Characterization of the RIFIN protein family of the malaria parasite Plasmodium falciparum (Welch, 1897)

Abstract

SUMMARY

Plasmodium falciparum, responsible for the most severe form of human malaria, replicates asexually in erythrocytes. The capacity to express and switch antigenically variant proteins on the surface of the host cell allows the parasite to evade host immune responses and contributes to its success in establishing long lasting and relapsing infections. An insight into the physiology of variant antigens may lead to better control measures against severe disease, ideally aiding in the generation of anti-malaria vaccines. The rif (repetitive interspersed family) gene family constitutes the largest of these variant antigen families and is encoded by more than 150 gene copies per haploid genome. In this thesis, different aspects of RIFIN biology were investigated. In the first part, I analyzed membrane association and topology of RIFINs using antisera directed against semiconserved regions of these proteins. Sequential extraction of proteins from infected erythrocytes indicated that RIFINs are membrane-spanning and anchored in a fashion similar to the major virulence protein of Plasmodium, PfEMP1. Results from protease protection assays revealed protein anchorage by one transmembrane domain, in contrast to the widely accepted model predicting a two-transmembrane topology. Moreover, with respect to domain exposure there appeared to be two topologically distinct pools of RIFIN proteins. In view of this variability between RIFIN proteins, I focused on classification and characterization of distinct RIFIN subgroups in the second and third parts of my thesis. Based mainly on the presence or absence of a 25 amino acid peptide, two structurally distinct families were evident. Immunofluorescence analysis of asexual and sexual blood stages showed that members of the larger and more diverse A-type RIFIN family were exported into the host cell and associated with host membranes, while variants of the smaller and more conserved B-type RIFIN subgroup displayed a parasite-restricted pattern. Most probably, this intriguing disparity in sub-compartmentalization reflects differences in protein functions. Importantly, it was demonstrated experimentally for the first time that RIFINs are associated with both the invasive stages and sexual forms of P. falciparum, identifying them as multistage antigens, thereby corroborating their biological significance in the parasite. Since A-type variants were shown to be differentially regulated throughout development, I investigated the upstream non-coding regions of rif genes thought to encode regulatory motifs. Phylogenetic analysis led to the definition of five major rif gene supgroups, members of which were found to share similar genomic organizations and protein features. Subclassification in such a way has previously allowed a correlation of PfEMP1 subgroups with disease phenotypes, and may now pave the way for a similar assignment of RIFIN subtypes. In conclusion, RIFINs were identified as a highly diverse family of structurally distinct members, likely to play different roles in malaria disease. The classification of RIFINs into subgroups now provides a solid foundation for gaining a better understanding of the physiological roles inherent to these variant antigens.