Title redacted
Abstract
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.
Type
Thesis, PhD Doctor of Philosophy
Collections
Items in the St Andrews Research Repository are protected by copyright, with all rights reserved, unless otherwise indicated.