Mycolactone subverts immunity by selectively blocking the Sec61 translocon

Mycolactone, an immunosuppressive macrolide released by the human pathogen Mycobacterium ulcerans, was previously shown to impair Sec61-dependent protein translocation, but the underlying molecular mechanism was not identified. In this study, we show that mycolactone directly targets the alpha subunit of the Sec61 translocon to block the production of secreted and integral membrane proteins with high potency. We identify a single-amino acid mutation conferring resistance to mycolactone, which localizes its interaction site near the lumenal plug of Sec61 alpha. Quantitative proteomics reveals that during T cell activation, mycolactone-mediated Sec61 blockade affects a selective subset of secretory proteins including key signal-transmitting receptors and adhesion molecules. Expression of mutant Sec61 alpha in mycolactone-treated T cells rescued their homing potential and effector functions. Furthermore, when expressed in macrophages, the mycolactone-resistant mutant restored IFN-gamma receptor-mediated antimicrobial responses. Thus, our data provide definitive genetic evidence that Sec61 is the host receptor mediating the diverse immunomodulatory effects of mycolactone and identify Sec61 as a novel regulator of immune cell functions.Peer reviewe

Mycolactone Diffuses into the Peripheral Blood of Buruli Ulcer Patients - Implications for Diagnosis and Disease Monitoring.

BACKGROUND: Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established. METHODOLOGY/PRINCIPAL FINDING: Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone. CONCLUSIONS/SIGNIFICANCE: Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies

Lipids of Pathogenic Mycobacteria: Contributions to Virulence and Host Immune Suppression

International audienceMycobacteria are characterized by a complex cell wall, the lipid nature of which confers to the bacilli resistance to drying, acid or alkaline conditions, and to chemical disinfectants and therapeutic agents. Pathogenic species, such as Mycobacterium tuberculosis, M. leprae and M. ulcerans, have evolved various strategies to establish residence in their hosts and provoke long-term infections. There is mounting evidence that the unique lipids composing their envelopes, strategically located at the host-pathogen interface, contribute to their escape from immune surveillance. Here, the chemical structure, host cell receptors and biological actions of this emerging class of mycobacterial virulence factors are reviewed

Mycolactone Purification from M. ulcerans Cultures and HPLC-Based Approaches for Mycolactone Quantification in Biological Samples

International audienceMycolactones are a family of polyketide synthase products made by the human pathogen Mycobacterium ulcerans that were recently identified as novel inhibitors of the host membrane translocation complex (Sec61). Here, we provide protocols for the purification of mycolactones from bacterial cultures, and for their quantitative assessment in biological samples

Pathogenesis of skin ulcers: lessons from the Mycobacterium ulcerans and Leishmania spp. pathogens

International audienceSkin ulcers are most commonly due to circulatory or metabolic disorders and are a majorpublic health concern. In developed countries, chronic wounds affect more than 1% of thepopulation and their incidence is expected to follow those observed for diabetes andobesity. In tropical and subtropical countries, an additional issue is the occurrence of ulcersof infectious origins with diverse aetiologies. While the severity of cutaneous Leishmaniasiscorrelates with protective immune responses, Buruli ulcers caused by Mycobacteriumulcerans develop in the absence of inflammation. Based on these two examples, this reviewaims to demonstrate how studies on microorganism-provoked wounds can provide insightinto the molecular mechanisms controlling skin integrity. We highlight the potential interestof a mouse model of non-inflammatory skin ulceration caused by intradermal injection ofmycolactone, an unusual lipid toxin with ulcerative and immunosuppressive propertiesproduced by M. ulcerans

Mycolactone: More than Just a Cytotoxin

International audienceFrom their observation of necrotic areas around bacterial foci in Buruli ulcers (BUs), Connor and co-workers were the first, back in 1965, to suggest that M. ulcerans may produce a diffusible cytotoxin [1]. This hypothesis was later confirmed by injecting mycobacterial culture filtrates into the skin of guinea pigs, showing that this causes focal necrosis resembling that of naturally occurring human infections [1–3]. In 1999, George et al. succeeded in isolating a cytotoxic factor from M. ulcerans lipid extracts, and deciphered its chemical nature [4]. The M. ulcerans toxin was named mycolactone, based on its mycobacterial origin and macrolactone structure: a 12-membered lactone ring, to which a C5-O-linked polyunsaturated acyl side chain and a C-linked upper side chain comprising C12–C20 are appended (Fig. 1). Follow-up studies showed that M. ulcerans-derived mycolactone was in reality a mixture of two stereoisomers called A and B [6, 7] (Fig. 1). Since the initial discovery of mycolactone A/B, eight additional mycolactone congeners have been identified that are either produced by M. ulcerans strains of different geographical origins or genetically related fish and frog pathogens, which were initially given different species designations such as M. pseudoshottsii and M. liflandii. Comparative genome analysis later revealed that all mycolactone-producing mycobacteria evolved from a common M. marinum-like progenitor, and are therefore ecovars of a single M. ulcerans species [8]. While the macrolide core structure and upper side chain are conserved, mycolactone populations from different M. ulcerans sub-lineages vary in the length, number and localization of hydroxyl groups and number of double bonds of the lower side chain. These modifications of the lower polyunsaturated acyl side chain cause pronounced changes in cytotoxicity [9–11]. The origin and chemistry of natural mycolactones, structure-activity relationships and the various approaches that were developed to generate synthetic mycolactones have been reviewed recently [12, 13]. These aspects will therefore not be further discussed here. Instead, this chapter provides an update on our understanding of mycolactone (A/B) biology, and discusses its proposed mechanisms of action in relation with BU pathogenesis

Modular total syntheses of mycolactone A/B and its [ 2 H]- isotopologue

International audienceA modular total synthesis of mycolactone A/B, the exotoxin produced by Mycobacterium ulcerans has been achieved through the orchestration of several Pd-catalyzed key steps. While this route leads to a mixture of the natural product and its C12 epimer (4:1 ratio), this was inconsequential from the biological activity standpoint. Compared to previously reported routes, this synthetic blueprint allows the late-stage modification of the toxin, as exemplified with the preparation of [22,22,22-

Immunosuppressive Signature of Cutaneous Mycobacterium ulcerans Infection in the Peripheral Blood of Patients with Buruli Ulcer Disease

International audienceBuruli ulcer disease (BUD) is an emerging human disease caused by infection with Mycobacterium ulcerans, which leads to the development of necrotic skin lesions. The pathogenesis of the ulcer is closely associated with the production of mycolactone, a diffusible cytotoxin with immunomodulatory properties. To identify immunological correlates of BUD, we performed a broad screen of inflammatory mediators in serum samples and stimulated whole-blood supernatants of patients. We found that patients with active ulcers displayed a distinctive profile of immune suppression, marked by the down-modulation of selected chemokines and an impaired capacity to produce Th1, Th2, and Th17 cytokines on stimulation with mitogenic agents. These immunological defects were induced early in the disease and resolved after anti-BUD therapy, establishing their association with the presence of M. ulcerans. Interestingly, some of the defects in cytokine and chemokine response could be mimicked in vitro by incubation of CD4(+) peripheral blood lymphocytes with mycolactone. Our findings support the hypothesis that mycolactone contributes to bacterial persistence in human hosts by limiting the generation of adaptive cellular responses. Moreover, we identified immunological markers of BUD, which may be helpful for confirmatory diagnosis of the disease and, especially, for monitoring the response to antibiotic treatment