Membrane Vesicles Of Mycobacterium Ulcerans And Their Role In Buruli Ulcer Pathogenesis

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2021-01

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University Of Ghana

Abstract

The release of bacterial extracellular membrane vesicles (EMVs) is essential for pathogen’s adaptation and virulence. Mycobacterium ulcerans, the causative agent of Buruli ulcer, remains with queries in its pathogenic mechanism. The current study interrogated biological functions and protein content of EMVs from viable M. ulcerans’ cells as potential medium of virulence in BU pathogenesis. Here, we demonstrate release of intact EMVs from the thick cell wall of log-phase M. ulcerans (Nm 209) as well as M. marinum (Sa 200695) in respective liquid cultures. Size distributions of isolated EMVs were similar between the two strains and did not differ from EMVs released by M. smegmatis used as a positive controlled strain. Mycolactone could not be detected in isolated EMVs from M. ulcerans (Nm 209). However, presence of M. ulcerans EMVs was associated with higher total intracellular reactive oxygen species which eventually compromised viability of RAW264.7 cells through oxidative stress. After 48 hours of co-incubation, native and UV-A irradiated EMVs induced 45% and 40% loss in viability of RAW264.7 cells, respectively. Moribund phagocytes exhibited apoptotic changes. Proteomic analysis on the isolated M.ulcerans EMVs revealed an enrichment of 32 unique proteins mostly localized in the pathogen’s cell wall/membrane. A conserved hypothetical protein (MUL_2313), had the highest log2 fold change (11.92) followed by Amidase amiC, a cell-wall remodeling hydrolase (4.19). Others included integral membrane indolylacetylinositol arabinosyltransferase EmbA/B, and many conserved hypothetical proteins. Direct contributions of these proteins to EMVs cytotoxicity could not be established. Yet, protein moon-lighting or possible cross-linking could have potentially contributed to EMV-associated toxicity on RAW264.7 cells. Our results suggest that M. ulcerans EMVs can elicit toxic response from host’s macrophage cells through yet to be established mediators. This potentially reveals new dimension on macrophage-M. ulcerans interactions with possible contribution to local immunosuppression in BU and paradoxical reactions observed in its treatment.

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MPhil. Molecular Cell Biology Of Infectious

Keywords

Mycobacterium Ulcerans, Buruli Ulcer Pathogenesis, Membrane Vesicles

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