Functional characterisation of a putative amino acid transporter in toxoplasma gondii
File(s)
Author(s)
Wallbank, Bethan
Type
Thesis
Abstract
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.
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.
Version
Open Access
Date Issued
2018-10
Date Awarded
2019-02
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Treeck, Moritz
Blackman, Mike
Baum, Jake
Publisher Department
Francis Crick Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)