Proteomics of Buruli Ulcer Disease Healing
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July, 2018
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Abstract
Background: Buruli ulcer (Bu) is a neglected tropical disease, caused by Mycobacterium ulcerans. The disease often presents itself as a painless subcutaneous nodule, plaque, or oedema that subsequently develops into an ulcer with undermined edges. The pathogenesis of Bu is linked to M. ulcerans secretion of a lipid toxin known as mycolactone. It serves as a biomarker for M. ulcerans and has been demonstrated to be present in all forms of Bu disease before or after antibiotic treatment. This toxin has been demonstrated to be responsible for immunosuppression and tissue necrosis which are characteristic of the Buruli ulcer. Bu disease is reported to downregulate the circulating levels of a large array of proteins involved in immune response. These proteins contribute to acute phase reaction, lipid metabolism, coagulation and tissue remodeling; processes involved in healing of mycobacteria disease and hence may interfere with wound healing. In this study novel and unique proteins capable of predicting fast and slow healers of M. ulcerans disease were analysed and validated. Methods: This study was a prospective hospital-based cohort study. Suspected Buruli ulcer cases were confirmed by dry reagent based standard Polymerase chain reaction (PCR). Protein profiling was done using mass spectrometry and bioinformatics. This part was an in-silico approach, where a high throughput data was generated by mass spectrometry for tissue samples obtained from fast and slow healing Buruli ulcer patients. Interferon Gama inducible Protein-30 (IFI30 / IP30), Cluster of Differentiation 74 (CD74), Proteasome activator subunit 3 (PSME3) and Classical component 4A (C4A) levels were investigated in sera of fast and slow healing Buruli ulcer patients, endemic controls and non-endemic controls using ELISA Results: The results from this study show that Buruli ulcer cases, endemic controls and nonendemic controls generally express IFI30 / IP30, CD74, PSME3 and C4A proteins. Fast healing Bu patients expressed higher levels of IFI30 with median of 1.5 ng/mL and (0.8 – 2.2) ng/mL v interquartile range compared to slow healing Bu with median of 1.2 ng/mL (0.2 - 2.0) ng/mL. Although fast healers expressed slightly higher levels of the protein, compared to slow healers, it was not statistically significant (p > 0.05). The expression levels of CD74 in fast healers (0.4 (0.07 – 2.8) ng/mL) were lower, compared with those of slow healers (1.2 (0.2 - 4.1) ng/mL) but the difference was not statistically significant (p > 0.05). The expression level of PSME3 in fast healers (0.35(0.21 – 0.56) ng/mL) were lower when compared with those of slow healers (0.39 (0.20 – 0.63) ng/mL) but the difference was not significant statistically (p > 0.05). The expression level of C4A in fast healers (7.0 (0.31 – 35.29) ng/mL) was higher, compared with those of slow healers (4.0 (0.09 – 22.13) ng/mL) but the difference was not significant statistically (p > 0.05). Conclusion: The study has shown that IFI30/IP30, CD74, PSME3 and C4A proteins were generally expressed by both Bu patients and control. Higher median level of IFI30 protein was associated with fast healing, in agreement with initial analyses of the three pathways of immune response. Though C4A was higher in fast healing, this was contrary to the initial immune pathway analysis. However, CD74 and PSME3 were of lower expression in fast healing. Interestingly, IFI30 showed opposing expression levels in plaques and nodules. In nodules, the higher the baseline concentration of IFI30, the longer the time to complete healing, but in plaques, the higher the baseline concentration of IFI30, the shorter the time to complete healing. This finding could be a useful guide for extending or shortening antibiotic therapy in nodules or plaques, based on their initial or baseline concentration of IFI30 if confirmed in larger studies.
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A thesis submitted in fulfillment of the requirements for the degree of Master of Philosophy, Biochemistry in the Department of Biochemistry and Biotechnology.