Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei

Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology

Role of STEVOR and its interacting partners in parasite virulence

The genomes of Plasmodium spp. encode a number of different multigene families that are thought to play a critical role for survival. However, with the exception of the P. falciparum var genes, very little is known about the biological roles of any of the other multigene families. The stevor family includes about 40 genes and is therefore the smallest family of variant surface antigens in P. falciparum. While some of its members have been shown to interact with glycophorin C, some others are presented at the apical tip of merozoites where they are involved in invasion. However, much of the diversity within the family is still understudied. The first part of this work describes our efforts to identify STEVOR with novel binding properties to red blood cells. To this end, amino acid sequence alignment provided information about the diversity within the family. In order to cover the entire diversity within the stevor multigene family, a large subset of different STEVOR were expressed on the surface of mammalian cells or recombinantly in bacteria. We successfully identified one stevor with atypical binding behaviour to red blood cells, providing us with a lead candidate for further studies. The second part of this work describes efforts to develop a platform to study members of multigene families in P. falciparum. Using the recently developed Selection Linked Integration method, we have been able to activate the expression of a single member of a multigene family of our choice from its endogenous promoter. We characterize parasites expressing specific members of the var, rifin and stevor multigene families in terms of protein localization, function and epigenetic control mechanisms underlying their expression. We show that endogenous tagging of a PfEMP1 does not disrupt transport of the protein to the surface of the host red blood cell, suggesting that the protein transport is not altered by the knock-in method. Furthermore, knock-in of two previously described RIFIN causes the parasites to become resistant to natural killer cell killing, proving functionality of the proteins. Finally, knock-in of three different STEVOR highlights the diversity observed in this multigene family. Each of the proteins shows a different subcellular localization, with one being exposed in the surface of the infected red blood cell, one remaining within the Maurer’s Clefts and one being expressed as a truncation. Using microarray analysis, we gain new insights into the regulatory mechanisms within and between multigene families. We can show that knock-in of stevor causes monoallelic expression, suggesting mutual exclusive control within this family. In contrast, rifin do not appear to have any level of control mechanisms. Furthermore, we observe cross-talk between multigene families as expression from a genomic locus causes upregulation of some, but not all, adjacent genes. We show that similar observations have been reported previously, suggesting true interaction instead of artefacts introduced by the knock-in Finally, we studied intracellular interaction partners of STEVOR utilizing two independent approaches: co-immunoprecipitation and proximity labelling by BioID. We show that both the surface exposed and the Maurer’s Cleft resident STEVOR interact with unique subsets of proteins. While the surface exposed protein appears to be incorporated into the new permeability complex together with other small variant antigens, the other one is associated with lipid rafts within the Clefts.Doctor of Philosoph

Rapid activation of distinct members of multigene families in Plasmodium spp

© 2020, The Author(s). The genomes of Plasmodium spp. encode a number of different multigene families that are thought to play a critical role for survival. However, with the exception of the P. falciparum var genes, very little is known about the biological roles of any of the other multigene families. Using the recently developed Selection Linked Integration method, we have been able to activate the expression of a single member of a multigene family of our choice in Plasmodium spp. from its endogenous promoter. We demonstrate the usefulness of this approach by activating the expression of a unique var, rifin and stevor in P. falciparum as well as yir in P. yoelii. Characterization of the selected parasites reveals differences between the different families in terms of mutual exclusive control, co-regulation, and host adaptation. Our results further support the application of the approach for the study of multigene families in Plasmodium and other organisms

Selective expression of variant surface antigens enables Plasmodium falciparum to evade immune clearance in vivo

AbstractPlasmodium falciparum has developed extensive mechanisms to evade host immune clearance. Currently, most of our understanding is based on in vitro studies of individual parasite variant surface antigens and how this relates to the processes in vivo is not well-understood. Here, we have used a humanized mouse model to identify parasite factors important for in vivo growth. We show that upregulation of the specific PfEMP1, VAR2CSA, provides the parasite with protection from macrophage phagocytosis and clearance in the humanized mice. Furthermore, parasites adapted to thrive in the humanized mice show reduced NK cell-mediated killing through interaction with the immune inhibitory receptor, LILRB1. Taken together, these findings reveal new insights into the molecular and cellular mechanisms that the parasite utilizes to coordinate immune escape in vivo. Identification and targeting of these specific parasite variant surface antigens crucial for immune evasion provides a unique approach for therapy.</jats:p