Silmon de Monerri, N.C.; (2010) Exiting the erythrocyte: functional and temporal analysis of a malarial subtilase. Doctoral thesis , UCL (University College London).

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Abstract

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.

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