Report

Mycolactone: A Polyketide Toxin from Mycobacterium ulcerans Required for Virulence

See allHide authors and affiliations

Science  05 Feb 1999:
Vol. 283, Issue 5403, pp. 854-857
DOI: 10.1126/science.283.5403.854

Abstract

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe human skin disease that occurs primarily in Africa and Australia. Infection with M. ulcerans results in persistent severe necrosis without an acute inflammatory response. The presence of histopathological changes distant from the site of infection suggested that pathogenesis might be toxin mediated. A polyketide-derived macrolide designated mycolactone was isolated that causes cytopathicity and cell cycle arrest in cultured L929 murine fibroblasts. Intradermal inoculation of purified toxin into guinea pigs produced a lesion similar to that of Buruli ulcer in humans. This toxin may represent one of a family of virulence factors associated with pathology in mycobacterial diseases such as leprosy and tuberculosis.

Most pathogenic bacteria produce toxins that are important in disease. However, none has been identified for Mycobacterium tuberculosis andMycobacterium leprae. The only mycobacterial pathogen for which there is any evidence of toxin production is Mycobacteria ulcerans, the causative agent of Buruli ulcer. Although Buruli ulcer is little known outside the tropics, it recently has been recognized as an emerging infection in western Africa (1).Mycobacterium ulcerans disease has several distinctive features. Infection results in progressive necrotic cutaneous lesions, which may persist for a decade if untreated and may extend to 15% of a patient's skin surface. Despite extensive necrosis, lesions are painless, symptoms of systemic disease are absent, and there is little histological evidence of an initial acute inflammatory response (2, 3). Finally, in contrast to other pathogenic mycobacteria, which are facultative intracellular parasites of macrophages, M. ulcerans occurs in lesions primarily as extracellular microcolonies.

A curious feature of Buruli ulcer pathology is that organisms lie in a necrotic focus with the necrosis extending some distance from the site of bacterial colonization. This observation led to the hypothesis thatM. ulcerans secreted a toxin (2). In 1974, Readet al. reported that a sterile filtrate of M. ulcerans had a cytopathic effect on cultured murine fibroblasts (4). Early efforts to isolate this toxin were not successful (5, 6). More recently, Pimsler et al. (7) reported that a sterile filtrate of M. ulcerans had immunosuppressive properties. Earlier this year, we reported cytotoxic activity associated with acetone-soluble lipids (ASL) present in an organic extract from M. ulcerans sterile filtrate (8). The cytopathic effect of M. ulcerans or ASL on L929 murine fibroblasts was further characterized by showing that M. ulcerans or ASL arrested cells in the G0/G1 stage of the cell cycle. In this paper, we report the purification of this toxin and present evidence for its role in the pathogenesis of Buruli ulcer.

Initial attempts to obtain sufficient toxin from M. ulcerans sterile filtrate for structural analysis were frustrated by low yield. To increase yield, we developed a method for isolating toxin from intact bacteria (9). ASL were prepared from an extract of M. ulcerans containing chloroform and methanol (2:1) and were separated by thin-layer chromatography (TLC) on silica gel plates. Lipid bands were eluted from TLC plates and tested for cytopathicity on L929 mouse fibroblast cells as described (8). Maximum toxic activity was associated with a light yellow, ultraviolet-active component (10) with a refractive index of 0.23 in a solvent system containing chloroform, methanol, and water (90:10:1) (Fig. lA). This compound was further purified by reversed-phase high-performance liquid chromatography and subjected to structural analysis. Mass spectral analysis of the toxin molecule under microspray conditions showed peaks at m/z 765 (strong), 743 (weak), and 725 (medium) (Fig. 1B). Accurate mass measurement of the peak at m/z 765 (M+ + Na: C44H70O9Na, observed 765.4912; calculated 765.4912; error <0.1 ppm) and the peak atm/z 725 (M+−OH: C44H69O8, observed 725.4988; calculated 725.4987; error = 0.1 ppm) gave the formula C44H70O9 for the compound. The compound was identified by two-dimensional nuclear magnetic resonance spectral analysis as a polyketide-derived 12-membered ring macrolide (Fig. 2) (11). The toxin was named mycolactone to reflect its mycobacterial source and chemical structure.

Figure 1

Isolation of mycolactone and effects on cultured fibroblasts. (A) Mycolactone as a major lipid species in ASL from M. ulcerans, run on silica TLC, and with chloroform, methanol, and water (90:10:1) used as a solvent system. Several mycolactone-derived degradation products appear between Rf 0.3 and 0.6. Lane WT, wild type; lane mycolactone, purified mycolactone; lane 1615A, ASL from isogenic tox mutant. Lipids were visualized by charging TLC plates with ceric ammonium molybdate. Rf, refractive index. (B) Mass spectrum of mycolactone. (C) L929 murine fibroblasts were treated for 48 hours with mycolactone at 25 pg/ml. (Right) Mycolactone-treated cells; (left) control monolayer. ×100. (D) Flow cytometric analysis of L929 cells treated with ASL or mycolactone from M. ulcerans (13). Cells exposed to M. ulcerans ASL (1 μg/ml), mycolactone (1 μg/ml), or control lipids (1 μg/ml) were lysed after 48 hours in the presence of RNase, stained with propidium iodide, and analyzed with FACStar for a cell cycle profile.

Figure 2

Mycolactone is composed of a 12-membered ring to which two polyketide-derived side chains (R1 and R2) are attached.

To determine whether mycolactone was cytopathic, we applied serial dilutions of sterile filtrate or mycolactone to an overnight culture of L929 cells. Within 24 hours after addition of mycolactone, the cells rounded up and by 48 hours most cells lifted off the plate (Fig. 1C). Washed cells were capable of regrowth, which indicated that the effect of toxin was reversible. Mycolactone at a concentration of 25 pg/ml was sufficient for cytopathicity. Chemical modification of mycolactone by acetylation of hydroxyl groups or hydrogenation that resulted in saturation of double bonds led to ablation of cytopathic activity (12). We had previously shown that M. ulcerans–derived ASL-treated cells were arrested in G0/G1 of the cell cycle (8). Flow cytometric analysis of the mycolactone-treated cell population showed that treatment of L929 cells with mycolactone arrested cells in G0/G1 of the cell cycle (Fig. 1D). These studies confirmed that the morphological changes and kinetics of cytopathicity reported previously with M. ulcerans ASL or sterile filtrate could be entirely reproduced by mycolactone alone.

To determine whether mycolactone-mediated cytopathicity was a correlate of in vivo virulence, Mycolactone was tested in a guinea pig model of virulence (14). The extensive necrosis in the absence of an acute inflammatory response is well preserved in the guinea pig model of infection (5). For these studies, we injected samples intradermally into the shaved back of six Hartley guinea pigs, and examined animals daily for evidence of gross pathological changes (15). Two guinea pigs were killed 2, 8, and 20 days after injection, and tissue was excised for histopathological analysis. Within 24 hours after injection, erythema was present at the site of highest mycolactone concentration (100 μg). Gross pathology was absent from all other injection sites at that time. The area injected with 100 μg of mycolactone increased in size until day 5 when the lesion appeared as a dark, necrotic, open wound. The lesion remained essentially the same until day 8 when tissue was harvested (Fig. 3A). Pressure applied to the necrotic area did not elicit a response from the guinea pig, which suggests that the lesion was painless. We observed slight erythema near the injection site of 10 μg of mycolac- tone on day 3 but a lesion did not develop. Injection of 1 μg of mycolactone was negative for gross pathology. Injection of 107 M. ulcerans produced slight erythema by day 3; by day 8 a small, open lesion was present (Fig. 3A). Although the upper dermis sloughed off the top of lesions where both mycolactone and M. ulcerans had been injected, resulting in a depressed ulcer, a purulent exudate was never present. In contrast, injection of 107 Mycobacterium marinum resulted in a purulent pus-filled lesion by day 5. No gross pathological changes resulted from the injection of 100 μl of mycobacterial medium or 100 μg of Red 77, an abundant lipid species present in the ASL fraction from which mycolactone was purified.

Figure 3

Pathology in guinea pig skin after intradermal injection of mycolactone and M. ulcerans 8 days after infection. (A) Injection of mycobacterial medium or tox mutant M. ulcerans 1615A did not produce a lesion, whereas injection of either viable M. ulcerans or 100 μg of mycolactone resulted in formation of a necrotic, noninflammatory lesion. Histopathology was evaluated from paraffin-embedded sections stained with hematoxylin and eosin. WT, wild type. (B) Injection of mycobacterial medium used as a control. (C) (left) Injection of M ulcerans. (Right) Microhemorrhage and necrosis throughout the fat layer extending into the muscle were frequent findings. (D) Zeihl-Neelson staining showing typical large clusters of predominantly extracellular M. ulcerans. (E) Injection of 10 μg of mycolactone. Occluded vessels (right) were frequently found in areas of necrosis. (F) Injection of M. marinum. (B) and (C) (left), (E) (left), and (F) (left), ×40; (D) and (C) (right), (E) (right), and (F) (right), ×400.

Histopathological findings (Fig. 3) demonstrated that the cellular damage resulting from injection of mycolactone and M. ulcerans bacteria was nearly identical (Fig. 3, C and E). An examination of paraffin-embedded sections stained with hematoxylin and eosin revealed the characteristic pathology of Buruli ulcer. Focal necrosis extended through the dermis and adipose tissue and into muscle. Despite considerable tissue destruction, few polymorphonuclear neutrophils were present. Vascular erosion, microhemorrhage, and thrombosed vessels were prominent features in these lesions (Fig. 3E). Although gross pathological changes did not result from injection of 1 μg of mycolactone, histopathology was present as a small area of necrosis near the injection site. No significant pathological changes resulted from injection of 100 μg of Red 77, an abundant lipid species present in the M. ulcerans ASL extract (Fig. 1A). In contrast, injection of M. marinum produced an acute inflammatory response consisting of large numbers of mononuclear and polymorphonuclear cells (Fig. 3F).

We also obtained genetic evidence to support the role of mycolactone in virulence. Mycobacterium ulcerans can become attenuated through laboratory passage (5). Two isolates ofM. ulcerans in our collection that were not cytopathic for fibroblasts and were avirulent in guinea pigs had aberrant colonial pigment. Whereas virulent strains produced yellowish tan colonies on Middlebrook 7H10 medium supplemented with oleic acid, albumin, dextrose, and catalase, avirulent strains formed colonies that were less pigmented. Because mycolactone is a pale yellow compound and polyketide production often leads to pigmentation inStreptomyces species, we thought that the lack of pigment might be a marker for toxin loss. Examining aged cultures, we isolated an isogenic mutant of M. ulcerans by selecting a less pigmented sector from a normal yellow colony of M. ulcerans1615. This tox mutant was designated 1615A. ASL were extracted from 1615A as described (8), separated by TLC, and found to be devoid of mycolactone (Fig. 1A). Sterile culture filtrate from 1615A as well as ASL extracted from M. ulcerans 1615A was not cytopathic for L929 cells (16). Finally, intradermal inoculation of either 108 or 107 M. ulcerans 1615A into guinea pigs did not produce an ulcer (Fig. 3A). Aside from slight erythema, no abnormalities resulted from these injections. Histopathological examination of these sites showed mild inflammation with no necrosis.

Mycolactone is the first toxin isolated from Mycobacteriaspecies as well as the first complex polyketide isolated from a pathogenic mycobacteria species (17). The discovery thatM. ulcerans toxin is a polyketide is highly significant. Polyketides are lipid-like molecules that, although relatively small compared with protein toxins, have potent biological activities. Well-known polyketides include antibiotics (erythromycin), immunosuppressants, (rapamycin, FK506), antifungal agents (amphotericin B), antihelmetic agents (avermectin), and cytostatins (bafilomycin). Most complex polyketides are made as secondary metabolites by soil bacteria in the order Actinomycetales (18–21). We speculate that mycolactone protects M. ulcerans from predatory eukaryotes in its natural habitat (22, 23).

The role of mycolactone as a virulence determinant may have implications far beyond those for Buruli ulcer. A major surprise from the M. tuberculosis genome project was the discovery that the genome contains a large number of polyketide synthesis (PKS I) genes, although no polyketides have been isolated from M. tuberculosis (24). This family of potent compounds could play a major role in both the tissue destruction and immunological modulation characteristic of diseases such as leprosy and tuberculosis. Further, because most pathogenic mycobacteria are intracellular pathogens, it is possible that they play an important role in intracellular survival. Complex polyketides such as Mycolactone may represent the first of a newly discovered class of virulence compounds.

REFERENCES AND NOTES

View Abstract

Navigate This Article