Mycobacterium ulcerans is a significant human pathogen and the causative agent of Buruli ulcer (BU), a disease with severe morbidity characterized by chronic skin ulceration and extensive necrosis of subcutaneous fat (
40). Cases of BU have been reported in many parts of the world, with the greatest burden of disease occurring in West and Central Africa (
16). 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). 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).
M. marinum is phenotypically distinct from
M. ulcerans, producing photochromogenic pigments and generally growing more quickly.
M. marinum causes granulomatous lesions in fish and other ectotherms and can also cause granulomatous skin lesions in humans. Comparisons between the 5.8-Mb genome of the
M. ulcerans African epidemic strain Agy99 and the 6.6-Mb genome of
M. marinum strain “M” confirmed this hypothesis and showed that
M. ulcerans has recently passed through an evolutionary bottleneck, evolving from the generalist
M. marinum to become a specialist bacterium, adapted to a more restricted (possibly host-specific) environment (
32,
34;
http://genolist.pasteur.fr/BuruList/ ). Like other niche-adapted pathogens, such as
Yersinia pestis and
Bordetella pertussis,
M. ulcerans has undergone extensive gene loss due to DNA deletions, DNA rearrangements, and pseudogene formation. Many of these changes have been mediated by some of the 213 copies of IS
2404 and 91 copies of IS
2606 (
34). Neither of these insertion sequence elements (ISE) is present in
M. marinum (
32).
It was assumed until recently that mycolactone production was restricted to
M. ulcerans. However, in 2004, a previously unreported mycobacterium that contained both IS
2404 and IS
2606 was recovered from cases of an unusual, lethal edematous disease in laboratory-housed
Xenopus laevis and
Xenopus tropicalis frogs (
37). Limited sequence comparisons of
hsp65, the 16S rRNA gene, the 16S-23S rRNA gene internal transcribed sequence spacer, and
rpoB gene fragments showed that this mycobacterium shared greater than 98% nucleotide identity with
M. ulcerans and
M. marinum (
37). In a subsequent investigation, the frog mycobacterium was shown to contain a version of the
M. ulcerans pMUM plasmid and to produce a new mycolactone, mycolactone E (
13,
19). It was also given the unofficial epithet
Mycobacterium liflandii (
19). In 2005, another new mycobacterium, isolated during an epizootic of mycobacteriosis from diseased striped bass (
Morone saxatilis) in Chesapeake Bay, was also shown to contain IS
2404 and IS
2606 and to share >98% nucleotide sequence identity with
M. ulcerans,
M. marinum, and
Mycobacterium shottsii (
28). Based on some phenotypic traits that distinguished it from
M. ulcerans, such as photochromogenicity, absence of growth at 37°C, and scant growth at 30°C on Middlebrook 7H10 agar, this mycobacterium was given a new species designation,
Mycobacterium pseudoshottsii. Subsequent analysis of the strain showed that it too contained a pMUM-like plasmid with the mycolactone
mls genes and that it made yet another mycolactone, called mycolactone F (
26). In the same study, a cluster of
M. marinum strains that had been isolated from diseased fish in the Red and Mediterranean Seas (
38) were also positive for
mls gene sequences, and they too produced mycolactone F (
26). One of these strains (DL240490) shared identical
hsp65 gene sequences with
M. pseudoshottsii and another fish pathogen,
Mycobacterium seriolae (
28).
Thus, the distribution of mycolactone-producing mycobacteria (MPM) appears to be much wider than first appreciated, and this raises several interesting questions regarding the mobility of pMUM and the evolution of M. ulcerans and M. marinum. In this study, we conducted a comprehensive genetic analysis of these strains by DNA-DNA hybridization (DDH), by sequence comparisons of eight chromosomal and four plasmid genes, and by comparing the distributions of pseudogenes and DNA deletions to better define the taxonomic status of all mycolactone-producing strains, including those that cause Buruli ulcer, and to gain insights into their evolution.
DISCUSSION
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-
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). In this report, we show by a systematic genetic approach that all MPM are very closely related to each other and have evolved, not by multiple exchanges of pMUM, but from a common
M. marinum progenitor that acquired the plasmid.
MLSA is widely used to understand the taxonomic relationships among bacterial populations (
8,
10,
11), and it was the method we employed in an earlier study to suggest that
M. ulcerans recently evolved from
M. marinum (
32). To improve genome coverage and increase resolution of the MLSA method for the present investigation, we added an eighth locus to create a 3,210-bp semantide (a large information-bearing molecule). We reanalyzed our original data set and then added sequences from 12 additional isolates that included 5 mycobacteria recently shown to contain pMUM and to produce mycolactones. There was significant sequence diversity among non-mycolactone-producing
M. marinum isolates, and as shown by others (
32,
39), the majority of isolates fell into two distinct clusters represented by ST1, -2, and -6 and ST 3, -4, and -5. The sample size in this study was too small to draw conclusions linking specific
M. marinum genotypes to virulence in humans, as has been proposed (
39); however, such a correlation seems unlikely, given that
M. marinum isolates of human origin spanned the spectrum of sequence diversity revealed by MLSA. Two
M. marinum isolates recovered from armadillos showed intermediate sequence types (ST7 and -8), as did
M. marinum strain “M” (ST22), whose genome has recently been sequenced (
http://www.sanger.ac.uk/Projects/M_marinum ).
MLSA unambiguously showed that, despite their varying phenotypes, all MPM have evolved from a single
M. marinum clone that has since expanded into at least two distinct lineages, and this was supported by the congruent tree topology derived from the four pMUM plasmid loci (Fig.
3). Together with equivalent levels of synonymous nucleotide substitution frequency between chromosome and episome sequences, these data suggest that plasmid acquisition was probably the principal event that enabled an
M. marinum progenitor to survive in a new environment.
The genetic homogeneity of MPM was also reflected in their high DDH values. DDH has been widely used for over 30 years in bacterial taxonomy to infer relatedness between genomes, and a DDH value greater than 70% is one criterion used to help define a bacterial species. The high DDH values among
M. ulcerans and the other MPM were further evidence of their common origin and contrasted with the low (<55% RBR) values when the same MPM were tested against non-mycolactone-producing
M. marinum strains. These data are consistent with an earlier investigation of
M. ulcerans and
M. marinum that showed intraspecies DDH values of >90% and interspecies DDH values of <50% (
36). The presence of pMUM-like plasmids and the multiple copies of IS
2404 in all MPM may explain, at least in part, the striking DDH results. These data also indicate that IS
2404 acquisition (and possibly its expansion to high copy numbers) occurred before radiation of MPM around the world. In contrast, IS
2606 is present in high copy numbers only in the lineage of
M. ulcerans strains that contain isolates from Africa, Australia, and Malaysia (ST17, -18, and -20) (
30,
32). The varied distribution of this ISE among other MPM and its absence from the MPM
M. marinum strain CC240299 (ST10) suggest it has been transferred independently to at least two different populations of MPM subsequent to IS
2404 and pMUM acquisition.
The
M. ulcerans genome project and a recent microarray-based study have both revealed extensive DNA deletion polymorphism among
M. ulcerans strains (
34; M. Käser, S. Rondini, T. Stinear, M. Tessier, C. Mangold, G. Dernick, M. Naegeli, F. Portaels, U. Certa, and G. Pluschke, submitted for publication). In the current work, the pattern of DNA deletion observed among MPM for three deletions was in good agreement with MLSA (Fig.
5). MURD12, a 10-kbp fragment containing CDS involved in secondary metabolism, was a marker for distinguishing between the ectotherm and endotherm lineages, as it was absent from
M. ulcerans isolates from both Mexico and Japan (ST15 and -19) and the African, Malaysian, and Australian cluster (ST17, -18, and -20) (Fig.
3C and
5C). The MURD54 and MURD152 deletions differentiate the ST17, -18, and -20 subcluster from other MPM and are indicative of more advanced genome reduction in these strains. MURD152 is a 2.8-kb DNA fragment deleted from
M. ulcerans strain Agy99 (ST17) that spans
esxA and
esxB, genes encoding key components of the ESX-1 secretion apparatus and virulence factor (
4,
34). The MURD152 deletion PCR assay confirmed earlier findings that showed that
esxA and
esxB are absent from
M. ulcerans strains from Africa, Australia, and Malaysia but present in other MPM (
19,
26). Both the MURD54 and MURD152 assays may have diagnostic applications in countries where Buruli ulcer is endemic, such as Africa and Australia, where it will be useful, particularly when screening environmental samples, to distinguish between
M. ulcerans strains that cause Buruli ulcer and other MPM.
The split of MPM into two distinct lineages, which include strains with different species names (e.g.,
M. marinum,
M. liflandii, and
M. pseudoshottsii) that typically cause disease in ectotherms but also have a high zoonotic potential and strains of
M. ulcerans (ST17, -18, and -20) that cause Buruli ulcer in humans and target other endotherms, is an important finding. Some insights into the genetic basis for this separation have been gleaned from this study and, combined with previous research showing that these strains have different optimal growth temperatures and produce mycolactones with varying potencies (
19,
26), they suggest MPM have evolved to occupy different ecological niches.
M. ulcerans is not known to infect fish, while the diseases caused by
M. marinum and
M. ulcerans in humans differ greatly in their clinical, histopathologic, and epidemiologic aspects (
32).
The large number of gene deletions and pseudogenes in the M. ulcerans Agy99 genome compared with M. marinum M is indicative of a bacterium adapting to a restricted and privileged environment, where mutations are tolerated in genes that are no longer required for survival. However, testing three MPM from the ectotherm lineage for 20 of these pseudogenes found only five inactivated CDS, indicating a much less advanced level of genome decay and metabolic streamlining, consistent with the hypothesis that they occupy different environments. Only one of the five mutations (an insertion in arsC) was conserved between the two MPM lineages (see Table S4 in the supplemental material), suggesting that a certain level of genome decay had begun before divergence. The other four mutations occurred at different positions in the same genes (sigJ, echA13, and accD1), and this may indicate that the products of these CDS are not only redundant but perhaps deleterious for survival of MPM and so have been subjected to independent, purifying selection.
The difference in pseudogene profiles may also help explain the phenotypic variation observed among MPM. For example,
crtI encodes phytoene dehydrogenase, an enzyme essential for the production of carotenoid pigments in
M. marinum (
25).
M. ulcerans Agy99 has the same
crt locus as
M. marinum, but it is nonpigmented, and this has been explained by a point mutation in
crtI that introduces a premature stop codon and truncates the gene (see Table S4 in the supplemental material) (
34). There was complete correlation between lack of pigment production and the disrupted
crtI gene, as only MPM of the ST17, -18, and -20 cluster are nonpigmented (
24,
26), and it was only this cluster that contained the mutation (Fig.
3C).
In this report, we have sought to clarify the genetic relationships among mycolactone- and non-mycolactone-producing mycobacteria, but this has in turn highlighted the recurring problem of assigning species status to highly related bacteria, as the question remains how much diversity is permissible in a genetically discrete cluster for it to be regarded as a distinct taxon. From a population genetics standpoint, the data presented here do not support the separation of MPM into different species. Employing a subspecies nomenclature might allow a more meaningful naming system that accurately reflects the common origin of MPM. A comprehensive polyphasic and multicenter study of MPM, as performed by Wayne et al. (
41), would help decide their taxonomic positions.
Defining the host specificity and natural ecology of MPM is also a research priority. It may be that there are many different MPM but the only strains isolated are those producing mycolactones with sufficient potency to cause disease in humans, fish, frogs, possums, and koalas (
23). A better understanding of MPM in the environment will be crucial to halting the spread of the diseases they cause, in particular Buruli ulcer.