Heterogeneity of mycolactones produced by clinical isolates of Mycobacterium ulcerans: implications for virulence

Abstract

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease endemic in tropical countries. Clinical evidence suggests that M. ulcerans isolates from Asia, Mexico, and Australia may be less virulent than isolates from Africa. In vivo studies suggest that mycolactone, a polyketide-derived macrolide toxin, plays a major role in the tissue destruction and immune suppression which occur in cases of Buruli ulcer. Mycolactones were extracted from 34 isolates of M. ulcerans representing strains from Africa, Malaysia, Asia, Australia, and Mexico. Thin-layer chromatography, mass spectroscopic analysis, and cytopathic assays of partially purified mycolactones from these isolates revealed that M. ulcerans produces a heterogeneous mixture of mycolactone variants. Mycolactone A/B, the most biologically active mycolactone species, was identified by mass spectroscopy as [M(+)Na](+) at m/z 765.5 in all cytotoxic isolates except for those from Mexico. Mycolactone C [M+Na](+) at m/z 726.3 was the dominant mycolactone species in eight Australian isolates, and mycolactone D [M+Na](+) m/z 781.2 was characteristic of two Asian strains. Mycolactone species are conserved within specific geographic areas, suggesting that there may be a correlation between mycolactone profile and virulence. In addition, the core lactone, [M+Na](+) m/z 447.4, was identified as a minor species, supporting the hypothesis that mycolactones are synthesized by two polyketide synthases. A cytopathic assay of the core lactone showed that this molecule is sufficient for cytotoxicity, although it is much less potent than the complete mycolactone.

FIG. 1.

Silica TLC of ASLs from Mycobacterium ulcerans isolates run in chloroform-methanol-water (90:10:1, vol/vol/vol) and visualized by oxidative charring in a ceric molybdate-10% sulfuric acid stain. (A) ASLs extracted from the bacterial CM. (B) ASLs extracted from sterile culture filtrate. ML_A/B, mycolactone A/B; Rf, refractive indices; lane M, ASLs extracted from Middlebrook 7H9 media with 10% oleic acid-albumin-dextrose enrichment. Strains are represented in lanes as follows: lane 1, 97-617; lane 2, 97-112; lane 3, 97-101; lane 4, 97-1112; lane 5, 97-110; lane 6, 98-700; lane 7, 98-1137; lane 8, 99-1563; lane 9, 99-1711; lane 10, 97-1116; lane 11, 98-239; lane 12, 97-616; lane 13, 98-156; lane 14, 99-742; lane 15, 97-684; lane 16, 99-1642; lane 17, 99-1722; lane 18, 99-1567; lane 19, 94-1324; lane 20, 94-1325; lane 21, 94-1326; lane 22, 94-1327; lane 23, 94-1328; lane 24, 5114; lane 25, 5143; lane 26, 98-912; lane 27, 8756; lane 28, 1615.

FIG. 2.

Comparison of ASLs extracted from six Australian isolates of M. ulcerans (TS-1 to TS-6) with ASLs extracted from M. ulcerans 1615. Silica TLC was run in chloroform-methanol-water (90:10:1, vol/vol/vol) and visualized by oxidative charring in a ceric molybdate-10% sulfuric acid stain (insert). Mass spectroscopic analysis of ASLs from TS-2, showing the presence of mycolactone C as a major peak at m/z 749.6 along with the presence of mycolactone A/B at m/z 765.0, is shown. ∗, mycolactone A/B; ∗∗, mycolactone C, core lactone; Abund., % abundance.

FIG. 3.

Cytopathicity of individual lipid species in ASLs from M. ulcerans 1615. Individual lipids from bands 1 to 8 were isolated using preparative silica TLC run in a chloroform-methanol-water (90:10:1, vol/vol/vol) solvent system. For cytopathicity assays, twofold dilutions of individual lipids from bands 1 to 8 were added to L929 fibroblasts and scored for CPA. CPA is defined as the least amount of lipid (in nanograms per milliliter) required to cause 90% cell rounding in 24 h and monolayer detachment after 48 h. Lane ASLs, ASLs extracted from M. ulcerans 1615 with chloroform-methanol (2:1, vol/vol); ML_A/B, mycolactone A/B; ML_C/core, mix of mycolactone C and core lactone.

FIG. 4.

Base hydrolysis of mycolactone. Mycolactone (left) was stirred with potassium carbonate in anhydrous methanol for 24 h at room temperature. Hydrolysis products include the core lactone and fatty acid side chain.

FIG. 5.

Mass spectroscopic analysis of the upper band containing the core lactone product. (Insert) Results of silica TLC, showing products of base hydrolysis of mycolactone and hydrolysis products, are shown. Lane 1, mycolactone starting material; lane 2, hydrolysis products; ∗, mycolactone; ∗∗, core lactone; Abund., % abundance.