Reconstructing ICAM-1-binding var genes of Plasmodium falciparum

Carrington, E (2016) Reconstructing ICAM-1-binding var genes of Plasmodium falciparum. PhD thesis, University of Liverpool.

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Abstract

Malaria is a major cause of morbidity and mortality globally. Severe malaria in patients infected with the most deadly species, Plasmodium falciparum, involves sequestration of P. falciparum-infected erythrocytes in the microvasculature, a process involving the interaction of numerous parasite and host factors. The highly variable gene family var encodes PfEMP1 proteins which are thought to be responsible for the majority of endothelial cell binding via receptors such as CD36, ICAM-1 and EPCR. To date, knowledge of the specific PfEMP1 domains that mediate binding to these receptors has emerged from the study of two reference parasite strains IT4 and 3D7. The aim of this study is to identify and characterise new ICAM-1 binding var genes from several culture-adapted ICAM-1-selected patient isolates. Sequencing var genes is complicated by their high variability within the 50-60 gene copies per genome and the absence of overlap between var repertoires of different isolates. We test the predictive powers of a new var gene database of > 62,000 var genes generated by the Pf3k genome sequencing project. We find database matches to be a useful starting point for primer design in this targeted sequencing approach. However, key differences between database genes and those amplified from the patient isolates highlights the need for experimental validation. We perform sequence analysis of var genes from three patient isolates, BC12, J1 and PCM7, and identify their PfEMP1 domain structures. We extensively characterise the DBLβ domains, which are responsible for ICAM-1 binding, of these genes by phylogenetic and homology block analysis and compare them to known ICAM-1 binding DBLβ domains. We express two of the newly identified DBLβ domains as recombinant proteins and analyse their binding to ICAM-1. Using SPR assays, we show that the recombinant DBLβ domains of J1a and BC12a bind ICAM-1D1D5 with nanomolar affinity. We compared their binding to the previously characterised IT4var13DBLβ and studied the binding of all three DBLβ domains to four ICAM-1 mutant proteins. Residues K29, L42 and L44 are essential for ICAM-1 binding of all three DBLβ domains, whereas S22 is essential for J1aDBLβ and BC12aDBLβ binding but not IT4var13DBLβ binding. These results have important implications for the design of anti-disease therapies targeting severe malaria.

Item Type:	Thesis (PhD)
Divisions:	Faculty of Health and Life Sciences
Depositing User:	Symplectic Admin
Date Deposited:	10 Aug 2016 08:07
Last Modified:	19 Jan 2023 07:35
DOI:	10.17638/03001685
Supervisors:	Craig, AG Urban, B
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3001685