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

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.     

Mycobacterium ulcerans toxic macrolide, mycolactone modulates the host immune response and cellular location of M. ulcerans in vitro and in vivo

Mycobacterium ulcerans produces an extracellular cutaneous infection (Buruli ulcer) characterized by immunosuppression. This is in stark contrast to all other pathogenic Mycobacteria species that cause intracellular, granulomatous infections. The unique mycobacterial pathology of M. ulcerans infection is attributed to a plasmid-encoded immunomodulatory macrolide toxin, mycolactone. In this article we explore the role of mycolactone in the virulence of M. ulcerans using mycolactone and genetically defined mycolactone negative mutants. In a guinea pig infection model wild-type (WT) M. ulcerans produces an extracellular infection whereas mycolactone negative mutants produce an intracellular inflammatory infection similar to that of Mycobacterium marinum. Although mycolactone negative mutants are avirulent, they persist for at least 6 weeks. Chemical complementation of M. ulcerans mutants with mycolactone restores WT M. ulcerans pathology. Mycolactone negative mutants are capable of growth within macrophages in vitro whereas macrophages are killed by WT M. ulcerans. The ability of mycolactone to caused delayed cell death via apoptosis has been reported. However, mycolactone also causes cell death via necrosis. In vitro mycolactone has antiphagocytic properties. Neither WT M. ulcerans nor mycolactone negative strains are strong neutrophil attractants. These results suggest that mycolactone is largely responsible for the unique pathology produced by M. ulcerans.     

Mycobacterium ulcerans causes minimal pathogenesis and colonization in medaka (Oryzias latipes): an experimental fish model of disease transmission

Mycobacterium ulcerans causes Buruli ulcer in humans, a progressive ulcerative epidermal lesion due to the mycolactone toxin produced by the bacterium. Molecular analysis of M. ulcerans reveals it is closely related to Mycobacterium marinum, a pathogen of both fish and man. Molecular evidence from diagnostic PCR assays for the insertion sequence IS2404 suggests an association of M. ulcerans with fish. However, fish infections by M. ulcerans have not been well documented and IS2404 has been found in other mycobacteria. We have thus, employed two experimental approaches to test for M. ulcerans in fish. We show here for the first time that M. ulcerans with or without the toxin does not mount acute or chronic infections in Japanese Medaka "Oryzias latipes" even at high doses. Moreover, M. ulcerans-infected medaka do not exhibit any visible signs of infection nor disease and the bacteria do not appear to replicate over time. In contrast, similar high doses of the wild-type M. marinum or a mycolactone-producing M. marinum "DL" strain are able to mount an acute disease with mortality in medaka. Although these results would suggest that M. ulcerans does not mount infections in fish we have evidence that CLC macrophages from goldfish are susceptible to mycolactones.     

Development and application of two multiplex real-time PCR assays for the detection of Mycobacterium ulcerans in clinical and environmental samples

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS2404 PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.     

The unusual macrocycle forming thioesterase of mycolactone

Mycolactone is a polyketide natural product secreted by Mycobacterium ulcerans, the organism responsible for the tropical skin disease Buruli ulcer. The finding that this small molecule virulence factor is sufficient to reconstitute the necrotic pathology associated with Buruli ulcer suggests that a better understanding of mycolactone biosynthesis, particularly the processes which are distinct from those in human metabolism, may provide a unique avenue for the development of selective therapeutics. In the present study we have cloned, expressed, and biochemically characterized the putative macrocycle forming thioesterase for mycolactone, MLSA2 TE. We have evaluated the enzyme both as the truncated thioesterase domain and as a carrier protein-linked didomain construct. The results of these analyses distinguish MLSA2 TE from traditional fatty acid and polyketide synthase TE-domains in terms of its sequence, kinetic parameters, and susceptibility to traditional active-site directed inhibitors. These findings suggest that MLSA2 TE utilizes a unique biochemical mechanism for macrocycle formation.     

Impact of Mycobacterium ulcerans biofilm on transmissibility to ecological niches and Buruli ulcer pathogenesis

The role of biofilms in the pathogenesis of mycobacterial diseases remains largely unknown. Mycobacterium ulcerans, the etiological agent of Buruli ulcer, a disfiguring disease in humans, adopts a biofilm-like structure in vitro and in vivo, displaying an abundant extracellular matrix (ECM) that harbors vesicles. The composition and structure of the ECM differs from that of the classical matrix found in other bacterial biofilms. More than 80 proteins are present within this extracellular compartment and appear to be involved in stress responses, respiration, and intermediary metabolism. In addition to a large amount of carbohydrates and lipids, ECM is the reservoir of the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, and purified vesicles extracted from ECM are highly cytotoxic. ECM confers to the mycobacterium increased resistance to antimicrobial agents, and enhances colonization of insect vectors and mammalian hosts. The results of this study support a model whereby biofilm changes confer selective advantages to M. ulcerans in colonizing various ecological niches successfully, with repercussions for Buruli ulcer pathogenesis.     

Common evolutionary origin for the unstable virulence plasmid pMUM found in geographically diverse strains of Mycobacterium ulcerans

The 174-kb virulence plasmid pMUM001 in Mycobacterium ulcerans epidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation of M. ulcerans Agy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine diverse M. ulcerans strains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in all M. ulcerans strains. Physical mapping of these plasmids revealed that like M. ulcerans Agy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis that M. ulcerans diverged from the closely related organism Mycobacterium marinum by acquiring pMUM. Our results suggest that pMUM is a defining characteristic of M. ulcerans but that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.     

Comprehensive proteome analysis of Mycobacterium ulcerans and quantitative comparison of mycolactone biosynthesis

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rapidly emerging human disease in which mycolactone, a cytotoxic and immunosuppressive macrocyclic polyketide, is responsible for massive skin destruction. The genome sequencing of M. ulcerans has recently been accomplished (http://genolist.pasteur.fr/BuruList/) enabling the first proteome study of this important human pathogen. Here, we present a comprehensive proteome analysis of different subcellular fractions and culture supernatant of in vitro grown M. ulcerans. By a combination of gel-based and gel-free techniques for protein and peptide separation with subsequent analysis by MS, we identified 1074 different proteins, corresponding to 25% of the protein-coding DNA sequence. Interestingly, new information was obtained about central metabolism and lipid biosynthesis, and as many as 192 conserved hypothetical proteins were found. Comparative analysis of the wild-type strain and an isogenic mycolactone-deficient mutant, by 2-DE and iTRAQ labeling of the cytoplasmic fraction, revealed differences in the expression profiles of proteins involved in lipid metabolism and information pathways, as well as stress responses.     

Colonization of the salivary glands of Naucoris cimicoides by Mycobacterium ulcerans requires host plasmatocytes and a macrolide toxin, mycolactone

Mycobacterium ulcerans was first identified as the causative agent of Buruli ulcer; this cutaneous tissue-destructive process represents the third most important mycobacterial disease in humans after tuberculosis and leprosy. More recently other life traits were documented. M. ulcerans is mainly detected in humid tropical zones as part of a complex ecosystem comprising algae, aquatic insect predators of the genus Naucoris, and very likely their vegetarian preys. Coelomic plasmatocytes could be the first cells of Naucoris cimicoides to be involved in the infection process, acting as shuttle cells that deliver M. ulcerans to the salivary glands as suggested by both in vitro and in vivo approaches. Furthermore, a key element for the early and long-term establishment of M. ulcerans in Naucoridae is demonstrated by the fact that only mycolactone toxin-producing M. ulcerans isolates are able to invade the salivary glands, a site where they proliferate. Later, the raptorial legs of Naucoris are covered by M. ulcerans-containing material that displays features of biofilms.     

Serological evaluation of Mycobacterium ulcerans antigens identified by comparative genomics

A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45 potential M. ulcerans specific proteins, of which we were able to express and purify 33 in E. coli. Sera from 30 confirmed Buruli ulcer patients, 24 healthy controls from the same endemic region and 30 healthy controls from a non-endemic region in Benin were screened for antibody responses to these specific proteins by ELISA. Serum IgG responses of Buruli ulcer patients were highly variable, however, seven proteins (MUP045, MUP057, MUL_0513, Hsp65, and the polyketide synthase domains ER, AT propionate, and KR A) showed a significant difference between patient and non-endemic control antibody responses. However, when sera from the healthy control subjects living in the same Buruli ulcer endemic area as the patients were examined, none of the proteins were able to discriminate between these two groups. Nevertheless, six of the seven proteins showed an ability to distinguish people living in an endemic area from those in a non-endemic area with an average sensitivity of 69% and specificity of 88%, suggesting exposure to M. ulcerans. Further validation of these six proteins is now underway to assess their suitability for use in Buruli ulcer seroepidemiological studies. Such studies are urgently needed to assist efforts to uncover environmental reservoirs and understand transmission pathways of the M. ulcerans.     

Suspension cultivation of Mycobacterium ulcerans for the production of mycolactones

Mycolactones are polyketide toxins produced by Mycobacterium ulcerans, the causative agent of the tropical skin disease known as Buruli ulcer. Development of novel therapeutic agents from mycolactones has been hindered by the difficulty of producing sufficient amounts of material. Here, we describe the successful adaptation of M. ulcerans to suspension cultivation and the development of a fed-batch fermentation process that was scaled up to 150 l. In addition to producing mycolactones A and B, a number of new mycolactone-related compounds were also observed.     

Early trafficking events of Mycobacterium ulcerans within Naucoris cimicoides

The severe skin-destructive disease caused by Mycobacterium ulcerans, named Buruli ulcer, is the third most important mycobacterial disease in humans after tuberculosis and leprosy. Recently we demonstrated that M. ulcerans could colonize the salivary glands of the water bug, Naucoris cimicoides. In this study, we report that M. ulcerans may be delivered from the digested prey aspirate to the coelomic cavity via a unique headspace, the head capsule (HC). During the infected meal, we observed that M. ulcerans clusters adhered to the stylets that were retracted in the HC at the end of the meal. M. ulcerans was able to translocate from the HC to the coelomic cavity where it is phagocytosed by the plasmatocytes. These cells are subverted as shuttle cells and deliver M. ulcerans to the salivary glands. At this early stage of its parasitic life style, two other important features of M. ulcerans can be documented: first, mycolactone is not required for translocation of M. ulcerans into the HC, in contrast to the next step, colonization of the salivary glands; second, M. ulcerans clusters bind a member of the serpin protein family present in the salivary gland homogenate.     

Aquatic plants stimulate the growth of and biofilm formation by Mycobacterium ulcerans in axenic culture and harbor these bacteria in the environment

Mycobacterium ulcerans is the causative agent of Buruli ulcer, one of the most common mycobacterial diseases of humans. Recent studies have implicated aquatic insects in the transmission of this pathogen, but the contributions of other elements of the environment remain largely unknown. We report here that crude extracts from two green algae added to the BACTEC 7H12B culture medium halved the doubling time of M. ulcerans and promoted biofilm formation. Using the 7H12B medium, modified by the addition of the algal extract, and immunomagnetic separation, we also demonstrate that M. ulcerans is associated with aquatic plants in an area of the Ivory Coast where Buruli ulcer is endemic. Genotype analysis showed that plant-associated M. ulcerans had the same profile as isolates recovered in the same region from both aquatic insects and clinical specimens. These observations implicate aquatic plants as a reservoir of M. ulcerans and add a new potential link in the chain of transmission of M. ulcerans to humans.     

Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence

Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ulcerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restriction fragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of M. tuberculosis, where species diversity is being driven mainly by the activity of mobile DNA elements.     

Modulation of the host immune response by a transient intracellular stage of Mycobacterium ulcerans: the contribution of endogenous mycolactone toxin

Mycobacterium ulcerans (Mu), the aetiological agent of Buruli ulcer, is an extracellular pathogen producing the macrolide toxin mycolactone. Using a mouse model of intradermal infection, we found that Mu was initially captured by phagocytes and transported to draining lymph nodes (DLN) within host cells. Similar to Buruli ulcers in humans, the infection site eventually became ulcerated with tissue necrosis and extracellular bacteria, at later stages. In contrast to Mycobacterium bovis BCG (BCG), Mu did not disseminate to the spleen. However, mice infected with Mu or BCG developed comparable primary cellular responses to mycobacterial antigens in DLN and spleen. The role of mycolactone in this sequence of events was examined with a mycolactone-deficient (mup045) mutant of Mu. Mup045 bacilli were better internalized than wild-type (wt) bacteria by mouse phagocytes in vitro. Moreover, infection with wt but not mup045 Mu led to inhibition of TNF-alpha expression, upregulation of MIP-2 chemokine, and host cell death within 1 day. Our results suggest that mycolactone expression during the intracellular life of Mu may contribute to immune evasion by inhibiting phagocytosis, provoking apoptosis of antigen presenting cells and altering the establishment of an appropriate inflammatory reaction.     

Aquatic insects as a vector for Mycobacterium ulcerans

Mycobacterium ulcerans is an emerging environmental pathogen which causes chronic skin ulcers (i.e., Buruli ulcer) in otherwise healthy humans living in tropical countries, particularly those in Africa. In spite of epidemiological and PCR data linking M. ulcerans to water, the mode of transmission of this organism remains elusive. To determine the role of aquatic insects in the transmission of M. ulcerans, we have set up an experimental model with aquariums that mimic aquatic microenvironments. We report that M. ulcerans may be transmitted to laboratory mice by the bite of aquatic bugs (Naucoridae) that are infected with this organism. In addition, M. ulcerans appears to be localized exclusively within salivary glands of these insects, where it can both survive and multiply without causing any observable damage in the insect tissues. Subsequently, we isolated M. ulcerans from wild aquatic insects collected from a zone in the Daloa region of Ivory Coast where Buruli ulcer is endemic. Taken together, these results point to aquatic insects as a possible vector of M. ulcerans.     

Distribution of Mycobacterium ulcerans in buruli ulcer endemic and non-endemic aquatic sites in Ghana

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is an emerging environmental bacterium in Australia and West Africa. The primary risk factor associated with Buruli ulcer is proximity to slow moving water. Environmental constraints for disease are shown by the absence of infection in arid regions of infected countries. A particularly mysterious aspect of Buruli ulcer is the fact that endemic and non-endemic villages may be only a few kilometers apart within the same watershed. Recent studies suggest that aquatic invertebrate species may serve as reservoirs for M. ulcerans, although transmission pathways remain unknown. Systematic studies of the distribution of M. ulcerans in the environment using standard ecological methods have not been reported. Here we present results from the first study based on random sampling of endemic and non-endemic sites. In this study PCR-based methods, along with biofilm collections, have been used to map the presence of M. ulcerans within 26 aquatic sites in Ghana. Results suggest that M. ulcerans is present in both endemic and non-endemic sites and that variable number tandem repeat (VNTR) profiling can be used to follow chains of transmission from the environment to humans. Our results suggesting that the distribution of M. ulcerans is far broader than the distribution of human disease is characteristic of environmental pathogens. These findings imply that focal demography, along with patterns of human water contact, may play a major role in transmission of Buruli ulcer.     

Potential role for fish in transmission of Mycobacterium ulcerans disease (Buruli ulcer): an environmental study

This study reports a potential role that fish may play in the transmission of Mycobacterium ulcerans disease (Buruli ulcer). Fish found positive for M. ulcerans DNA all appear to feed on insects or plankton and are believed to concentrate M. ulcerans from this usual food source. These observations provide additional data supporting our previous hypothesis on sources of M. ulcerans and modes of transmission.