An Antigen Capture Assay for the Detection of Mycolactone, the Polyketide Toxin of Mycobacterium ulcerans.

Mycolactone is a cytotoxin responsible for most of the chronic necrotizing pathology of Mycobacterium ulcerans disease (Buruli ulcer). The polyketide toxin consists of a 12-membered lactone ring with a lower O-linked polyunsaturated acyl side chain and an upper C-linked side chain. Mycolactone is unique to M. ulcerans and an immunological Ag capture assay would represent an important tool for the study of Buruli ulcer pathogenesis and for laboratory diagnosis. When testing sets of mycolactone-specific mouse mAbs, we found that Abs against the hydrophobic lower side chain only bind mycolactone immobilized on a solid support but not when present in solution. This observation supports previous findings that mycolactone forms micellar structures in aqueous solution with the hydrophobic region sequestered into the inner core of the aggregates. Although an Ag capture assay typically requires two Abs that recognize nonoverlapping epitopes, our search for matching pairs of mAbs showed that the same mAb could be used both as capture and as detecting reagent for the detection of the mycolactone aggregates. However, the combination of a core-specific and a core/upper side chain-specific mAb constituted the most sensitive ELISA with a sensitivity in the low nanogram range. The results of a pilot experiment showed that the sensitivity of the assay is sufficient to detect mycolactone in swab samples from Buruli ulcer lesions. Although the described capture ELISA can serve as a tool for research on the biology of mycolactone, the assay system will have to be adapted for use as a diagnostic tool.

Development of an ELISA for the quantification of mycolactone, the cytotoxic macrolide toxin of Mycobacterium ulcerans.

Mycolactones, macrolide cytotoxins, are key virulence factors of Mycobacterium ulcerans, the etiological agent of the chronic necrotizing skin disease Buruli ulcer. There is urgent need for a simple point-of-care laboratory test for Buruli ulcer and mycolactone represents a promising target for the development of an immunological assay. However, for a long time, all efforts to generate mycolactone-specific antibodies have failed. By using a protein conjugate of a truncated non-toxic synthetic mycolactone derivative, we recently described generation of a set of mycolactone-specific monoclonal antibodies. Using the first mycolactone-specific monoclonal antibodies that we have described before, we were able to develop an antigen competition assay that detects mycolactones. By the systematic selection of a capturing antibody and a reporter molecule, and the optimization of assay conditions, we developed an ELISA that detects common natural variants of mycolactone with a limit of detection in the low nanomolar range. The mycolactone-specific ELISA described here will be a very useful tool for research on the biology of this macrolide toxin. After conversion into a simple point-of-care test format, the competition assay may have great potential as laboratory assay for both the diagnosis of Buruli ulcer and for the monitoring of treatment efficacy.

Configurationally Stabilized Analogs of M. ulcerans Exotoxins Mycolactones A and B Reveal the Importance of Side Chain Geometry for Mycolactone Virulence.

Mycolactones A/B (1a/b) are exotoxins of Mycobacterium ulcerans that are the molecular cause of Buruli ulcer. 1a/b represent a rapidly equilibrating mixture of Z/E isomers about the C4'═C5' double bond of the C5-side chain. Here, we describe the syntheses of mycolactone analogs with configurationally stable C5-side chains (2a, E mimetic; 2b/c, Z mimetics). Based on the cytotoxicity of 2a-c, the Δ4',5'-trans isomer of mycolactones A/B (1b) appears to be the major virulence factor.

The Macrolide Toxin Mycolactone Promotes Bim-Dependent Apoptosis in Buruli Ulcer through Inhibition of mTOR.

Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, is central to the pathogenesis of the chronic necrotizing skin disease Buruli ulcer (BU). Here we show that mycolactone acts as an inhibitor of the mechanistic Target of Rapamycin (mTOR) signaling pathway by interfering with the assembly of the two distinct mTOR protein complexes mTORC1 and mTORC2, which regulate different cellular processes. Inhibition of the assembly of the rictor containing mTORC2 complex by mycolactone prevents phosphorylation of the serine/threonine protein kinase Akt. The associated inactivation of Akt leads to the dephosphorylation and activation of the Akt-targeted transcription factor FoxO3. Subsequent up-regulation of the FoxO3 target gene BCL2L11 (Bim) increases expression of the pro-apoptotic regulator Bim, driving mycolactone treated mammalian cells into apoptosis. The central role of Bim-dependent apoptosis in BU pathogenesis deduced from our experiments with cultured mammalian cells was further verified in an experimental M. ulcerans infection model. As predicted by the model, M. ulcerans infected Bim knockout mice did not develop necrotic BU lesions with large clusters of extracellular bacteria, but were able to contain the mycobacterial multiplication. Our findings provide a new coherent and comprehensive concept of BU pathogenesis.

Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by Mycobacterium ulcerans.

BACKGROUND: Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone. METHODOLOGY/PRINCIPAL FINDINGS: Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone. CONCLUSIONS/SIGNIFICANCE: The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target.

Mycobacterial toxin induces analgesia in buruli ulcer by targeting the angiotensin pathways.

Mycobacterium ulcerans, the etiological agent of Buruli ulcer, causes extensive skin lesions, which despite their severity are not accompanied by pain. It was previously thought that this remarkable analgesia is ensured by direct nerve cell destruction. We demonstrate here that M. ulcerans-induced hypoesthesia is instead achieved through a specific neurological pathway triggered by the secreted mycobacterial polyketide mycolactone. We decipher this pathway at the molecular level, showing that mycolactone elicits signaling through type 2 angiotensin II receptors (AT2Rs), leading to potassium-dependent hyperpolarization of neurons. We further validate the physiological relevance of this mechanism with in vivo studies of pain sensitivity in mice infected with M. ulcerans, following the disruption of the identified pathway. Our findings shed new light on molecular mechanisms evolved by natural systems for the induction of very effective analgesia, opening up the prospect of new families of analgesics derived from such systems.

Structure-activity relationship studies on the macrolide exotoxin mycolactone of Mycobacterium ulcerans.

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.

A ring-closing metathesis (RCM)-based approach to mycolactones†A/B.

The total synthesis of the mycobacterial toxins mycolactones A/B (1 a/b) has been accomplished based on a strategy built around the construction of the mycolactone core through ring-closing metathesis. By employing the Grubbs second-generation catalyst, the 12-membered core macrocycle of mycolactones, with a functionalized C2 handle attached to C11, was obtained in 60-80 % yield. The C-linked upper side chain (comprising C12-C20) was completed by a highly efficient modified Suzuki coupling between C13 and C14, while the attachment of the C5-O-linked polyunsaturated acyl side chain was achieved by Yamaguchi esterification. Surprisingly, a diene containing a simple isopropyl group attached to C11 could not be induced to undergo ring-closing metathesis. By employing fluorescence microscopy and flow cytometry techniques, the synthetic mycolactones A/B (1 a/b) were demonstrated to display similar apoptosis-inducing and cytopathic effects as mycolactones A/B extracted from Mycobacterium ulcerans. In contrast, a simplified analogue with truncated upper and lower side chains was found to be inactive.