Export determinants and pathways in protein trafficking in the human malaria parasite Plasmodium falciparum
Final Report Abstract
Blood stages of the human malaria parasite P. falciparum cause the symptoms of the severest form of malaria that kills nearly half a million people every year. The parasite grows within red blood cells (RBCs), where it multiplies in a 2 day cycle to produce daughter cells. These are released under destruction of the host cell to invade fresh RBCs. RBCs are highly specialised, containing almost exclusively hemoglobin and no cellular organelles or protein trafficking system. In order to survive in this environment, the parasite induces profound changes to the host cell. This is achieved through the export of a large number of parasite proteins into the host cell and for instance leads to changed nutrient permeability of the RBC or the display of virulence factors on the RBC surface (essential for parasite survival in the human host and a major cause of malaria associated death due to the resulting cerebral malaria). It is therefore important to understand how the parasite exports proteins into the host RBC, which proteins are exported and what the function of these proteins are. These questions were addressed in this project. Firstly a common export domain shared by different classes of exported proteins was analysed for the amino acid sequences needed for export. This revealed that there is a complex interplay of multiple regions that are however usually confined to the very N-terminus (the 'front' part) of the protein. Interestingly we also identified a protein which had an additional export domain that surprisingly is functional in an internal position. This region turned out to be a protein-protein interaction domain and was needed for attachment of this protein to parasite-induced structures in the host cell termed Maurer's clefts. Maurer's clefts are believed to be sorting stations for the transport of virulence proteins to the RBC surface. We used this attachment domain to identify its interactors at the Maurer's clefts and identified a Maurer's clefts complex found in late stage parasites. Deletion of some of the proteins of this complex led to a fragmentation and deterioration of the Maurer's clefts. Hence, we have here identified a complex needed for the maintenance of Maurer's clefts in the host cell. In addition we identified virulence specific trafficking proteins in mouse model malaria parasites. In a second part of the project we studied how the different classes of exported proteins are transported into the host cell. Within the RBC the parasite is contained in a compartment termed the parasitophorous vacuolar membrane (PVM) that separates the parasite from its host cell. For export, proteins need to pass through this membrane. Soluble proteins of the most common class of exported proteins were suspected to go through a translocation machine at the PVM termed PTEX. Vesicular trafficking would release these proteins into the vacuolar space around the parasite where they can become substrates of PTEX at the inner face of the PVM. However, integral membrane proteins are delivered embedded in the parasite plasma membrane (PPM). It was unclear if and how these proteins could become substrates of PTEX. It was also unclear if all classes of exported proteins pass through PTEX. We generated protein constructs that can be conditionally arrested in the transport step across the PVM that for the first time remained stuck in the translocon, thereby blocking passage for all other exported proteins. Using this system, we demonstrated that all classes of exported pass through PTEX and that - depending on the size of their C-terminal domain - transmembrane proteins either are handed over directly from the PPM to the PVM translocon are first released into the vacuolar space between parasite and PVM. This is reminiscent of protein translocation into organelles such as the mitochondria where matrix proteins can also be handed over from the outer to the inner membrane, although in that case the proteins do not arrive membrane embedded. We also demonstrated that proteins arrested in the translocation step are in contact with the suspected translocation pore of PTEX, giving functional evidence for the role of this protein. Finally this work also provided evidence for the (now confirmed) suspicion that protein export is essential for parasite survival. Together with other publications that appeared in recent years this work established that PTEX is essential for the export of all types of exported proteins and provides a snapshot of the mechanism of transmembrane protein export.
Publications
- (2015) Critical Steps in Protein Export of Plasmodium falciparum Blood Stages. Trends Parasitol., 31(10):514-25
Spielmann T, Gilberger TW
(See online at https://doi.org/10.1016/j.pt.2015.06.010) - (2016) Stable translocation intermediates jam global protein export in Plasmodium falciparum parasites and link the PTEX component EXP2 with translocation activity. PLOS Pathog 12: e1005618.5
Mesén-Ramírez P, Reinsch F, Blancke Soares A, Bergmann B, Ullrich A-K, Tenzer S, and Spielmann T
(See online at https://doi.org/10.1371/journal.ppat.1005618) - (2016) The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites. Nat Commun 7: 11659
De Niz M, Ullrich A-K, Heiber A, Blancke Soares A, Pick C, Lyck R, Keller D, Kaiser G, Prado M, Flemming S, del Portillo H, Janse CJ, Heussler V, Spielmann T
(See online at https://doi.org/10.1038/ncomms11659)
