An investigation into the (2-aminoethyl)phosphonate pathway in Trypanosoma cruzi - etiological agent of Chagas' disease
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The neglected tropical disease, American Trypanosomiasis (also known as Chagas’ disease) is the most important parasitic infection in Latin America, and the most lethal endemic infectious disease in the Western Hemisphere. 8‐9 million individuals are currently infected with the disease, and a further 25 million are at risk. Currently available chemotherapies approved for the treatment of Chagas’ disease are ageing, ineffective, and exhibit severe side effects ‐ new treatments are urgently required. One possible drug target against Trypanosoma cruzi ‐ the protozoan etiological agent of Chagas’ disease ‐ is the biosynthesis of the parasitic glycosylphosphatidylinositol (GPI) anchor and GPI‐related molecules such as glycosylinositolphospholipids (GIPLs), which dominate the organism’s cell surface. Despite structural variations, these complex structures are ubiquitously decorated with an unusual (2‐aminoethyl) phosphonate (AEP) moiety on the O‐6 of glucosamine in the GPI core motif, and is hypothesized to play a role in both host‐infection and parasitic persistence. Entirely absent in higher eukaryotes ‐ including humans ‐ a greater understanding of the Trypanosoma cruzi AEP biosynthetic / biodegradative pathway may allow for the development of novel, parasite‐specific chemotherapeutics. This study determined the essentiality of the T. cruzi AEP biosynthetic / biodegradative pathway through both chemical and genetic methodologies, including a classical two‐ step gene replacement and ectopic reintroduction strategy. Validation was provided through both qRT‐PCR and Southern blot analysis. The resulting genetically modified cell‐line phenotypes were extensively described through a number of techniques including: cell growth studies, radiolabelling experiments, gas chromatography mass‐spectrometry (GC‐MS), multiple reaction monitoring mass‐spectrometry (MRM), and lipidomic analysis. Furthermore, the three enzymes of the poorly characterised T. cruzi AEP biosynthetic pathway (i.e. phosphoenolpyruvate mutase, phosphonopyruvate decarboxylase, and (2‐aminoethyl)phosphonate transaminase) were recombinantly expressed and purified for characterization through a series of biochemical assays. Immunofluorescence microscopy studies additionally were performed to determine the subcellular localization of these enzymes. Significant progress was also made in generating a crystal structure of the T. cruzi PEP mutase enzyme, whilst in silico modeling efforts provided catalytic insights into the TcAEP pathway enzymes in the interim. An interrogation of the T. cruzi genome provided evidence that the parasite does not encode for phosphonoacetaldehyde hydrolase (phosphonatase) ‐ an enzyme present in a number of other organisms capable of AEP biosynthesis. High throughput screening of compounds by differential scanning fluorimetry (DSF) was additionally performed against the T. cruzi AEP transaminase. Putative hits were further screened against T. cruzi epimastigotes in vivo. These efforts identified a number of fragments with EC₅₀ values in‐line with the current frontline treatment against Chagas’ disease. Finally, this body of work also reported the putative identification of a novel, high‐energy donor of AEP, and other AEP‐containing metabolites through both lipidomic and mass‐spectrometric techniques.
Thesis, PhD Doctor of Philosophy
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