Actomyosin motors and malaria parasite invasion of the host cell

In the blood stream the merozoite is the invasive form of the malaria parasite that must enter a new erythrocyte. Merozoites lack structure for locomotion such as cilia and flagella, but use an actomyosin motor complex that provides the force to drive them into the cell. This molecular motor is located at the pellicle membrane of the parasite. The motor protein complex or glideosome includes a class XIV myosin heavy chain (MyoA), Myosin Tail Interacting Protein (MTIP) and two anchoring proteins known as Glideosome Associated Protein 45 (GAP45) and GAP50. A ternary complex of MyoA-MTIP-GAP45 is first formed and this later associates with GAP50. Using a GFP-tagged MyoA transgenic parasite line the expression of MyoA was examined in both Plasmodium falciparum and P. knowlesi. The protein was expressed at the periphery of the parasite and associates with MTIP, GAP45 and GAP50. In time course studies it was shown that recruitment of GAP50 to the complex occurs late relative to the onset of GAP50 synthesis, likely reflecting a role for GAP50 in cellular processes prior to host cell invasion. A second class XIV myosin, Myosin B (MyoB) was also tagged with GFP in a transgenic parasite line. Whilst the timing of MyoB expression followed that of MyoA, with clearly detectable protein in segmenting schizonts at 40 hours post invasion, MyoB did not associates with MyoA or any of the other invasion complex proteins. The localisation of MyoB-GFP in P. falciparum was at the apical end of the merozoite, in front of other apical organelle markers such as Rhoptry neck protein 4 (RON4). During erythrocyte invasion, MyoB -GFP remained in the same location, whilst RON4 moved out onto the parasite surface as part of the moving junction and migrated to the rear of the parasite. Although the exact role of MyoB is unknown, it is suggested that it may be involved in the invasion process but within its own protein complex that is distinct from the glideosome

Exiting the erythrocyte: functional and temporal analysis of a malarial subtilase

Plasmodium falciparum is an obligate intracellular parasite, which causes 95% of worldwide malaria cases annually. Malarial symptoms occur during replication of parasites inside erythrocytes. Multiple cycles of host cell invasion, replication inside a parasitophorous vacuole (PV) and escape from the host cell result in gradually increasing parasitaemia. Escape from the host cell (egress) is regulated by proteases and may involve perforin-like proteins. PfSUB1, a subtilisin-like serine protease, is essential to P. falciparum blood stage development and egress. Just before cell rupture, the protease is discharged into the PV, where it is processes multiple parasite surface proteins and PV proteins. The main aim of this project was to analyse the function of PfSUB1 by three approaches which relied on in vitro biochemical analyses and P. falciparum transfections. Firstly, a conditional knockdown approach was used to analyse the function of PfSUB1 using the FKBP regulatable system. Two complementary strategies were used: down-regulation of PfSUB1 levels using a C-terminal FKBP domain and inhibition of PfSUB1 activity using an N-terminal FKBP fusion with the PfSUB1 prodomain (a potent inhibitor of recombinant PfSUB1). Expression of recombinant PfSUB1-FKBP in Sf9 insect cells demonstrated that FKBP does not interfere with PfSUB1 activity, FKBP was successfully integrated into the endogenous pfsub1 gene. In the second approach, in vitro studies showed that recombinant E. coli-derived FKBP-prodomain fusion protein inhibits recombinant PfSUB1. Strong evidence was obtained which indicates that episomal expression of a non-regulatable prodomain in P. falciparum is not tolerated by the parasite. Secondly, to further characterise the enzyme, an in silico approach was used to predict new SUB1 substrates, and a proteomic approach was taken to validate substrates in vitro. Several putative new substrates were identified, which suggest that PfSUB1 is a multifunctional enzyme with numerous roles in invasion and egress. Finally, attempts were made to establish a PfSUB1-sensitive FRET-based system to monitor PfSUB1 activity in vivo. A recombinant FRET reporter was expressed in E. coli; this was shown to exhibit FRET and to be PfSUB1-sensitive in vitro. Preliminary in vivo data are presented, which suggest that protease-sensitive FRET is possible in P. falciparum