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Title: Functional characterisation of a putative amino acid transporter in Toxoplasma gondii
Author: Wallbank, Bethan
ISNI:       0000 0004 7963 7505
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2019
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To establish infection and disseminate within its host, the obligate intracellular parasite, Toxoplasma gondii, must undergo cycles of invasion, replication, and egress from the host cell. These recurring cycles rely upon a network of finely tuned signalling pathways that allow the parasite to sense and respond to its environment. Phosphoproteomic analysis of Calcium Dependent Protein Kinase 3 (TgCDPK3), a key moderator of these processes, has allowed for the study of the proteins acting downstream of this kinase. Intriguingly, the phosphoproteome revealed several proteins involved in metabolism, including the branched chain amino acid dehydrogenase (BCKDH). This revealed a potential link between TgCDPK3 and metabolic processes. Three candidates identified from the TgCDPK3 phosphoproteome analysis were chosen for functional characterisation. These were a lipid-binding protein (phosphorylated lipid binding protein; PLBP), a hypothetical protein (putative TgCDPK3-phosphorylated protein; PCPP), and a putative branched chain amino acid (BCAA) transporter (ApiAT5-3; Apicomplexan amino acid transporter 5- 3). As TgCDPK3 was hypothesised to mediate BCKDH phosphorylation, potentially regulating BCAA metabolism, the ApiAT5-3 became the main focus of this study. Ectopictagging demonstrated that all three candidates localise to the parasite periphery, the same subcellular location as TgCDPK3. Upon conditional knockout (cKO) of ApiAT5-3, growth was completely ablated. Complementation of these cKOs with an ectopic copy of the WT gene rescued this phenotype. A combination of ectopic expression in Xenopus laevis and metabolomic analysis of the cKOs revealed this protein to be a transporter of the essential amino acid tyrosine, and not BCAAs. Mutation of the TgCDPK3-dependent phosphorylation site, serine 56, revealed that it is important for parasite fitness. However, phosphorylation of this site alone is not essential for tyrosine import into the parasite. Together this study advances our understanding of the TgCDPK3 signalling pathway and the way in which T. gondii scavenges nutrients from its host.
Supervisor: Treeck, Moritz ; Blackman, Mike ; Baum, Jake Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral