Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans

Abstract

Mycobacterium ulcerans (MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized example of plasmid-mediated virulence in a Mycobacterium, and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster (>97% amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.

Fig. 1.

Demonstration of the mycolactone plasmid. Pulsed-field gel electrophoresis (A) and Southern hybridization (B) analyses of MU Agy99 (lanes 1 and 2) and MU1615 (lanes 3 and 4), showing the presence of the linearized form of the plasmid in nondigested genomic DNA (lanes 1 and 3) and after digestion with XbaI (lanes 2 and 4), hybridized to a combination probe derived from mlsA, mlsB, mup038, and mup045. Lane M is the lambda low-range DNA size ladder (NEB).

Fig. 2.

Circular representation of pMUM001. The scale is shown in kilobases by the outer black circle. Moving in from the outside, the next two circles show forward and reverse strand protein-coding DNA sequences, respectively, with colors representing the functional classification (red, replication; light blue, regulation; light green, hypothetical protein; dark green, cell wall and cell processes; orange, conserved hypothetical protein; cyan, insertion sequence elements; yellow, intermediate metabolism; gray, lipid metabolism). These circles are followed by the GC skew (G – C)/(G + C) and finally the G + C content by using a 1-kb window. The arrangement of the mycolactone biosynthetic cluster has been highlighted, and the locations of all XbaI sites are indicated.

Fig. 3.

Domain and module organization of the mycolactone PKS genes. Within each of the three genes (mlsA1, mlsA2, and mlsB), different domains are represented by a colored block. The domain designation is described in the key. White blocks represent interdomain regions of 100% identity. Module arrangements are depicted below each gene, and the modules are color-coded to indicate identity both in function and sequence (>98%). For example, module 5 of MLSA1 is identical to modules 1 and 2 of MLSB. The crosses through four of the dehydratase domains indicate that they are predicted to be inactive based on a point mutation in the active site sequence. The structure of mycolactone has also been color-coded to match the module responsible for a particular chain extension.

Fig. 4.

Mycolactone transposon mutants. Mycolactone negative mutants were identified as nonpigmented colonies (Inset). Bacteria (1 × 10⁷) and 50 μl of culture filtrate were added to a semiconfluent monolayer of L929 fibroblasts for detection of cytotoxicity. Treated cells shown at 24 h. (A) MU1615::Tn104 containing an insertion in mlsB. (B) WT MU1615. (C) Untreated control cells. (D) MU1615::Tn141 containing an insertion in mlsA (×20).

Fig. 5.

MS analyses of the mycolactone transposon mutants. (A) MU1615::Tn104 containing an insertion in mlsB, showing the absence of the mycolactone ion m/z 765 and the presence of the lactone core ion at m/z 447. (B) WT MU1615 showing the presence of the mycolactone ion m/z 765. (C) Control mutant MU1615::Tn99 containing a non-MLS insertion, showing the presence of the mycolactone ion m/z 765. (D) MU1615::Tn141 containing an insertion in mlsA, showing the absence of both the mycolactone ion m/z 765 and the lactone core ion at m/z 447.