Dataset Information

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

ABSTRACT: 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.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.The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target.

SUBMITTER: Dangy JP

PROVIDER: S-EPMC4924874 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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

Dangy Jean-Pierre JP Scherr Nicole N Gersbach Philipp P Hug Melanie N MN Bieri Raphael R Bomio Claudio C Li Jun J Huber Sylwia S Altmann Karl-Heinz KH Pluschke Gerd G

PLoS neglected tropical diseases 20160628 6

<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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 conjuga ...[more]

PMID: 27351976

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Project description:Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer.

Project description: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.

Dataset Information

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

Publications

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

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