Abstract
Buruli ulcer (BU) is a progressive necrotising skin disease caused by Mycobacterium ulcerans. The bacteria produce a lipid virulence factor, mycolactone which is responsible for the tissue necrosis seen in the disease but the trigger for this is not known. Endothelial cells play a critical role in maintaining tissue homeostasis but prior to this thesis, the effect of mycolactone on endothelial cells had not been investigated. Mycolactone caused a reduction in the expression of proteins involved in the regulation of clotting as well as adhesion molecules in endothelial cells. The mycolactone-mediated inhibition of thrombomodulin (TM) and endothelial cell protein C receptor (EPCR) is of particular interest because of the role these proteins play in promoting protein C activation. Defects in the protein C anticoagulant pathway have been shown to cause excessive fibrin clot formation and in BU patient tissue a profound decrease in TM abundance was also observed. This suggests indicates that mycolactone impairs the anti-coagulant functions of the vascular endothelium which can trigger thrombus formation resulting vascular occlusion, ischemia and subsequently tissue necrosis. In addition to this indirect mechanism of cell death, another well-established effect of mycolactone is its direct cytotoxicity. Translational microarray profiling revealed that mycolactone increases the translation of the integrated stress response (ISR) genes, ATF4 and CHOP. The ISR is an evolutionarily conserved pathway that can cause cell death following chronic exposure to stress and I determined that this pathway is activated in cells exposed to mycolactone. While the precise trigger of the ISR is yet to be identified, my working model is that it is mediated by GCN2. Furthermore, genetic deletion of ATF4 revealed that this pathway plays an important role in mycolactone-mediated cell death. Taken together, my results indicate that both direct (involving the ISR) and indirect (involving altered haemostasis) mechanisms likely bring about the tissue necrosis observed in BU.