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Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is unique amongst human pathogens in its capacity to produce a lipid toxin called mycolactone. While previous studies have demonstrated that bacterially-released mycolactone diffuses beyond infection foci, the spatiotemporal distribution of mycolactone remained largely unknown. Here, we used the zebrafish model to provide the first global kinetic analysis of mycolactone’s diffusion in vivo, and multicellular co-culture systems to address the critical question of the toxin’s access to the brain. Zebrafish larvae were injected with a fluorescent-derivative of mycolactone to visualize the in vivo diffusion of the toxin from the peripheral circulation. A rapid, body-wide distribution of mycolactone was observed, with selective accumulation in tissues near the injection site and brain, together with an important excretion through the gastro-intestinal tract. Our conclusion that mycolactone reached the central nervous system was reinforced by an in cellulo model of human blood brain barrier and a mouse model of M. ulcerans-infection. Here we show that mycolactone has a broad but heterogenous profile of distribution in vivo. Our investigations in vitro and in vivo support the view that a fraction of bacterially-produced mycolactone gains access to the central nervous system. The relative persistence of mycolactone in the bloodstream suggests that assays of circulating mycolactone are relevant for BU disease monitoring and treatment optimization., Author summary Mycolactone is the major virulence factor of Mycobacterium ulcerans, the human pathogen causing Buruli ulcer (BU) disease. While it is now established that mycolactone is able to diffuse from infected tissues to exert immunomodulatory and analgesic effects at the systemic level, the in vivo spatiotemporal distribution of mycolactone remained largely unknown. Here, using the zebrafish larva, we describe the spatiotemporal distribution of a fluorescent derivative of mycolactone in vivo. We show that fluorescent mycolactone quickly diffuses from the blood circulation into various organs, and accumulates in muscles and brain. Mycolactone’s diffusion into the central nervous system was further confirmed in the mouse model of M. ulcerans infection. We reported previously that mycolactone suppresses the production of inflammatory mediators by primary microglia at nanomolar concentrations in vitro [1]. In the present study, we provide evidence suggesting that bacterially-produced mycolactone is able to reach the brain. While additional in vivo investigations will be required, we can speculate that the amount of mycolactone reaching the brain in the context of M. ulcerans infection is sufficient to modulate inflammation and pain transmission.

Mycobacterium ulcerans is a human pathogen that causes a necrotizing skin disease known as Buruli ulcer. Necrosis of infected skin is driven by bacterial production of mycolactone, a diffusible exotoxin targeting the host translocon (Sec61). By blocking Sec61, mycolactone prevents the transport of nascent secretory proteins into the endoplasmic reticulum of host cells. This triggers pro-apoptotic stress responses partially depending on activation of the ATF4 transcription factor. To gain further insight into the molecular pathways mediating the cytotoxic effects of mycolactone we conducted the first haploid genetic screen with the M. ulcerans toxin in KBM-7 cells. This approach allowed us to identify the histone methyltransferase SETD1B as a novel mediator of mycolactone-induced cell death. CRISPR/Cas9-based inactivation of SETD1B rendered cells resistant to lethal doses of the toxin, highlighting the critical importance of this gene’s expression. To understand how SETD1B contributes to mycolactone cytotoxicity, we compared the transcriptomes of wild-type (WT) and SETD1B knockout KBM-7 cells upon exposure to the toxin. While ATF4 effectors were upregulated by mycolactone in both WT and SETD1B knockout cells, mycolactone selectively induced the expression of pro-apoptotic genes in WT cells. Among those genes we identified CHAC1, which codes for a major glutathione (GSH)-degrading enzyme, and whose strong upregulation in mycolactone-treated WT cells correlated with a marked reduction in GSH protein level. Moreover, GSH supplementation conferred cells with substantial protection against the toxic effects of mycolactone. Our data thus identify SETD1B/CHAC1/GSH as a novel, epigenetic mechanism connecting Sec61 blockade with apoptotic cell death. They suggest that GSH-based treatments might have the capacity to limit skin necrosis in Buruli ulcer., Author summary The human pathogen Mycobacterium ulcerans causes a necrotizing skin disease known as Buruli ulcer. The major toxin of the mycobacteria, mycolactone, prevents the transport of secretory proteins into the endoplasmic reticulum, and thereby triggers a deadly stress response. We conducted the first haploid genetic screen to identify host factors with impact on mycolactone toxicity. This enabled us to identify the histone methyltransferase SETD1B as a novel mediator of mycolactone-induced cell death. RNA analyses of wild-type cells and resistant SETD1B knockout cells treated with mycolactone then showed a selective induction of genes implicated in programmed cell-death only in wild-type cells. This was accompanied by a marked reduction of the antioxidant glutathione, which might cause the mycolactone induced cell death.

Buruli Ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. Although the current antibiotic treatment for BU is effective, daily administrations for a prolonged period of time, combined with potential risk of severe side effects, negatively impact on patient adherence. In that sense, we tested the efficacy of an alternative strategy based on Lysin B (LysB), a phage encoded lipolytic enzyme that degrades the mycolylarabinogalactan-peptidoglycan complex present in the mycobacterial cell wall. In this study, we show that LysB not only displays lytic activity against M. ulcerans isolates in vitro, but also leads to a decrease of M. ulcerans proliferation in infected mouse footpads. These findings highlight the potential use of lysins as a novel therapeutic approach against this neglected tropical disease., The projectwas developed withinthescopeof the projectsNORTE-01-0145-FEDER-000013and NORTE-01-0145-FEDER-000023,supported by the Northern Portugal Regional Operational Programme (NORTE2020),under the Portugal2020 Partnership Agreement through FEDER.This work was also supported by BioTecNorte operation (NORTE-01-0145-FEDER -000004) funded by the European Regional Development Fund under the scope of NORTE2020.This study was supportedby the Portuguese Foundation for Scienceand Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit; the Competitiveness Factors Operational Programme (COMPETE 2020) projectsPOCI-01-0145-FEDER-006684 and POCI-01-0145-FEDER-007038; and the project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678). This study was also supported by Infect-ERA grant Infect-ERA/0002/2015 :BU_SPONT_HEAL. AGF,GT, and HO wouldlike to acknowledge FCT for the individual fellowships SFRH/BPD/112903/2015, SFRH/BPD/64032/2009,and SFRH/BPD/111653/2015,respectively. CMG received an individual QRENfellowship (UMINHO/BPD/15/2014). GangaGen acknowledges CSIR/ OSDD,Govt of India,for funding this project.The funders had no role in study design,data collection and analysis, decision to publish, or preparation of the manuscript, info:eu-repo/semantics/publishedVersion

Addressing the transmission enigma of the neglected disease Buruli ulcer (BU) is a World Health Organization priority. In Australia, we have observed an association between mosquitoes harboring the causative agent, Mycobacterium ulcerans, and BU. Here we tested a contaminated skin model of BU transmission by dipping the tails from healthy mice in cultures of the causative agent, Mycobacterium ulcerans. Tails were exposed to mosquito (Aedes notoscriptus and Aedes aegypti) blood feeding or punctured with sterile needles. Two of 12 of mice with M. ulcerans contaminated tails exposed to feeding A. notoscriptus mosquitoes developed BU. There were no mice exposed to A. aegypti that developed BU. Eighty-eight percent of mice (21/24) subjected to contaminated tail needle puncture developed BU. Mouse tails coated only in bacteria did not develop disease. A median incubation time of 12 weeks, consistent with data from human infections, was noted. We then specifically tested the M. ulcerans infectious dose-50 (ID50) in this contaminated skin surface infection model with needle puncture and observed an ID50 of 2.6 colony-forming units. We have uncovered a biologically plausible mechanical transmission mode of BU via natural or anthropogenic skin punctures., Author summary Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans. Unfortunately, how people contract this disease is not well understood. Here we show for the first time using experimental infections in mice that a very low dose of M. ulcerans delivered beneath the skin by a minor injury caused by a blood-feeding insect (mosquito) or an experimental needle puncture is sufficient to cause Buruli ulcer. This research provides important laboratory evidence to advance our understanding of Buruli ulcer disease transmission.

Background Mycobacterium ulcerans causes necrotising infections of skin and soft tissue mediated by the polyketide exotoxin mycolactone that causes cell apoptosis and immune suppression. It has been postulated that infection can be eradicated before the development of clinical lesions but spontaneous resolution of clinical lesions has been rarely described. Methodology/Principal findings We report a case series of five Australian patients who achieved healing of small M. ulcerans lesions without antibiotics or surgery. The median age of patients was 47 years (IQR 30–68 years) and all patients had small ulcerative lesions (median size 144mm2, IQR 121-324mm2). The median duration of symptoms prior to diagnosis was 90 days (IQR 90–100 days) and the median time to heal from diagnosis without treatment was 68 days (IQR 63–105 days). No patients recurred after a median follow-up of 16.6 months (IQR 16.6–17.9 months) from the development of symptoms and no patients suffered long-term disability from the disease. Conclusions We have shown that healing without specific treatment can occur for small ulcerated M. ulcerans lesions suggesting that in selected cases a robust immune response alone can cure lesions. Further research is required to determine what lesion and host factors are associated with spontaneous healing, and whether observation alone is an effective and safe form of management for selected small M. ulcerans lesions., Author summary Mycobacterium ulcerans causes a destructive infection of skin and soft tissue known as Buruli ulcer that when severe can lead to serious long-term deformity and disability. It is currently not well documented whether people with Mycobacterium ulcerans disease can cure themselves without treatment. In our study we describe five people with small ulcers who cured their disease without specific medical or surgical treatment. This suggests that a proportion of people can develop an immune response sufficient enough to eradicate the disease without the help of medical intervention. This is an important step, as recognition of this possibility provides important further insights into the human immune response against the disease. It also opens the possibility to further studies that may determine characteristics of the organism and hosts that favour spontaneous healing of lesions. This knowledge may in turn improve efforts to prevent and control the disease which are currently lacking.

Background Buruli ulcer (BU) is a subcutaneous skin disease listed among the neglected tropical diseases by the World Health Organization (WHO). Early case detection and management is very important to reduce morbidity and the accompanied characteristic disfiguring nature of BU. Since diagnosis based on clinical evidence can lead to misdiagnosis, microbiological confirmation is essential to reduce abuse of drugs; since the anti-mycobacterial drugs are also used for TB treatment. The current WHO gold standard PCR method is expensive, requires infrastructure and expertise are usually not available at the peripheral centers where BU cases are managed. Thus one of the main research agendas is to develop methods that can be applied at the point of care. In this study we selected aptamers, which are emerging novel class of detection molecules, for detecting mycolactone, the first to be conducted in a BUD endemic country. Methods Aptamers that bind to mycolactone were isolated by the SELEX process. To measure their affinity and specificity to mycolactone, the selected aptamers were screened by means of isothermal titration calorimetry (ITC) and an enzyme-linked oligonucleotide assay (ELONA). Selected aptamers were assessed by ELONA using swab samples from forty-one suspected BU patients with IS2404 PCR and culture as standard methods. ROC analysis was used to evaluate their accuracy and cutoff-points. Results Five out of the nine selected aptamers bound significantly (p< 0.05) to mycolactone, of these, three were able to distinguish between mycolactone producing mycobacteria, M. marinum (CC240299, Israel) and other bacteria whilst two others also bounded significantly to Mycobacterium smegmatis. Their dissociation constants were in the micro-molar range. At 95% confidence interval, the ROC curve analysis among the aptamers at OD450 ranged from 0.5–0.7. Using this cut-off for the ELONA assay, the aptamers had 100% specificity and sensitivity between 0.0% and 50.0%. The most promising aptamer, Apt-3683 showed a discernible cleavage difference relative to the non-specific autocatalysis over a 3-minute time course. Conclusion This preliminary proof-of-concept indicates that diagnosis of BUD with RNA aptamers is feasible and can be used as point of care upon incorporation into a diagnostic platform., Author Summary Buruli ulcer disease is a subcutaneous necrotizing skin disease caused by ecological Mycobacterium ulcerans. The availability of antibiotic treatment makes it imperative for early detection of the disease to avoid the disfiguring and morbid effects associated with the disease. Currently available detection methods include: microscopy for detecting acid-fast bacilli, culture to isolate viable organism, and PCR for detecting pathogen specific DNA which is usually IS2404 and histopathology. However, most of these methods have major drawbacks, they are done in referenced laboratories and cannot be used in the field where the disease is most prevalent. M. ulcerans produces a lipid chemical called mycolactone, which is accountable for the virulent nature of the disease and has been postulated as a diagnostic target. The lipid nature of the mycolactone however makes it difficult for the body to produce antibodies against it. We therefore designed nucleic acid detection molecules that have high affinity to its target called aptamers. This aptamers were selected to detect mycolactone and hence M. ulcerans. The selected aptamers were tested with clinical samples and concluded preliminarily that, aptamers can be raised against mycolactone and this be used as point of care upon optimization and subsequent incorporation into a diagnostic platform.

Mycolactone is the exotoxin produced by Mycobacterium ulcerans and is the virulence factor behind the neglected tropical disease Buruli ulcer. The toxin has a broad spectrum of biological effects within the host organism, stemming from its interaction with at least two molecular targets and the inhibition of protein uptake into the endoplasmic reticulum. Although it has been shown that the toxin can passively permeate into host cells, it is clearly lipophilic. Association with lipid carriers would have substantial implications for the toxin’s distribution within a host organism, delivery to cellular targets, diagnostic susceptibility, and mechanisms of pathogenicity. Yet the toxin’s interactions with, and distribution in, lipids are unknown. Herein we have used coarse-grained molecular dynamics simulations, guided by all-atom simulations, to study the interaction of mycolactone with pure and mixed lipid membranes. Using established techniques, we calculated the toxin’s preferential localization, membrane translocation, and impact on membrane physical and dynamical properties. The computed water-octanol partition coefficient indicates that mycolactone prefers to be in an organic phase rather than in an aqueous environment. Our results show that in a solvated membrane environment the exotoxin mainly localizes in the water-membrane interface, with a preference for the glycerol moiety of lipids, consistent with the reported studies that found it in lipid extracts of the cell. The calculated association constant to the model membrane is similar to the reported association constant for Wiskott-Aldrich syndrome protein. Mycolactone is shown to modify the physical properties of membranes, lowering the transition temperature, compressibility modulus, and critical line tension at which pores can be stabilized. It also shows a tendency to behave as a linactant, a molecule that localizes at the boundary between different fluid lipid domains in membranes and promotes inter-mixing of domains. This property has implications for the toxin’s cellular access, T-cell immunosuppression, and therapeutic potential., Author summary Mycolactone is a macrolide exotoxin secreted by Mycobacterium ulcerans, which causes a skin disease called Buruli ulcer, a neglected emerging disease. It is the third most common mycobacterial disease after tuberculosis and leprosy. Studies have shown how mycolactone plays a pivotal role in Buruli ulcer pathogenesis, and identified it as an attractive therapeutic target. This multifunctional cytotoxin exerts multiple local and global responses, including ulcerative, analgesic, and anti-inflammatory effects. Prompted by its lipid-like structure, we used extensive multi-resolution simulations to probe mycolactone’s interactions with model membranes. Our results suggest that mycolactone is sequestered in membranes where it alters several dynamical, physical, and mechanical properties. It also behaves as a linactant, localizing at the interface between lipid domains and decreasing the inter-domain line tension. Our results shed light on how mycolactone permeates host cell membranes and is distributed between lipid and aqueous environments. These findings have significant implications for the toxin’s distribution in the host environment and mechanisms of pathogenicity. Understanding the toxin’s distribution and mechanism of trafficking will have ramifications for targeted diagnostics, therapeutic approaches, and our understanding of Buruli ulcer pathogenesis.

Introduction Buruli ulcer (BU) caused by Mycobacterium ulcerans is effectively treated with rifampicin and streptomycin for 8 weeks but some lesions take several months to heal. We have shown previously that some slowly healing lesions contain mycolactone suggesting continuing infection after antibiotic therapy. Now we have determined how rapidly combined M. ulcerans 16S rRNA reverse transcriptase / IS2404 qPCR assay (16S rRNA) became negative during antibiotic treatment and investigated its influence on healing. Methods Fine needle aspirates and swab samples were obtained for culture, acid fast bacilli (AFB) and detection of M. ulcerans 16S rRNA and IS2404 by qPCR (16S rRNA) from patients with IS2404 PCR confirmed BU at baseline, during antibiotic and after treatment. Patients were followed up at 2 weekly intervals to determine the rate of healing. The Kaplan-Meier survival analysis was used to analyse the time to clearance of M. ulcerans 16S rRNA and the influence of persistent M ulcerans 16S rRNA on time to healing. The Mann Whitney test was used to compare the bacillary load at baseline in patients with or without viable organisms at week 4, and to analyse rate of healing at week 4 in relation to detection of viable organisms. Results Out of 129 patients, 16S rRNA was detected in 65% of lesions at baseline. The M. ulcerans 16S rRNA remained positive in 78% of patients with unhealed lesions at 4 weeks, 52% at 8 weeks, 23% at 12 weeks and 10% at week 16. The median time to clearance of M. ulcerans 16S rRNA was 12 weeks. BU lesions with positive 16S rRNA after antibiotic treatment had significantly higher bacterial load at baseline, longer healing time and lower healing rate at week 4 compared with those in which 16S rRNA was not detected at baseline or had become undetectable by week 4. Conclusions Current antibiotic therapy for BU is highly successful in most patients but it may be possible to abbreviate treatment to 4 weeks in patients with a low initial bacterial load. On the other hand persistent infection contributes to slow healing in patients with a high bacterial load at baseline, some of whom may need antibiotic treatment extended beyond 8 weeks. Bacterial load was estimated from a single sample taken at baseline. A better estimate could be made by taking multiple samples or biopsies but this was not ethically acceptable., Author summary Buruli ulcer (BU) caused by Mycobacterium ulcerans is effectively treated with rifampicin and streptomycin for 8 weeks but some lesions take several months to heal. We have shown previously that some slowly healing lesions contain the M. ulcerans toxin, mycolactone, suggesting continuing infection after completion of antibiotic therapy. In the present study we have determined how soon M. ulcerans was killed during antibiotic treatment using the M. ulcerans 16S rRNA assay combined with qPCR for IS2404 to detect live bacilli in clinical samples and investigated its influence on healing. This assay is more sensitive than culture for the organism. Using samples collected from one hundred and twenty-nine BU patients prior to antibiotic treatment, viable organisms were detected by culture in 34% but the 16S rRNA assay was positive in 65%. The 16S rRNA remained positive in 78% of patients with unhealed lesions at 4 weeks, 52% at 8 weeks, 23% at 12 weeks, and 10% at week 16. Lesions with positive 16S rRNA after antibiotic treatment also contained a higher number of bacteria at baseline, had a lower rate of healing at week 4 and took a longer time to heal compared with those in which the organism was undetectable at baseline or by week 4. Positive 16S rRNA was less likely in ulcerative compared with nodular forms of disease 4 weeks after antibiotic treatment. It may be possible to shorten the treatment to 4 weeks in patients with low numbers of bacteria at baseline. Since persistent infection appears to contribute to slow healing, some patients with a high bacterial load at baseline may need antibiotic treatment for longer than 8 weeks.

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis., Author Summary Buruli Ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans infection. It has been recognized for many years that BU pathogenesis is mediated by the potent exotoxin mycolactone; however, the molecular action of this toxin on the host cell biology that drives its pathogenesis is not fully understood. Here we present a proteomic-based study that explores the molecular action of mycolactone on host cells biology. Our results provide further molecular evidence for the cytoskeleton-disarrangement induced by mycolactone, and unveil its impact on cytoskeleton-dependent cellular functions. Moreover, we extend the field of action of this toxin to the biosynthesis of collagen, implicating mycolactone on the decrease of dermal collagen found on BU lesions. Given the dependence of M. ulcerans virulence on its toxin, these findings on mycolactone's molecular action on host cells and tissues are of major importance for the understanding of BU pathogenesis.

Diagnosis of the neglected tropical disease, Buruli ulcer, can be made by acid-fast smear microscopy, specimen culture on mycobacterial growth media, polymerase chain reaction (PCR), and/or histopathology. All have drawbacks, including non-specificity and requirements for prolonged culture at 32°C, relatively sophisticated laboratory facilities, and expertise, respectively. The causative organism, Mycobacterium ulcerans, produces a unique toxin, mycolactone A/B (ML) that can be detected by thin layer chromatography (TLC) or mass spectrometric analysis. Detection by the latter technique requires sophisticated facilities. TLC is relatively simple but can be complicated by the presence of other lipids in the specimen. A method using a boronate-assisted fluorogenic chemosensor in TLC can overcome this challenge by selectively detecting ML when visualized with UV light. This report describes modifications in the fluorescent TLC (F-TLC) procedure and its application to the mouse footpad model of M. ulcerans disease to determine the kinetics of mycolactone production and its correlation with footpad swelling and the number of colony forming units in the footpad. The response of all three parameters to treatment with the current standard regimen of rifampin (RIF) and streptomycin (STR) or a proposed oral regimen of RIF and clarithromycin (CLR) was also assessed. ML was detectable before the onset of footpad swelling when there were 105 CFU per footpad. Mycolactone concentrations increased as swelling increased whereas CFU levels reached a plateau. Treatment with either RIF+STR or RIF+CLR resulted in comparable reductions of mycolactone, footpad swelling, and CFU burden. Storage in absolute ethanol appears critical to successful detection of ML in footpads and would be practical for storage of clinical samples. F-TLC may offer a new tool for confirmation of suspected clinical lesions and be more specific than smear microscopy, much faster than culture, and simpler than PCR., Author Summary The diagnosis of Buruli ulcer, caused by infection with Mycobacterium ulcerans, is complicated by its resemblance to other diseases that may also cause ulcers in the skin. Clinical diagnosis can be supported by microscopic detection of acid-fast bacilli in the skin, by prolonged culture of at least 8 weeks, in a dedicated incubator set at 32°C, or by the polymerase chain reaction in a well-equipped laboratory usually far from the clinic where the patient comes for treatment. The treatment involves taking two drugs, one requiring injections, every day for two months, a burden for patients and their families. Since all drugs may have side effects, it is important that the treatment be appropriate for the patient's disease. We describe a new technique to rapidly and inexpensively detect the presence of the unique toxin produced by M. ulcerans in the mouse footpad model of Buruli ulcer. We show that the toxin can be detected in footpads before the development of signs of the disease, that more toxin is produced as the disease progresses, and that toxin levels decline in mice treated with either the current standard regimen of rifampin and streptomycin or a proposed all-oral drug regimen of rifampin and clarithromycin.

Background Mycobacterium ulcerans, a slow-growing environmental bacterium, is the etiologic agent of Buruli ulcer, a necrotic skin disease. Skin lesions are caused by mycolactone, the main virulence factor of M. ulcerans, with dermonecrotic (destruction of the skin and soft tissues) and immunosuppressive activities. This toxin is secreted in vesicles that enhance its biological activities. Nowadays, it is well established that the main reservoir of the bacilli is localized in the aquatic environment where the bacillus may be able to colonize different niches. Here we report that plant polysaccharides stimulate M. ulcerans growth and are implicated in toxin synthesis regulation. Methodology/Principal Findings In this study, by selecting various algal components, we have identified plant-specific carbohydrates, particularly glucose polymers, capable of stimulating M. ulcerans growth in vitro. Furthermore, we underscored for the first time culture conditions under which the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, is down-regulated. Using a quantitative proteomic approach and analyzing transcript levels by RT-qPCR, we demonstrated that its regulation is not at the transcriptional or translational levels but must involve another type of regulation. M. ulcerans produces membrane vesicles, as other mycobacterial species, in which are the mycolactone is concentrated. By transmission electron microscopy, we observed that the production of vesicles is independent from the toxin production. Concomitant with this observed decrease in mycolactone production, the production of mycobacterial siderophores known as mycobactins was enhanced. Conclusions/Significance This work is the first step in the identification of the mechanisms involved in mycolactone regulation and paves the way for the discovery of putative new drug targets in the future., Author Summary Mycolactone, a polyketide cytotoxic toxin, is the key virulence factor responsible for large skin ulcers in Buruli ulcer. This disease, mainly occurring in humid tropical zones, especially in West African countries, is due to infection by Mycobacterium ulcerans, a slow-growing environmental mycobacterium. The toxin has destructive effects on the skin, soft tissues and bones. In this study, we brought out for the first time that addition of specific carbohydrate to culture medium induces the down-regulation of the toxin. Furthermore, this decrease in toxin production is correlated with the activation of the iron acquisition pathway, especially by siderophore production. These results show that M. ulcerans adapts its metabolism to culture conditions, which probably reflect its adaptation in its natural habitats. This work is the first step in toxin regulation understanding which is a key issue for a better comprehension of Buruli ulcer physiopathology and the identification of putative therapeutic targets.

Buruli ulcer (BU), caused by infection with Mycobacterium ulcerans, is a chronic necrotizing human skin disease associated with the production of the cytotoxic macrolide exotoxin mycolactone. Despite extensive research, the type of immune responses elicited against this pathogen and the effector functions conferring protection against BU are not yet fully understood. While histopathological analyses of advanced BU lesions have demonstrated a mainly extracellular localization of the toxin producing acid fast bacilli, there is growing evidence for an early intra-macrophage growth phase of M. ulcerans. This has led us to investigate whether interferon-γ might play an important role in containing M. ulcerans infections. In an experimental Buruli ulcer mouse model we found that interferon-γ is indeed a critical regulator of early host immune defense against M. ulcerans infections. Interferon-γ knockout mice displayed a faster progression of the infection compared to wild-type mice. This accelerated progression was reflected in faster and more extensive tissue necrosis and oedema formation, as well as in a significantly higher bacterial burden after five weeks of infection, indicating that mice lacking interferon-γ have a reduced capacity to kill intracellular bacilli during the early intra-macrophage growth phase of M. ulcerans. This data demonstrates a prominent role of interferon-γ in early defense against M. ulcerans infection and supports the view that concepts for vaccine development against tuberculosis may also be valid for BU., Author Summary Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a slow progressing ulcerative skin disease. The mode of transmission of M. ulcerans remains unknown and only little is known about the early stages of the disease and the nature of protective immune responses against this pathogen. Given the increasing evidence for an early intracellular growth phase of M. ulcerans, we aimed at evaluating the impact of cell-mediated immunity for immunological defense against M. ulcerans infections. By comparing wild-type and interferon-γ-deficient mice in a BU mouse model, we could demonstrate that interferon-γ is a critical regulator of early host immune defense against M. ulcerans infections, indicative for an important role of early intracellular multiplication of the pathogen. In mice lacking interferon-γ the bacterial burden increased faster, resulting in accelerated pathogenesis. The observed differences between the two mouse strains were most likely due to differences in the capacity of macrophages to kill intracellular bacilli during the early stages of infection.

Background Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic macrolide exotoxin called mycolactone, which causes extensive necrosis of infected subcutaneous tissue and the development of characteristic ulcerative lesions with undermined edges. While cellular immune responses are expected to play a key role against early intracellular stages of M. ulcerans in macrophages, antibody mediated protection might be of major relevance against advanced stages, where bacilli are predominantly found as extracellular clusters. Methodology/Principal Findings To assess whether vaccine induced antibodies against surface antigens of M. ulcerans can protect against Buruli ulcer we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as recombinant proteins with the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion. The candidate vaccines elicited strong antibody responses without a strong bias towards a TH1 type cellular response, as indicated by the IgG2a to IgG1 ratio. Despite the cross-reactivity of the induced antibodies with the native antigens, no significant protection was observed against progression of an experimental M. ulcerans infection in a mouse footpad challenge model. Conclusions Even though vaccine-induced antibodies have the potential to opsonise the extracellular bacilli they do not have a protective effect since infiltrating phagocytes might be killed by mycolactone before reaching the bacteria, as indicated by lack of viable infiltrates in the necrotic infection foci., Author Summary Buruli ulcer is a slow progressing ulcerative disease of the skin and subcutaneous tissue that is most prevalent in West African rural communities. Mycobacterium ulcerans, the causative agent of the disease, produces a toxin called mycolactone, which is held responsible for the extensive tissue damage seen in advanced Buruli ulcer lesions. To date, no effective vaccine against the disease exists and it is unclear to what extent antibodies against cell surface antigens of M. ulcerans play a role in protection. To assess whether vaccine induced antibodies against cell surface proteins can protect against Buruli ulcer, we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as adjuvanted recombinant proteins and investigated their protective potential in a mouse model of M. ulcerans infection. Despite the induction of strong antibody responses against the surface molecules and cross-reactivity of the induced antibodies with the antigens in their native context, we did not observe protection against the disease. While the vaccine-induced antibodies could opsonize the extracellular bacilli, infiltrating phagocytes might be killed early by mycolactone.

Background Mycobacterium ulcerans disease (Buruli ulcer) is a neglected tropical disease common amongst children in rural West Africa. Animal experiments have shown that tissue destruction is caused by a toxin called mycolactone. Methodology/Principal Findings A molecule was identified among acetone-soluble lipid extracts from M. ulcerans (Mu)-infected human lesions with chemical and biological properties of mycolactone A/B. On thin layer chromatography this molecule had a retention factor value of 0.23, MS analyses showed it had an m/z of 765.6 [M+Na+] and on MS:MS fragmented to produce the core lactone ring with m/z of 429.4 and the polyketide side chain of mycolactone A/B with m/z of 359.2. Acetone-soluble lipids from lesions demonstrated significant cytotoxic, pro-apoptotic and anti-inflammatory activities on cultured fibroblast and macrophage cell lines. Mycolactone A/B was detected in all of 10 tissue samples from patients with ulcerative and pre-ulcerative Mu disease. Conclusions/Significance Mycolactone can be detected in human tissue infected with Mu. This could have important implications for successful management of Mu infection by antibiotic treatment but further studies are needed to measure its concentration., Author Summary Skin infection with bacteria called Mycobacterium ulcerans causes Buruli ulcer, a disease common in West Africa and mainly affecting children. M. ulcerans is the only mycobacterium to cause disease by production of a toxin. This lipid molecule called mycolactone diffuses from the site of infection, killing surrounding cells and, at low concentration, suppressing the immune response. The aim of this study was to show that mycolactone can be detected among lipids extracted from human M. ulcerans lesions in order to study its role in the pathogenesis of M. ulcerans disease. Lipids were extracted from skin biopsies and tested for the presence of mycolactone using thin layer chromatography and mass spectrometry. The extracts were shown to kill cultured cells in a cytotoxicity assay. Mycolactone was detected in both pre-ulcerative and ulcerative forms of the disease and also in lesions during antibiotic treatment but with reduced bioactivity, suggesting a lower concentration compared to untreated lesions. These findings indicate that there is mycolactone in affected skin at all stages of M. ulcerans disease and it could be used as a biomarker for monitoring the clinical response to antibiotic treatment.

BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+), completely prevented mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.

Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans., Author Summary Buruli ulcer (BU) is a serious skin infection of humans predominantly occurring in West and Central Africa. The disease is caused by infection with Mycobacterium ulcerans, a bacterium that produces an unusual toxin called mycolactone. There are many unanswered questions surrounding BU, particularly regarding the role of mycolactone in disease and how M. ulcerans is transmitted to humans. Here, we have partly addressed these questions by identifying genetic factors controlling the transcription of the mycolactone genes. Using a variety of experimental approaches, including green fluorescent protein (GFP) as a reporter of gene expression, we have identified strong promoters that drive transcription of the mycolactone genes in M. ulcerans. We then used our GFP reporters to produce highly fluorescent M. ulcerans-GFP that were readily visualized by microscopy. Mosquitoes have been proposed as a potential vector of M. ulcerans so we used M. ulcerans-GFP in feeding experiments with mosquito larvae. M. ulcerans-GFP accumulated within the insects, whereas other mycobacteria did not. This is the first report of the mycolactone gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a powerful tool for studying Buruli ulcer.

Although several studies have associated Mycobacterium ulcerans (MU) infection, Buruli ulcer (BU), with slow moving water bodies, there is still no definite mode of transmission. Ecological and transmission studies suggest Variable Number Tandem Repeat (VNTR) typing as a useful tool to differentiate MU strains from other Mycolactone Producing Mycobacteria (MPM). Deciphering the genetic relatedness of clinical and environmental isolates is seminal to determining reservoirs, vectors and transmission routes. In this study, we attempted to source-track MU infections to specific water bodies by matching VNTR profiles of MU in human samples to those in the environment. Environmental samples were collected from 10 water bodies in four BU endemic communities in the Ashanti region, Ghana. Four VNTR loci in MU Agy99 genome, were used to genotype environmental MU ecovars, and those from 14 confirmed BU patients within the same study area. Length polymorphism was confirmed with sequencing. MU was present in the 3 different types of water bodies, but significantly higher in biofilm samples. Four MU genotypes, designated W, X, Y and Z, were typed in both human and environmental samples. Other reported genotypes were only found in water bodies. Animal trapping identified 1 mouse with lesion characteristic of BU, which was confirmed as MU infection. Our findings suggest that patients may have been infected from community associated water bodies. Further, we present evidence that small mammals within endemic communities could be susceptible to MU infections. M. ulcerans transmission could involve several routes where humans have contact with risk environments, which may be further compounded by water bodies acting as vehicles for disseminating strains., Author Summary Buruli ulcer is a skin disease, which is endemic in over thirty countries, mostly in West Africa, with affected populations being largely rural. The causative organism, Mycobacterium ulcerans (MU), is an environmental mycobacterium and although transmission is unclear, frequent exposure to these MU-contaminated environments have been suggested as risk factors. We conducted this study on the premise that if patients are infected from MU-contaminated water bodies, then the genotype of MU strains in these patients should be identical to those in their community associated water bodies and wetlands. Using Variable Number Tandem Repeat (VNTR) as a genetic tool, we determined the genotypes of MU from both water bodies and patient samples. Comparison and overlap of these genotypes, within each community, suggest that patients were possibly infected from at least one water body. Additionally, we present evidence that small mammals within endemic communities could be susceptible to MU infections and may be acting as reservoirs. Our findings suggest that future ecological and molecular studies in the hope of elucidating a definite transmission route, should focus on source-tracking MU infections to community associated risk environments while employing a OneHealth approach in the process.

Background Mycolactones are a family of polyketide-derived macrolide exotoxins produced by Mycobacterium ulcerans, the causative agent of the chronic necrotizing skin disease Buruli ulcer. The toxin is synthesized by polyketide synthases encoded by the virulence plasmid pMUM. The apoptotic, necrotic and immunosuppressive properties of mycolactones play a central role in the pathogenesis of M. ulcerans. Methodology/Principal Findings We have synthesized and tested a series of mycolactone derivatives to conduct structure-activity relationship studies. Flow cytometry, fluorescence microscopy and Alamar Blue-based metabolic assays were used to assess activities of mycolactones on the murine L929 fibroblast cell line. Modifications of the C-linked upper side chain (comprising C12–C20) caused less pronounced changes in cytotoxicity than modifications in the lower C5-O-linked polyunsaturated acyl side chain. A derivative with a truncated lower side chain was unique in having strong inhibitory effects on fibroblast metabolism and cell proliferation at non-cytotoxic concentrations. We also tested whether mycolactones have antimicrobial activity and found no activity against representatives of Gram-positive (Streptococcus pneumoniae) or Gram-negative bacteria (Neisseria meningitis and Escherichia coli), the fungus Saccharomyces cerevisae or the amoeba Dictyostelium discoideum. Conclusion Highly defined synthetic compounds allowed to unambiguously compare biological activities of mycolactones expressed by different M. ulcerans lineages and may help identifying target structures and triggering pathways., PLoS Neglected Tropical Diseases, 7 (3), ISSN:1935-2727, ISSN:1935-2735

The authors would like to thank Luís Martins and Miguel Carneiro for laboratory assistance. Author Contributions Conceived and designed the experiments: AGF AGC JP. Performed the experiments: AGF TGM ET. Analyzed the data: AGF. Contributed reagents/materials/analysis tools: FP. Wrote the paper: AGF KH FP MTS AGC JP., BACKGROUND: Buruli ulcer (BU) is an emerging infectious disease caused by Mycobacterium ulcerans that can result in extensive necrotizing cutaneous lesions due to the cytotoxic exotoxin mycolactone. There is no specific vaccine against BU but reports show some degree of cross-reactive protection conferred by M. bovis BCG immunization. Alternatively, an M. ulcerans-specific immunization could be a better preventive strategy. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used the mouse model to characterize the histological and cytokine profiles triggered by vaccination with either BCG or mycolactone-negative M. ulcerans, followed by footpad infection with virulent M. ulcerans. We observed that BCG vaccination significantly delayed the onset of M. ulcerans growth and footpad swelling through the induction of an earlier and sustained IFN-γ T cell response in the draining lymph node (DLN). BCG vaccination also resulted in cell-mediated immunity (CMI) in M. ulcerans-infected footpads, given the predominance of a chronic mononuclear infiltrate positive for iNOS, as well as increased and sustained levels of IFN-γ and TNF. No significant IL-4, IL-17 or IL-10 responses were detected in the footpad or the DLN, in either infected or vaccinated mice. Despite this protective Th1 response, BCG vaccination did not avoid the later progression of M. ulcerans infection, regardless of challenge dose. Immunization with mycolactone-deficient M. ulcerans also significantly delayed the progression of footpad infection, swelling and ulceration, but ultimately M. ulcerans pathogenic mechanisms prevailed. CONCLUSIONS/SIGNIFICANCE: The delay in the emergence of pathology observed in vaccinated mice emphasizes the relevance of protective Th1 recall responses against M. ulcerans. In future studies it will be important to determine how the transient CMI induced by vaccination is compromised., The research leading to these results has received funding from the European Community’s Seventh Framework Program (FP7/2007–2013) under grant agreement Nu 241500 (BuruliVac). This work was additionally supported by a Grant from the Health Services of Fundação Calouste Gulbenkian, and by thePortuguese Science and Technology Foundation (FCT) fellowships SFRH/BPD/68547/2010, SFRH/BD/41598/2007 to A. G. Fraga and T. G. Martins.

Background Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) is widely used to reduce the risk of childhood tuberculosis and has been reported to have efficacy against two other mycobacterial diseases, leprosy and Buruli ulcer caused by M. ulcerans (Mu). Studies in experimental models have also shown some efficacy against infection caused by Mu. In mice, most studies use the C57BL/6 strain that is known to develop good cell-mediated protective immunity. We hypothesized that there may be differences in vaccination efficacy between C57BL/6 and the less resistant BALB/c strain. Methods We evaluated BCG vaccine efficacy against challenge with ∼3×105 M. ulcerans in the right hind footpad using three strains: initially, the Australian type strain, designated Mu1617, then, a Malaysian strain, Mu1615, and a recent Ghanaian isolate, Mu1059. The latter two strains both produce mycolactone while the Australian strain has lost that capacity. CFU of both BCG and Mu and splenocyte cytokine production were determined at intervals after infection. Time to footpad swelling was assessed weekly. Principal Findings BCG injection induced visible scars in 95.5% of BALB/c mice but only 43.4% of C57BL/6 mice. BCG persisted at higher levels in spleens of BALB/c than C57BL/6 mice. Vaccination delayed swelling and reduced Mu CFU in BALB/c mice, regardless of challenge strain. However, vaccination was only protective against Mu1615 and Mu1617 in C57BL/6 mice. Possible correlates of the better protection of BALB/c mice included 1) the near universal development of BCG scars in these mice compared to less frequent and smaller scars observed in C57BL/6 mice and 2) the induction of sustained cytokine, e.g., IL17, production as detected in the spleens of BALB/c mice whereas cytokine production was significantly reduced, e.g., IL17, or transient, e.g., Ifnγ, in the spleens of C57BL/6 mice. Conclusions The efficacy of BCG against M. ulcerans, in particular, and possibly mycobacteria in general, may vary due to differences in both host and pathogen., Author Summary Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) is used to reduce the risk of childhood tuberculosis and is reported to have efficacy against two other diseases also caused by mycobacteria, leprosy and Buruli ulcer caused by M. ulcerans. We hypothesized that there may be differences in the effectiveness of BCG vaccination in different mouse strains. We vaccinated two mouse strains with BCG eight weeks before infection with three different strains of M. ulcerans. Two of the bacterial strains make a toxin that is critical for Buruli ulcer disease and the third does not. We observed the progression of disease in vaccinated and mock-vaccinated mice and also evaluated the immune response of the mice. We found that the BALB/c mice respond to BCG vaccination with prominent scars, a vigorous immune response, and delayed or no manifestations of M. ulcerans infection. C57BL/6 mice, on the other hand, usually do not have vaccination scars, make a relatively short-lived and/or weaker immune response, and all show disease at the site of M. ulcerans infection. We conclude that the efficacy of BCG against M. ulcerans, and possibly other diseases, depends on the nature of the host and of the infecting strain of the bacteria.

Background Buruli ulcer (BU) is a progressive disease of subcutaneous tissues caused by Mycobacterium ulcerans. The pathology of BU lesions is associated with the local production of a diffusible substance, mycolactone, with cytocidal and immunosuppressive properties. The defective inflammatory responses in BU lesions reflect these biological properties of the toxin. However, whether mycolactone diffuses from infected tissues and suppresses IFN-γ responses in BU patients remains unclear. Methodology/Principal Findings Here we have investigated the pharmacodistribution of mycolactone following injection in animal models by tracing a radiolabeled form of the toxin, and by directly quantifying mycolactone in lipid extracts from internal organs and cell subpopulations. We show that subcutaneously delivered mycolactone diffused into mouse peripheral blood and accumulated in internal organs with a particular tropism for the spleen. When mice were infected subcutaneously with M. ulcerans, this led to a comparable pattern of distribution of mycolactone. No evidence that mycolactone circulated in blood serum during infection could be demonstrated. However, structurally intact toxin was identified in the mononuclear cells of blood, lymph nodes and spleen several weeks before ulcerative lesions appear. Importantly, diffusion of mycolactone into the blood of M. ulcerans–infected mice coincided with alterations in the functions of circulating lymphocytes. Conclusion In addition to providing the first evidence that mycolactone diffuses beyond the site of M. ulcerans infection, our results support the hypothesis that the toxin exerts immunosuppressive effects at the systemic level. Furthermore, they suggest that assays based on mycolactone detection in circulating blood cells may be considered for diagnostic tests of early disease., Author Summary Mycolactone is a lipophilic molecule produced by Mycobacterium ulcerans, the causative agent of the skin disease Buruli ulcer (BU). Mycolactone displays unique cytocidal and immunosuppressive properties that are reflected locally by massive tissue necrosis and minimal inflammation. Here we investigated whether mycolactone diffuses from infected tissues and exerts immunosuppressive properties at the systemic level. We used both a radiolabeled form of the toxin and a direct LC-MS/MS analysis of lipid extracts from internal organs and cell subpopulations to investigate the pharmacodistribution of mycolactone in vivo. Using a mouse model of subcutaneous infection with M. ulcerans, we show that mycolactone distributes far beyond the sphere of its cytocidal action. The toxin diffused from infected tissues into the blood and the spleen, where it concentrated in mononuclear cell subsets. Importantly, mycolactone was detected in circulating blood several weeks before ulcerative lesions develop. The presence of mycolactone in blood cells was associated with a decreased capacity of circulating lymphocytes to produce interleukin-2 upon stimulation. In addition to providing the first evidence that mycolactone targets key immune cell populations in infected hosts, this work suggests that mycolactone detection in peripheral blood cells may form the basis of diagnostic tests of early disease.

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., Author Summary Mycobacterium ulcerans is the etiologic agent of Buruli ulcer, a necrotic skin disease affecting humans living close to wetlands in tropical countries. This mycobacteria resides in water where it could colonize many ecological niches such as aquatic plants, herbivorous animals, and water bugs. The latter were shown to be able to transmit the bacteria to mammalian hosts. Here, we described that the bacilli could be structured with a thick envelope called the extracellular matrix (ECM). This peculiar coat contains in small vesicles a toxin named mycolactone, the main virulence factor of M. ulcerans. The ECM confers to the mycobacterium increased resistance to antimicrobial agents and plays a role in virulence. Indeed, a bacteria with ECM is more potent for colonization of insect vectors and mammalian hosts compared to bacteria. Unraveling the regulation of the production of the ECM together with the export of mycolactone will be an important step in developing new pharmacological approaches for the treatment of Buruli ulcer, which has been greatly handicapped by the lack of effectiveness of the current antibiotics.