Antioxidant protection against mycolactone cytotoxicity.

<p>(A) Antioxidants were added 30 min before mycolactone (300 ng/ml) and the incubation was continued for 48 h when cell numbers were determined by measuring WST-1color at 450 nm. Data represent means of triplicate determinations and standard deviations from one representative experiment out of two performed (*, p<0.05, one way ANOVA and students t-test). (B) Deferoxamine (D) and TEMPOL were added at different concentrations alone or in combination 30 min before mycolactone (300 ng/ml) and the incubation was continued for 48 h when cell numbers were determined by measuring neutral red uptake Concentrations are in µM for all substances except catalase which is in U/ml. Data shown are means and SEM of four experiments (*, p<0.05, ANOVA and students t-test). TEMPOL 1600 µM was not included in the statistical analysis (N = 2).</p

Mycolactone increases ROS production.

<p>Intracellular ROS production was detected by intracellular CM-H₂DCFDA fluorescence in keratinocytes after 45 min incubation in control medium or with 100 and 300 ng/ml mycolactone. Parallel cultures were pretreated for 30 min with a combination of deferoxamine (100 µM) and TEMPOL (200 µM) (D+T) before CM-H₂DCFDA and addition of mycolactone. Asterisks indicate significant (p<0.05) differences in fluorescence between treatments and their corresponding control in one representative experiment with four to six replicates (means and SD).</p

Mycolactone is cytotoxic for keratinocytes.

<p>Mycolactone was added to sub-confluent cultures of keratinocytes and (A) cell numbers were measured as optical density of WST-1after 24, 48 and 72 h of treatment with 1000 ng/ml of mycolactone, or (B) after 72 h at different concentrations of mycolactone. An asterisk indicates a significant (p<0.05) reduction in WST-1 labeling as compared to the corresponding medium control. Cell cultures of untreated controls (C) and cultures treated with 300 ng/m mycolactone (D) were photographed after 48 h. Data are from one representative experiment showing means and SD of triplicates.</p

Cytotoxicity of ASL from human Mu lesions on human embryonic lung fibroblasts.

<p>Cytotoxicity after 48 h culture was assessed using an MTT assay. Negative control 1 is untreated cells, negative control 2 is ASL from uninfected skin. Positive control was purified mycolactone at a concentration of 5 µg/ml. Significant cytotoxicity was observed with all patient samples with ***p<0.001 compared to negative control 1. The apparent difference in percentage cytotoxicity between 5 untreated and 5 antibiotic treated lesions was not statistically significant. HELF cells were treated in quadruplicates and cytotoxicity determined in at least 2 independent experiments. Data are shown as a percentage of untreated cells (negative control 1). Error bars are ±SEM of duplicate assays.</p

Detection of mycolactone by mass spectrometry.

<p>A. MS analysis of ASL from Mu infected human skin showing a molecule with m/z 765.5 which represents the sodium adduct of mycolactone A/B [M+Na⁺]. B. MS-MS analysis of this ion produced the core lactone ring of mycolactone with m/z 429.4 and the polyketide side chain of mycolactone A/B with m/z 359.2.</p

Patient characteristics, diagnostic data and treatment status.

*<p>Antibiotic treatment was with streptomycin injection15 mg/kg and oral rifampicin 10 mg/kg daily.</p

Detection of mycolactone A/B by thin layer chromatography.

<p>A. 20 µl of two-fold serial dilutions of mycolactone A/B at concentrations from 125 to 1 µg/ml were spotted and examined under UV-light and by oxidative charring. The detection limit on TLC was at a concentration of 2–8 µg/ml (8 µg corresponding to 160 ng of mycolactone A/B). B. Each track represents one sample. M is purified mycolactone A/B; tracks 1 and 2 are positive controls with100 µg of purified mycolactone; tracks 3 and 4 are samples extracted from human skin spiked with 100 µg of purified mycolactone; tracks 5 and 6 are negative controls from healthy human skin; tracks 7 to 16 are extracts from infected human skin samples. Mycolactone A/B was the predominant UV-active band with an Rf of 0.23 in positive controls and in ASLs from infected human skin. There were perceptible signals from patients 7, 8, 11, 14 and 15 whereas samples from 9, 10, 12, 13 and 16 were below the detection limit.</p

Chemical structure of mycolactone A/B showing the lactone core ring and polyketide side chains.

<p>Chemical structure of mycolactone A/B showing the lactone core ring and polyketide side chains.</p

The effect of acetone soluble lipids from human Mu lesions on TNF-α release by J774 macrophages.

<p>J774 macrophages were stimulated with 0.5 µg/ml of LPS. Negative control 1 is untreated J774 macrophages, negative control 2 is J774 treated with ASL from uninfected skin. Positive control refers to purified mycolactone at a concentration of 500 ng/ml and patient samples refers to all 10 patient lesions. ASL from infected lesions significantly inhibited TNF-α release compared to both negative controls with ***p<0.001. Error bars are ±SEM of duplicate assays. Although TNF-α release by J774 macrophages was significantly inhibited by purified mycolactone and lipid extracts from patient lesions, this occurred without significant cytotoxicity.</p