| Zusammenfassung: | Malaria is one of the world’s leading causes of death due to infectious diseases. When Plasmodium infects a red blood cell, it exports many proteins beyond its own cellular boundaries. This leads to extensive remodelling of the host cell, a process that ensures parasite survival and is a direct mediator of parasite virulence. The transmembrane domain (TMD) protein PfEMP1 is important for virulence... Malaria is one of the world’s leading causes of death due to infectious diseases. When Plasmodium infects a red blood cell, it exports many proteins beyond its own cellular boundaries. This leads to extensive remodelling of the host cell, a process that ensures parasite survival and is a direct mediator of parasite virulence. The transmembrane domain (TMD) protein PfEMP1 is important for virulence. PfEMP1 mediates the cytoadherence of infected red blood cells to the endothelium of blood vessels. While the transport step leading to the export of proteins across the PVM into the host cell is well characterized and appears to involve the Plasmodium translocon of exported proteins (PTEX) for all so far analyzed proteins, the transport of PfEMP1 is much less clear and in general the process of extraction of exported TMD proteins out of the parasite plasma membrane (PPM) to be further transported into the host cell, remains elusive.
The aim of this thesis was to identify translocation factors involved in the extraction of exported TMD proteins out of the PPM, enabling the delivery to PTEX to continue their way to the erythrocyte cytosol. Inactivation of the protein translocation step could prevent the export of PfEMP1, and thus abolish the parasite's virulence and adhesion properties, leading to a more efficient clearance of it by the host immune system. This export step was addressed by two approaches in this thesis, firstly by a general quest for proteins that aid the PPM extraction step and secondly, by a direct analysis of PfEMP1 trafficking.
For the first aim, two constructs containing an mDHFR-BirA* sequence were designed. One fused with SBP1 as a control arrested at the PVM and the other with REX2 that is arrested at the PPM. This aimed to biotinylate proteins that contribute to the extraction of proteins out of the PPM. By arresting constructs on both membranes, it was hoped that the transport factors at each membrane would be tagged to be able to distinguish the proteins specifically involved in the extraction at the PPM. By this approach a total of 2000 different proteins were identified. The fact that the SBP1-mDHFR-L-BirA*-3xHA detected mostly PTEX components confirmed that this construct was arrested deeply in the translocon in the PVM, whereas REX2-mDHFR-L-BirA*-3xHA detected a much wider range of proteins, suggesting an arrest of at least part of the population of this molecule in the PPM. Ten promising proteins were cloned, revealing the location and function of previously unknown proteins in P. falciparum. As several of these proteins were located in the parasite periphery, this suggested that the approach successful. However, a detailed characterization of the other analyzed hits revealed that none of the ten hits aided the extraction of TMD proteins out of the PPM towards the PV. This indicates that either the mechanism of removal out of the PPM depends entirely on PTEX which was detected and could provide force for the PPM extraction from the PVM and interacts with the protein only on the outer face of the PPM, or the proteins contributing to extraction were not available for BirA* mediated biotinylation because they were not in reach, for instance if buried deeply in the PPM.
In second part of this work PfEMP1 was directly analyzed. This was not possible in previous work as this protein is very large and part of a gene family subject to mutually exclusive expression, leading to parasite cultures consisting of mix of parasites expressing a different PfEMP1 molecule. These problems were here solved for the first time by generating a SLI activated PfEMP1 that was tagged with a small HA epitope and expressed from its own genomic locus. All parasites expressed this PfEMP1, which was conveniently detectable using an anti-HA serum. These cells were used to assess the trafficking of PfEMP1 through the pathway other exported proteins are known to take through PTEX. For this it was assessed if blocking PTEX would inhibit PfEMP1 transport. The data provided in this thesis indicates that PfEMP1 may not depend on PTEX, congruent with previously published ideas. The PfEMP1 SLI line could also serve as tool to study PfEMP1 transport. Therefore, a new version of SLI was developed that can be used in parasites that already have a SLI genome modification. This system was termed SLI2 and permitted to disrupt genes in the PfEMP1 SLI line. Unexpectedly, this revealed that PfEMP1 transport to the Maurer´s clefts (MCs) was not disturbed after disruption of PTP1, contradicting previous findings. While these findings do not with certainty reveal the exact pathway PfEMP1 takes to reach its final destination, it provides critical tools for the field to (i) identify factors in PfEMP1 transport, (ii) to monitor expression and trafficking of this protein and (iii) crucially, to test the binding properties of individual PfEMP1 molecules that can be activated using SLI as done for the first time in this thesis.
Malaria is one of the world’s leading causes of death due to infectious diseases. When Plasmodium infects a red blood cell, it exports many proteins beyond its own cellular boundaries. This leads to extensive remodelling of the host cell, a process that ensures parasite survival and is a direct mediator of parasite virulence. The transmembrane domain (TMD) protein PfEMP1 is important for virulence... Malaria is one of the world’s leading causes of death due to infectious diseases. When Plasmodium infects a red blood cell, it exports many proteins beyond its own cellular boundaries. This leads to extensive remodelling of the host cell, a process that ensures parasite survival and is a direct mediator of parasite virulence. The transmembrane domain (TMD) protein PfEMP1 is important for virulence. PfEMP1 mediates the cytoadherence of infected red blood cells to the endothelium of blood vessels. While the transport step leading to the export of proteins across the PVM into the host cell is well characterized and appears to involve the Plasmodium translocon of exported proteins (PTEX) for all so far analyzed proteins, the transport of PfEMP1 is much less clear and in general the process of extraction of exported TMD proteins out of the parasite plasma membrane (PPM) to be further transported into the host cell, remains elusive.
The aim of this thesis was to identify translocation factors involved in the extraction of exported TMD proteins out of the PPM, enabling the delivery to PTEX to continue their way to the erythrocyte cytosol. Inactivation of the protein translocation step could prevent the export of PfEMP1, and thus abolish the parasite's virulence and adhesion properties, leading to a more efficient clearance of it by the host immune system. This export step was addressed by two approaches in this thesis, firstly by a general quest for proteins that aid the PPM extraction step and secondly, by a direct analysis of PfEMP1 trafficking.
For the first aim, two constructs containing an mDHFR-BirA* sequence were designed. One fused with SBP1 as a control arrested at the PVM and the other with REX2 that is arrested at the PPM. This aimed to biotinylate proteins that contribute to the extraction of proteins out of the PPM. By arresting constructs on both membranes, it was hoped that the transport factors at each membrane would be tagged to be able to distinguish the proteins specifically involved in the extraction at the PPM. By this approach a total of 2000 different proteins were identified. The fact that the SBP1-mDHFR-L-BirA*-3xHA detected mostly PTEX components confirmed that this construct was arrested deeply in the translocon in the PVM, whereas REX2-mDHFR-L-BirA*-3xHA detected a much wider range of proteins, suggesting an arrest of at least part of the population of this molecule in the PPM. Ten promising proteins were cloned, revealing the location and function of previously unknown proteins in P. falciparum. As several of these proteins were located in the parasite periphery, this suggested that the approach successful. However, a detailed characterization of the other analyzed hits revealed that none of the ten hits aided the extraction of TMD proteins out of the PPM towards the PV. This indicates that either the mechanism of removal out of the PPM depends entirely on PTEX which was detected and could provide force for the PPM extraction from the PVM and interacts with the protein only on the outer face of the PPM, or the proteins contributing to extraction were not available for BirA* mediated biotinylation because they were not in reach, for instance if buried deeply in the PPM.
In second part of this work PfEMP1 was directly analyzed. This was not possible in previous work as this protein is very large and part of a gene family subject to mutually exclusive expression, leading to parasite cultures consisting of mix of parasites expressing a different PfEMP1 molecule. These problems were here solved for the first time by generating a SLI activated PfEMP1 that was tagged with a small HA epitope and expressed from its own genomic locus. All parasites expressed this PfEMP1, which was conveniently detectable using an anti-HA serum. These cells were used to assess the trafficking of PfEMP1 through the pathway other exported proteins are known to take through PTEX. For this it was assessed if blocking PTEX would inhibit PfEMP1 transport. The data provided in this thesis indicates that PfEMP1 may not depend on PTEX, congruent with previously published ideas. The PfEMP1 SLI line could also serve as tool to study PfEMP1 transport. Therefore, a new version of SLI was developed that can be used in parasites that already have a SLI genome modification. This system was termed SLI2 and permitted to disrupt genes in the PfEMP1 SLI line. Unexpectedly, this revealed that PfEMP1 transport to the Maurer´s clefts (MCs) was not disturbed after disruption of PTP1, contradicting previous findings. While these findings do not with certainty reveal the exact pathway PfEMP1 takes to reach its final destination, it provides critical tools for the field to (i) identify factors in PfEMP1 transport, (ii) to monitor expression and trafficking of this protein and (iii) crucially, to test the binding properties of individual PfEMP1 molecules that can be activated using SLI as done for the first time in this thesis. |
