Date Published: April 3, 2014
Publisher: Public Library of Science
Author(s): Belinda S. Hall, Kirsti Hill, Michael McKenna, Joy Ogbechi, Stephen High, Anne E. Willis, Rachel E. Simmonds, Vojo Deretic.
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.
Mycolactone is a lipid-like polyketide macrolide virulence factor produced by Mycobacterium ulcerans, the infectious agent of Buruli ulcer (BU) , . This progressive, necrotizing, cutaneous lesion is common in West Africa but also found in other regions, including Australia, Asia and South America. Mycolactone is a key factor in BU pathology: possession of a plasmid carrying enzymes involved in mycolactone synthesis is essential for virulence and injection of mycolactone alone can reproduce many characteristics of the infection, including ulceration, necrosis and suppression of immune responses , . Mycolactone has been shown to have diverse effects on a range of cells and tissues but a unifying mechanism underlying its pleiotropic actions has remained elusive.
In this manuscript we have identified an important new activity for the M. ulcerans virulence factor, mycolactone. By investigating the inhibition of cytokine production as a model system to examine its basic cell biology, we have shown that mycolactone effectively blockades the translocation of nascent proteins across the ER membrane in a mechanism that seems to involve the Sec61 translocon. This finding is remarkable because previous data suggested that mycolactone was probably inhibiting the translation of inflammatory mediators such as TNF, IL-6 and Cox-2 , . However, our detailed investigation refutes this. We showed conclusively that the transcripts were actively translating in vitro and in a RAW264.7 cell model in both the absence and presence of mycolactone. The polysomal location of each of these transcripts in treated cells is remarkably similar to that seen in untreated cells and their relocation in response to inhibitors of translation is also unaffected. Likewise the demonstration of an association between these mRNAs and the membrane fraction in mycolactone-treated cells provides independent evidence of sufficient translation having occurred to allow signal peptide-directed targeting to the ER.