Subcellular localisation of the cir multigene family in plasmodium chabaudi
File(s)
Author(s)
Amis, Sarah
Type
Thesis
Abstract
The pir genes are a multigene family found in every species of the malaria parasite Plasmodium so far sequenced. Their preservation in large numbers in the parasite genome implies they fulfill a function or functions of evolutionary importance. Other Plasmodium multigene families, most notably the P. falciparum var family, have been linked to immune evasion and pathogenesis, and it has been speculated that pirs may have similar functions. Previous RNA Sequencing (RNA Seq) data has demonstrated that pir expression both varies with parasite lifecycle stage, and changes dramatically between mosquito transmitted and serially blood passaged infections – concurrent with changes in parasite virulence.
This thesis uses the P. chabaudi murine model of malaria to investigate the localisation of both specific candidate CIRs (PIR in P. chabaudi) identified from prior RNA Seq data, and that of CIR subfamilies. Robust localisation may shed light on the potential PIR functions that have been hypothesized. A twofold approach was taken, generating transgenic parasite lines expressing epitope-tagged candidate CIRs, and designing peptide motifs characteristic of CIR subfamilies which were used to produce anti-CIR antisera. Candidate CIRs were also expressed as recombinant proteins for use in assays in this and future projects.
Peptides conserved in the two major CIR subfamilies were designed and synthesized. Antisera were generated and tested against recombinant CIR proteins and whole parasite lysates. Although there was some cross-reactivity, two antisera successfully recognized their target subfamily. Immunofluorescence assays with these antisera showed the two subfamilies have different localisations. Parasite lines expressing tagged candidate CIRs from three different subfamilies were successfully generated and characterised. These showed each CIR has a different pattern of localisation across the asexual lifecycle stages, verified when probed with the anti-CIR antisera. This provides further evidence for the differential localisation of PIRs, and the hypothesis that the superfamily may have multiple functions.
This thesis uses the P. chabaudi murine model of malaria to investigate the localisation of both specific candidate CIRs (PIR in P. chabaudi) identified from prior RNA Seq data, and that of CIR subfamilies. Robust localisation may shed light on the potential PIR functions that have been hypothesized. A twofold approach was taken, generating transgenic parasite lines expressing epitope-tagged candidate CIRs, and designing peptide motifs characteristic of CIR subfamilies which were used to produce anti-CIR antisera. Candidate CIRs were also expressed as recombinant proteins for use in assays in this and future projects.
Peptides conserved in the two major CIR subfamilies were designed and synthesized. Antisera were generated and tested against recombinant CIR proteins and whole parasite lysates. Although there was some cross-reactivity, two antisera successfully recognized their target subfamily. Immunofluorescence assays with these antisera showed the two subfamilies have different localisations. Parasite lines expressing tagged candidate CIRs from three different subfamilies were successfully generated and characterised. These showed each CIR has a different pattern of localisation across the asexual lifecycle stages, verified when probed with the anti-CIR antisera. This provides further evidence for the differential localisation of PIRs, and the hypothesis that the superfamily may have multiple functions.
Version
Open Access
Date Issued
2018-09
Date Awarded
2019-03
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Langhorne, Jean
Muller, Ingrid
Publisher Department
Francis Crick Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Master of Philosophy (MPhil)