The pathogenic mechanism of the Mycobacterium ulcerans virulence factor, mycolactone, depends on blockade of protein translocation into the 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.
Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.
Belinda S
BS
Hall
The Babraham Institute, Babraham, Cambridge, United Kingdom.
Kirsti
K
Hill
Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
Michael
M
McKenna
Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.
Joy
J
Ogbechi
Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
Stephen
S
High
MRC Toxicology Unit, Leicester, United Kingdom.
Anne E
AE
Willis
Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.
Rachel E
RE
Simmonds
D000818:Animals; D054312:Buruli Ulcer / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000473:pathology; D018836:Inflammation Mediators / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D018942:Macrolides / Q000378:metabolism*; D051379:Mice; D019911:Mycobacterium ulcerans / Q000378:metabolism / Q000472:pathogenicity*; D014176:Protein Biosynthesis / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D015815:Cell Adhesion Molecules; D018836:Inflammation Mediators; D015850:Interleukin-6; D008070:Lipopolysaccharides; D018942:Macrolides; D014409:Tumor Necrosis Factor-alpha; C117218:mycolactone; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2