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

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

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