Proteomic Analysis of the Action of the Mycobacterium ulcerans Toxin Mycolactone: Targeting Host Cells Cytoskeleton and Collagen

Abstract

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

IL-17A Promotes Intracellular Growth of Mycobacterium by Inhibiting Apoptosis of Infected Macrophages

“…Protein extracts were prepared, resolved in 12% SDS-PAGE, and western blot performed as described previously ( 24 ). Primary antibodies were actin (JLA20, developed by Lin JJC and obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biology), Bax, Bcl2, and caspase-3 (all Cell Signaling).…”

Sec61 blockade by mycolactone: A central mechanism in Buruli ulcer disease

“…Short‐term (4–16 h) exposure to mycolactone induced rapid alterations in the actin cytoskeleton of HeLa cells, coinciding with a defective capacity of the cells to establish adhesive contacts and migrate directionally in wound‐healing assays in vitro [Guenin‐Mace et al., ]. In all skin cells studied, longer treatments (>48 h) induced cell retraction followed by detachment and apoptosis, albeit with slight differences in time‐to‐death across cell types [Bieri et al., ; Dangy et al., ; Gama et al., ; George et al., ; Guenin‐Mace et al., ; Ogbechi et al., ; Snyder and Small, ]. In human dermal microvascular endothelial cells, mycolactone treatment also resulted in the depletion of the blood coagulation regulator thrombomodulin from the cell surface [Ogbechi et al., ].…”

Clinical Epidemiology of Buruli Ulcer from Benin (2005-2013): Effect of Time-Delay to Diagnosis on Clinical Forms and Severe Phenotypes

“…Buruli ulcer (BU), caused by Mycobacterium ulcerans , is the third most common mycobacteriosis worldwide, after tuberculosis and leprosy [ 1 ]. BU pathogenesis is mediated by mycolactone, a potent polyketide-derived macrolide that triggers apoptotic cell death [ 2 ] and is associated with the necrotic nature of the disease [ 3 ]. BU mostly affects people in tropical countries in Africa [ 4 ], America [ 5 ], Asia [ 6 ] and Australia [ 7 ].…”