Molecular cytopathogenesis induced by the Mycobacterium ulcerans toxin, mycolactone
Year: 2014
Type: dissertation
Abstract: Buruli ulcer (BU) is a tropical neglected, necrotizing skin disease caused by Mycobacterium ulcerans infection. The tissue damage that characterizes the disease is driven by the lipidic exotoxin mycolactone, which is known to play a central role in the pathogenesis of BU. However, the mechanisms by which mycolactone exerts its cytotoxicity are still unknown. Uncovering these mechanisms could lead to new forms of BU treatment, based on the neutralization of the mycolactone cytotoxic effects. The research conducted in the scope of this thesis aimed at increasing our understanding of the molecular cytopathogenicity of mycolactone, focusing on its potent cytotoxicity. To this end, two complementary experimental approaches were followed: in vitro studies on L929 mouse fibroblasts incubated with purified mycolactone and; in vivo studies in an experimental model of BU disease, the mouse footpad infection with M. ulcerans. Using two-dimensional gel electrophoresis, we found several regulators and constituents of both actin- and tubulin-cytoskeleton (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) altered in mycolactone-exposed L929 cells, providing new molecular evidences for the impact of mycolactone upon cytoskeleton, which present conformational disarrangements, as observed by immunofluorescence. Consistently, our study of autophagy showed that autophagosomelysosome fusion, a process dependent on microtubule and dynein-driven transport, is inhibited by mycolactone. In agreement with the impairment of cytoskeleton dynamics and functions in mycolactone-exposed cells, we found that both cytoskeleton-associated BH3-only proteins (BIM and BMF) are responsive to the cellular stress induced by mycolactone, being up-regulated in L929 cells exposed to purified toxin, as well as in mycolactone-producing M. ulcerans-infected mouse footpads. Importantly, the knockdown of Bim protected L929 cells from mycolactoneinduced cytotoxicity, demonstrating that the toxin triggers an intrinsic apoptotic pathway mediated by BIM. The second larger group of proteins found down-regulated in mycolactone-exposed L929 cells comprises collagen-modifying enzymes (Plod1, Plod3, P4ha1) essential for the formation and stabilization of collagen fibers. Consistent with a mycolactone impact upon the collagen fibers stability, in vivo analyses in the BU mouse model revealed a mycolactone-dependent degeneration of dermal collagen fibers upon infection with M. ulcerans.VII Overall, this work provides new insights on the cytotoxic mechanisms of mycolactone, the key pathogenic factor of M. ulcerans. We propose that mycolactone induces nonfunctional cytoskeletal alterations, affecting cytoskeleton-dependent functions and, consequently, cellular homeostasis, triggering a BIM-mediated apoptosis. Moreover, we unveil the action of the M. ulcerans toxin on collagen biosynthesis. Our findings provide new perspectives on BU pathogenesis and pave the way for future therapeutic approaches
Author: José B. Gama
Language: English
Subfield: Epidemiology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W76946078