University of Cambridge
Investigation of the interactions between Trypanosoma congolense invariant surface glycoproteins and components of the mammalian immune system
Abstract
dc:description.abstractAfrican trypanosomes are single-celled eukaryotic pathogens of humans and animals. As they are extracellular in vertebrate hosts, their cell surface forms the primary host-pathogen interface. Invariant surface glycoproteins (ISGs) are a large family of cell surface proteins with recent expansion and diversification in *Trypanosoma congolense*. *Trypanosoma brucei* ISG65 is known to be a receptor for complement C3, and *T. brucei* ISG75 has been implicated in trypanocidal drug uptake mechanisms. The function of other ISGs including all ISGs in *T. congolense* is unknown. The aim of the work here was to identify ligands for ISGs in *T. congolense*. First, the *T. congolense* ISG gene repertoire was investigated as a whole. Analyses of the sequences and synteny of ISGs in the available genome sequences were used to define a list of *T. congolense* ISGs. A phylogenetic analysis of *T. congolense*, *T. brucei* and *T. vivax* ISGs indicated that diversification of ISGs occurred before and after speciation events in the African trypanosome clade, and gave estimations of which ISGs are most closely related. *T. congolense* ISGs were found to be generally upregulated in the mammalian life stage (bloodstream form), supporting the hypothesis that these proteins may function as receptors for ligands in the mammalian host. Selected *T. congolense* ISGs were produced as recombinant proteins using bacterial and mammalian expression systems. Pulldown assays and mass spectrometry were used to identify ISG binding partners in goat serum. Putative ligands for two ISGs were identified. Protein-protein interactions were confirmed by surface plasmon resonance with purified ligands. ISG 64/65C was identified as a putative receptor for complement C3. ISG LBA was identified as a putative receptor for complement C4. The binding of ISG LBA to C4 did not disrupt the interaction between C4 and C4b binding protein *in vitro*. The putative C4 receptor, ISG LBA, was found to be non-essential in bloodstream form cells in culture, as demonstrated by successful production of a cell line in which both alleles were deleted by targeted homologous recombination. A polyclonal rabbit antiserum raised against recombinant ISG LBA was used to confirm presence of the putative receptor in wild type *T. congolense* protein lysates, and absence in lysates from the ISG LBA -/- cell line. Immunofluorescence assays were not conclusive, but were suggestive of surface localisation of ISG LBA in wild type *T. congolense*. This work represents the first characterisation of large cell surface protein receptors of bloodstream form *T. congolense*, and the first characterisation of a putative C4 receptor in an African trypanosome. This represents a major advance in our understanding of the interactions between trypanosomes and the mammalian immune system. This thesis provides a useful tool for the further study of ISGs, and is a significant step towards understanding the role of this protein family in African trypanosomes.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- McDowell, Alice
- Advisor dc:contributor.advisor
-
- Carrington, David
Subjects
dc:subject × 5Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.103279
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/359989