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

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

Cytotoxicity of truncated mycolactone variants.

<p>Cells were treated for 48 hours with different concentrations of PG-119 or PG-120 and stained with annexin-V-FITC and PI. Flow cytometry was used to determine the annexin-positive (A⁺) and PI-positive (PI⁺) cell populations. Triplicate samples were analyzed and mean values as well as standard deviations are shown.</p

Cytotoxicity of mycolactone variants with modifications in the lower side chain.

<p>Cells were treated for 48 hours with different concentrations of mycolactone A/B, mycolactone F, mycolactone C or PG-155 and stained with annexin-V-FITC and PI. Flow cytometry was used to determine annexin-positive (A⁺) and PI-positive (PI⁺) cell populations. Triplicate samples were analyzed and mean values as well as standard deviations are shown.</p

Comparison of LC₅₀ and IC₅₀ values of the mycolactone variants.

<p>Comparison of LC₅₀ and IC₅₀ values of the mycolactone variants.</p

Inhibition of proliferation of L929 fibroblasts upon treatment with PG-120, PG-119 and mycolactone A/B.

<p>Cell proliferation was measured after 0, 24, 48 and 72 h by determining the number of cells treated with 60 ng/ml of PG-120, PG-119 or mycolactone A/B. Mean values and standard deviations of triplicates are shown.</p

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. METHODOLOGYPRINCIPAL 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

Two types of reactivity patterns.

<p>All mAbs displayed a similar fine specificity pattern, except for mAbs 5.9 and 5.11, which, in contrast to the other mAbs, showed inhibition with PG-157, a derivative with a slightly extended upper side chain (the difference is depicted for the prototype mAbs JD5.1 and JD5.11).</p

The anti-mycolactone mAbs JD5.1 to JD5.12 bind to PG-204, but not to PG-183.

<p>The biotinylated mycolactone derivatives PG-204 and PG-183 were bound to NeutrAvidin plates and incubated with serial dilutions of the mAbs. Bound anti-mycolactone mAbs were detected using alkaline phosphatase-conjugated goat anti-mouse IgG antibodies.</p