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Abstract

In many malaria endemic regions Plasmodium falciparum is seasonally transmitted, since parasite transmission is interrupted by the absence of anopheline mosquitoes during the dry season. P. falciparum persists at low parasitaemia during the dry season months through mechanisms involving longer time in circulation within each replicative cycle of infected erythrocytes without adhering to the endothelium, compared to clinical cases in the wet season. Cytoadhesion of infected erythocytes is mediated by parasite ligands trafficked via de-novo formed sorting organelles (Maurer’s cleft) to the surface of infected erythrocytes and anchored in knob structures. The adhesion ligands bind to endothelial cell receptors, sequestering infected erythrocytes in the vasculature and thus protecting parasites from splenic clearance.

This thesis aimed to understand the mechanisms leading to decreased cytoadhesion of infected erythrocyte, as altered formation of knobs, impaired trafficking of adhesion molecules through the host iRBC, or reduced presentation of adhesion molecules on the iRBC surface, as well as the possible contribution of the host environment. Additionally, it investigates whether, as a consequence of deceased adhesion, the longer-circulationg iRBC are at higher risk of splenic clearance. To address this, we collected infected erythrocytes from asymptomatic individuals at the end of the dry season and from individuals at their first febrile malaria episode during the transmission season. Using an artificial spleen, we found that circulating iRBCs during the dry season are more efficiently filtered in the spleen.

Erythrocyte remodeling and knob density were analyzed by transmission and scanning electron microscopy in developmental stage-matched samples from asymptomatic individuals at the end of the dry season and from individuals at their first febrile malaria episode during the transmission season. Knobs on the surface of the infected erythrocyte were detected at equal densities in both groups but with slightly smaller diameter in the dry season. Maurer’s clefts were formed in the host cells in all samples, but were found more frequently in infected erythrocytes in the dry season and were localized at slightly higher distance to the erythrocyte plasma membrane. However, the differences found were small and of unknown biological significance. Further, we quantified the transcription of parasite genes involved in host cell remodeling by qRT-PCR found differential expression of three genes coding for Maurer’s cleft proteins which are involved in adhesin trafficking, however possibly as a result of imperfect developmental stage matching. Hence, to test whether low parasite adhesion during the dry season stems from decreased expression of parasite ligands on the surface of erythrocytes, we used flow cytometry to compare the binding of hyperimmune plasma to the surface of P. falciparum-infected erythrocytes. We did not observe different labeling efficiencies in infected erythrocytes collected from asymptomatic individuals during the dry season vs. individuals presenting with acute febrile malaria during the rainy season. On the host side, we investigated how different inflammatory states in asymptomatic and clinical malaria cases alter the availability of host adhesion receptors on endothelial cells. Compared to plasmas from clinical malaria cases, we found lower levels of endothelium-stimulating cytokines and soluble adhesion receptors in plasmas of donors in the dry season, independent of if they carried asymptomatic P. falciparum infection or not. We explored whether this difference in endothelial receptor expression affected cytoadhesion efficiency, but in a preliminary test we found no differences in cytoadhesion of infected erythrocytes to endothelial cells stimulated with plasmas from donors with clinical malaria in the transmission season or asymptomatic donors or healthy controls in the dry season.

In summary, the presence of only minor differences in the ultrastructure and no differential labeling by hyperimmune pooled plasma suggests that the contribution of altered host cell remodeling to decreased cytoadhesion in the dry season is only minimal. However, we have first indications that the host environment in the dry season provides less receptors for adhesion. Further, through more efficient splenic clearance of longer-circulating infected erythrocytes, parasitemias are maintained low in asymptomatic infections in the dry season, allowing P. falciparum to persist for several months.