Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions

Abstract

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin’s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells’ ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone’s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.

Recent advances: role of mycolactone in the pathogenesis and monitoring ofMycobacterium ulceransinfection/Buruli ulcer disease

“…Summary of findings of multiple publications that have reported the effect of mycolactone directly on the production of various cytokines, chemokines and other proteins by monocytes (Simmonds et al ., ), macrophages (Hall et al ., ), dendritic cells (Coutanceau et al ., ), primary T‐cells (Phillips et al ., , Guenin‐Mace et al 2011), T‐cell lines (Pahlevan et al ., , Boulkroun et al ., ) and endothelial cells (Ogbechi et al ., ). Colour coding is as follows: Red, profound inhibition; orange, some inhibition; yellow, little inhibition; green, no inhibition; dark green, increased expression; white, not studied…”

“…These cells have a central role in wound healing and repair, forming a protective epithelial barrier over the wound bed. Thrombomodulin expression by keratinocytes in the epidermis of some BU patients was found to be decreased (Ogbechi et al ., ), although it is not yet clear what the relevance of this is.…”

“…Furthermore, cessation of production of N-linked glycosylated proteins was demonstrated by metabolic labelling, indicating that potentially hundreds of proteins are influenced by this mechanism in every cell. Therefore, while the purpose of this study was to explain the lack of inflammation in BU, the identified mechanism probably impacts the pathogenesis in multiple ways (Hall et al, 2014;Ogbechi et al, 2015).…”

Mycolactone reveals the substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis

“…Mycolactone is linked to the underproduction of several key proteins involved in the inflammatory response (Pahlevan et al, 1999; Simmonds et al, 2009; Torrado et al, 2007) and the control of blood coagulation (Ogbechi et al, 2015) as a direct result of its effect on the Sec61 complex at the endoplasmic reticulum (ER) (Baron et al, 2016; Hall et al, 2014; Ogbechi et al, 2015; McKenna et al, 2016). …”

Buruli ulcer: wound care and rehabilitation

“…45,46 Recent evidence has revealed that mycolactone exerts a profound effect on protein secretion by blocking the co-translational translocation of a plethora of proteins that pass through the endoplasmic reticulum for secretion or placement in cell membranes. 47,48 It is noteworthy that many of these proteins are important in wound healing, and we hypothesize that the rate of healing of ulcers under antibiotic treatment may be significantly impacted by the rate of clearance of mycolactone from tissues. Studies are underway to evaluate the synergy between tissue levels of mycolactone, bacterial load, and rate of wound healing in BUD in humans.…”