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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 2 ng/ml and as early as 8 hrs 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.

SUBMITTER: Ogbechi J

PROVIDER: S-EPMC4504485 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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

Ogbechi Joy J Ruf Marie-Thérèse MT Hall Belinda S BS Bodman-Smith Katherine K Vogel Moritz M Wu Hua-Lin HL Stainer Alexander A Esmon Charles T CT Ahnström Josefin J Pluschke Gerd G Simmonds Rachel E RE

PLoS pathogens 20150716 7

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 complicatio ...[more]

PMID: 26181660

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Project description:BackgroundBuruli ulcer (BU) is a subcutaneous skin disease listed among the neglected tropical diseases by the World Health Organization (WHO). Early case detection and management is very important to reduce morbidity and the accompanied characteristic disfiguring nature of BU. Since diagnosis based on clinical evidence can lead to misdiagnosis, microbiological confirmation is essential to reduce abuse of drugs; since the anti-mycobacterial drugs are also used for TB treatment. The current WHO gold standard PCR method is expensive, requires infrastructure and expertise are usually not available at the peripheral centers where BU cases are managed. Thus one of the main research agendas is to develop methods that can be applied at the point of care. In this study we selected aptamers, which are emerging novel class of detection molecules, for detecting mycolactone, the first to be conducted in a BUD endemic country.MethodsAptamers that bind to mycolactone were isolated by the SELEX process. To measure their affinity and specificity to mycolactone, the selected aptamers were screened by means of isothermal titration calorimetry (ITC) and an enzyme-linked oligonucleotide assay (ELONA). Selected aptamers were assessed by ELONA using swab samples from forty-one suspected BU patients with IS2404 PCR and culture as standard methods. ROC analysis was used to evaluate their accuracy and cutoff-points.ResultsFive out of the nine selected aptamers bound significantly (p< 0.05) to mycolactone, of these, three were able to distinguish between mycolactone producing mycobacteria, M. marinum (CC240299, Israel) and other bacteria whilst two others also bounded significantly to Mycobacterium smegmatis. Their dissociation constants were in the micro-molar range. At 95% confidence interval, the ROC curve analysis among the aptamers at OD450 ranged from 0.5-0.7. Using this cut-off for the ELONA assay, the aptamers had 100% specificity and sensitivity between 0.0% and 50.0%. The most promising aptamer, Apt-3683 showed a discernible cleavage difference relative to the non-specific autocatalysis over a 3-minute time course.ConclusionThis preliminary proof-of-concept indicates that diagnosis of BUD with RNA aptamers is feasible and can be used as point of care upon incorporation into a diagnostic platform.

Dataset Information

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

Publications

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

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