Detection of Mycolactone A/B in Mycobacterium ulcerans–Infected Human Tissue

Skin infection with bacteria called Mycobacterium ulcerans causes Buruli ulcer, a disease common in West Africa and mainly affecting children. M. ulcerans is the only mycobacterium to cause disease by production of a toxin. This lipid molecule called mycolactone diffuses from the site of infection, killing surrounding cells and, at low concentration, suppressing the immune response. The aim of this study was to show that mycolactone can be detected among lipids extracted from human M. ulcerans lesions in order to study its role in the pathogenesis of M. ulcerans disease. Lipids were extracted from skin biopsies and tested for the presence of mycolactone using thin layer chromatography and mass spectrometry. The extracts were shown to kill cultured cells in a cytotoxicity assay. Mycolactone was detected in both pre-ulcerative and ulcerative forms of the disease and also in lesions during antibiotic treatment but with reduced bioactivity, suggesting a lower concentration compared to untreated lesions. These findings indicate that there is mycolactone in affected skin at all stages of M. ulcerans disease and it could be used as a biomarker for monitoring the clinical response to antibiotic treatment

Using ants to monitor changes within and surrounding the endangered Monsoon Vine Thickets of the tropical Dampier Peninsula, north Western Australia

The 79 naturally fragmented and localised Monsoon Vine Thicket (MVT) patches on the coast of the Dampier Peninsula, Kimberley region, Western Australia, are listed as: a culturally significant ‘Threatened Ecological Community’ (TEC), ‘Endangered’ under the Commonwealth (EPBC Act, 1999) and ‘Vulnerable’ under Western Australian legislation. Fire and introduced species are significant disturbance factors affecting the MVT patches. Focussing on five patches, this study aimed to: assess ant diversity and composition; determine relationships between environmental variables, fire history and invasive ants; determine the value of ants as bioindicators of environmental change; and involve indigenous communities in planning and data collection. Three habitats, inside (I) and edge (E) of five MVT patches (11 ha–56 ha) and immediately outside (O) in the surrounding Pindan woodland (PW), were sampled over two seasons; 7,342 ants from 7 subfamilies and 81 species were collected by pitfall trapping and hand collecting. A gradation of species richness from I (37 spp.), to E (48 spp.), to O (62 spp.) occurred, reflecting trends in environmental variables of decreasing litter volume, litter depth and canopy cover, and increasing fire frequency across habitat types. NMDS ordination identified a separation of ant composition between I, E and O, while CCA revealed a clear separation between I, E and O driven by litter volume and depth, canopy cover and fire history.Distinctly different ant assemblages were found within the MVT, with species inside being those that prefer cool relatively dense vegetation and those at the edges preferring more open habitat. The degree of openness of the edge of the MVT edges is related to fire frequency, while species within the PW were typically arid-adapted species that prefer a more open habitat. Two invasive ant species, namely Paratrechina longicornis and Monomorium destructor, were found to be present in the patches and, in common with many invasive ant species, P. longicornis was characterised by high numbers, comprising 43% of total individuals across the study. As little data previously existed on the ant communities of the dry MVT patches of the Dampier Peninsula, the results add significant new information on the ant fauna of the MVT patches and illustrate how using ants as bioindicators can assist interpreting the impact of fire, invasive organisms and management on the conservation status of MVT patches. This study also serves as a model for collaborating with indigenous people to undertake data gathering, interpretation and management of such areas

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

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

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

Patient characteristics, diagnostic data and treatment status.

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