p>A. RAW264.7 cells were incubated for 1+/−various concentrations of mycolactone (MYC as indicated), 0.5 µg/ml Actinomycin D (Act D) or 0.0125% DMSO then stimulated or not with LPS for 4 hr. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays). B–D. RAW264.7 cells were incubated for 1 hr+/−125 ng/ml mycolactone (MYC) then stimulated or not with LPS. After 4 hrs cells were harvested and lysed in the presence of CHX. B and C. Polysomes were separated on a 10–50% sucrose gradient and the profiles measured by absorbance at 254 nm. Note the increase in the 60S peak and reduced height of the polysome peaks in MYC and LPS+MYC samples. RNA was purified from gradient fractions and transcripts were detected by Northern blotting using full coding region cDNA probes for the genes indicated. D. Signal intensity was quantified by ImageJ analysis of non-saturated phosphorscreen images. Values are presented as percentage of total signal for control (dashed line), LPS (solid black line) and LPS+MYC (red line) cells. All are representative of 3 independent experiments. PABP; poly A-tract binding protein.</p

Clinical features of the BU patients from whom punch biopsy samples were analysed in this work.

<p>The WHO category for each lesion was as follows: Category 1; a single small lesion or ulcer <5 cm in diameter, category 2; a single lesion 5–15 cm in diameter, category 3 a single lesion > 15 cm, multiple small lesions or lesions on the face. Data are n (%) or median (IQR).</p><p>Clinical features of the BU patients from whom punch biopsy samples were analysed in this work.</p

Histopathological features and biomarker expression in the skin of Buruli ulcer patients.

<p>The distribution of the data were assessed using the D'Agostino & Pearson normality test with a cut-off value of P<0.05. Semi-quantitative scoring was carried out as described in Materials and Methods. TM is normally expressed in the keratinocytes of the epidermis and the endothelial cells of the dermis and subcutis. Necrosis was scored according to the appearance of the tissue in H&E staining.</p><p>¹ TM staining was only scored in areas of lesions that had stained positively for SMA (endothelial cell marker), and a combined score derived from both the coverage and intensity was used.</p><p>²A major effect was defined as a score <1 for SMA and keratinocyte TM, <4 for endothelial TM combined score and >2 for fibrin staining and appearance of necrosis.</p><p>³A moderate effect was defined as a score of 1–2.5 inclusive for SMA and keratinocyte TM, combined endothelial TM score of 4–8 inclusive and 0.5–2 inclusive for fibrin staining and appearance of necrosis.</p><p>⁴No effect was defined as patients with maximum (SMA and TM) or zero (fibrin staining and appearance of necrosis) scores.</p><p>⁵In 11 (25%) of patients, TM was considered to be absent (combined TM score <1).</p><p>⁶Four of these patients did not show evidence of SMA staining in the subcutis and so TM was not quantified in these biopsies due to lack of intact endothelium.</p><p>⁷In 18 (50%) of patients, TM was considered to be absent (combined TM score <1)</p><p>Histopathological features and biomarker expression in the skin of Buruli ulcer patients.</p

Mycolactone causes detachment of endothelial cells, apoptosis in 3–4 days, and depletes CD31 (PECAM-1) and VE-cadherin the surface.

<p>Human dermal microvascular endothelial cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml TNF, 1μM staurosporin or 0.025% DMSO (solvent control) as appropriate. A. Both attached and detached cells were subjected to Calcein/EtBr staining for live/dead cells. The proportion of cells that were either attached or detached and alive (Calcein +/EtBr-), and attached or detached and dead (Calcein-/EtBr +) are expressed as a % of the total population of cells. mean±SEM, n = 3. B. HDMVECs were exposed to 7.8ng/ml mycolactone over a timecourse. Cells were then analysed by confocal microscopy following staining of cells with no-wash reagents. CellEvent caspase-3/7 green detection reagent identified cells undergoing apoptosis, alongside PI and DRAQ5. The number of cells in late apoptosis (positive for both active caspase 3/7 and PI) were counted in 3 fields and expressed as a proportion of total cells (DRAQ5 stained) Mean±SEM (n = 3). C. Cells were harvested and subjected to flow cytometry for VE-Cadherin and CD31 (PECAM-1). MFIs are expressed as % of untreated cells (mean±SEM, n = 4). Unstained and isotype bars are for untreated cells. **, P<0.01; ***, P<0.001.</p

Abundant fibrin deposition in the skin of patients with Buruli ulcer.

<p>A and B. Histological sections were stained with an α-fibrin antibody and counterstained with Haematoxylin. Slides were analyzed with a DM2500 Microscope (Leica). Pictures were taken with an Aperio scanner. Comparative staining of a healthy skin sample from an unaffected donor (A) or 4mm punch biopsies from 8 different laboratory confirmed BU patients (B) showing the variability in fibrin staining observed ranging from small isolated fibrin depositions (B1-B2), to large deposition seen only in the dermis or subcutis (B3-B6) and finally to extensive depositions covering the whole tissue sample (B7-B8). C and D. Scoring was carried out as described in Materials and Methods and are relative to a maximum of 3. The score for each individual biopsy analysed is shown, consisting of 31 patients with ulcers (red circles) and 9 patients with plaque lesions (green circles). The expected score for healthy tissue was 0 for both fibrin and necrosis (see A). In all cases, error bars show the median and 25–75% percentile scores. C. Fibrin staining in the dermis and subcutis. D. The degree of necrosis in each biopsy was scored according to appearance of the entire biopsy.</p

Mycolactone causes a profound depletion of thrombomodulin (TM) endothelial cell surfaces.

<p>Human dermal microvascular endothelial cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml IL-1β or 0.025% DMSO (solvent control, equivalent to that in 125ng/ml MYC) for 24 hours (or other times as indicated). Cells were harvested and subjected to flow cytometry for TM and EPCR. A. Forward and side scatter plots. B. Histogram plots for TM and EPCR. Untreated unstained cells; filled grey, untreated cells with isotype control; dotted black line, untreated cells with TM or EPCR-PE; black line; MYC treated cells (7.8ng/ml) with TM or EPCR-PE, red line; IL-1β treated cells with TM or EPCR-PE, green line. C. Quantitation of TM and EPCR surface expression. MFIs are expressed as % of untreated cells with TM or EPCR-PE (mean±SEM, n = 3). Unstained and isotype bars are for untreated cells. D Quantitation of TM and EPCR surface expression over time in the presence of 7.8ng/ml mycolactone. MFIs are expressed as % of untreated cells with TM or EPCR-PE. DMSO control (0.08%) is after 48hrs exposure (mean±SEM, n = 3). *, P<0.05; **, P<0.01; ***, P<0.001.</p