Evolution of Mycobacterium ulcerans and other mycolactone-producing mycobacteria from a common Mycobacterium marinum progenitor

Abstract

It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.

FIG. 1.

Distributions and nucleotide positions of the eight loci on the 5.6-Mbp chromosome of M. ulcerans Agy99 (inner circle) and the 6.6-Mbp chromosome of M. marinum M (outer circle).

FIG. 2.

Representative DNA-DNA hybridization result showing using whole-genome probes from three MPM against a panel of nine strains representing different MLSA sequence types.

FIG. 3.

Multilocus sequence analysis. (A) Species designations for the 20 sequence types, their origins, and (in parentheses) the numbers of isolates tested. (B) Alignment of the 3,210-bp sequences derived from the eight concatenated chromosomal CDS fragments among 20 different sequence types. Only variable nucleotides are shown. A period indicates identity with M. ulcerans ST17 (African type). (C) Split-decomposition representation of the phylogenetic relationships among M. ulcerans, other mycolactone-producing mycobacteria, and M. marinum strains. The circles at the vertices are labeled with the sequence types, and their sizes are proportional to the numbers of isolates within the groups, following a layout described previously (8). M. marinum ST1, -2, -3, -4, and -5 correspond to STI, -II, -III, -IV, and -V as previously reported (32). All edges had 100% bootstrap support (1,000 replicates). (D) Alignment of the 1,266-bp sequences derived from the four concatenated pMUM CDS fragments among 11 different sequence types. Only variable nucleotides are shown. A period indicates identity with M. ulcerans ST17 (African type). (E) Split-decomposition graph of the phylogenetic relationships among M. ulcerans and other mycolactone-producing mycobacteria, derived from the plasmid gene sequences. All edges had >60% bootstrap support (1,000 replicates).

FIG. 4.

Pulsed-field gel electrophoresis and Southern hybridization analysis of MPM showing the presence in all isolates of pMUM-like plasmids and the distributions of IS2404 and IS2606.

FIG. 5.

PCR deletion analysis for MURD12, MURD55, and MURD152 of strains representing each MLSA sequence type. (A) Arrangement of the oligonucleotides used for PCR of MURD152, showing the 471-bp product predicted from the genome sequence of M. marinum strain M and the 353-bp product predicted from the genome sequence of M. ulcerans Agy99. Also shown for reference is the alignment of MURD152 with the RD1 region absent from Mycobacterium bovis BCG. (B, C, and D) Results of deletion PCR analysis for MURD152, MURD54, and MURD12, respectively. Lanes 1 to 11, M. marinum (1, 99/84; 2, JKD2394; 3, 471; 4, 99/87; 5, 99/89; 6, 2000-372; 7, 1717; 8, M; 9, DL240490; 10, CC240299; 11, DL045); lane 12, M. pseudoshottsii L15; lane 13, M. liflandii 128FXT; lane 14, M. ulcerans Mexico 5114; lane 15, M. ulcerans Japan 753; lane 16, M. marinum Surinam 842; lane 17, M. ulcerans Agy99; lane 18, M. ulcerans 1615; lane 19, M. ulcerans 13822/70; lane 20, M. ulcerans 19423; lane 21, no template control. On the left is a 100-bp DNA molecular size ladder (Promega).