Anderson L thesis 07

267 Pages·2007·7.32 MB·English

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Abstract
The invasive and transmission stages of the malaria parasite Plasmodium
falciparum express several proteins with domains implicated in host-parasite
interactions, that are potential vaccine candidates or drug targets. The expression
patterns of two proteins PfTRAMP (Plasmodium Related Apical Merozoite Protein)
and PCRAGS (Plasmodium cysteine related antigen of gametocytes and schizonts),
containing such putative domains, are examined and their potential roles in
merozoite invasion and egress are discussed. PfTRAMP has a possible role in
merozoite invasion. Transcription occurs in mature schizonts as shown by Northern
blot. Recombinant protein was successfully expressed in insect cells indicating that
this eukaryotic system can be utilised for the expression of Plasmodium proteins.
PCRAGS is a member of the Plasmodium Cysteine Repeat Modular Proteins
(PCRMPs), a family of high molecular weight proteins with highly conserved,
cysteine-rich regions and multipass transmembrane domains. The gene encodes a
signal sequence, a truncated extracellular domain, an EGF-like domain and a
multipass-transmembrane domain. PCRAGS is highly conserved in Plasmodium spp
and other Apicomplexa. The extracellular domain has been under purifying selection,
suggesting that the sequence or the structure of this domain is important for function.
The gene is transcribed throughout the asexual erythrocytic cycle and is expressed in
both gametocytes and schizonts in P. falciparum and in the rodent malaria P.berghei.
Antibodies raised against a short peptide in the C-terminus detect PfCRAGS during

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schizont rupture and in mature segmenting schizonts but not in merozoites. Western
blotting showed that PfCRAGS is present in the membrane fraction. Co-localisation
studies showed that PfCRAGS is associated with the infected erythrocyte membrane,
suggesting a role in merozoite egress. PfCRAGS is also expressed in stage II-IV
male gametocytes in association with a membrane and is the earliest known male
specific protein expressed. Gene knock-out of pbcrags in P. berghei showed that
PbCRAGS is not essential for asexual development. In vivo evaluation of phenotype
showed that pbcrags knock-out parasites are less virulent than wildtype parasites and
have an increased gametocyte production in a non-susceptible host. The unique
expression and localisation pattern of PfCRAGS in combination with putative host-
parasite binding domains implicate this novel protein as a potential vaccine candidate
or drug target.
Declaration
I, Laura Fay Anderson, declare that the content of this thesis is all my own work
apart from the following:
RNA extracted for the PfTRAMP Northern blot was carried out by Sally Moore.
FIGE blots and extraction and transfer of P.berghei RNA for Northern blot was
carried out by Jai Ramesar at the LUMC.
All P.berghei transfections were performed by Dr Chris Janse at the LUMC.

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Acknowledgements
I would like to thank the following people for their help and advice
throughout my PhD in various parts of the project: From the University of
Edinburgh, Dr Mark Blaxter, Dr Tom Little and Dr Darren Obbard for helping me to
learn to use the computer programmes for phylogenetic and evolutionary analysis. Dr
Sarah Reece, Derek Sim, Brian Chan, Henry McSorley and Ronnie Mooney for
technical assistance in the in vivo studies. Les Steven and Ronnie Mooney for help
with the maintenance of mosquitos. Andrew Wargo and Dr Andy Bell for assistance
with qPCR and probe design. Grainne Long and Andrew Wargo for advice on
statistical analysis. Henry Mcsorley for advice with maintenance of insect cells and
Karen Gilmore for her assistance in protein purification. Dr Jean-Phillipe Semblat for
his help with His-tag Westerns and for driving me to work everyday for 1 1/2 years!
Josefin Fernius for her advice on TAP-tag systems and for lending me her antibody.
Dr Richard Carter for advice on gametocyte production and gametocyte antigen
detection. Thank you to Alex Hall, Helen Kyriacou and Henry McSorley for proof
reading.
From the LUMC group I would like to thank Professor Andrew Waters for
giving me the opportunity to work in the Netherlands, which I thoroughly enjoyed,
Dr Chris Janse for performing the transfections, Jai Ramesar for the initial selection
of P.berghei knock-outs and Dr Kevin Augustijn for his involvement in generating
GFP and TAP-tag parasite lines and teaching me relevant protein expression
techniques.
From my laboratory I would like to thank Dr Joanne Thompson for her
supervision and giving me the opportunity to carry out such a varied PhD which
allowed me to develop a wide range of skills and hopefully has turned me into a
competent scientist. I would especially like to thank Sally Moore for her technical
assistance throughout the 3 years, in particular with in vivo work and experiments
involving radioactivity, both of which caused many sleepless nights! She also helped
me to drastically improve my organisational and time-keeping skills and provided
emotional support and daily encouragement. She is a great friend and an excellent
researcher to work with. I would also like to give a special thanks to Helen
McElhenney and Dr Balaz Szoor who put up with all my cursing during the write up

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and made me laugh everyday and also to Alex Hall for helping me to stay motivated,
focused and optimistic and for making my writing up time in Edinburgh fun. Thank
you also to the MRC for funding this project. Finally I would like to thank my sister
Melanie Anderson for giving me continuous encouragement, support, advice and
always believing I would finish.
This thesis is dedicated to my parents Francis and Alison Anderson who
never doubted my ability and taught me that with dedication, hard work, self-
confidence and determination any goal can be met and that I should always be proud
of my achievements.

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Table of contents
Chapter 1 1
Introduction 2
1.1 Malaria and the Plasmodium falciparum life cycle 2
1.2 Commitment to gametocytogenesis and sex determination 4
1.3 Proteins of gametocytes and transmission blocking vaccines 9
1.4 Mechanisms of merozoites egress 13
1.5 Host cell invasion and the Apical complex 16
1.6 Interesting binding motifs important in host-parasite interactions 22
1.6.1 Thrombospondin structural homology repeat domain (TSR) domain 22
1.6.2 Epidermal growth factor (EGF)-like domain 23
1.6.3 The Cysteine Repeat Modular domain 23
1.7 Invasion proteins of merozoites 24
1.8 Invasion proteins of sporozoites 28
1.9 Plasmodium Thrombospondin-Related Apical Merozoite Protein (PTRAMP): A
novel protein of merozoites. 30
1.10 Plasmodium Cysteine Repeat Modular Proteins (PCRMPs) 31
1.11 The current situation of malaria vaccine development 32
Pre-erythrocytic stage vaccines 33
Erythrocytic stage vaccines 34
Transmission blocking vaccines 35
1.12 Aims 35
Chapter 2 37

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Bioinformatics of Plasmodium Cysteine Related Antigen of Gametocytes and
Schizonts (PCRAGS): the fifth member of the PCRMP family that has putative
features implicating it in host-parasite interactions. 37
2.1 Introduction 38
2.2 Results 39
2.2.1 Identification of Plasmodium Cysteine Related Antigen of Gametocytes
and Schizonts (PCRAGS). 39
2.2.2 Conformation of the Intron/Exon boundaries of pfcrags and pbcrags 41
2.2.3 Putative features of PCRAGS 45
The CRM domain 46
The EGF-like domain 48
The Transmembrane domain 49
2.2.3 A comparison of PfCRAGS between laboratory and field isolates. 50
2.3 Discussion 50
Chapter 3 57
Phylogenetics and the Molecular Evolution of PCRAGS 57
3.1 Introduction 58
3.2 Results 62
3.2.1 Phylogenetic relationships of PCRAGS between Plasmodium species
and C.parvum. 58
3.2.2 PCRAGS is under a purifying selection between seven Plasmodium
species. 65
3.3 Discussion 71
Chapter 4 74
Transcription, expression and localisation patterns of PfCRAGS 74
4.1 Introduction 75

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4.2 Results 76
4.2.1 PfTRAMP is expressed as a single 2.3 kb transcript in mature schizont
76
4.2.2 PfCRAGS is transcribed at low levels throughout the asexual life-cycle
but is fully spliced in schizonts. 77
4.2.3 Pbcrags is transcribed in asexual and gametocyte stages. 80
4.2.4 PfCRAGS is expressed in schizonts and gametocytes with a molecular
weight greater than 250 kDaltons. 83
4.2.5 PfCRAGS is expressed in very mature schizonts immediately prior to
and during rupture. 85
4.2.6 PfCRAGS is expressed in stage II-V gametocytes 101
4.2.7 PfCRAGS is a male specific gametocyte marker 104
4.3 Discussion
4.3.1 Transcription and Expression of PCRAGS 106
4.3.2 Expression and localisation pattern of PfCRAGS in schizonts. 111
4.2.3 PfCRAGS expression and localisation in gametocytes. 114
Chapter 5 117
Evaluation of pbcrags gene knock-outs in the mosquito and the rodent host 117
5.1 Part one: Generation of pcrags knock-outs in Plasmodium berghei and
preliminary evaluation of phentoype. 118
Introduction 118
5.2 Results 119
5.2.1 Generation of pbcrags knock-out parasite 119
5.2.2 Evaluation of knock-out phenotype in blood and mosquito stages 122
5.3 Part two: In vivo evaluation of pbcrags knock-out 125
Introduction 125

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5.4 Results 127
5.4.1 Infection parameters and counting methods 127
5.4.2 Experiment 1: Infection parameters of pbcrags KO parasites in BALB/c
mice during blood stage development. 131
Experiment 1 summary 137
5.4.3 Experiment 2: Infection parameters of pbcrags KO parasites in a non-
susceptible and susceptible host in blood stage development. 138
Experiment 2 Summary 145
5.4.4 Experiment 3: Infection parameters of pbcrags wild type and pbcrags
knock-outs in a susceptible and non-susceptible host following transmission
from the mosquito. 145
Experiment 3 Summary 153
5.4.4 Experiment 4: Infection parameters of pbcrags KO in a non susceptible
and susceptible host following passage through the mosquito examined by
microscopy and qPCR. 153
5.5 Discussion 162
Differences in virulence 162
Significant phenotypes and trends 163
Reduced parasitaemia 164
Gametocytes 165
Limitations of the P. berghei in vivo system 166
Chapter 6 168
Green Fluorescent Protein (GFP) and Tandem Affinity Purification (TAP) tagging of
pbcrags: Methods of detection throughout the various life cycle stages. 168
6.1 Introduction 169
6.1.1 Green Fluorescent Protein Tagging 169
6.1.2 Tandem Affinity Purification tagging 170

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6.2 Results 171
6.2.1 Generation of GFP and TAP pbcrags fusion constructs 171
6.2.2 Generation of PbCRAGS-GFP tagged parasites. 174
6.2.3. Generation of PbCRAGS-TAP-tag parasites 179
6.3 Discussion 179
Chapter 7 182
Expression of Plasmodium falciparum thrombospondin-related apical merozoite
protein (PfTRAMP) as recombinant protein in a eukaryotic expression system 182
7.1 Introduction 183
7.1.1 Expression of Recombinant Protein 183
7.2 Results 186
7.2.1 Generation of PfTRAMP and PfCRAGS contructs for expression of
recombinant protein in High Five Insect cells 186
7.2.2 PfTRAMP was transiently expressed in High Five insect cells 188
7.2.3 PfTRAMP is not secreted from High Five cells but is constitutively
expressed in stable cells lines. 190
7.2.4 PfTRAMP heterologous protein can be solubilised and partially purified
from Insect Cells using the 6xHis-tag. 192
7.2.5 PfCRAGS could not be successfully detected in High Five Cells 196
7.3 Discussion 196
Chapter 8 200
Final Discussion 200
Chapter 9 206
Materials and Methods 206
References 237

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