Solute uptake into B. divergens and P. falciparum infected erythrocytes: same theme but different mechanisms https://doi.org/10.17192/z2015.0466 Philipps-Universität Marburg Parasit Der Transport von Lösung in von B. divergens und P. falciparum infizierten Erythrozyten Biologie Infektion Blutzelle 2015 urn:nbn:de:hebis:04-z2015-04663 Life sciences Biowissenschaften, Biologie 2015-10-29 Sharma, Jyotsna Sharma Jyotsna doctoralThesis English opus:6420 136 application/pdf monograph red blood cell infection by malaria parasite Plasmodium falciparum and Babesia divergens are parasites of the same phylogeny apicomplexa and they invade and replicate in the human erythrocytes. Both the parasites differently modify the host cell membrane during and after infection, providing an excellent example of parallel but distinct adaptations of the parasites to survive in the RBC. During invasion of the host erythrocyte, the parasite targets specific entry sites on the RBCs membrane, which is also the initial step in the biogenesis of the parasitophorous vacuole (PV). The parasitophorous vacuolar membrane (PVM) surrounds the parasite and thus P. falciparum remains surrounded by the vacuolar membrane for most of the parasite’s development. On the other hand in B. divergens, vacuolar membrane fate is unclear, owing to the lack of suitable markers. The electron microscopic studies suggest the disintegration of the PVM in the later stages of parasite maturation. As the differentiated erythrocyte does not endocytose or phagocytose, events leading to the formation of the PVM, in particular the contribution of the erythrocyte membrane (RBCM) are unknown. In order to understand better the internalization of RBCM proteins by parasites, we wanted to determine whether the lipid rafts, which are concentrated patches of lipids and proteins on the erythrocyte membrane, play any such role as specific entry site´ in the biogenesis of PV and also determine whether both parasites respectively internalize the same type of lipids/proteins or not. Recent work has shown that there is no internalization of major RBCM proteins, but the proteins which were found internalized are comparatively present in low abundance in the RBCM and are associated with the lipid rafts. In this work I used B. divergens and also P. falciparum infected erythrocytes to do a comparative investigation of the host cell lipids/proteins, whether they are included or excluded in PV. As a result (i) we found that both parasites recruit and exclude the same set of proteins. (ii) we used cholera toxin B subunit which binds to the ganglioside GM1, to follow the fate of this lipid raft associated glycophingolipid during PVM formation and disintegration. (iii) we observed that GM1 remains in the vicinity of P. falciparum during maturation but disappears from the vicinity of B. divergens as the parasite matures, consistent with a disintegration of the PVM. In conclusion, it appears that there is similarity between the early events in the PVM formation in both the parasites. All the biochemical pathways are dependent on the balance of the transport mechanisms and counteractions like the import of extracellular metabolites, export of the intracellular metabolites and proper waste disposal of unused metabolites. Thus the second part of my work was focused on the increased permeability of the host cell membrane after infection and appearance of new permeability pathways (NPP) in the infected erythrocyte. The erythrocyte which is save heaven´ for the parasite have limited resources and metabolism, that the parasite can use. The intracellular parasite is far more active inside the erythrocyte than outside cell. After invasion the small metabolically active merozoite stage of each genus (Plasmodium and Babesia) grows rapidly into other developmental stages (ring form and trophozoite form) inside the erythrocyte and thereby synthesizing DNA, RNA, proteins and lipids in large amounts. In order to fulfil all the essential requirements, the growing parasite induces changes in the membrane permeability of the erythrocyte and takes up solutes from the extracellular media via NPP. One among the other important amino acids required by the parasite is L-glutamate. The main aim of my work was to observe and characterize the transport of L- glutamate into B. divergens infected erythrocytes, and compare it with P. falciparum infected erythrocytes. As it is known that P. falciparum infected erythrocytes have both low- and high-affinity (EAAT3) glutamate transporter activated. The transport system present in B. divergens infected erythrocytes is unclear, therefore it is interesting to know the details of transport mechanism and see if it is parasite specific mechanism. Like P. falciparum, after infection of erythrocytes, B. divergens also induces the uptake of several solutes which are not taken up by non-infected erythrocytes. We found that B. divergens infected erythrocytes show uptake of glutamate via a low-affinity transport system. The other important characteristics of the glutamate transport observed was Na+-independency, non-saturability and non-stereo selectivity. There was also enhanced uptake of glutamate in the presence of choline. In conclusion, it appears that both parasites induce different mechanisms or transport systems for glutamate uptake and also the activation of the transport system is parasite specific. https://archiv.ub.uni-marburg.de/diss/z2015/0466/cover.png Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg ths Prof. Dr. Przyborski Jude M. Przyborski, Jude M. (Prof. Dr.) Plasmodium falciparum und Babesia divergens sind Parasiten der selben phylogenetischen Apicomplexa, die Erythrozyten befallen und sich replizieren. Beide Parasiten modifizieren die Zellmembran während und nach der Infektion und geben so ein hervorragendes Beispiel für parallele aber unterschiedliche Adaption von Parasiten um im RBC zu überleben. In dieser Arbeit werden Aufnahme und Transport verschiedener Lösungen durch die veränderte Zellmembran untersucht. Fachbereich Biologie 2016-05-25