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.A single Buruli ulcer, which can cover Ͼ15% of a person's skin surface, contains huge numbers of extracellular bacteria. Despite their abundance and extensive tissue damage, there is a remarkable absence of an acute inflammatory response to the bacteria, and the lesions are often painless (1). This unique pathology is attributed to mycolactone, a macrolide toxin consisting of a polyketide side chain attached to a 12-membered core that seems to have cytotoxic, analgesic, and immunosuppressive activities. Its mode of action is unclear, but, in a guinea pig model of the disease, purified mycolactone injected s.c. reproduces the natural pathology, and mycolactone negative variants are avirulent, implying a key role for the toxin in pathogenesis (2).Mycobacterium ulcerans (MU) and Mycobacterium marinum (MM) share over 98% DNA sequence identity, they occupy aquatic environments, and both cause cutaneous infections (3). However, MM produces a granulomatous intracellular lesion, typical for pathogenic mycobacteria and totally distinct from Buruli ulcer in which MU are mainly found extracellularly. The fact that MM does not produce mycolactone suggested that it might be possible to identify genes for mycolactone synthesis by performing genomic subtraction experiments between MU and MM. Fragments of MU-specific polyketide synthase (PKS) genes were identified from these experiments (4). The subsequent investigation of these sequences led to the discovery of the MU virulence plasmid pMUM001 and the extraordinary PKS locus it encodes. Plasmid Sequence Determination....

Evolution of Mycobacterium ulcerans and Other Mycolactone-Producing Mycobacteria from a Common Mycobacterium marinum Progenitor

“…Initial MLSA analyses and subsequent whole-genome comparisons have shown that M. ulcerans has recently evolved from M. marinum by acquisition of the pMUM plasmid and reductive evolution (31)(32)(33)(34). The recent discovery of MPM that are phenotypically distinct from M. ulcerans in diseased fish and frogs has highlighted the possibility that pMUM is being transferred among different mycobacterial species (19,26).…”

“…However, other than travelers returning from countries where the disease is endemic, no cases of BU have ever been reported in the United States or Europe. M. ulcerans strains are characterized by the presence of a large circular virulence plasmid called pMUM (31,33). This plasmid harbors three large genes (mlsA1, mlsA2, and mlsB) encoding polyketide synthases that are required for the synthesis of the lipid toxin mycolactone, which is the primary virulence factor for the pathogen (33).…”

“…M. ulcerans strains are characterized by the presence of a large circular virulence plasmid called pMUM (31,33). This plasmid harbors three large genes (mlsA1, mlsA2, and mlsB) encoding polyketide synthases that are required for the synthesis of the lipid toxin mycolactone, which is the primary virulence factor for the pathogen (33). Comparisons of multiple plasmid and chromosomal genes among 10 M. ulcerans clinical isolates from diverse origins have suggested that plasmid acquisition was probably the key event that marked and permitted the recent emergence of M. ulcerans from a common Mycobacterium marinum progenitor (31).…”

Transfer, stable maintenance and expression of the mycolactone polyketide megasynthase mls genes in a recombination-impaired Mycobacterium marinum

“…pMUM001 also has three accessory genes that are involved, or have a predicted role, in mycolactone synthesis. These genes include mup038, encoding a putative type II thioesterase, mup045, encoding a potential acyltransferase that might catalyse the C-O bond between the mycolactone core and side-chain, and mup053 (cyp140A7), encoding a P450 monooxygenase that hydroxlates C129 of the side-chain (Stinear et al, 2004(Stinear et al, , 2005a. The mlsA1, mlsA2 and mlsB genes span 105 kb and encode 11 different functional domains that are repeated across the two load modules (LMs) and 16 extension modules that comprise these PKS.…”

The local immune response in ulcerative lesions of Buruli disease

“…The major virulence factor in M. ulcerans is a necrotizing toxin called mycolactone, a macrolide consisting of a polyketide side chain attached to a 12-membered core encoded by genes harboured in a large 174-kb plasmid [7]. This toxin has cytotoxic, analgesic and immunosuppressive activities [8][9][10].…”