Funktionelle Charakterisierung der wichtigsten integralen Proteinkomponenten des Parasiten-Wirtszellinterfaces der Blutstadien des menschlichen Malariaerregers Plasmodium falciparum
Zusammenfassung der Projektergebnisse
Allgemeinverständliche Darstellung der wichtigsten wissenschaftlichen Fortschritte und ggf. ihrer Anwendungsaspekte P. falciparum parasites proliferate exponentially in the human blood, leading to the symptoms of malaria. The parasites do not develop freely in the circulation but within human red blood cells, undergoing continuous cycles of invasion, multiplication and destruction of the host cell, each cycle producing new parasites that immediately invade new red blood cells. In the red blood cell the parasite develops inside a vacuole that is formed by a membrane separating the parasite from the host cell, the parasitophorous vacuolar membrane (PVM). The PVM holds key functions needed for the survival of the parasite in the red blood cell. It controls the access of nutrients to the surface of the parasite from where they are transported into the parasite by specific transporters. The so called nutrient permeable channel (NPC), a pore permitting the passive passage of small molecules, is responsible for the nutrient passage function of the PVM. Here we identified the integral PVM protein EXP1 as essential for the NPC activity. To our knowledge this is the first protein solely affecting this function, which permitted us to use it as a tool to study the PVM NPC. We found that the growth of the parasite depends on both, passage of glucose and amino acids through the NPC. Interestingly, we found that also many antimalarial drugs pass through the NPC and that reducing NPC levels reduced the effectivity of these drugs. Further our experiments indicated that the parasite increases NPC levels when subjected to amino acid restriction. This was particularly relevant in parasites resistant to the current frontline drug artemisinin (ART) because, due to the resistance mechanism, these parasites obtain less amino acids from the host cell than ART sensitive parasites. Our data indicated that ART resistant parasites in the field upregulate EXP1 to compensate this deficit. This indicated that a reason for the fitness cost in ART resistant parasites is amino acid restriction and that these parasites show compensatory in response to this. Taken together his part of the work revealed a critical function of EXP1 for the parasite and provided insights into the NPC and the role of the PVM in nutrient access, including insights important for drug resistance and for our understanding how drugs access the parasite. Knowledge about the properties needed for efficient NPC passage could inform medicinal chemistry approaches to optimise drug access, although it should be noted that only a limited number of chemicals were tested here (so far mainly enforcing the existing knowledge that small size and hydrophilicity are key parameters for chemicals to pass the NPC). However, the EXP1low parasites provide the means for more systematic analyses of the finer physiochemical properties needed for NPC passage which could be relevant for drug leads with low passive membrane-permeability. In further work in this study we found that fusing larger domains to the C-terminus of EXP1 or other integral PVM proteins prevents the insertion of the tagged protein into the PVM. In addition we found that all so far tested (most) members of the ETRAMPs (a family of highly abundant integral PVM proteins) were dispensable for the growth of blood stage parasites. While these findings did not lead to direct insights into parasite biology, they are nevertheless important for the field and have relevance for the design of future projects. "Überraschungen" im Projektverlauf und bei den Ergebnissen It was surprising to find that EXP1 is required for NPC function, as previous work showed that the NPC function depends on the channel component EXP2 of the protein translocon PTEX (EXP2 has a dual role in protein export and nutrient acquisition). However, the patch clamp and nutrient access experiments in this work are strong evidence that EXP1 is indispensable for the PVM NPC function. Loss of EXP1 influenced the distribution of EXP2 but this only affected NPC function but not PTEX function. It is unclear if the EXP2 misdistribution was the cause of the loss of NPC activity (particularly because PTEX function was not affected) or if it was a downstream effect of EXP1 loss. Overall these findings indicate that the current model of the connection between the PTEX pore component EXP2 and the nutrient pore function might be incomplete and warrants further consideration. A further surprise was that none of the ETRAMPs we targeted were important for the growth of blood stage parasites. It is puzzling why the parasite invests so much (investment in terms of high expression levels of ETRAMPs) if there is no critical role. One explanation could be redundancy between members of this family or a role that is only relevant in vivo. Hinweise auf mögliche Erfolgsberichte in den Publikumsmedien The second publication arising from this work was highlighted in Nature Reviews in Microbiology, in the appearing issue of Cell Host & Microbe and was chosen as editor's pick for that journal in 2021.
Projektbezogene Publikationen (Auswahl)
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EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites. PLOS Biology, 17(9), e3000473.
Mesén-Ramírez, Paolo; Bergmann, Bärbel; Tran, Thuy Tuyen; Garten, Matthias; Stäcker, Jan; Naranjo-Prado, Isabel; Höhn, Katharina; Zimmerberg, Joshua & Spielmann, Tobias
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The parasitophorous vacuole nutrient channel is critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost. Cell Host & Microbe, 29(12), 1774-1787.e9.
Mesén-Ramírez, Paolo; Bergmann, Bärbel; Elhabiri, Mourad; Zhu, Lei; von Thien, Heidrun; Castro-Peña, Carolina; Gilberger, Tim-Wolf; Davioud-Charvet, Elisabeth; Bozdech, Zbynek; Bachmann, Anna & Spielmann, Tobias
