REVIEWS Buruli ulcer: reductive evolution enhances pathogenicity of Mycobacterium ulcerans Caroline Demangel*||, Timothy P. Stinear‡|| and Stewart T. Cole§ Abstract | Buruli ulcer is an emerging human disease caused by infection with a slow-growing pathogen, Mycobacterium ulcerans, that produces mycolactone, a cytotoxin with immunomodulatory properties. The disease is associated with wetlands in certain tropical countries, and evidence for a role of insects in transmission of this pathogen is growing. Comparative genomic analysis has revealed that M. ulcerans arose from Mycobacterium marinum, a ubiquitous fast-growing aquatic species, by horizontal transfer of a virulence plasmid that carries a cluster of genes for mycolactone production, followed by reductive evolution. Here, the ecology, microbiology, evolutionary genomics and immunopathology of Buruli ulcer are reviewed. virulence plasmid Virulence plasmid The human skin disease that results from infection through horizontal gene transfer of a . An autonomously replicating with Mycobacterium ulcerans is commonly known Here, we review the current state of our knowledge DNA element that is found in as Buruli ulcer (BU), but would have been called and comment on prospects for disease control. some pathogenic Bairnsdale ulcer if microbiological history had been microorganisms and harbours Physiopathology and medical interventions genes that are essential for strictly respected. In 1935, a series of unusual, painless that microorganism to cause ulcers in patients from a remote farming community M. ulcerans produces mycolactone, a macrolide cyto- disease. in the Bairnsdale district of south-east Australia was toxin with immunosuppressive properties that is present reported1. Some 13 years later, Australian research- in the extracellular matrix surrounding large clusters of ers discovered the aetiological agent of Bairnsdale M. ulcerans cells organized as a biofilm4. BU begins as ulcer, a previously unknown mycobacterium that they a small, painless, raised skin papule, nodule, plaque or named M. ulcerans2. During the 1960s, many cases of oedema. Osteomyelitis may occur in bone adjacent to a infection with M. ulcerans were reported in Uganda, cutaneous lesion. Later, destruction of the subcutane- particularly in Buruli County (now known as the ous adipose tissue leads to collapse of the epidermis and Nakasongola district), and thus the disease became formation of a characteristic ulcer with undermined more generally known as BU. Today, the disease is edges5–7 (FIG. 1). Advanced lesions display massive tissue far more widespread in West and Central Africa, destruction and minimal inflammation, with extracel- especially among impoverished rural communities, lular microcolonies of M. ulcerans in the superficial *Institut Pasteur, 8 UP Pathogénomique although other parts of the world are also affected. necrotic areas . Despite some anti-phagocytic activity Mycobactérienne Intégrée, Thirty countries, mainly in the tropics, have reported of mycolactone, phagocytes can internalize M. ulcerans 28 Rue du Dr Roux, 75724 cases of BU and in some settings, such as Ghana or in vitro, although this culminates in host cell death9,10. Paris, France. Benin, BU is now more prevalent than leprosy3. In contrast to wild-type strains, mycolactone-deficient ‡Global Health Institute, Ecole Polytechnique Fédérale de During the past 10 years, there has been consid- mutants generate inflammation in the guinea pig 11 Lausanne, Station number 15, erable progress in our understanding of the ecology, model , suggesting that production of mycolactone by CH-1015 Lausanne, aetiology and microbiology of BU, which has led to intracellular bacteria can suppress innate inflammatory Switzerland. better definition of risk factors and awareness of the responses to infection in vivo. || These authors contributed potential role of insects in transmission of the disease. The physiopathology of BU is closely associated equally to this work. Correspondence to S.T.C. Comparative mycobacterial genomics has under- with diffusion of the toxin from bacterial foci, as first e-mail: [email protected] pinned these advances and provided compelling evi- suspected from histopathological studies of infected tis- doi:10.1038/nrmicro2077 dence for the emergence of M. ulcerans as a pathogen sues12. Injection of mycolactone is sufficient to induce 50 | JANUARY 2009 | VOLUME 10 www.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Local tests. However, this hydrophobic molecule is not immuno- inflammation genic, and attempts to raise anti-mycolactone antibodies Infection and systemic Bacterial Tissue interferon-γ have been unsuccessful. load necrosis responses Treatment of BU remains complicated and generally requires both surgery, sometimes accompanied by skin Pre-ulcerative grafts, and prolonged courses of antibiotics. WHO rec- lesion ommends that a combination of rifampicin (oral) and streptomycin (intramuscular) be administered daily for 8 weeks19. However, although results from this treatment are encouraging (4 weeks of treatment leads to culture negativity), most patients need to be hospitalized owing to the frequency, duration and route of antibiotic admin- istration. New drugs are clearly needed to treat this Ulcerative lesion disease and better biomarkers are required to monitor the therapeutic response of patients. There is no vaccine against M. ulcerans, although significant, but short-lasting, protection against BU has been reported following immunization with the Mycobacterium bovis bacille Calmette–Guérin (BCG) Spontaneous 20 healing, vaccine . Prospects for vaccine development include treatment modified BCGs and subunit-based vaccines21. DNA vaccines that encode antigen 85A and the heat-shock protein Hsp65 have been shown to protect mice against M. ulcerans infection22–25. However, these vac- cines remain less protective than BCG in mice, even Figure 1 | Disease progression. The progression from the pre-ulcerative to the when administered in DNA-prime–boost protocols. Nature Reviews | Microbiology ulcerative and healing stages of Buruli ulcer disease is shown. The width of the green, red Comparative genomics could open new avenues for and yellow shapes denotes the extent of the progression for bacterial load, necrosis and vaccine research and improved diagnostics. inflammatory responses, respectively. Epidemiology, risk factors and insect vectors The epidemiology of BU is poorly understood and necrotic lesions in the skin of guinea pigs13, by an outbreaks are sporadic and unpredictable; however, apoptosis-dependent mechanism14. Consistent with the proximity to, or contact with, slowly flowing or stag- extensive tissue damage observed, mycolactone kills a nant watercourses is a recognized risk factor26–28. range of mammalian cells15, but the molecular basis of Furthermore, disease is often confined to specific areas mycolactone-induced apoptosis nevertheless remains and has sometimes appeared following major environ- unclear. Indeed, studies that used mouse fibroblasts mental changes, such as deforestation, flooding, or the in vitro indicate that apoptosis follows cytopathic effects, introduction of dams and irrigation systems29. In Benin, including cytoskeletal rearrangements and cell-cycle for example, the incidence of BU is tenfold greater in arrest13,14. areas that have undergone environmental perturbation Schwann cell Schwann 29 A cell that surrounds neurons, Although M. ulcerans displays no tropism for compared with control areas . produces myelin in nerves in cells, nerve invasion occurs at the perineural and endone- In these settings, M. ulcerans is associated with the peripheral nervous system ural levels, causing degeneration16. Similar alterations are aquatic vegetation, such as plants and algae30, whereas and is the preferred niche for induced by injecting purified toxin into mice17, suggest- snails and other organisms that feed on this vegetation the leprosy bacillus, a relative 31 of Mycobacterium ulcerans. ing that mycolactone diffuses into Schwann cells in vivo can serve as passive hosts . Portaels and colleagues were Schwann cells were named to cause the painlessness that characterizes the disease. A the first to use PCR-based detection methods to inves- after their discoverer, Theodor surprising feature of BU is the scarcity of secondary infec- tigate a role for insect vectors in transmission32. Other Schwann. tions of these immunosuppressed open wounds, which groups have performed similar environmental surveys raises the possibility that M. ulcerans produces secondary to uncover likely niches of M. ulcerans. M. ulcerans Secondary infection An infection that occurs as a metabolites with anti-microbial activity (WHO provides from environmental specimens is notoriously difficult consequence of a weakened or a method for diagnosing secondary bacterial infection in and unreliable to culture owing to the long generation perturbed immune response M. ulcerans disease; see Further information). time of the pathogen (>48 hours) and overgrowth by following a primary infection by BU is usually diagnosed on the basis of clinical find- contaminants, including other mycobacteria. PCR is a different pathogen. ings, occasionally confirmed by microscopy, but to treat more reliable but is not foolproof, as one of the diag- Insertion sequence disease more effectively it is essential to develop novel, nostic PCR targets, insertion sequence (IS) 2404, is not A mobile DNA element that