Exiting the Erythrocyte: Functional and Temporal Analysis of a Malarial Subtilase Natalie Clare Silmon de Monerri Division of Parasitology MRC National Institute for Medical Research London, NW7 1AA A thesis submitted in part fulfilment of the requirements of University College London for the degree of Doctor of Philosophy September 2007-September 2010 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. 2 I, Natalie Clare Silmon de Monerri, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 3 Preface I duly acknowledge the contribution of individuals at The National Institute for Medical Research (NIMR) and elsewhere as stated who assisted in work presented in this thesis: Mike Blackman for performing the Western blots on PfSUB1-treated schizont proteins using anti-MSP1 and anti-SERA5 antibodies shown in Figure 37, for RP- HPLC fractionation of PfSUB1-treated schizont proteins and preparation of gel slices for LC/MS/MS analysis, help with computational analysis of LC/MS/MS data. Fiona Hackett for production of recombinant PfSUB1 in baculovirus-infected Sf9 cells used for studies in results chapters 1, 2 and 3. Marta G. Campos for digestion of schizont proteins with recombinant PfSUB1 shown in Figure 37. Steve Howell for performing ESI/MS on PfSUB1-treated RAP1 and MSRP2 peptides discussed in results chapter 3. Helen R. Flynn and Mark Skehel (Protein Analysis and Proteomics Laboratory, Clare Hall Laboratories, Cancer Research UK London Research Institute, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, United Kingdom) for the trypsinisation and LC/MS/MS analysis of PfSUB1-treated schizont protein samples in results chapter 3. 4 Acknowledgements First and foremost, enormous thanks to my supervisor, Mike Blackman, who has been a brilliant mentor throughout the PhD. He is an inspirational scientist: he follows through on ideas and leaves no stone unturned. He encouraged me to do better science, keep asking questions and come up with ideas and theories. Many, many thanks to Mike, Sharon, Chris, Fiona and Mike S., who helped make sure the thesis didn’t (all) sound like it was written by Yoda for the Daily Mirror. It has been a pleasure to be a part of this lab. I have received so much advice and support, had many useful and interesting discussions, and made valuable friendships. Thanks to everyone for making it such a great environment to be in, full of camaraderie. I would especially like to thank Chris and Fiona, without whom I think the lab would fall apart, and who taught me how to culture and transfect parasites. Chris, who made the hours we spent in tissue culture seem shorter and I’m indebted to her for the advice and encouragement throughout the PhD, especially towards the end; Fiona, for her patience and diplomatic (particularly with students) and I’m grateful for her kindness, support and keeping the lab ticking along with her amazing organisation! I’d like to thank Chrislaine and Sharon for their guidance and ideas all along the way. Thanks to Andrea for all her support, in so many ways in and out of the lab. To the boys Matt and Mike S., thank you for not tiring of my many questions and for (almost) always being in a good mood. Thanks also to Robert, Caty and Malcolm, and I can’t forget our old lab members Anna and Kostas who got me started on the right track. I am grateful to Dr. Heinrich Bruno for his straight-faced wit and good humour. Thanks to all members of our parasitology volleyball team, Fever, the (one) win, the losses, and the laughs were a good distraction from endless westerns and minipreps. A special thanks to Oniz, Claire and Rob in the Holder lab for discussions and good times (arr). Thanks also to Ellen for useful discussions, frank advice (and plasmids). I am very grateful to our collaborators at Cancer Research UK, Mark Shekel and Helen Flynn, and to all those whose kindly gave me reagents for this work. Thanks to my university friends Lin, Tasha, Gemma, who are all PhD students, we’ve shared so many tears and joy over the last few years! I would also like to thank my parents, my sister Simone, for your neverending support and trying to understand. Finally and most of all, thank you to my Andy, who was so patient, never complained and cheered me on through the lows, the middles, then celebrated with me at the highs. It would have been impossible to have done it all without him. 5 Table of contents Abstract .............................................................................................................................2 Preface ..............................................................................................................................4 Acknowledgements ...........................................................................................................5 Table of contents...............................................................................................................6 List of figures and tables .................................................................................................10 List of Abbreviations ........................................................................................................13 1. Introduction ..........................................................................................17 1.1. Malaria: a devastating worldwide burden ..............................................17 1.1.1. Malaria throughout history ................................................................17 1.1.2. The complexities of the Apicomplexa .................................................18 1.1.3. Introducing the Plasmodium genus....................................................21 1.1.4. Clinical symptoms and pathology ......................................................22 1.1.5. Geographic prevalence and epidemiological studies ...........................22 1.1.6. The socioeconomic burden of malaria................................................23 1.2. Malaria combat and control strategies...................................................24 1.2.1. Vector control ..................................................................................24 1.2.2. Vaccine stumbling blocks..................................................................25 1.2.3. Antimalarial drugs and resistance......................................................28 1.3. The life cycle of the malaria parasite .....................................................30 1.4. Malariology and genetic manipulation in the post-genomic era .............32 1.5. Transfection of asexual P. falciparum parasites ....................................32 1.6. Conditional knockdown and inducible systems in P. falciparum ...........34 1.7. The asexual erythrocytic cycle ..............................................................37 1.7.1. Invasion of erythrocytes by merozoites ..............................................37 1.7.2. Development after invasion: trophozoite stages ..................................41 1.7.3. Schizogony: formation of 16-32 merozoites ........................................43 1.8. Proteases have diverse functions .........................................................48