Investigating the Kennedy pathway : phosphatidylcholine and phosphatidylethanolamine biosynthesis in Trypanosoma cruzi

Abstract

The Kennedy pathway is a ubiquitous pathway in eukaryotes that synthesises two major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Numerous studies have found that the enzymes of the Kennedy pathway are essential for the survival of many organisms, including important human parasites. In this study, we wanted to understand the importance of the Kennedy pathway in the protozoan parasite Trypanosoma crusi, which causes Chagas disease, a neglected tropical disease in humans. Understanding the biology of Trypanosoma cruzi will help to develop novel,tolerable drugs.
This study focused on the characterisation of three enzymes in the Kennedy pathway: choline kinase (CK), ethanolamine kinase (EK) and the ethanolamine phosphate-cytidyltransferase (ECT). Kinetic parameters and substrate range and preferences were determined through activity assays. To understand the enzyme in vivo, knockout and overexpression cell lines were generated. These cell lines were biochemically phenotyped by various means, including quantitative and qualitative lipidomics. Stable isotope labeling experiments were performed to elucidate effects on phospholipid biosynthesis.
This work has shown that the putative CK and EK in Trypanosoma cruzi are bifunctional and produce both choline phosphate and ethanolamine phosphate; these enzymes are now denoted as C/Ek and E/CK, respectively. Furthermore, C/EK and E/CK are promiscuous and can accept various "non-natural" substrate analogues. A double knockout of E/CK was not possible without an ectopic copy of E/CK, suggesting that it is essential. Biochemical phenotyping indicate that removal of one E/CK allele does not grossly affect PC or PE biosynthesis, suggesting the remaining allele and C/EK homologue might compensate. Radiolabelling experiments revealed a link between the Kennedy pathway and phosphatidylserine and ceramide biosynthesis, where reduced biosynthesis was observed in E/CK single knockout cells. Other work in this thesis revealed that ECT can accept both ethanolamine phosphate and 2-aminoethylphosphonate, suggesting that ECT may play important roles in both phosphonolipid and glycosylphosphatidylinositol biosynthesis in T. cruzi.

Date of Award	29 Nov 2022
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Terry K Smith (Supervisor)