A newly discovered mycobacterial pathogen isolated from laboratory colonies of Xenopus species with lethal infections produces a novel form of mycolactone, the Mycobacterium ulcerans macrolide toxin

Abstract

Mycobacterium ulcerans, the causative agent of Buruli ulcer, produces a macrolide toxin, mycolactone A/B, which is thought to play a major role in virulence. A disease similar to Buruli ulcer recently appeared in United States frog colonies following importation of the West African frog, Xenopus tropicalis. The taxonomic position of the frog pathogen has not been fully elucidated, but this organism, tentatively designated Mycobacterium liflandii, is closely related to M. ulcerans and Mycobacterium marinum, and as further evidence is gathered, it will most likely be considered a subspecies of one of these species. In this paper we show that M. liflandii produces a novel plasmid-encoded mycolactone, mycolactone E. M. liflandii contains all of the genes in the mycolactone cluster with the exception of that encoding CYP140A2, a putative p450 monooxygenase. Although the core lactone structure is conserved in mycolactone E, the fatty acid side chain differs from that of mycolactone A/B in the number of hydroxyl groups and double bonds. The cytopathic phenotype of mycolactone E is identical to that of mycolactone A/B, although it is less potent. To further characterize the relationship between M. liflandii and M. ulcerans, strains were analyzed for the presence of the RD1 region genes, esxA (ESAT-6) and esxB (CFP-10). The M. ulcerans genome strain has a deletion in RD1 and lacks these genes. The results of these studies show that M. liflandii contains both esxA and esxB.

FIG. 1.

Identification of mycolactone biosynthesis genes in M. liflandii. (A) Schematic arrangement of the mycolactone gene cluster in M. ulcerans. repA, plasmid replication region; p450, CYP140A2 (p450 monooxygenase); MUP045, FabH-like ketosynthase; mlsA, Pks (lactone core); MUP037, thioesterase (TE II); mlsB, Pks (fatty acid side chain). (B) PCR evidence for mycolactone and plasmid genes in M. liflandii. Lanes 1 to 4, 7, and 9, M. liflandii from X. tropicalis; lanes 5 and 8, M. liflandii from X. laevis; lane 6, 6F; lane 10, M. marinum 1218; lane 11, M. ulcerans 1327; lane 12, M. ulcerans 1615; lane 13, water. Lane M contained a 1-kb DNA ladder (Invitrogen).

FIG. 2.

Mycolactone is plasmid encoded in M. liflandii. (A) Pulsed-field electrophoresis; (B) Southern hybridization analysis. Lane 1, M. ulcerans 1615; lane 2, M. ulcerans Agy99; lane 3, M. liflandii xt128; lane 4, M. liflandii xl5; lane 5, M. ulcerans V2; lane 6, pMUMP-1, showing the presence of a large plasmid hybridized to a combination probe derived from M. ulcerans 1615 plasmid-specific enoyl reductase (mlsA) and the origin of replication (repA). Lane M contained the lambda PFG size ladder (New England Biolabs).

FIG. 3.

Comparison of mycolactone E with mycolactone A/B. (A) MS-MS of mycolactones A/B (m/z 765.488) (M. ulcerans) and mycolactone E (m/z 737.498) (M. liflandii), showing the presence of core lactone at m/z 429 and a side chain at m/z 359.4 (mycolactone A/B) and m/z 331.4 (mycolactone E). (B) Structure of mycolactone A and mycolactone E. MW, molecular weight.

FIG. 4.

M. ulcerans and M. liflandii on Middlebrook 7H10 medium with oleic acid-albumin-dextrose supplement, showing the characteristic pigmentation. Clockwise from the top: M. ulcerans 1615 and M. liflandii xt3, xt4, xl5, xt6808, and xl7281 (xt, X. tropicalis; xl, X. laevis).

FIG. 5.

Cytopathic activity of 100 ng mycolactone on L929 fibroblasts after 36 h of incubation. (A) Untreated cells; (B) mycolactone A/B (100 ng); (C) mycolactone E (100 ng). Total magnification, ×200.

FIG. 6.

PCR amplification of esxA (ESAT-6) and esxB (CFP-10) in M. liflandii and geographically diverse isolates of M. ulcerans. Positive samples: lane 1, M. marinum 1218 (positive control); lane 5, M. ulcerans Valente; lane 6, M. ulcerans Gaillon; lane 7, M. ulcerans 01G897; lane 8, M. ulcerans 842; lane 9, M. ulcerans 7922; lane 10, M. ulcerans 5114; lane 11, M. ulcerans 5143; lane 12, M. ulcerans 98-912; lane 13, M. ulcerans 8756; lane 15, M. marinum 00-1026; lane 16, M. liflandii xt128; and lane 17, M. liflandii xl7281. Negative samples: lane 2, M. ulcerans 1615; lane 3, M. ulcerans Agy99; lane 4, M. ulcerans 1327; lane 14, M. ulcerans 00-524; and lane 18, water (negative control). Lanes M contained a 100-bp DNA ladder (Promega).