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.
Buruli ulcer (BU) is the third most common mycobacterial disease in immunocompetent hosts. BU is caused by Mycobacterium ulcerans, which produces skin ulcers and necrosis at the site of infection. The principal virulence factor of M. ulcerans is a polyketide-derived macrolide named mycolactone, which has cytotoxic and immunosuppressive activities. We determined the severity of inflammation, histopathology and bacillary loads in the subcutaneous footpad tissue of BALB/c mice infected with 11 different M. ulcerans isolates from diverse geographical areas. Strains from Africa (Benin, Ghana, Ivory Coast) induced the highest inflammation, necrosis and bacillary loads, whereas the strains collected from Australia, Asia (Japan, Malaysia, New Guinea), Europe (France) and America (Mexico) induced mild inflammation. Subsequently, animals were infected with the strain that exhibited the highest (Benin) or lowest (Mexico) level of virulence in order to analyse the local immune response generated. The Mexican strain, which does not produce mycolactone, induced a predominantly T helper type 1 (Th1) cytokine profile with constant high expression of the anti-microbial peptides beta defensins 3 and 4, in co-existence with low expression of the anti-inflammatory cytokines interleukin (IL)-10, IL-4 and transforming growth factor (TGF)-beta. The highly virulent strain from Benin which produces mycolactone A/B induced the opposite pattern. Thus, different local immune responses were found depending on the infecting M. ulcerans strain.