The Pathogenic mechanism of the mycobacterium ulcerans virulence factor, mycolactone, deends on blockade of protein translocation intot he ER

Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer. Author Summary Buruli ulcer is a progressive necrotic skin lesion caused by infection with the human pathogen Mycobacterium ulcerans. Mycolactone, a small compound produced by the mycobacterium, is the root cause of the disease pathology, but until now there has been no unifying mechanism explaining why. We have been using a model system to investigate the reason for the selective loss of protein that is a common feature of mycolactone exposure. Specifically, this involves identifying the point at which it stops immune cells making inflammatory mediators. In this work, we demonstrate that mycolactone inhibits production of such proteins by blocking the first step of protein export: translocation into a cellular compartment called the endoplasmic reticulum (ER). Proteins due for export are instead made in the cell cytosol where they are recognised as being in the wrong place and are rapidly degraded, causing a general cessation of the production of proteins that have to travel through the ER, including almost all secreted and surface proteins. This has a profound effect on basic cell functions such as growth, adhesion and survival. Therefore, we have identified the molecular basis underlying the key features of Buruli ulcer, and this will transform our understanding of disease progression.