Plasmodium Berghei
SEXUAL AND SPOROGONIC DEVELOPMENT OF PLASMODIUM BERGHEI
Anne Louise Dearsly
A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy of the University of London and Diploma of Imperial College
Imperial College Department of Pure and Applied Biology Prince Consort Road London
October 1990 THE SEXUAL AND SPOROGONIC DEVELOPMENT OF PLASMODIUM BERGHEI.
Plasmodium berghei infections were studied in vivo and in vitro and the production of gametocytes and ookinetes following 3, 8 and 14 sequential blood passages monitored. This allowed the definition of optimal conditions for the collection of parasite material for the study of the gametocyte and ookinete stages. The study also led to the identification of reliable indicators of ookinete production.
A method was developed using mitomycin Cto obtain enriched/purified gametocyte populations. Giemsa stained smears of infected mouse blood showed an immediate drop in the asexual parasitaemia while the immediate numbers of gametocytes remained unaffected. Treated gametocytes went on to produce ookinetes in culture that were infective to mosquitoes and readily purified by Nycodenz centrifugation.
Ookinete cultures were treated with metabolic inhibitors of DNA, RNA and protein synthesis and the effect on the morphological development was studied at both the light and electron microscope level. The effect of the inhibitors on protein synthesis was also studied by radiolabelling in the presence of the drugs and the differences between asexual and sexual stage protein synthesis determined.
The synthesis of the 21kD transmission blocking protein was followed from the early asexual trophozoite to the mature ookinete. The time of the initial synthesis and expression on the surface of the parasite was established. Attempts to identify the trigger were made and indicated that synthesis is induced by gametogenesis and not fertilization. Glucosamine/mannose labelling of the protein and preliminary detergent extraction with triton X-114 were carried out to further characterise the protein.
2 Contents Page Acknowledgements ...... 13 Abbreviations ...... 14
Chapter 1 General Introduction 1.1 Malaria and its control ...... 16 1.2 The Parasite ...... 16 1.3 The Plasmodial Life Cycle ...... 16 1.3.1 Gametocyte Density...... 23 1.3.2 Blood Factors...... 23 1.3.3 Host Immune Factors...... 24 1.4 Antimalarial Vaccines ...... 26 1.5 Plasmodium berghei ...... 27 1.6 The Biochemistry of Plasmodium ...... 27 1.7 Overview and Aims ...... 30
Chapter 2 The Sexual Development of Plasmodium berghei in vivo and in vitro 2.1 Introduction ...... 33 2.2 Materials and Methods ...... 36 2.2.1 In vivo Infection ...... 36 2.2.2 Red blood cell count ...... 36 2.2.3 Parasitaemia ...... 36 2.2.4 Exflagellation ...... 37 2.2.5 Culture Medium ...... 37 2.2.5.1 Complete Ookinete Culture Medium ...... 37 2.2.5.2 Methionine-free RPMI 1640 ...... 37 2.2.5.3 Serum-free Ookinete Medium ...... 37 2.2.5.4 Gametocyte Culture Medium ...... 38 2.2.5.5 Serum-free Gametocyte Medium ...... 38 2.2.6 Ookinete Production ...... 38 2.3 Results...... 39 2.3.1 Comparison of Asexual and Mixed Clones of P.berghei ...... 39 2.3.2 Asexual Parasitaemia ...... 39 2.3.3 Red Blood Cell Count ...... 39
3 Page 2.3.4 Sexual Parasitaemia ...... 43 2.3.5 Production of Gametocytes During the Course of an Infection ...... 43 2.3.6 The Effect of Blood Passage on Gametocyte Production 37 and Activity ...... 43 2.3.7 Ookinete Production ...... 53 2.3.8 Sexual Stage Parasites as Indicators of Ookinete Production ...... 56 2.3.9 The Effect of Red Blood Cell Count on Ookinete Formation in vitro ...... 56 2.3.10 The Effect of Asexual Parasites on Ookinete Formation in vitro ...... 58 2.4 Discussion ...... 59 2.4.1 Conclusions ...... 67
Chapter 3 The Enrichment of Plasmodium berghei Gametocytes in vivo and ookinetes in vitro 3.1 Introduction ...... 69 3.2 Materials and Methods ...... 70 3.2.1 Parasites ...... 70 3.2.2 Gametocyte Production ...... 70 3.2.3 The Effect of the Duration of Mitomycin C Treatment ...... 70 3.2.4 Ookinete Culture ...... 71 3.2.5 Ookinete Purification ...... 71 3.2.6 Mosquito Feeds ...... 72 3.2.7 Radiolabelling of the Nucleic Acids ...... 72 3.3 Results ...... 74 3.3.1 Mitomycin C treatment of P.berghei Parasites ...... 74 3.3.1.1 Effect of Time ...... 74 3.3.1.2 Effect of Mitomycin C Concentration ...... 74 3.3.2 Further Purification of Treated Parasites ...... 79 3.3.3 Viability of Mitomycin C Treated Gametocytes ...... 79 3.3.4 Effect of Mitomycin C on the Incorporation of 32p into Nucleic Acids ...... 79 3.4 Discussion ...... 84 3.4.1 Conclusions ...... 87
4 Page Chapter 4 The Effect of Metabolic Inhibitors on the Development of Plasmodium berzhei Gametocvtes and Ookinetes 4.1 Introduction ...... 89 4.2 Materials and Methods ...... 93 4.2.1 Micro Drug Culture Technique ...... 93 4.2.2 Electron Microscopy ...... 95 4.2.3 35S-Methionine Labelling of Ookinete Cultures ...... 96 4.2.4 Analysis of Protein Samples ...... 96 4.2.5 SDS-PAGE ...... 99 4.2.6 Coomassie Blue Staining of Gels ...... 100 4.2.7 Drying and Autoradiography of Gels ...... 100 4.2.8 Densitometry of Autoradiographs ...... 101 4.3 Results ...... 102 4.3.1 Total Protein Synthesis ...... 102 4.3.2 Exflagellation ...... 102 4.3.3 Ookinete Development ...... 112 4.3.3.1 Electron Microscopy of Untreated Control Cultures ...... 112 4.3.3.2 DNA Synthesis Inhibitors ...... 112 4.3.3.2.1 Light Microscopy ...... 112 4.3.3.2.2 Electron microscopy ...... 112 4.3.3.3 RNA Synthesis Inhibitors ...... 113 4.3.3.3.1 Light Microscopy ...... 113 4.3.3.3.2 Electron Microscopy ...... 113 4.3.3.4 Protein Synthesis Inhibitors ...... 122 4.3.3.4.1 Light Microscopy ...... 122 4.3.3.4.2 Electron Microscopy ...... 122 4.3.3.5 Inhibitors of Microtubule Assembly ...... 122 4.3.3.5.1 Light Microscopy ...... 122 4.3.3.5.2 Electron Microscopy ...... 123 4.3.4 The Inhibition of Protein Synthesis ...... 123 4.3.4.1 DNA Synthesis Inhibitors ...... 123 4.3.4.2 RNA Synthesis Inhibitors ...... 125 4.3.4.3 Protein Synthesis Inhibitors ...... 125 4.3.4.4. Inhibitors of Microtubule Assembly ...... 125
5 Page 4.4 Discussion ...... 127 4.4.1 Exflagellation ...... 128 4.4.2 Ookinete Development ...... 129 4.4.3 Comments on Methodology and Future Directions of Analysis ...... 131 4.4.4 Conclusions ...... 133
Chapter 5 Studies on the 21kD. Sexual Stage Transmission Blocking Protein of Plasmodium berghei 5.1 Introduction...... 135 5.2 Materials and Methods...... 137 5.2.1 35$.Methionine Labelling of Asexual and Gametocyte Stages of P. berghei ...... 137 5.2.2 35s_jvlethionine Labelling of Ookinete Cultures ...... 139 5.2.3 Immunoprecipitation ...... 141 5.2.4 W estern Blotting ...... 141 5.2.5 Autoradiography of Western Blots ...... 142 5.2.6 SSs-Methionine Labelling in the Presence of Microtubule Assembly Inhibiting Drugs ...... 142 5.2.7 ImmunoRuorescence Assay (IFA) of Ookinetes Cultured in the Presence and Absence of Tunicamycin ...... 143 5.2.8 Glucosamine/Mannose Labelling of Ookinete Cultures .... 143 5.2.9 Surface Labelling of Ookinete Cultures ...... 144 5.2.10 Triton X-114 Extraction of ^^S-Methionine Labelled Proteins ...... 144 5.3 Results ...... 147 5.3.1 Characterization of the Monoclonal Antibody 13.1 ...... 147 5.3.1.1 The 36/27/33 kD proteins ...... 147 5.3.1.2 The 45kD protein ...... 152 5.3.1.3 The 66kD Protein ...... 152 5.3.2 Synthesis of the 21kD protein - Pbs 21 ...... 155 5.3.3 Trigger of 21kD Protein Synthesis ...... 159 5.3.4 125I Surface Labelling of Ookinete Cultures ...... 159 5.3.5 IFA of Ookinetes Cultured in the Presence and Absence of Tunicamycin ...... 162 5.3.6 Glucosamine/Mannose Labelling of Ookinete Cultures .... 162
6 Page 5.3.7 Triton X-114 Extraction of 35s-methionine Labelled Proteins ...... 162 5.4 Discussion ...... 172 5.4.1 Conclusions ...... 180
Chapter 6 General Discussion 6.1 Background to this Study ...... 183 6.2 Pbs 21, A Transmission Blocking Protein of P. berghei ...... 184 6.3 Strategies for Vaccine Design ...... 188 6.4 Further Studies on Pbs 21 ...... 190 6.5 Treatment of Malaria...... 192
References ...... 195 Appendix I (Data from Chapter 2) ...... 216 Appendix II (Data from Chapter 3)...... 229
7 List of Figures
Figure Page 1.1 Life Cycle of Plasmodium ...... 18 2.1 Graph showing the mean asexual and sexual parasitaemias obtained from groups of three mice observed in Giemsa stained blood films for a P3 mixed (2.34L) and asexual (2.33L) P.berghei infection...... 40 2.2 Graphs showing the number of red blood cells per ml of blood (A) and the ookinete production observed in Giemsa stained blood films (B) during the course of a P3 P. berghei infection ..... 41 2.3 Graph showing the calculated total number of asexual P.berghei parasites per ml of blood throughout the course of a P3 infection...... 42 2.4 Graph showing the calculated number of P.berghei gametocytes per ml of blood throughout the course of a P3 infection...... 44 2.5 Graphs showing the mean conversion rates for three mice of trophozoites to gametocytes throughout the course of P3, P 8 and P14 P.berghei infections...... 45 2.6 Graph showing the mean sexual parasitaemias observed in Giemsa stained blood films throughout the course of P3, P 8 and PI 4 P.berghei infections...... 47 2.7 Graphs differentiating the sexual parasitaemia into the the microgametocytaemia and the macrogametocytaemia observed in Giemsa stained blood films throughout the course of P3, P 8 and PI4 P.berghei infections...... 48 2.8 Histogram summarising the sex ratio (F/ M) observed in Giemsa stained smears from P. berghei infections ...... 49 2.9 Graph showing the mean ookinete production observed in Giemsa stained blood films throughout the course of a P3 P.berghei infection...... 54 2.10 Graph showing the loss of mean ookinete production observed in Giemsa stained blood films by a P.berghei infection with passage...... 55
8 Figure Page 2.11 Graph showing the correlation between the mean exflagellation index and the mean ookinete production for a P3 P.berghei infection...... 57 2.12 Summaiy of known and projected factors influencing the infection of mosquitoes from gametocyte infected vertebrate hosts...... 61 3.1 The total number of P. bergheiparasites per 104 red blood cells in Giemsa stained blood smears for a mouse treated with 15fig mitomycin Cg_1 body weight and an untreated control mouse at different times after treatment ...... 75 3.2 The number of macrogametocytes and microgametocytes per 104 red blood cells in Giemsa stained blood smears for a mouse treated with 15jig mitomycin Cg*l body weight and an untreated control mouse at different times after treatment ...... 76 3.3 The number of exflagellation centres in 20 fields (mag 300x) for a mouse treated with 15|jg mitomycin Cg"l body weight and an untreated control mouse at different times after treatment ...... 77 3.4 Giemsa stained blood films made from material collected from Nycodenz cushions of 10% and 12% after centrifugation at 1600gfor 10 min...... 81 4.1 Flow diagram showing micro drug dulture technique ...... 94 4.2 Giemsa stained blood film made from 24 hour P. berghei ookinete cultures after lysis with ammonium chloride...... 97 4.3 Autoradiograph of an asexual P. berghei infection labelled with 35s-methionine during ookinete culture in the presence of selected metabolic inhibitors...... 108 4.4 Autoradiographs of a mixed P. berghei infection labelled with 35S-methionine during ookinete culture in the presence of selected metabolic inhibitors...... 110 4.5 Densitometer traces of 3^s-methionine labelled ookinete cultures of an asexual infection (A) and a mixed infection (B) ...... Ill 4.6 Electron micrographs of P. berghei ookinete cultures ...... 115 4.7 Electron micrographs of P. berghei in ookinete cultures in the presence of DNA synthesis inhibitors ...... 117 4.8 Electron micrograph of P. berghei in ookinete cultures in the presence of RNA synthesis inhibitors...... 119
9 F igure Page 4.9 Electron micrographs of P. berghei in ookinete cultures in the presence of the metabolic inhibitors cycloheximide and colchicine...... 121 4.10 Densitometer traces of aphidicolin (A) and hydroxyurea (B) treated ookinete cultures ...... 124 4.11 Densitometer traces of 35s-methionine labelled parasites showing the different sensitivities of an asexual (A) and a mixed (B) infection in ookinete culture to cycloheximide ...... 126 5.1 Flow diagram showing method for the synchronization of asexual and gametocyte stages of P berghei and the collection of protein samples ...... 138 5.2 Row diagram showing the method for the culture of P. berghei ookinetes and the collection of protein samples ...... 140 5.3 Row diagram showning triton X-114 extraction of proteins ...... 145 5.4 Western blots of material collected from synchronised gametocyte infections and ookinete culture after varying time durations blotted against monoclonal antibody 13.1 ...... 149 5.5 Western blots on material taken at different times during synchronised gametocyte production and ookinete culture of P.bergheiin which the monoclonal antibody has been replaced by normal mouse serum (A) and omitted (B) ...... 151 5.6 Autoradiograph of 35s-methionine labelled, material immunoprecipitated with monoclonal antibody 13.1 and collected at different times during synchronised, in vivo gametocyte and ookinete culture of P. berghei...... 154 5.7 Autoradiograph of total protein samples labelled with 35s_ methionine during early ookinete culture ...... 158 5.8 Autoradiograph of SDS-PAGE gel of total 35s radiolabelled protein from ookinete cultures treated with anti microtubule agents prior to the start of ookinete culture...... 161 5.9 Ruoresence micrograph showing the typical reaction of monoclonal antibody 13.1 with a retort form P. berghei ookinete in an indirect immunofluoresence assay...... 164 5.10 Autoradiograph of the immunoprecipitated 21kD protein radiolabelled in the absence and presence of tunicamycin ...... 166
10 Figure Page 5.11 Autoradiograph of total protein material collected at different times during in vitro ookinete formation and surface labelled with 125i using the iodobead technique ...... 168 5.12 Densitometer trace of a 24h ookinete culture surface labelled with 125I using the Iodobead method...... 169 5.13 Autoradiograph of proteins labelled with 35s_methionine during in vitro ookinete formation, extracted with Triton X- 114 and then immunoprecipitated with mAb 13.1 ...... 171
11 List of Tables
Table Page 2.1 Showing the mean number of counts ± standard deviation obtained from three mice infected with a P3 P. berghei infection. The counts were determined on a daily basis from Giemsa stained and live preparations ...... 50 2.2 Showing the mean number of counts ± standard deviation obtained from three mice infected with a P 8 P. berghei infection. The counts were determined on a daily basis from Giemsa stained and live preparations ...... 51 2.3 Showing the mean number of counts ± standard deviation obtained from three mice infected with a PI4 P. berghei infection. The counts were determined on a daily basis from Giemsa stained and live preparations...... 52 3.1 Mean number of parasites per 104 rbc 24h after treatment with different concentrations of mitomycin C ...... 78 3.2 Incorporation of 32P into parasite DNA when treated with mitomycin C and absorbed on DE81 paper and TCA precipitated onto GF/A filters...... 83 4.1 Computer identified proteins from densitometer scans of 35S- methionine labelled proteins from ookinete cultures of mixed and asexual infections...... 103 4.2 The effect of metabolic inhibitors on exflagellation of Plasmodium berghei, ...... 104 4.3 The effect of metabolic inhibitors on ookinete production in Plasmodium berghei, as assessed by the rapid assay method...... 105 4.4 The effect of metabolic inhibitors on ookinete production subsequent to exflagellation in Plasmodium berghei, as assessed by the rapid assay method...... 106 5.1 Comparison of the biosynthesis of transmission blocking proteins of zygotes/ookinetes of P. falciparum, P. gallinaceum and P. berghei...... 173
12 Acknowledgements
I would like to thank the following people for all their help and assistance throughout the course of this project.
My supervisor, Professor R E Sinden for his help and advice throughout this project, and particularly for his help with the electronmicroscopy.
Drs B Mons and C Janse and their colleagues for the invitation to work in their laboratory on the gametocyte synchronization studies and for all the assistance given to me.
Dr L Winger for the supply of mAb 13.1.
Dr O Dolly for the use of his scanning densitometer.
My husband for his help, advice and encouragement, particularly while writing up.
The many people who have given me the benefit of their advice and technical expertise.
The Medical Research Council of Great Britain for financial support.
13 Abbreviations cAMP cyclic adenosine monophosphate BPB Bromophenol blue DMSO Dimethylsulphoxide EDTA NNN'N'-Ethylenediaminetetraacetic acid El Exflagellation index HI-FCS Heat inactivated foetal calf serum HEPES N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid] i.p. Intraperitoneal IFA Immunofluorescence assay LP Lower phase L.S.B Laemmli sample buffer L. S.C Liquid scintillation counting Ma Macrogametocyte mAb Monoclonal antibody Mi Microgametocyte M. wt Molecular weight NMS Normal mouse serum OP Ookinete production P Passage PBS Phosphate buffered saline pH -loglO[H+] PMSF Phenylmethylsulphonylfluoride R Ring rbc Red blood cells S Schizont STE buffer Sodium, tris, EDTA buffer SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis S/P Subpassage TBS Tris buffered saline TCA Trichloroacetic acid TE buffer Tris EDTA buffer T.O. mice Theiler's Original mice Tris Tris (hydroxy methyl) methylamine T Trophozoite UP Upper phase
14 Chapter 1
General Introduction
15 1.1 Malaria and its Control
Malaria is one of the major problems faced by the third world today. The disease is reported to continue in some 100 countries or areas (WHO, 1990) causing an estimated incidence of 200 million cases per annum and an estimated one million child deaths each year (WHO report, 1985). This death and illness places severe restraints on any development programmes planned for the areas affected by the disease. After World War II pilot projects achieved the control of malaria by indoor spraying with DDT and this together with the development of safe, effective and inexpensive antimalarial drugs led to the consideration of global eradication of malaria (Houghton, 1984). Success from these early programmes met severe setbacks in the 1960s due to political, economic and operational problems. In addition mosquito vectors began to show resistance to DDT and is some areas multiple insecticide resistance was also seen. Resistance of P. falciparum to the drugs proguanil and pyrimethamine was also reported at this time. However, chloroquine still remained effective. Great concern followed the first reports of chloroquine resistance in Thailand and Columbia and today resistance is spreading over Eastern Asia, South America, Oceania and sub-Suharan. There now exists the situation in parts of the world where multiple drug resistance has been reported, for example in Thailand the only effective treatment for malaria is mefloquine. As a result of these events attention is now being turned towards vaccine development in order to try to combat the problems caused by malaria (Houghton, 1984).
1.2 The Parasite
The causative agent is a parasitic protozoan of the genus Plasmodium, which not only affects Man but many other mammals, birds and reptiles. Four species infect man: P. falciparum, P. vivax, P. ovale and P.malariae each of which shows it own morphology, biology and clinical characteristics (Miller, 1983). Of the four, P. falciparum causes the most morbidity and mortality and presents the therapeutic problem of drug resistance. The differences in the seriousness of the disease are predominantly due to the populations of red blood cells that each invades. P. falciparum infects red cells of all ages. The resultant high parasitaemia accounts for the high morbidity and mortality of falciparum malaria. P. vivax and P. ovale invade primarily reticulocytes; P.
16 malariae is limited to older red cells. Thus because these three malarias are limited to a subpopulation of red cells, they rarely reach a high parasitaemia and usually do not cause serious disease or death (Miller, 1983). Malaria is usually spread from one host to the next by the female mosquito which acts as a vector upon feeding on an infected host and there is a strict relationship between the mosquito species the parasite. One exception to the mosquito transmission is in the case of the lizard malarial infection which is transmitted by both midge and sandfly vectors (Kreier & Barker, 1987). The degree to which each of the different species of malarial parasite is related one to another is unknown (Weber, 1988). However, the transmission and growth of the malarial parasite follows a common path which is described below, although variations do occur between the different species and these included the red blood cell populations each species infected (see above); the time taken for gamete development eg, 10-12 days in P. falciparum as compared to 28 hours in P. berghei and 46 hours in P. gallinaceum (Garnham, 1966); the nature of the infection, whether it is asynchronous as is seen with species like P. berghei or synchronous as in the case of P. chaubaudi and whether or not the infection is cyclical, recurring at regular intervals as with P. chaubaudi.
1.3 The Plasmodia! Life Cycle
The mammalian malarial life cycle, illustrated in fig 1.1, starts when the haploid sporozoite stage 1() is injected into the mammalian host during feeding by an infected anopheline mosquito. The sporozoites then circulate in the blood until they reach the liver. Some then make their way into the hepatocytes, to differentiate into exo-erythrocytic or liver stages (2), and undergo a period of schizogony and maturation to form the liver schizont (3). At maturation the schizont ruptures (4) to release between 10,000 and 50,000 merozoites into the blood stream. It is now imperative that the merozoites rapidly invade red blood cells if further development is to occur (5). Once this has been achieved the merozoite transforms into the erythrocytic ring form (6), which grows to form the trophozoite (7). At some point during the growth phase the trophozoite undergoes a selective differentiation that determines whether future development will follow an asexual path or sexual.
17 which penetrate salivary gland
FIG 1.1 Life Cycle of Plasmodium. Adapted from Vickerman & Cox, 1972. The result of asexual development is a mature schizont ( 8), which forms after a series of mitotic divisions known as schizogony. The mature schizont contains between 8 and 32 daughter merozoites that are released into the blood stream when the host erythrocyte bursts and the cell debris that is released at this time is the cause of the recurring bouts of fever seen in the host (Garnham, 1966). This is followed by further rounds of merozoite invasion and erythrocytic development.
Those trophozoites that become committed to the sexual path undergo three distinct phases of development: the first of these is gametocyte formation which takes place in the vertebrate host. Following the ingestion of these stages by the mosquito the second, gametogenesis and third, zygote formation can take place. Each of these three phases may be considered as being distinct from one another and yet very closely related (Sinden, 1983).
Gametocytogenesis starts with the terminal differentiation of the trophozoite into a gametocyte. Initially sexual dimorphism is not visible (Mons, 1986), but as these stages mature they can be distinguished into macrogametocyte (female) (9) and microgametocyte (male) (10). Once maturation is complete the gametocytes circulate in the blood for variable time periods, depending on the species of Plasmodium . No further development can occur until the gametocytes that are ingested by a feeding mosquito. Those gametocytes that are not taken up into the mosquito midgut become senescent and are removed from the circulation.
Gametogenesis occurs once the gametocytes have been ingested by the mosquito. Fortunately these events can also be triggered in vitro. Initiation of gametogenesis in vitro involves the influence of several factors including the presence of bicarbonate ions (Bishop & McConnachie, 1956; Carter & Nijhout, 1977), pH (Micks et al, 1948; Kawamoto et al, 1990), a drop in temperature (Sinden, 1983; 1983a) and a mosquito exflagellation factor (Nijhout, 1979). These in vitro factors are not necessarily the same as those involved in the in vivo event, however these factors have been likened to the conditions found in the mosquito midgut (Bishop & McConnachie, 1956; 1960). Increased cAMP levels have also been implicated in triggering gametogenesis in P. gallinaceum (Martin et al, 1978). Recently however, Kawamoto et al (1990) have shown that increased mobilization of internal Ca2+ resources is also a prerequisite of exflagellation (the formation and
19 release of 8 microgametes) in P. falciparum and P. berghei as are increased levels of cGMP and suggest that cAMP may not be involved in exflagellation as has been suggested for P. gallinaceum and P.yoelii. . In the same study Kawamoto et al (1990) also find that pH may not be a critical factor, but is simply a convenient way in which gametogenesis can be triggered in vitro.
The formation of both macro- and microgametes involves the escape of the gametocytes from the host erythrocyte mediated by osmiophilic bodies fusing with the parasite plasmalemma. Recently Quakyi et al (1989) have shown that the Pfl55/RESA protein is found associated with the parasitophorous vacuole and later along the erythrocytic membranes. Pfl55/ RES A was first described in P. falciparum merozoites, located in micronemes found at the apical end of the parasite (Perlmannet al, 1984). The function of RES A is thought to be the perturbation of the erythrocytic membrane allowing the merozoite to invade. Quakyi et al (1989) have therefore concluded that this protein serves the same function in the gametocyte stages and by disrupting the membrane allows gametocyte escape. Whether gametogenesis comes about by either the breakdown of the host cell membrane or by the imbibition of fluids and thus swelling of the gametocytes the result is the release of gametes into the bloodmeal.
Macrogamete formation (11) involves few visible changes to the gametocyte, however during this time the macrogametocyte does increase its surface area and an associated increase in the assembly of lipids and proteins is seen (Sinden, 1983; 1983a). By contrast microgamete formation is an extremely dramatic event, with gamete formation and escape from the host cell, an event known as exflagellation ( 12), occurring within approximately 10 min of activation.
Zygote formation is the third and final stage of sexual development. It occurs when the microgamete and macrogamete meet and fuse to form the fertilized zygote. The zygote then undergoes a series of changes in shape to form first the retort form ookinete and then the mature banana-shaped motile ookinete (13). This penetrates the midgut wall to lie under the basal lamina where it rounds up to form an oocyst (14). This matures, undergoing a series of mitotic divisions to form thousands of sporozoites, which are released into the mosquito haemolymph when the oocyst bursts (15). The sporozoites
20 migrate from here to the salivary glands (16), where they wait until they are inoculated into a new host during the next feed, thus completing the cycle.
The functions of the gametocyte stage of the malarial parasite are sexual development, meiotic recombination and the transmission of the parasite from the vertebrate host to the mosquito vector and it is therefore the stage that is responsible for the spread of the disease.
The biological aspects of gametocyte development until recently have remained poorly understood owing to difficulties encountered in their study (Carter & Gwadz, 1980). In the case of P. falciparum the gametocytes, although easily recognised, develop in inaccessible, deep tissues while the asynchronous development of murine malarias, such as P. berghei make early identification of the gametocytes difficult. However, following the development of in vitro cultivation of P. falciparum asexual stages (Trager & Jensen, 1976), the culture of synchronised sexual stages of P. falciparum in vitro (Vermeulen et al, 1986) and the synchronization of in vivo infections of P. berghei (Mons et al, 1985) a great deal of new information on the subject has been gained.
The onset of gametogenesis starts immediately the blood infection begins (Killick-Kendrick & Warren, 1968) with gametocytes arising directly from the exoerythrocytic stages and from the asexual erythrocytic stages. At the ultrastructural level gametocyte development in rodent malarial parasites is first discernible in the young trophozoite (Ladda, 1969; Kilby & Silverman, 1971) and recently Mons (1986) has identified the commitment to sexual development as being complete by this time. The differentiation of the sexual stages is not determined by major differences in karyotype, as is the case in man, and this has been demonstrated by the ability of a single haploid asexual parasite to give rise to a complete infection including micro- and macrogametocytes (Walliker et al, 1973).
The ability of a clone of parasites to produce gametocytes varies greatly depending on the surrounding environment and its history. A parasite clone that has been mechanically passaged will loose its ability to produce gametocytes (see Chapter 2). This loss of gametocyte production has been associated with a decrease in the parasite DNA content (Birago et al, 1982), but could be attributed to the suppression of the genes responsible. This ability
21 however is easily 'restored* by transmitting infections in which low gametocyte production has been induced through mosquitoes (Wery, 1968). This could act by selecting for small number of functional gametocytes (ie those with the full DNA complement) that are present, so that the infection passed on to the vertebrate host consists of only parasites with the full gene complement. Alternatively, it has been reported that treatment with low temperatures (Bafort, 1965) or treatment with chemotherapeutic agents (Peters, 1970) will induce increased gametocyte production by releasing restrictions placed on the genes controlling gametocyte production (Ichimori et al, 1990). However, drug treatment may also have the effect of selectively killing the asexual stages, thereby increasing the proportion of gametocytes by comparison. It has also been shown that the release of restrictions placed on the parasite can be achieved by transferring parasites to uninfected animals (Dearsly et al, 1990) indicating the role of the host in the suppression of gametocyte production. The topic of the parasitein vivo and infectivity will be dealt with more fully in Chapter 2.
The time taken for the development of gametocytes to reach maturity is in general approximately equal to the time taken for asexual development to be achieved, 40 hours (sexual) as compared to 46 hours (asexual) for P. gallinaceum and 28 hours (sexual) against 26 hours (asexual) for P. berghei (Gamham, 1966). The one exception to this is the case of P. falciparum where the development time of gametocytes is 10-12 days compared to the 45-48 hours for asexual development (Gamham, 1966). There is also a difference between the time taken for P. falciparum to reach morphological maturity and functional maturity (Jeffery & Eyles, 1955) but it is unknown whether this difference extends to other species. The differences in the development of falciparum malaria highlight the need for caution when extrapolating data obtained from models using other malarias to falciparum malaria.
In order to achieve their function the mature gametocytes need to be taken up by a mosquito and successfully produce gametes the result of which culminates in an oocyst infection of the midgut and subsequent sporozoite production. The processes involved in this are highly dependent on many factors pertaining to the vertebrate host and the mosquito vector. The stimulus that triggers gametogenesis has attracted much attention in the past and is reviewed by Bishop & McConnachie (1956) but factors implicated include pH, loss of CO2, bicarbonate ions and plasma factors. The relative
22 importance of these factors varies but gametogenesis will only occur at a pH of between 7.7 and 8.0 in the presence of Na+, Cl“ and HC03‘ ions (Carter & Nijhout, 1977). Temperature also appears to be involved, acting as a limiting factor, and must fall in the case of P. y. nigeriensis to below 30°C (Sinden & Croll, 1975) before gametogenesis occurs. It has been proposed that gametogenesis is triggered by the interaction of simple environmental stimuli reacting with the gametocyte surface (Micks et al, 1948).
Gametogenesis is the preliminary to producing an oocyst infection in susceptible mosquitoes and success is a result of the interaction of many factors related to both the host and the parasite and these have been reviewed by Vanderberg et al (1977). The factors involved in infectivity include 1) gametocyte density, 2) blood factors and 3) the immune response of the host (reviewed Carter & Gwadz, 1980).
1.3.1 Gametocyte Density
The gametocyte density in the circulation shows a positive relationship to the oocyst numbers found in the mosquito (Eyles, 1951). There are however cases where low gametocyte counts produce mosquito infections (Jeffery & Eyles, 1955), which has led to the suggestion that not all gametocytes are infective, and for P. falciparum the delay in reaching functional maturity could account for this (Carter & Graves, 1988). It has also been suggested that gametocyte maturation follows a circadian rhythm that coincides with the peak mosquito biting activity (Hawking et al, 1968; 1971; 1972). There is however evidence against this theory (Bray et al, 1976) and it therefore requires further study before it can be accepted, but it must be considered when establishing studies on infectivity and the parasite in vivo.
1.3.2 Blood Factors
Blood factors also play a major part in determining the infectiousness of an infection to a mosquito. The importance of blood factors was first described by Eyles (1951; 1952; 1952a) for P. gallinaceum infections to Aedes aegypti and resulted in the conclusion that serum was an essential component if full development of the parasite was to occur. Subsequently, it has been shown
23 that the 'serum factor' is released from the erythrocytes upon digestion of the blood meal (Rosenberg & Koontz, 1984). In the same study Rosenberg & Koontz showed that peak oocyst numbers in the mosquito were obtained 24 hours before peak gametocyte counts were observed in the vertebrate host although this may be a result of counting errors or the loss of the serum factor as a result of the "nature of the infection". While the nature of the serum factor is unknown Rosenberg & Koontz (1984) also showed that oocyst production is directly proportional to the haematocrit, implying that the better the blood meal quality the better the chances of oocyst formation.
Associated with the ability of the mosquito to feed is the observation that the increasing asexual parasitaemia serves to passify the animal. This allows the mosquito to feed for longer without interuption and should allow a better blood meal to be taken (Rossignol et al, 1985).
1.3.3 Host Immune Factors
The loss of infectivity to mosquitoes has been known for many years and immunity to the sexual stages could protect human populations against malaria by reducing the transmission rates (Carter et al 1988).. Early studies on the loss of infectivity (Huff & Marchbank, 1955; Huff et a/, 1958; Micks et at, 1948; Eyles, 1951) suggested that the rapid rise in the asexual parasitaemia reduced the availability of necessary nutrients for gametocyte formation or the production of toxins that inactivated the sexual stages. In recent years this latter suggestion has received considerable support and recently it has been shown that the rates of transmission implicate a natural factor in the host population that places a restriction on the rates of transmission (De Zoysa, 1988). Immunity to the sexual stages of the parasite has been shown to be via both cell-mediated and antibody-mediated mechanisms and have been reviewed by Carter et al (1988). Cell-mediated factors that have been demonstrated include: T-cell mediated immunity in P. y. nigeriensis (Harte et al, 1985) and in vitro for P. falciparum (Good et al, 1987); phagocytosis (Sinden & Smalley, 1976); C 3 complement (Grotendorst et alt 1986; Grotendorst & Carter, 1987); the production of cytokines by the host at a time that coincides with the loss of infectivity has also been implicated Weidanz & Long, 1988; Troye-Blomberg & Perleman, 1988; Bate et al, 1989; Mendis & Targett, 1982; Mendis et al 1989). The involvement of y-interferon in the
24 induction of crisis-forms has also been reported (Bastien et al, 1987). Recently, tumour necrosis factor has also been implicated as an inhibitory factor in P. cynomologi (Mendis et al, 1989) and most recently it has been suggested that TNF and y-IFN are dependent on an additional unknown factor in order to mediate killing of P. cynomolgi in torque monkeys (Mendis et at, 1990)
Antibody-mediated immunity against the sexual stages has been seen in many cases in hosts that show sustained or repeated infections. In humans antibodies that are induced by natural infection and show transmission blocking effects have also been found (Mendis et at, 1987; Ranawaka et al 1988). In patients infected with P. falciparum infections these antibodies have been shown to recognise proteins with Mrs of 230kD, 48kD and 45kD that are located on the surface of gametes and newly fertilised zygotes (Graves et al, 1988). Targets of transmission-blocking antibodies have not only been identified for P. falciparum (Quakyi et al, 1987; Graves et al, 1988a; Carter et al, 1988) and P. vivax (Premawansa et al, 1990) but also P. gallinaceum at 230kD, 210kD, 48kD, 45kD, 40kD, 20kD and 18kD on female gametes (Kaushal & Carter, 1984) and 25kD on ookinetes (Carter et al, 1984), P.y. nigeriensis (at 42kD on male gametes)(Harte et al, 1985a) and P. berghei (21kD on zygotes/ookinetes)(Winger et al, 1988). The manner in which antibodies against these sites act is unclear, although possibilities include blocking fertilisation, lysis of the young zygote or prevention of the ookinete passing through the mosquito midgut wall. Independent of their mode of action however is the fact that such antigenic sites provide ideal targets against which to aim the so call altruistic vaccines (see below).
Host immune factors in addition to having an inhibitory effect have recently been shown to have the ability to enhance transmission at low antibody concentrations (Mendis et al, 1987; Peiris et al, 1988; Tirawanchai, 1989). Naotunne et al (1990) found that serum samples taken at various times during P. cynomolgi infections would enhance transmission. This occurred early in infections at a time when antibody titres were low but rising and again some months after parasitaemias ceased patency. During mosquito feeding experiments, the best oocyst infections are obtained from feeds performed early (day 3 in P. berghei), and this fits with the suggestion of Naotunne et al (1990) that low early antibody titres enhance transmission at this time.
25 The factors mentioned so far that have a role in controlling infectivity have all been naturally occuring. On the other hand transmission blocking vaccine artificially alters the infectivity of the parasite and is therefore of great importance to attempts to prevent the spread of the disease.
1.4 Antimalarial Vaccines
There are three major vaccine strategies offered for the direct control of malaria parasite number, all of which act against an extracellular stage of the parasite life cycle. The sporozoite vaccine (Nussenzweig & Nussenzweig 1989; 1989a ) is based on immune response to the major surface protein, the CS protein, that is synthesised by the sporozoite. Sporozoite induced immunity is species specific and does not extend to the erythrocytic stages (Clyde et al, 1975).. It has now been shown that protective immunity is the result of a complex set of immune reactions against not only the sporozoite but also the action of CD 8 cells against infected liver cells (Nussenzweig & Nussenzweig 1989; 1989a), The sporozoite vaccine does however have certain drawbacks reviewed by Ravetch et al, (1985) and Miller et al (1986) that suggest its primary use will be for the short term protection of non- immune individuals entering an endemic area and in curbing epidemics. However to be effective a high level of immunity must be induced.
The second vaccine approach is against the asexual erythrocytic parasite. Since it is this stage that causes the clinical manifestations of the disease any inhibition of the asexual parasite would be useful in reducing the morbidity and mortality associated with malaria. Even partially effective vaccines would be of some use in reducing the severity of the disease (Ravetch et al, 1985). Antigens that have so far been identified as possible targets of such vaccines are the Plasmodium falciparum Pf 195 (Holder, 1988) and Pf 155 (Martinez, 1987) proteins that are located on the merozoite surface.
The third of the potential vaccines are the transmission blocking or altruistic vaccines, so called because the infected person derives no direct benefit from them. The transmission blocking vaccine aims to prevent the transmission and thus spread of the disease from the vertebrate host to the mosquito vector by interrupting the life cycle. Possible targets for the altruistic vaccine are the blocking of fertilization of gametocytes, lysing gametocytes and/or zygotes and
26 by altering zygote/ookinete development. The possible targets identified so far are the female gamete, the male gamete, the zygote and ookinete (Mendis & Targett, 1979; 1982; Grotendorst et al, 1984; Carter et al, 1984; Sinden et al, 1987; Carter et al, 1988).
1.5 Easmodium berghei
The rodent malaria, Plasmodium berghei, was first described in detail in the 1940s and the potential of this species ofPlasmodium, which shows many of the characteristics of human malaria while infecting one of the most common laboratory animals, such as the mouse, hamster and rat, was soon realised (Killick-Kendrick & Peters, 1978). After early unsuccessful attempts to transmit the parasite cyclically under controlled conditions this was finally achieved on a regular basis by Vanderberg & Yoeli (1965). Following its isolation from the wild, Plasmodium berghei soon became established as an extremely useful laboratory model, that lends itself easily to both in vivo and in vitro studies and the work described in this thesis was carried out on cloned lines of the P. berghei ANKA strain with a view to characterizing the course of the infection, thus enabling the routine culture and purification of the sexual stage parasites for further biochemical studies.
1.6 The Biochemistry ofEasm odium
Many studies on the vast area of the biochemistry of the rodent species of the malarial parasite Easmodium have been reported and are extensively reviewed in Killick-Kendrick & Peters (1978). The majority of these studies concentrate on the intraerythrocytic stages of the parasite life-cycle, however, of relevance to this study is the work relating to the synthesis of DNA, RNA and protein in the gametocyte, gamete and ookinete stages.
Until recently the study of sexual stages has been hampered by the inability to obtain pure gametocyte, zygote and ookinete stages. However, the recent development of techniques to purify these stages; the synchronisation of infection to give pure gametocytes in P. falciparum (Sinden et al, 1986; Ponnudurai et al, 1986) and in P. berghei (Mons, 1986) means that more detailed biochemical studies on these parasites are now feasible.
27 At the start of this study the consensus of opinion was that DNA synthesis did not occur during microgametogenesis as the 10 minutes that elapse between initiation and exflagellation was not sufficiently long to allow the microgametocyte to undergo the three rounds of mitotic division necessary to produce the 8 haploid microgamete nuclei. This idea was further supported by studies based on the drug mitomycin C, an inhibitor of DNA synthesis, which failed to block exflagellation in P. yoelii nigeriensis (Toy6 et al, 1977). Furthermore, drug studies have suggested that DNA synthesis occurs in the young gametocyte (Sinden & Smalley, 1979; Toy 6 et al, 1977) but cannot be detected in the mature gametocyte and in drug studies, as the gametocyte matures a loss in sensitivity to DNA synthesis inhibitors is seen (Sinden & Smalley, 1976). This led to the view that the mature gametocyte is octaploid and fully prepared to undergo rapid gamete formation. It has however recently been shown using microfluorometry, that the microgametocyte does actually show an 8-fold increase in its DNA content in the 8-10 minutes between activation and exflagellation (Janse, 1987). Following fertilization the DNA content of the zygote, as expected, shows a diploid value, which rises to a tetraploid value during ookinete formation. The haploid value is restored during oocyst development in that the sporozoite produced is haploid.
While DNA synthesis in the sexual stages of Plasmodium is well documented little is known about the synthesis of RNA in these stages and the data that exist are contradictory. Toye et al (1977) used inhibitors of RNA synthesis to study the process during gametogenesis and while they showed that actinomycin D inhibited exflagellation they also showed that 8- azaguanine did not. However since de novo protein synthesis was also shown the synthesis of messenger RNA seems likely. The incorporation of precursors of nucleic acids by both the zygote and ookinete imply the occurrence of DNA and RNA synthesis at this stage of the life cycle. The localisation of ^H-adenosine within the nucleolus by autoradiography could be either DNA or RNA labelling. The incorporation of ^H-adenosine into endoplasmic reticulum indicates that RNA synthesis certainly takes place in the zygote (Sinden, 1983). Likewise the same study showed regions of label over the nucleus and nuclear envelope which suggested that either DNA or messenger RNA synthesis was also underway.
28 Protein synthesis certainly occurs throughout the sexual development of Plasmodium. In P. falciparum inhibitors of protein synthesis have been shown to block gametocyte development and radiotracer studies confirm that protein synthesis continues in the mature gamete (Simm, 1984). Further drug studies using inhibitors of protein synthesis, this time in P. y. nigeriensis have demonstrated that protein synthesis is also essential for gametogenesis to occur (Toy6 et al, 1977). As with RNA synthesis, radiolabelled amino acids are also actively incorporated by the zygote and radiolabelling with 35s_ methionine has confirmed that de novo protein synthesis occurs in the zygote/ookinete stage (Kaushal et al, 1983). Studies on the synthesis of proteins have led to the identification of stage specific proteins. Simm (1984) showed the existence of six gametocyte specific proteins in P. falciparum and Kumar & Carter (1984) have described the synthesis of sexual stage proteins in the zygote stage of P. falciparum. Kaushal & Carter (1981) and Howard et al (1982) have identified at least 15 zygote specific proteins in P. gallinaceum and Winger et al (1988) have similarly identified a 21kD sexual stage protein in P. berghei.
The 21kD P. berghei zygote /ookinete protein shows similarities to those found in P. gallinaceum (Kumar & Carter, 1984; Vermeulen et al, 1985;1986) and P. falciparum (Kaushal & Carter, 1984) in terms of the biosynthesis of the protein, which starts around the time of exflagellation/fertilization and the location on the cell. In P. gallinaceum the trigger for synthesis has been identified as fertilization (Carter & Kaushal, 1984). The trigger for the 21kD P. berghei protein has yet to be identified but it would be expected to be associated with the events surrounding exflagellation and fertilization.
The 21kD protein is recognised by a monoclonal antibody designated 13.1, which blocks the transmission of the parasite to the mosquito. This transmission blocking ability of monoclonal antibodies that recognise the zygote and ookinete proteins has also been seen for P. falciparum (Amerongen et al, 1987), P. yoelii (Mendis & Targett, 1982; Harte et al, 1985), P. berghei (Kaushik et al, 1982, Sinden et al, 1987; Winger et al, 1988), P. vivax (Pieris et al, 1988) and P. gallinaceum (Rener et al, 1980; 1981).
Therefore these proteins become important from the point of view of vaccine development and have thereby become the subject of attention of the latter part of this thesis.
29 1.7 Overview and Aims
In view of the current situation where the malarial parasite is becoming increasingly resistant to antimalarial drugs (Moore & Lanier, 1961; WHO, 1978; Peters, 1970; Rieckmann et al, 1983; Reacher et al, 1981; McNamara et al, 1967; Smith and MacKenzie, 1985; Anonymous, 1987) and the mosquito vector has either adapted to a way of life in which it succeeds in avoiding insecticides or has even become resistant to them (Bruce-Chwatt, 1986), the overall objective of this study was to investigate the basic science surrounding the parasite, its biochemistry and its transmission to the mosquito. The hope was that by identifying new scientific data surrounding these areas of interest it would be possible to underpin studies on vaccine development that may aid the containment of the disease.
More specifically, and from a practical point of view, the aim initially was to characterise the 2.34L clone of Rberghei in my hands, identifying the times at which material for biochemical studies on the sexual stages of parasites could be harvested with reliability. It was also hoped that this study would be able to determine indicators for the production of the different parasite stages (Chapter 2) and shed light on the intriguing problems of the natural variation in infectivity of gametocytes in the naturally infected host. Having described the production of parasites for the 2.34L and 2.33L clone in detail the aim was then to use this to study the biochemical events occuring during zygote and ookinete development.
Owing to asynchronous pattern of parasite growth in the mouse it became necessary to develop a method whereby the stages of interest, namely gametocytes and ookinetes, could be purified, and this work is described in Chapter 3 and Dearsly et al (1987).
The study of DNA, RNA and protein synthesis events underlying the morphological events of ookinete formation was the next step and Chapter 3 describes how P. berghei ookinete cultures were treated with various drugs that show inhibitory effects against metabolic events in an attempt to show which events were occuring at any one time.
30 Following on from this I embarked on a study of a specific protein, the P. berghei 21kD protein, a transmission blocking determinant and therefore of great interest in terms of vaccine development, with the aim of to describing the biosynthesis and the preliminary characterisation of this protein.
By studying such events in the laboratory it may then be possible to apply such specific findings to results obtained from studying the parasite in the field and to ultimately obtain a full picture interrelating many aspects of the parasite, its life cycle, biochemistry and morphology.
31 Chapter 2
The Sexual Development of Plasmodium berghei in vivo and in vitro
Published as: Sexual development in Malarial Parasites: Gametocyte Production, Fertility and Infectivity to the Mosquito Vector. Parasitology 100, 359-368
32 2.1 Introduction
Gametocyte production during the course of Plasmodial infections is highly variable. The numbers of gametocytes produced by a single strain of the parasite will even vary between cloned lines of the same strain, as has been shown with clones of P. falciparum taken from the same infection producing either high numbers of gametocytes or low numbers (Graves & Carter, 1982). Mons (1986) has shown the ANKA isolate of P.berghei to be a mixture of high and low producers and has suggested that differences in commitment to sexual development may occur soon after cloning as a result of "mutation" and/or gene suppression. An alternative explanation is that the process of cloning leads to the mechanical separation of parasites that are "genetically programmed" as either high or nil producers, and will therefore bring about the observed clonal differences. This however can only apply to clones resulting in high or low producers and does not account for those lines that are intermediate producers of gametocytes. In addition to the variation between strains/lines of infection variations can also occur between infections by a single clone as a result of the host environment, changes brought about as a result of the infection itself or as a result of the laboratory management of the infection.
A P.berghei infection will ultimately kill the host mouse and an easy and routine way of maintaining the infection is via mechanical or syringe passage. Unfortunately sustained mechanical passage leads to a progressive decline in the numbers of sexual stages produced in both P. berghei (Mons, 1986) and P. vivax (Korteweg, 1930; Bylmer & Kraan, 1948) infections. This decline in the sexual capacity of P. berghei has been associated with the loss of reitterated DNA (Birago et al, 1982; Casaglia, 1985). However, Janse (Personal Communication) has not been able to show that gene amplification (reitteration) is necessary for sexual commitment and have suggested that changes in karyotype are responsible for the loss of gametocyte production.
As the work described later in this study centres on the gametocyte and ookinete stages it was necessary to be able to guarantee a supply of these parasites considering the many influences that affect their production. The first aim of this study was therefore to set the limits within which experimental material could be collected. It was therefore necessary to
33 determine the number of times an infection could be passaged without loss of gametocyte and ookinete numbers, the optimum time for collection and if possible to try to identify useful indicators for their production. Therefore, the effect of mechanical passage on the production of the sexual stages was studied by monitoring the course of infection followed by the 2.34L clone in passage 3 (P3), passage 8 (P8) and passage 14 (P14) infections, with the aim of finding the number of times this clone could be passaged before the loss of sexual development occurred.
The second aim of the study was to investigate the relationship between asexual and sexual stage development and the mechanisms through which gametocyte development is controlled, which is a complex and poorly understood area (Carter & Gwadz, 1980; Sinden, 1983). Factors that have been implicated in inducing gametocyte production include: innate parasite and environmental factors (Carter & Miller, 1979), clonal potential (Graves et al, 1984) and the effects of chemicals and drugs (Shute & Maiyon, 1954; Ono & Nakabayashi, 1989; Ono et al, 1986).
Also associated with this is the transmission of the parasite to the mosquito, or the infectivity of the parasite, which for species like P. berghei, P. yoelii and P. gallinaceum is lost throughout the course of an infection. This loss has been correlated with the rising asexual parasitaemia in P. gallinaceum (Carter & Gwadz, 1980) and is thought to be due to host immune factors (Huff & Marchbank, 1955; Huff et al, 1958; Micks et al, 1948). More recently loss of infectivity has been correlated with antibodies against gametes, zygotes and ookinetes (Mendis & Target, 1979; Grotendorst etal, 1984; Carter et al, 1984; Winger et al, 1988) and in the case of rodent and simian malarias with cytokine release (Mendis & Targett, 1982; Mendis etal, 1989; Bastien et al, 1987; Petit et al, 1982). Also identified as important factors are erythrocytic factors (Rosenberg et al, 1984) and the modification of host behaviour by the parasite (Rossignol et al, 1984; 1985; 1986).
For this study two sources of parasite material were used. Both were cloned lines of infection; the first, 2.34C gives a mixed infection of both asexual and sexual parasites while the second, 2.33L> is reportedly purely asexual (D.Walliker, Personal Communication). To establish the difference between the two lines of infection the course that each took in vivo was followed, by monitoring the asexual and sexual parasitaemias on a daily basis. In addition
34 to this the ability or the microgametocytes to exflagellate was also studied. From the macrogametocyte count, microgametocyte count and the exflagellation index (number of exflagellation centres per 10^ rbc) an indicator for the best time to collect parasitized blood for ookinete production was sought.
To summarise, the purpose of the work was to study the asexual and sexual parasitaemia, and to correlate the capacity of the gametocytes by observing ookinete production in vitro and to try to relate these observations to those seen for the infectivity of P. berghei to the mosquito. The parasite was also studied across several mechanical passages in order to investigate the change brought about by maintenance of the parasite in the laboratory.
35 2.2 Materials and Methods
2.2.1 In vivo Infection
For each of the 3 passages monitored, three Theiler's Original (TO) mice were inoculated via the intraperitoneal (i.p.) route with parasitised blood in RPMI 1640 (see 2.2.5.1) (0.2ml), which contained approximately 104 parasites of mixed stages. The number of parasites in the inoculum was determined by counting the number of red blood cells (rbc) per ml (2.2.2) using a Neubauer haemocytometer and the number of parasites per 104 rbc in Giemsa stained blood films (2.2.3). Material was collected on a daily basis. In light of evidence to suggest that there circadian pattern controlling gametocyte development material was collected at approximately the same time each day to overcome any variation due to such factors.
2.2.2 Red Blood Cell Count
Blood was taken from the tail of each mouse and diluted 1:200 in ookinete culture medium (2.2.5.1). This was then loaded onto a modified Neubauer haemocytometer and after the red blood cells had settled (30 mins) they were counted in a total volume of 5 x KHmI. The number of red blood cells per 104.n.y ml of blood could then be calculated using the formula — where n= the counted number of rbc and y= the fold dilution.
2.2.3 Parasitaemia
A drop of blood was taken from the tail of each mouse and smears made. They were then stained in Giemsa for 10 min, washed briefly under running water and dried. The numbers of parasites per 104 rbc were then counted along the length of the smear. Counting along the full length of the smear compensates for the uneven distribution of the parasites, with the large parasites being located in the tail of the smear and the small parasites at the start. A similar phenomenon has been reported by Mirkova & Ashby (1987) for the uneven distribution of normal erythrocytes, polychromatic erythrocytes and micronucleated erythrocytes in bone marrow smears.
36 2.2.4 Exflagellation
Blood taken from the tail of each mouse was diluted 1:10 with complete ookinete medium (2.2.5.1) at 19°C. A drop of this was then placed on a microscope slide and sealed under a coverslip sealed with vaseline. It was then repeatedly examined for exflagellation. Ten minutes after exflagellation had started the number of exflagellation centres per 104 rbc were counted, to give the exflagellation index.
2.2.5 Culture Medium
2.2.5.1 Complete Ookinete Culture Medium RPMI 1640 powder (Gibco) was dissolved in 1 litre distilled water. Hypoxanthine (50mg/l), which has been reported to increase gamete production (Ifedeba & Vanderberg, 1982), HEPES (5.94g/l) and NaHCOs (40ml of a 4% w /v soln), were added to buffer the system. Penicillin (50mg/l), streptomycin (50mg/l), neomycin (100mg/l) (PSN) and heat inactivated foetal calf serum (HI-FCS) (20%v/v), which is necessary for exflagellation to occur (Janse et al, 1985) were then added. For ookinete culture the medium was adjusted to pH8.0 by the addition of 1M NaOH
This medium will from now on be referred to in the text either as ookinete medium or RPMI 1640. Any other medium used will be given the titles used below.
2.2.5.2 Methionine-free RPMI 1640 Methionine-free RPMI 1640 (Gibco Special Recipe) was prepared by Gibco to include hypoxanthine, HEPES, NaHCOs and PSN as described in 2.2.5.I. HI- FCS was added to 20% and adjustment to pH8.0 with 1M NaOH were carried out as required.
2.2.5.3 Serum-free Ookinete Medium Ookinete culture medium was prepared as described in 2.2.5.1 with the omission of the 20% HI-FCS.
37 2.2.5.4 Gametocyte. Culture. Medium Methionine-free RPMI 1640 for the radiolabelling of gametocyte cultures was prepared as described in 2.2.5.2 with the single difference of the pH being adjusted to pH7.2 and not 8.0 as stated above.
2.2.5.5 Serum-free Gametocyte Medium Serum-free gametocyte medium was prepared as described in 2.2.5.3, differing only in the adjustment of the pH to 7.2.
2.2.6 Ookinete Production
Ookinete production in vitro was assessed on a daily basis after exflagellation was first seen in an infection. Parasitised blood (20jil) was diluted 1:10 in ookinete culture medium at 19°C in a 96-well microtitre plated and incubated at this temperature for 24h; after which the excess medium was removed and blood smears made. These were Giemsa stained and counted along the length of the smear as described in 2.2.3.
38 2.3 Results
2.3.1 Comparison of Asexual and Mixed Clones of P.berghei
The mean daily number of parasites in 10 4 rbc for the group of 3 mice, expressed as the total asexual and total sexual stages in Giemsa stained blood films for P3 infections for the mixed (2.34L) and asexual (2.33L) clones of P.berghei are shown in fig 2.1. Large statistical variation was seen due to difficulties in the identification of young gametocytes and trophozoites. However this was treated as a constant error across all the counts made.
2.3.2 Asexual Parasitaemia
The asexual parasitaemia in both 2.33L and 2.34L rises exponentially until it reaches approximately 20%. The only obvious difference is the time it takes to reach this level, 20-25 days post-infection for the P3 mixed infection and 5- 7 days post-infection for the asexual clone, with the death of the mouse following shortly afterwards (fig 2.1). The time taken for the asexual parasitaemia to reach these levels in the higher passage numbers, P8 and PI4, falls somewhere between those of the two extremes, P3 and asexual infections, being around day 20 for P 8 and day 7 for PI4. However on calculating the absolute numbers of parasites per ml of blood in the mixed infection, by taking into account the severe anaemia induced by the disease and seen in the haematocrit (see Appendix I), a maximum parasite burden is found at about day 7 (fig 2.3) and thereafter decreases.
2.3.3 Red Blood Cell Count
As a result of erythrocytic schizogony seen during a malarial infection, with parasite development leading to cell lysis, acute anaemia is induced in the host as the disease progresses. The red blood cell count for P3 infected (fig 2.2) animals fell from a mean starting value of ~400 x 10 6 rbc to a count of ~36 x 106 rbc per ml of blood, just prior to death. A peak in the red blood cell numbers was observed at day 7 and coincides with a major influx of reticulocytes, triggered as a result of the initial anaemia.
39 from groups of three mice observed in Giemsa stained blood films for a P3a for films blood stained Giemsa in observed mice three of groups from mixed (2.34L) and asexual (2.33L) asexual (2.34L) and mixed FIG FIG 2.1 number of parasltes/10000 rbc Graph showing the mean asexual and sexual parasitaemias obtained obtained parasitaemias sexual and asexual mean the showing Graph P.berghei time infection. a- asexual 2.34Lasexual a- ♦ — sexual 2.34L sexual ♦— r o sexual 2.33L sexual asexual 2.33Lasexual B
O S i
o o o o
0) CL (0o *-•o JSC oo
Time (days)
FIG 2.2 Graphs showing the number of red blood cells per ml of blood (A) and the ookinete production observed in Giemsa stained blood films (B) during the course of a P3 P. berghei infection parasites per ml of blood throughout the course of a P a of course the throughout blood of ml per parasites FIG 2.3 Graph showing the calculated total number of asexual asexual of number total calculated the showing Graph 2.3 FIG parasite/ml blood x105 time (days) time 3 infection. P.berghei
2.3.4 Sexual Parasitaemia
No sexual stage parasites were found in the smears of the mice infected with the 2.33L clone. Allowing for one parasite being missed in the count, a sexual stage parasitaemia of less than 0.0001% was awarded. The sexual parasitaemia, consisting of microgametocytes and macrogametocytes, was only observed in the mixed clone 2.34L reaching a maximum of 1.8% at about 10 days post-infection then falling off (fig 2.1). This sexual parasitaemia is however only a very small percentage of the total parasitaemia. In terms of absolute numbers of parasites the numbers of gametocytes peak around day 6 and then maintain more or less steady numbers until the mouse dies (fig 2.4).
2.3.5 Production of Gametocytes During the Course of an Infection
Recognising the 24-26 hour gametocyte maturation period (Mons, 1986) the rate at which the conversion of trophozoites into gametocytes takes place can be calculated as a percentage by dividing the number of gametocytes on one day by the number of trophozoites counted 24h earlier and multiplying this number by 100. When this is plotted against time, for P3 infections, an initial burst in conversion is followed by a negative logarithmic decay in the conversion rate (fig 2.5A). A similar pattern was followed by all three infections observed, ie P3, P 8 and PI4.
While the correlation coefficient values are comparatively low (R2=0.75 for P3) they were the best achieved at any time throughout the experiment. In support of the correlation values the situation has now been computer modelled, independently of the experimental data, and the same pattern has been observed, and includes the initial rise seen at the start of each passage (fig 2.5).
2.3.6 The Effect of B lood Passage on Gametocyte Production and Activity
The reported loss of sexual potential of P.berghei on blood passage makes it necessary to know how long a particular clone can be maintained in the
43 o lo truhu te ore fa 3 infection. P3 a of course the throughout blood of l m FIG 2.4 Graph showing the calculated number of of number calculated the showing Graph 2.4 FIG
metocytes/ml blood x104 \ time (days) time P.berghei a tcts per etocytes gam
time (days)
FIG 2.5 Graphs showing the mean conversion rates for three mice of trophozoites to gametocytes throughout the course of P3 (A), P8 (B) and P14 (C) P.berghei infection laboratory by mechanical passage without loss of gametocyte and therefore ookinete production. Therefore, the course of P 8 and PI4 infections were monitored in addition to that of the P3 infection described earlier, with special interest being shown with regard to the macro gametocyte and microgametocyte count, the exflagellation index and ookinete production.
Comparison of the mean gametocytaemias from groups of three mice for the passage numbers 3, 8 and 14 shows that while, even in P3, the percentage of the sexual stages compared to the asexual stages is small, it is much reduced in subsequent passages falling to approximately half its P3 figure by P 8 (fig 2.6) . For clone 2.34L this figure does not drop further between P 8 and PI4 (fig 2.6) but longevity of the infection is reduced. Fig 2.6 also reveals that the sexual parasitaemia peaks at about day 10 post-infection in P3, gradually trailing off towards the end of the infection. A similar pattern is seen for the very short life of a PI4 infection, however P8 shows no early peak of gametocyte production and the numbers towards the end, while improving, are variable. Calculating the standard errors of the mean indicated a significant difference in the counts observed between P3 and P8/P14 between day 7 and day 12 of the infection (tables 2.1,2.2,2.3).
The gametocytaemia figures consist of two counts; the macrogametocyte (Fig 2.7A) and the microgametocyte (Fig 2.7B) numbers. When looked at separately a similar pattern to that for the joint figures is found. Again the numbers are much reduced in later passages with P3 producing a distinct peak at around day 9. P 8 in this case would also appear to have lost the ability to produce large numbers of macrogametocytes as has PI4. The situation for the microgametocytes is similar with P3 producing large numbers early in the course of infection while P 8 and P14 only begin to produce higher numbers towards the end of the infection and these are at much lower levels than seen before. Calculation of the standard error of the means showed that observed differences between P3 and P 8 and PI4, while slight are outside the limits of error and therefore can be considered as being statistically different between day 7 and 10 for macrogametocytes and day 7 to day 11 for microgametocytes.
The ratio of macrogametocytes to microgametocytes was found to show a normal distribution of values (Fig 2.8), ranging from 1:0.07 to 1:6.3 with a modal value of 1:1 (F:M), suggesting that this is the normal female to male
46 200
time (days)
FIG 2.6 Graph showing the mean sexual parasitaemias observed in Giemsa stained blood films throughout the course of P3, P8 and P14P.bcrghci infections -----□---- P3 P8 — A — P14
B
----□— P3 P8 — •A— P14
FIG 2.7 Graphs differentiating the sexual parasitaemia into the the microgametocytaemia (A) and the macrogametocytaemia (B) observed in Giemsa stained blood films throughout the course of P3, P8 and P14P.berghei infections. The counts are the mean numbers obtained from the observation of infections in three mice Count en 17 GoercMa = 1.4 = Mean Geometric 1.7; = Mean i . Hsorm u rsn te e rto FM) bevd n Giemsa in observed ) (F/M ratio sex the arising m sum Histogram 2.8 Fig ieec st ncltd ih ihr 3 P o P4 netos Moa Vle 1; = Value odal M infections. P14 or P8 P3, either with inoculated set each mice stained smears from from smears stained 14, . berghci P. netos band rm he st o three of sets three from obtained infections e Ratio Sex
Mean Numbers Day Trophozoitess Macrogametocytes Microgametocytcs Gamctocytcs Exflagellation Ookinetes Sex ratio Index F:M 1 20±13.5 0.711.2 010 0.711.2 010 010 - 2 41.7±42 5.717.4 2.714.6 4.518.6 213.5 111.7 - 3 111.3±137.2 16.7117.6 3.315.8 20122.9 9112.3 0.310.6 - 4 289.3±263.9 20121.3 4.715 24.7125.3 5.315.6 0.310.6 1:0.2 5 3841318.2 19.7114.4 8.7111.7 25123.6 20121.3 314.4 1:0.5 6 624.71309.4 40136.7 1919 67.7133.5 34.3112.7 4.714.2 1:0.4 7 9661258.9 84164.1 51.3110.2 135.3168.4 202185.1 715.2 1:1.9 8 10251352.9 64.7154.9 52.7138.2 117.3168.7 168192.2 8.717.6 1:0.7 9 1108.71467.1 55.314.2 66124.3 121.3128.4 224.31109.6 1515 1:1.2 10 868.7172.6 65.7154.9 66125.6 131.7179.2 188.3171.2 17.312.5 1:1.2 11 957.31596.9 36.3115.9 50.3114 86.7127.3 2131146.1 27.7115.7 1:1.5 12 1376.31438.4 36117.3 68141.6 104156.4 308.796.9 2212.6 1:1.7 13 980.7124.5 2319.5 55.7129.4 66.315.5 240.31146.3 21115.7 1:2.1 14 817.3140.8 39128.8 88.7140.4 127.7150.1 215.3154.9 35118.7 1:5.7 15 11111557.7 46.7137.5 61.3132.3 108168.4 186.3127.6 30.3114.5 1:1.5 16 1012.31219.9 28121 51117.5 79136.1 120144.4 1916 1:2.3 17 1127.71187.2 32.2122.2 31.3112.1 68.7127.1 87.714 010 1:1.2 18 1252.71247 21.314.6 42.616.4 64110 64.7111 010 1:2.1 19 1265.31187.1 22.6114.7 27114.9 49.7126 35117.3 111.7 1:2 20 12761207.6 11.318.4 13.317.5 24.7111.4 23.7110.7 1.111.6 1:1.5 21 10981223.4 23.3111.7 22.719.9 46118.3 2818.7 2.713.8 1:1.3 22 1335.3199.1 26.7115 15.717.6 42.3122.6 22.318.5 111 1:0.6 23 14281283.2 15.313.1 14.318.3 29.7111.4 47.3142.6 0.310.6 1:0.9 24 1579.71224.4 26.6128.3 44.3127 74.3160.2 76.7130.9 010 1:2.3 25 1733.71166.1 1814.4 49.7115.4 67.7119.7 29.319.2 111.7 1:2.7 26 1625.71232.6 11.718.1 32.7113.4 44.3121.4 52134.9 010 1:3.2 27 16771215.4 36129.8 30115.9 86.7157.2 314.4 010 1:3.5 28 1735.7174.8 51.3141.3 27.7118.8 79129.2 1.712.9 010 1:2.2 29 169710 1610 1110 2710 010 010 1:0.7
Table 2.1 Showing the mean number of counts ± standard deviation obtained from three mice infected with a P3P. berghei infection. The counts were determined on a daily basis from Giemsa stained and live preparations (see sections 1.2.3 & 1.2.4). Mean Numbers Day Trophozoites Macrogametocytes Microgamctocytcs Total Exflagellation Ookinetes Sex Ratio Gametocytes Index F:M 1 16.7±3.1 010 010 010 010 - 1:1 2 51±73.6 4+6.9 010 416.9 010 - 1:1 3 196±164.4 14.3113.6 6+5.6 20.3118 4.718.1 010 1:0.7 4 135.3±219.7 7+12.1 6110.4 13122.5 8.7+15 010 1:1 5 201+323.2 10.7116.8 7.3+12.7 18129.5 9.3116.2 010 1:0.9 6 182.3±302.8 7.3112.7 6.7111.5 14124.2 9.6115 0.310.6 1:1 7 227±376.7 2.614.6 416.9 6.6111.5 18.3131 111.7 1:1.1 8 228±381.1 2.614.6 416.9 6.7111.5 20134.6 010 1:1.1 9 332±562.9 3.315.8 11.6+19.3 15125.1 26139 5+7.8 1:1.6 10 271+444.2 3.315.8 9.5+12 9.7+15.8 36.7+47.2 4.6+8 1:1.4 11 164± 100.9 19.3115.1 8.314.9 27.6118.9 49+22.6 8.3114.4 1:1.1 12 474.7±335.6 32116.4 23.2111 55.3127.3 123.3+22.5 13.3+6.1 1:0.8 13 7811617.2 24.7111 34+13.1 58.7+23.2 42.6+36 4.315.1 1:1.6 14 764.41444.1 29.3117 126.3+13.4 53.6+30 40+40 2+3.5 1:1 15 820.31203.5 14.7+11 19+16.5 33.6126.1 33.7+27.2 14.3+20.6 1:1.2 16 1048+99 17.3+17 25.3121.4 42.6+35.2 40.7138.7 18129.5 1:1.7 17 15301382.3 24112 39.7+34.6 63.7141.1 80+77.2 4+3.6 1:1.6 18 12261331 26.3+27.5 33.3+22.7 59.6148.5 60157.7 18.6+30.6 1:1.7 19 1584+191.4 19.3111 44.7147.7 64+46.8 49+60.1 9.619.5 1:2.9 20 15811354.5 14+6.6 25.6126 39.7+18.9 71182.9 10110.8 1:2.4 21 1582.5+112.4 27.5+0.7 42.5+21.9 70+22.6 80.5+84.1 22.5112 1:2.1 22 1626192.6 21.513.5 46139.6 67.5+43.1 67.5+84.1 4+5.7 1:2 23 1420.5+579.1 3010 57+63.6 87+63.6 617.1 010 1:1.9 24 204010 16+0 45+0 6110 010 010 2.8
Table 2.2 Showing the mean number ±standard deviation obtained from three mice infected with a P8P. berghei infection. The counts were determined on a daily basis from Giemsa stained and live preparations (see sections 1.2.3 & 1.2.4) Mean Numbers Day Trophozoites Macrogamctocytes Microgametocytes Total Exflagellation Ookinetes Sex ratio Gamctocytes Index F:M 1 23.7±3.8 1±1 0±0 1±1 0±0 0+0 - 2 70±14 4.6±4.6 0±0 4.6±4.6 0±0 010 - 3 353.3±163 38±7.8 8±4.4 46±3.6 11.7±10.5 010 6.3 4 816±248.2 34.7±2.3 15.3±12.1 50±12.5 23.217.6 1+1 1.8 5 938.3±586.6 66.7±60 20.3±13.9 87±70.9 67.6156.7 617.9 4 6 1552.3±457.9 25±10.4 66±49 91±54.6 18.3122.5 0.711.2 0.6 7 1755±352.1 31.5±26.5 51.3±40.3 81.5±68.6 30.5134.6 0.510.7 0.6 8 1299±386.1 8±0 20±17 28±17 15.5120.5 0.510.7 1.3 9 1780±17 8±11.3 5±7.1 13+18.4 0.510.7 0.510.7 -
10 1788±0 6±0 0±0 6±0 0+0 -
Table 2.3 Showing the mean number of counts ± standard deviation obtained from three mice infected with a P14P. berghei infection. The counts were determined on a daily basis from Giemsa stained and live preparations (see sections 1.2.3 & 1.2.4) ratio. Owing to difficulties in parasite identification counting errors may be responsible for the large variation in observed ratios. However, since the mean (1.7) and the geometric mean (1.4) are of similar values it tends to support this proposal as there is no obvious trend in favour of either macro- or microgametocytes, although the average values would suggest that in any given population one would expect to find a slightly higher number of macrogametocytes than microgametocytes.
The exflagellation index for P3 starts to increase between day 7-8, then increases rapidly reaching a maximum at day 12, after which it decreases just as dramatically, showing little change for the rest of the study (see Appendix I). This pattern that was mirrored by the observed micro gam etocyte counts. Again P8 and P14 show a much reduced response with no peak production being seen in the P 8 counts.
2.3.7 Ookinete Production
Passage 3 infections proved to be good producers of ookinetes in vitro. Ookinete yield is seen to increase rapidly over a period of 10 days starting 7 days into the course of infection. Ookinete production occurs in a single wave, with counts falling to zero 3 days after the maximum is reached at day 14 (fig 2.9). Contrasting to this is the ookinete yield obtained from the P 8 infection, where successive waves of reduced numbers of ookinetes are formed towards the end of the infection and for PI4 the numbers of ookinetes produced are reduced to minimal levels, with a maximum count of 6 ookinetes per 104 red blood cells being recorded (fig 2.1 OB). This pattern of ookinete production follows that seen with gametocyte production as can be seen by comparison of figures 2.9 A and 2.9B. While the differences are not statistically significant, it does not eliminate the possibility of a biological importance. In hindsight the small group size (3 mice) does not allow for the wide variations in the counts seen between the individual animals and one way of partially overcoming this biological variation is to repeat the experiments using larger group sizes, giving the chance of obtaining a better estimate in spite of the biological variation.
53 stained blood films throughout the course of a P3 P3 a of course the throughout films blood stained a nmes ee band rm ons d o gop o tre mice three of groups on ade m counts from obtained were numbers ean m I 29 rp so n te a oknt pouto osre i Giemsa in observed production ookinete ean m the ing show Graph 2.9 FIG
ooklnetes/1 OOOOrb time (days) time P.berghei neto. The infection.
A 40
time (days)
Time (days)
FIG 2.10 (A) Graph showing the loss of mean ookinete production observed in Giemsa stained blood films by aP.bcrghei infection with passage. Means were obtained by counting the numbers of parasites in three mice. (B) Graph showing the mean numbers of gametocytes produced by each of three infections (P3, P8 and PI4). Means were again obtained from three mice. 2.3.8 Sexual Stage Parasites as Indicators of Ookinete Production
The proposed biochemical studies of P.berghei zygotes and ookinetes rely upon the in vitro production of these stages and it is therefore necessary to be able to determine the time at which maximum numbers can be harvested. To investigate whether the numbers of either of the sexual stage parasites or the exflagellation index would provide a good indicator, the figures for each were plotted against the corresponding ookinete yield and the regression coefficient calculated While none of the tested factors gave a good correlation, the best indicator of ookinete production is the exflagellation index with an R2 value across all passage numbers of 0.794 (fig 2.11). Microgametocyte counts showed a correlation of 0.509, and somewhat surprisingly the macrogametocyte counts, since each macrogamete goes on to produce one ookinete, showed a very poor correlation giving a value for R 2 of 0.083. The exflagellation index as a measure of ookinete production across all the passage numbers tested also gave a comparatively good correlation with a value for R 2 of 0.619. The graph in fig 2.11 also suggests from the y- axis intercept that a minimum number of 40 exflagellations per 104rbc is required before ookinete formation will on average take place. In practice this means, that when blood diluted 1:10 and examined at a magnification of 200x, 4 or 5 exflagellation centres per field should be visible, before blood is taken for ookinete formation and this simple screening procedure has saved time and resources.
2.3.9 The Effect of Red Blood Cell Count on Ookinete Formation in vitro
The poor correlation of ookinete production to the observed gametocyte number is consistent with the notion that ookinete production is additionally related to other criteria; blood factors (Eyles, 1951,1952,1952a), serum factors (Rosenberg & Koontz, 1984) and host immune factors (reviewed Carter et al, 1988).
As other workers have shown the importance of blood related factors in ookinete formation a comparison of ookinete yield and red blood cell count was made for P.berghei in vitro, however the peak of ookinete production.
56 index and the mean ookinete production for a P3 P3 a for production ookinete mean the and index r otie fo osrain maeo he mie R = 0.794). = (R2 ice m three on ade m observations from obtained ere w i 21 Gah hwig h creain ewen h men exflagellation ean m the een betw correlation the ing show Graph 2.11 Fig
exflagellation index ookinetes/IOOOOrbc P.berghei neto. Mear infection.
observed here does not appear to correlate (R2=0.011) with any noticeable change in the haematocrit (fig 2.2B).
2.3.10 The Effect of Asexual Parasites on Ookinete Formation in vitro
It has been suggested that the rising asexual parasitaemia is responsible for inducing the onset of sexual development in Plasmodium (Sinden, 1983). This study shows a very low correlation (R2=0.003) between ookinete formation in vitro and the observed asexual parasitaemia.
58 2.4 Discussion
The characterisation of clones from a single isolate has allowed P. berghei to be described as being a mixture of parasites that vary in their ability to produce gametocytes (Mons, 1986). Cloning of P. falciparum has produced lines that vary in their ability to produce gametocytes (Graves et a\ 1984), of which high and low gametocyte producers have been identified and mechanically separated, resulting in lines of infection that vary greatly from one another and from the parent line. It is therefore important to characterise the course on an infection under the conditions of use in the laboratory.
In this study the two clones, 2.34L and 2.33L* derived from the ANKA isolate were primarily differentiated on their ability to produce gametocytes. Both of the clones used show a similar asynchronous spectrum of asexual stages, however they vary in the gametocyte production and this difference will be used to attempt to describe biochemical features attributable to the sexual stages (see Chapter 4).
The aim of the work described in this chapter was to study the course of a P. berghei infection in vivo with a view to identifying when best to isolate parasites for the study of sexual differentiation, fertilization and ookinete development and thereby attempt to identify indicators for the successful completion of in vitro ookinete formation. It must at this point be said that no statistically good correlations were obtained for any of the parameters measured. However, within the limitations of the analysis of the properties measured the exflagellation index was found to have the highest correlation and therefore to be the most reliable indicator of ookinete development
Gametocytes are the source of infectiousness to mosquitoes (Carter & Gwadz, 1980) and a linear relationship between gametocytes and ookinetes in vitro has been described (reviewed Vanderberg et al, 1977). Similarly, a linear relationship in P. falciparum between gametocytes and oocysts has also been reported (Eyles, 1951). There are however, cases where relatively low numbers of P. gallinaceum gametocytes are capable of producing mosquito infections (Jeffery & Eyles, 1955). Conversely high gametocyte counts have not, as expected, produced an oocyst infection in the mosquito. This variation reflects the complex set of conditions that have been implicated in
59 suppressing oocyst formation in vivo (Huff & Marchbank, 1955; Huff et al, 1958; Mendis & Targett, 1982; Mendis et al, 1989; Bastien et al, 1987; Rossignol, 1984;1985;1986). Figure 2.12 illustrates the factors that are thought to influence the successful completion of transmission to the vertebrate host.
This study was able to demonstrate a correlation between gametocytes in vivo and ookinetes in vitro, however, the in vitro ookinete formation data contrasts markedly with the observed oocyst formation in vivo. In the mosquito situation the best oocyst infections are seen early in an infection ie, in P. berghei at or, prior to day 4, often before the first gametocytes are seen in the blood in routine bloodfilms and then as the gametocyte numbers increase the mosquito infections are lost (Vanderberg & Gwadz, 1980; Huff & Marchbank, 1955; Huff et al, 1958; Gwadz & Green, 1978). The situation in vitro however shows that ookinete formation increases with the age of the infection peaking at around days 10 -13 as compared to day 3 for oocyst formation. Responsibility for this loss of oocyst formation has been placed on host immune responses (reviewed by Carter et al,1988) bloodmeal quality and nutrient factors (Rosenberg & Koontz, 1984; Rosenberg et al, 1985) and host modification (Rossignol, 1984; 1985; 1986). Recently the study presented here has been taken further by following the in vivo oocyst production for the clone 2.34L across the course of an infection (Self, 1989). These results confirm that there is some factor(s) that restrict the successful development of oocysts in vivo, but is not active in the in vitro system used, possibly due to a dilution effect.
The loss of infectivity to mosquitoes is not associated with any apparent change in the moiphology of the gametocyte and it has been shown by taking late produced gametocytes and transferring them to a 2.33L infected mouse that gametocytes remain physically capable of inducing mosquito infection throughout the course of the disease in the vertebrate host (Mendis, 1980; Self, 1989). Since the evidence shows that the gametocytes do not loose their ability to induce mosquito infection it follows that some other reversible or transient factor must be responsible for the modification of the functional capacity of the gametocyte, and thus gametocyte numbers observed in blood films are not a useful indicator of ookinete production and subsequent mosquito infections (Munderloh & Kutti, 1987).
60 Number adequate Infective Mature Number Inadequate
PARASITE Non - infective (s te rile )
------Immature
INFECTIVITY HOST
------VECTOR
Fig 2.12 Summary of known and projected factors influencing the infection of mosquitoes from gametocyte infected vertebrate hosts. Bold type, - enhancing factors; Italics, - inhibiting factors; Normal type, - unknown or ambivalent effects. The factors reportedly involved are many and varied but include: the vertebrate host immune responses such as the humoral components of the system (Eyles, 1951; Bastien et al, 1987), the production of anti-sexual stage antibodies (Carter et al, 1988), blood factors (Rosenberg & Koontz, 1984), toxins (Gwadz, 1976) and even host behavioural modification (Rossignol et al, 1985; 1986; 1987).
The involvement of a factor that is produced by the host as a result of the infection has been demonstrated in several ways. Firstly, as mentioned above the transfer of late-produced gametocytes into 2.33L infected mice releases the observed suppression of infectivity. Conversely, early-produced gametocytes when transferred to heavily parasitised blood from late infections will only give rise to poor mosquito infections, further implicating the presence of a factor(s) that appears throughout the course of an infection (Mendis, 1980; Self, 1989). It has also been suggested by Self (1989) that this factor is not produced in response to the gametocytes but simply as a result of the infection since the transfer of gametocytes into mice infected with the 2.33L clone, the asexual clone, also leads to the suppression of infectivity. The fact that suppression of infectivity occurs 5 days into an infection (Petit et al, 1982; Dei-Cas, 1980; Bastien et al, 1987), the approximate minimum time taken for an immune response to be mounted (Vos et al, 1984), lends weight to the suggestion that the observed suppression is immune response mediated.
Further evidence for the involvement of an immune response in the suppression of infectivity exists. Eyles (1952) demonstrated that humoral factors serve to reduce gametocyte infectivity in P. gallinaceum and Bastien et al (1987) suggested that a number of humoral agents, including y- interferon (y-IFN), were responsible for the induction of crisis forms and thereby a reduction in infectivity. The involvement of the cytokines in responses to parasite infection have also been shown by Taveme et al (1990) and Clarke et al, (1989). The production of cytokines as a cause of the reduction in infectivity has also be suggested by Mendis & Targett (1982), Mendis et al (1989), Bastien et al (1987), Petit et al (1982), Bate et al (1989). Other immune responses that have been implicated are complement C3 (Grotendorst et al, 1986; Grotendorst & Carter, 1987), tumour necrosis factor (Mendis et al, 1989; Miller et al, 1989). Most recently it has been suggested
62 that TNF and y-IFN are dependent on an additional unknown factor in order to mediate killing of P cynomologi in torque monkeys (Mendis et al, 1990; 1990a). The production of antibodies against gametocytes and gametes (Mendis & Targett, 1982) has been also been identified as being involved in suppressing infectivity, and the timing of the loss of infectivity corresponds directly with the earliest time at which an antibody response has been found to any source of invasion (5 days) (Vos et al, 1984).
In addition to the immune response of the host, blood factors are said to play a major part in determining the infectiousness to a mosquito. The importance of blood factors was first described by Eyles (1951; 1952; 1952a) for P. gallinaceum infections to Aedes aegypti and resulted in the conclusion that serum was an essential component if full development of the parasites was to occur. Subsequently, it has been shown that the 'serum factor' is released from the erythrocytes upon digestion of the blood meal (Rosenberg & Koontz, 1984). In the same study Rosenberg & Koontz (1984) showed that for P. gallinaceum peak oocyst numbers in the mosquito were obtained 24 hours before peak gametocyte counts were observed in the vertebrate host. This difference may he due to the fact that young (12 hour) gametocytes may be confused with trophozoites in the smears and an abnormally low gametocyte count is obtained. Alternatively the loss of erythrocytes from the host due to the course of infection must dilute the essential erythrocyte factor necessary for oocyst formation, thereby decreasing the infectivity despite the rise in gametocyte count. Since it is an increase in the asexual parasites that brings about the chronic anaemia, this might explain the observed correlation between infectivity and the asexual burden. It has been suggested that a nutrient factor may be involved in oocyst formation. It has been shown that certain protein components are concentrated from the bloodmeal during oogenesis and that the underlying "good quality" bloodmeal is essential for successful oogenesis (Friend & Smith, 1977; Briegel, 1985) and it has been shown that the mosquito is able to concentrate host blood proteins during feeding (Briegel & Rezzonico, 1985). If a similar situation was involved in oocyst production as has been suggested by Gooding (1966; 1972) where ookinetes are dependent on blood nutrients and with late infections potentially having to support many parasites, in the mosquito vector the situation may occur whereby essential nutrients are not available in high enough concentrations. It is also possible that the nutrient
63 depletion could be overcome in vitro by the addition of nutrients in the culture medium, giving the sustained ookinete production.
The "quality" of the bloodmeal therefore plays an important role in infectivity. It was originally thought that the diuresis of the mosquito during feeding resulted in the concentration of erythrocytes (Beards, 1983) and thus the protein components required. Recently it has been shown that the bloodmeal shows a parallel fall in haematocrit to that seen in the mouse (Self, 1989) and it therefore supports the view that the bloodmeal "quality" is important. While the nature of the serum factor(s) is unknown Rosenberg and Koontz (1984) showed that oocyst production is directly proportional to the haematocrit, and is said to be a red blood cell associated factor. By the very nature of its lytic erythrocytic infection, malaria is an inducer of anaemia in the host animal (Seed & Krier, 1980), thus levels of possible red blood cell factors would naturally become reduced. However, loss of infectivity occurs long before severe anaemia has set in, although concommittant with the loss of infectivity is a decrease in the red blood cell volume by 28% (Dearsly et al, 1990) and such a dramatic change could conceivably contribute to the loss of infectivity. The quality of the bloodmeal may also play a critical role in buffering the developing zygotes and ookinetes from attack by the digestive enzymes, eg proteases to to which the ookinete is very sensitive (Gass, 1977; Gass & Yates, 1979; Yates and Steiger, 1981) and complement C3 (Grotendorst et al, 1986; Grotendorst & Carter, 1987).
On the other hand it has been shown that dilution of the parasite concentration can increase infectivity (Petit, 1984; Janse, 1987; Ponnudurai, 1989; Self, 1989) and propose that in membrane feeds fertilization is a result of contact and that dilution increases the chances of two gametocytes of the opposite sex meeting by lowering the red blood cell concentration (Kumar & Carter, 1985). Another explanation for the reduction of inhibition due to dilution in vitro could easily result from the dilution of inhibitory factors that are present, and since their effect is reversible, thus release the block on mosquito stage development. Similarly, in vitro culture for ookinete development could also have the effect of diluting any such factors, again with the effect of releasing the in vivo block.
64 Another possible explanation if the differences seen between ookinete production in vitro and oocyst production in vivo is a result of the experimental method, which assumes that gametocytes collected from tail blood for in vitro ookinete formation are identical to those ingested by the mosquito from the capillary beds. The differences have been attributed to parasite distribution being under the control of a circadian rhythm (Dei-Cas, 1980; Landau et al, 1979). Similarly, Hawking et al (1971; 1972) have described the control of exflagellation and gametocyte production as following a circadian pattern. In L. simondi, Gore & Pitman-Noblet (1978) suggest this is responsible for a pituitary mediated control, that affects hormone production. In turn this controls the host temperature cycle that correlates with changes in gametocyte distribution. A similar pattern of distribution has been suggested for microfilarae (Hawking et al, 1968). A cyclical pattern of gametocyte distribution has been suggested to explain the cyclical infectivity Plasmodium (Sinden, 1983a). It has been suggested that this synchronised, cyclical production probably occurs in all periodic malaria and that the purpose is to produce a supply of infective gametocytes at a time when mosquito transmission is most likely to occur (Hawking et al, 1968). In contrast to the evidence of Hawking et al (1968) are observations made on P. falciparum, where Bray et al (1976) failed to find any evidence of a circadian pattern of gametocyte production in P. falciparum.
Another aspect of the transmission of Plasmodium to mosquitoes is the phenomenon of enhancement, which is seen under two sets of conditions. The first is described by Gass (1977) where mosquitoes that have taken two non-overlapping bloodmeals show increased rates of transmission. This observation has also been reported by Terzian et al (1956) for Aedes aegypti and P. gallinaceum. Gass (1977) suggested that it occurred as a result of lower levels of the digestive enzymes responsible for the destruction of young zygotes being present at the time of the second bloodmeal and hence the increased rates of transmission. The second instance is in the presence of low concentrations of mAb against gamete (Mendis et al, 1987a; Peiris et al, 1988) or zygote (Tirawanchai, 1989) stages and such target antigens have been identified on the surface of both gametes (Harte et al, 1985) and zygotes/ookinetes (Winger et al, 1988; Carter et al, 1984). In view of the identification of naturally occurring antibodies against gamete surface proteins in the sera of infected hosts (Mendis et al, 1987; Graves et al, 1988) it is possible that the parasite has developed a method by which to boost its
65 transmission in the natural situation. Recently, it has been shown that low levels of naturally occurring anti-gamete antibodies are found early and late in and infection and this lends support to this view. Such a system could be of particular importance from the point of view of the parasite in endemic areas where many of the local population are likely to be recovering from previous attacks and as such show low antibody titres against parasite antigens.
The work in this chapter aimed to characterise the production of sexual stage parasites under conditions of laboratory maintenance. Mechanical passage is a very easy and convenient method of maintaining P. berghei in the laboratory, but it does have the drawback of bringing about the loss of sexual stage production (Mons, 1986; this study), which has been associated with the loss of DNA from the parasite (Birago et al, 1982). The loss of sexual potential is however is easily overcome by mosquito passage, which inevitably acts by the selection of the gametocyte producing clones.
It is also noticeable that within a single infection gametocytaemias decrease, but on passage will increase partially for a short time before further loss. This decrease in the production of gametocytes may be due to anti- gametocyte antibodies or other humoral responses of the host, that release their inhibitory pressure due to a dilution effect that occurs with passage. Likewise infectivity is lost during an infection again due the action of immune responses of the host. An explanation for this could be that there are two forms of suppression at work: genotypic suppression (the loss of genetic material resulting in decreases in infectivity) and phenotypic suppression (the suppression of gametocyte production and infectivity as a result of host responses to the infection). The overall effect however is that due to laboratory maintenance reduced numbers of gametocyte, ookinetes, and presumably oocysts are produced with time, however for the purpose of using this parasite for studies on the sexual stages exflagellation was established as the most reliable indicator of ookinete production and was used to determine when to collect material. It was also decided that because of the loss of gametocyte and ookinete production not to collect material beyond P7 and in that way obtain a steady and reliable supply of sexual stage parasites for further study.
66 2.4.1 Conclusions
The work described in this chapter has shown:
1. For gametocyte and ookinete production the 2.34L clone should not be maintained by blood passage beyond P7.
2. The best indicator of ookinete production in this system was the exflagellation index.
3. There is a considerable difference between the time of peak ookinete production in vitro and peak oocyst numbers in vivo. This has since led to a further study on this variation (Self, 1989; Dearsly et al, 1990).
4. The conversion rate of trophozoites to gametocytes within a single infection decays logarithmically, following an initial burst.
5. The normal sex ratio of macrogametocytes to microgametocytes is 1:1.
6. No correlation between either the red blood cell count or the asexual parasitaemia and the decline in the sexual stage parasitaemia or the loss of ookinete production.
67 Chapter 3
The Enrichment of Plasmodium berghei Gametocytes in vivo and Ookinetes in vitro
Published as: Sexual Development in Plasmodium berghei: the Use of Mitomycin Cto Separate Gametocytes In V/voand In Vitro. International Journal of Parasitology 17,1307-1312
68 3.1 Introduction
Plasmodium berghei in vivo shows an asynchronous mode of parasite growth (Landau & Boulard, 1978) which means that at the time of harvest, whether it be gametocytes from the host or ookinetes from in vitro culture the resulting population consists of mixed blood stage parasites, thus making biochemical studies on the sexual stages difficult. The aim of the work described in this chapter was to establish a simple method by which populations consisting of predominantly gametocytes and/or ookinetes could be obtained with the plan to use this material for stage specific biochemical studies.
It has been shown that mitomycin C, an inhibitor of DNA synthesis, will inhibit the in vitro growth of asexual stages of Plasmodium yoelii nigeriensis when used at concentrations of 10|ig/ml (Toy 6 et alt 1977). In the same study it was shown that this concentration of mitomycin C also kills immature gametocytes, but did not affect the mature sexual stages, as judged by its effect on the microgametocyte, only inhibiting exflagellation at far higher concentrations (>250p.g/ml). It was therefore assumed that no DNA synthesis occurred during gametogenesis nor in the mature gametocyte. Irrespective of its mode of action the potential of mitomycin C selection as a mechanism for in vitro purification of the sexual stages in Plasmodium falciparum has been successfully applied by Sinden et al (1984). It has since been shown that DNA synthesis does occur during gametogenesis (Janse, 1987) which thus casts doubt upon the viability of mitomycin C purified gametocytes.
This chapter describes the use of mitomycin C to select in vivo sexual stages of Plasmodium berghei and the production of highly enriched gametocyte and ookinete preparations from the treated cultures. The viability of these mitomycin C treated stages, as shown by their ability to develop normally in the mosquito host, was investigated and the apparent inactivity of mitomycin C on the biosynthetic incorporation of 32P into the nucleic acids demonstrated.
69 3.2 Materials and Methods
3.2.1 Parasites
This section of the study was in part to test the viability of ookinetes produced from mitomycin C treated gametocytes, and their ability to infect mosquitoes. The full analysis of chapter 2 was not completed at the time of this study thus the collection of material was based on the existing data, which indicated that gametocytes were maximally infective to the mosquito 3-4 days following blood passage. Despite the observations in chapter 2 that maximum ookinete counts were obtained much later in an infection in vivo studies suggest that after day 5 infectivity of gametocytes to mosquitoes is lost. Three days prior to infection Theiler's Original (TO) mice were treated with 0.2ml of phenylhydrazine solution (1.2% (w/v) in physiological (0.9% w/v) saline), to induce reticulocytosis and thus stimulate rapid proliferation of the parasite. Plasmodium berghei gametocytes for ookinete formation in vitro were obtained for this study from mice 3 days after intraperitoneal (i.p.) infection with 0.2ml parasitised blood (10-15% parasitaemia), following the method of Janse et al, (1985).
3.2.2 Gametocyte Production
Mitomycin C for injection (Sigma) was dissolved in distilled water to a final concentration of 2mg/ml. This was given to infected mice by i.p. injection to achieve final concentrations of 15-30jig/g body weight. The blood from these mice was collected, by cardiac puncture into syringes containing 0.1ml heparin solution (15,000 USP units/ml)(Sigma) 24h later, while under terminal ether anathesia and before any clinical effects of mitomycin C became manifest.
3.2.3 The Effect of the Duration of Mitomycin C Treatment
To study the effect of the duration of mitomycin C treatment on the parasitaemia of P.berghei infected mice the animals were given 15^xg/g body weight, via the ip route. The effect on the parasite growth was monitored by studying several different parameters. 1) The percentage parasitaemia in
70 Giemsa stained smears (see section 2. 2.3). 2) The viability of the treated asexual parasites was tested by subpassage. Two drops of tail blood were collected in 0.2ml RPMI 1640. Half was injected via the i.p. route into each of two uninfected mice. A week later blood films were made from each of the mice, Giemsa stained and examined for parasites. 3) The exflagellation index (section 2.2.4) was monitored by counting the number of exflagellation centres in 20 microscope fields (mag 400x) for blood diluted 1:10.
3.2.4 Ookinete Culture
Three days after infection mice were treated with 25jig/g body weight mitomycin C, administered via the ip route. Parasitised blood from these phenylhydrazine/mitomycin C treated animals (3.2.1) was collected by cardiac puncture in heparinised syringes (3.2.1) 24 hours later as it is at this point the asexual parasitaemia reaches its lowest level. Blood was also collected from control animals that had undergone the phenylhydrazine treatment but not the mitomycin C treatment. This was immediately passed through a Whatmann CF11 cellulose columns (5cm x 2cm) equilibrated with ookinete culture medium (2.2.5.1) at 19°C to remove the white blood cells. The eluate from each column was then diluted with ookinete culture medium to give a final blood dilution of 1:10 in a total volume of lOmls. This was maintained in 25ml Falcon culture flasks for 24h at 19°C.
3.2.5 Ookinete Purification
Nycodenz powder (Nyegard, UK Ltd) was dissolved in nycodenz buffer (5mM Tris-HCl pH7.5, 3mM KC1) to make a stock solution of 20% (w/v). The stock solution was then further diluted with ookinete culture medium to a concentration of 10% and 12%, for use as nycodenz cushions for density centrifugation.
Mature ookinete cultures (24h old) were then carefully loaded onto the cushions using pasteur pipettes, so as to give a sharp interface between the nycodenz and the culture. These were then centrifuged at l,600g in a swing out rotor for lOmin. Material was collected from the interface using pasteur pipettes. The cells collected from the interface were then pelleted by
71 centrifugation at 14,000# and resuspended in RPMI1640, washed and then repelleted. This was followed by resuspension and final washing in serum- free RPMI 1640 (section 225.3).
3.2.6 Mosquito Feeds
Mature ookinete cultures were centrifuged at 150# for 10 min at 19°C and the supernatant discarded. The pellet was reconstituted to a 40% haematocrit in FCS and membrane fed to Anopheles stephensi mosquitoes that had been starved for 24h. Membrane feeding was carried out according to Wade (1976). Parafilm membrane was stretched to breaking point over the feeder and the blood loaded. Feeding was carried out at room temperature, with no heating of the membrane feeder and the mosquitoes allowed to feed for as long as they would take food (approximately 2 hrs). Ten days later, during which time the mosquitoes were kept at 19°C and fed on 5% fructose in 0.0005% PABA solution, they were dissected and the gut examined for oocyst formation.
3.2.7 Radiolabelling of the Nucleic Acids
Ookinete cultures were established using parasitised blood from animals that had not previously been treated with mitomycin C. Mitomycin C was added to the cultures in the range of 15-50{ig/ml and were incubated at 19°C for 2h. At the end of this time 20jiCi 32P as inorganic phosphate (Amersham International - PBS13) were added and the culture returned to 19°C for a further 22h incubation, after which all the cells were pelleted and NH 4CI treated to lyse the red blood cells (Winger et al, 1988). To achieve purification the cultures were centrifuged at 1600# for 5 min, the excess medium removed, the cells resuspended in 0.17M NH 4 CI (lOmls NH4CI solution/lml packed cells) and then incubated on ice for 10 min. At the end of this time an equal volume of complete RPMI 1640 was added to halt the lysis reaction and the cells pelleted by centrifugation at 1600# The supernatant was again removed and the pellet resuspended and washed in phosphate buffered saline (PBS) and repelleted. This wash step was then repeated a second time. The remaining parasites were then lysed in STE buffer (lOmM NaCl, lOmM Tris, lOOmM EDTA, ph7.5), freeze-thawed 2 times in liquid nitrogen and the
72 membrane/particle debris removed by centrifugation at 14,000g. The RNA was then removed by treatment with RNAse (rendered DNAse-free by boiling at 100°C for 5 min) for 45 min at 37°C.
Two methods were used to test the incorporation of 32P into macromolecular DNA; 1) trichloroacetic acid (TCA) precipitation and 2) adsorption onto Whatmann DE81 paper.
1) For TCA precipitation 10}il of the lysate was added to a 'carrier' in the form of 10|il lmg/ml solution of salmon sperm DNA (Sigma). The total DNA was then precipitated by the addition of 2mls of ice-cold 10% TCA and incubated for 15 min on ice. The precipitated nucleic acid was then collected by filtering through Whatman GF/A filters. The filters were washed in an excess of TCA followed by a final wash with ethanol at -20°C. After drying incorporation of the radiolabel was determined by liquid scintillation counting (LSC). Samples were counted in glass vials containing lOmls of Cocktail T scintillation fluid (BDH) to an accuracy of 0.7% as determined by the Beckman 520 scintillation counter.
2) The incorporation was also assessed by the absorption of DNA on to Whatmann DE81 paper (Maniatis et al, 1982). After RNAse treatment lO^il aliquots of the lysate were added to the filters. The filters were then washed 6 times in 0.5 M sodium phosphate buffer (5mls/wash) followed by one wash in absolute ethanol at -20°C. After drying the filters were counted as described above for the TCA precipitated samples.
73 3.3 Results
3.3.1 Mitomycin C treatment of P .berghei Parasites
3.3.1.1 The Effect of Time The required duration of mitomycin C treatment (at 15 \Lgf g body weight) to achieve selection of the sexual stages was established in preliminary experiments. The viability of asexual parasites upon subpassage, and gametocyte production shown by the exflagellation index were compared between experimental and untreated control mice. The results of this study, are shown in fig 3.1.
The mitomycin C treatment did not appear to interrupt gametocyte maturation from the younger untreated parasites. Gametocyte numbers were maintained at levels comparable to the controls throughout the first 24h of treatment (figs 3.2a & 3.2b). Similarly mitomycin C failed to affect the functional capacity of the microgametocyte during this first 24h period of treatment, with the number of exflagellations being maintained throughout (fig 3.3). The 24 hour period of treatment was therefore the time of exposure to drug selected in subsequent steps of the study. A reduction in gametocyte numbers was seen to result from the successive waves of schizogony, stemming from the inhibition of early schizont development in successive rounds of asexual development following treatment resulting in the accumulation of small trophozoite stages.
3.3.1.2 Effect of Mitomycin C Concentration The dose administered in the above experiment failed to kill the asexual stages as was expected based on the results obtained by Sinden eta], (1984). Thus a range of higher doses of mitomycin C were administered. The results after 24h treatment are given in table 3.1. These showed that at no concentration tested did the mitomycin C kill all the asexual stages, but a single dose of 25jig/gbody weight reduced the asexual parasitaemia to a minimum without the loss of gametocyte numbers or ookinete production.
74 TOTAL ASEXUAL PARASITAEMIA
1200 ■■ ■O 1000
800 ••
Parasites in Mitomycin 600 10,000 rbc. Control
200
0 -I----1----r 1------1------1------1------1------1------1------1------1------1------1 0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time in hours
Fig 3.1 The total number of P. berghei parasites per 104 red blood cells in Giemsa stained blood smears for a mouse treated with 15pg mitomycin Cg _1 body weight and an untreated control mouse at different times after treatment GAMETOCYTAEMIA-CONTROL
120 -r
Parasites in 10,000 rbc.
0 H----- 1------1------1------1------1------1------1---- 1------1------1------1------1------1 0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time in hours
GAMETOCYTAEMIA-MITOMYCIN
*♦" Macro
•o- Micro
0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time in hours
Fig 3.2 The number of macrogametocytes and microgametocytes per 104 red blood cells (rbc) in Giemsa stained blood smears for a mouse treated with 15}ig mitomycin C g“l body weight and an untreated control mouse at different times after treatment EXFLAGELLATION
Exflagellation centres in 20 microscope fields
0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time in hours
Fig 3.3 The number of exflagellation centres in 20 fields (mag 300x) for a mouse treated with 15mg mitomycin Cg"^ body weight and an untreated control mouse at different times after treatment Concentration Ring Trophozoite Schizont Macrogametocyte Microgametocyte Exflagellation Ookinete Sub passage (fig/g body weight) Index Production 0 9 207 4 38 26 + + 2 /2 15 7 31 5 51 58 + + 2 /2 20 9 34 5 53 22 + + 2 /2 25 0 12 6 57 30 + + 2 /2
30 0 25 0 38 23 + - 2 /2
Table 3.1 Mean number of parasites per 10 4 rbc 24h after treatment with different concentrations of mitomycin C 3.3.2 Further Purification of Treated Parasites
Parasites that had been treated in vivo for 24h with mitomycin C (25|ig/g body wt) were cultured in vitro for ookinete production. The significant difference in the size of the gametocytes and the homogeneous population of small arrested asexual parasites suggested the possibility of purifying the sexual stages by density centrifugation. Nycodenz centrifugation was performed on these cultures (3.2.5). The material collected from the culture/ Nycodenz interface was smeared and Giemsa stained. Nycodenz cushions of 10% and 12% clearly separated the two populations of small (asexual) and large (sexual) parasites and successfully yielded pure gametocyte preparations. Material collected from the 10% interface consisted almost entirely of macro gametes and zygotes (fig 3.4a), while from the 12% cushion the material contained numerous ookinetes in addition (fig 3.4b). This material was therefore ideal for subsequent molecular analysis, subject to the confirmation of parasite viability.
3.3.3 Viability of Mitomycin C Treated Gametocytes
The functional capacity of ookinetes from mitomycin C treated gametocytes has not previously been demonstrated and hence logically questioned. Therefore groups of 25 mosquitoes were fed on cultures of ookinetes derived from parasites treated (25)ig mitomycin C/g body wt) and control (untreated) mice with similar ookinete densities and were subsequently examined for oocyst formation. The results showed no difference between the mean (Xt) number of oocysts (Xt = 40 ±75) and untreated (Xt = 25163). Thus these parasites may be assumed to be of normal viability and therefore useful for subsequent analysis. Unfortunately it is not possible to present the individual counts for the mosquito dissections as they were one of several sets of data that were lost during the laboratory move.
3.3.4 Effect of Mitomycin C on the Incorporation of 32P into Nucleic Adds
To test the effect of the incorporation of 32P into parasite DNA in the presence of mitomycin C (0-50jig/ml) parasites were labelled by the addition of 32P-
79 Fig 3.4 Giemsa stained blood films made from material collected from Nycodenz cushions of 10% (A), showing almost total purity of extracellular macrogametocytes/ zygotes and retort form ookinetes (the occasional uninfected (u) and asexual infected (a) erythrocyte are indicated; and 12% (B) showing banana shaped ookinetes and macrogamete/zygote (m) after centrifugation at 1600# for 10 min. labelled inorganic phosphate to the ookinete culture medium (Section 3.3.7). Following cell lysis samples were treated such that contaminating RNA and protein would be removed from the final preparation (Section 3.3.7). The DNA was then extracted from the parasite either by absoiption onto DE81 paper (Whatman) or by TCA precipitation onto glass fibre filters (Whatman - GF/A) (Maniatis et al, 1982). While the labelled phosphate would be incoiporated in RNA and proteins in addition to the DNA these counts were removed from the samples by the RNAse treatment and washing procedures described in section 3.2.7. Incorporation was measured by liquid scintillation counting (3.2.7). Mitomycin C concentrations up to 50jig/ml culture made no difference to the numbers of counts detected, and it was therefore concluded that mitomycin C, within the concentration range studied did not affect nucleic acid synthesis in the sexual stages of P.berghei (table 3.2). The results obtained by the two different methods show a marked difference in the number of counts detected. However, the samples were not taken from a single radiolabelled culture and it is highly probable that the absolute levels of 32P incorporation were different in the two experiments. Although the two sets of data cannot be directly compared they both show that mitomycin C has very little effect on the incorporation of 32P into the parasite DNA within the range of concentrations tested.
82 Table 3.2 Incorporation of 32P into parasite DNA when treated with mitomycin C and absorbed on DE81 paper and TCA precipitated onto GF/A filters. All counts were performed to an accuracy of 0.7%
Mean number of incorporated counts Mitomycin C TCA Precipitated DE81 Absorption Concentration (}ig/ml) Counts 0 3120 ± 1550 212 ±8 10 3083 ± 508 183 ± 35 15 3054 ± 402 201 ± 48 20 2532 ±147 173 ± 22 25 3102 ± 247 139 ±8 30 3205 ± 203 206 ± 38 35 1382 ± 62 145 ± 17 40 2994 ± 219 167 ± 33 45 2884 ± 447 237 ± 55 50 2663 ± 513 147 ± 0.5
83 3.4 Discussion
Methods for the purification of P.falciparum gametocytes have been reported by Sinden etal (1984) who successfully treated mixed cultures of asexual and sexual stages with mitomycin C, and Ponnudurai et al (1986) who used N- acetylglucosamine to inhibit reinvasion of the red blood cells by the asexual merozoites during the 7 -12 day period of gametocyte development. Unfortunately, neither method can be applied to P. berghei in vivo as the time for gametocyte maturation (24h) does not allow a long enough treatment period to intervene because the growth time of the asexual and sexual stages, 24 and 26 hours respectively, is not significantly different from that of the longevity of the mature sexual parasite (26 hrs). Thus at the latest time of harvesting (52 hrs post treatment) a mixed population of parasites would still be present.
The mitomycin C/Nycodenz treatment described here for P. berghei provides an alternative to the very time consuming and elaborate system of gametocyte enrichment by synchronization /centrifugation described by Mons et al (1985) (see section 5.2.1). These very different methods of purification each has its advantages and disadvantages. The synchronization method produces parasites that show a high degree of synchronicity from the early trophozoite stage to the mature gametocyte stage. This synchronization breaks down with the onset of subsequent rounds of schizogony, at approximately 26 hours. However, in the early part of the synchronization it does allow the study of specific parasite stages at a known time into their development. The method also excludes drug treatment and therefore there is no possibility of altering the natural path of development due to the effects of such chemicals. The method is however, labour intensive, going through two stages in vivo and one stage of in vitro culture and only yields low numbers of parasites, and the procedures used induce their own metabolic artefacts and parasite death due to their concentration on gradient interfaces and the repeated manipulation.
On the other hand the method described here, using mitomycin C has the major disadvantage of involving drug treatment. However, it was confirmed that the sexual stages do not loose their viability, remaining infective to mosquitoes, thus any effects are either minimal or rapidly reversible. An additional disadvantage is that it does not yield purified parasites at an early
84 stage in development as was seen with the "Mons-technique". It does however, yield material that may be described as being synchronised at the ookinete stage. The main advantage of this mitomycin C based purification is that it has been easily incorporated into routine gametocyte and ookinete cultures and yields material on a scale suitable for biochemical analysis. Parasites produced by this method therefore have been used in preference to the "Mons-technique" in experiments described in subsequent chapters.
Mitomycin C is a rapidly acting inhibitor of DNA synthesis that acts by covalently cross-linking the two DNA strands due to its action as a bifunctional alkylation agent (Gale et al, 1981). Unlike the observations of Sinden et al, (1984) on P.falciparum mitomycin C treatment of P. berghei in vivo did not have the immediate and devastating effect described by them in vitro and at no point were all the asexual stages completely killed, as was shown by the positive subpassage at all times. However mitomycin C did retard the growth of these asexual stages, reducing them to minimal numbers after 24h treatment. This difference in efficiency between the two systems however may be due to a reduced bioavailability of the drug in the in vivo system presented here compared to thein vitro systems of Toy6 et al (1977) and Sinden et al (1984). The immediate decline in the numbers of asexual parasites following treatment was followed by a subsequent rise in the asexual parasitaemia, with the trophozoite number peaking at 36h. The activity of mitomycin C shows a high degree of specificity, acting against the early erythrocytic stages (rings and trophozoites) of the life cycle, as demonstrated by the block development of in vivo parasites beyond the trophozoite stage while still allowing schizont and gametocyte development to continue. As mitomycin C is an inhibitor of DNA synthesis it would be expected that this was the method by which parasite development was inhibited. Because mitomycin C arrested, but did not kill asexual parasites, the arrested parasites persisting beyond the effective mitomycin C blockade, would subsequently re enter the asexual schizogonic cycle as the drug level declines. A similar observation has been made for P.falciparum cultures treated with aphidicolin, another DNA synthesis inhibitor (Inselburg & Banyal, 1984).
It has been shown here that in P. berghei mitomycin C does not block the biosynthetic incorporation of 32P into nucleic acids, and therefore it would appear that it does not block DNA synthesis in this species of Plasmodium. Although the evidence is consistent with this view it is possible that the 32P is
85 not incorporated into the DNA and that the counts detected have come about as a technical artefact. This seems unlikely, as two different methods, each designed to obtain DNA that was free of contamination from other likely sources of phosphate, eg, RNA and phospholipids, were used. However, the purity of the DNA samples were not demonstrated experimentally, but phenol/chloroform extraction followed by separation of gels would be able to confirm whether this was the case.
The idea that mitomycin C is inactive against DNA synthesis is further supported by the observation that exflagellation of gametocytes continues in the presence of mitomycin C, a time at which DNA synthesis has been demonstrated by microfluorometric analysis (Janse et al, 1986a). Janse et al (1986) have also shown that mitomycin C is also inactive against other DNA synthesising stages and have similarly concluded that the drug does not act directly against DNA synthesis. Alternatively, DNA synthesis in Plasmodium follows an alternative pathway which is resistant to mitomycin C, as has been suggested for E. coli infected with the phage T2r (Sekiguchi & Takagi, 1959), where infection with T2r can prevent/restore inhibition by mitomycin C. There are however alternative explanations, which are 1) that the drug is active but does not reach its site of action (the DNA), or 2) that the drug is inactive because the parasite metabolic pathways prevent it from existing in its active (reduced) state or 3) that its stage specific activity is achieved via some unknown route.
1) It has been suggested that the failure of mitomycin C to act against all DNA synthesising stages is due to altered membrane permeability to the drug (Janse et al, 1986 ). Permeability of the membrane would however need to be stage specific allowing the entry of mitomycin C to some but not all stages. This hypothesis could be tested using either a fluorescence- or radiolabelled form of the drug to determine the distribution of mitomycin C within a mixed parasite population.
2) The activity of mitomycin C is also dependent upon the drug being in its reduced form (Moore, 1977). It has been suggested that certain stages of the plasmodial life cycle may lack all the NADPH-generating activity of the pentose phosphate pathway, the necessary quinone reductase and malic enzyme, thus preventing the reduction step (Janse et al, 1986). In addition to these enzymes any reductase or reducing agent would be able to activate
86 the mitomycin C (Tomasz et a\ 1987). Separation of the parasites in sensitive and non-sensisitive stages followed by testing for these reducing enzymes in each population may determine whether these enzymes were present in rings and trophozoites while being absent in the schizont and gametocyte stages. Whilst the reduction of the drug is necessary it has been observed additionally that mitomycin C toxicity decreases at high oxygen concentrations. As the parasites are erythrocytic in origin it is possible that they have evolved mechanisms to exist in an environment in which there is a high oxygen partial pressure. To avoid the toxic effects of oxygen these parasites may show highly efficient reducing pathways. These pathways might be expected to bring about the highly efficient reduction of mitomycin C. If however, as a result of parasite growth, the O2 carrying capacity of the infected erythrocyte were lost or reduced and these parasites (schizonts and gametocytes) would be living under conditions of lower oxygen concentrations. Under these conditions the efficiency of these parasite reductive pathways may also be reduced, thus decreasing the efficiency of the reduction (activation) of mitomycin C, causing the apparent difference in the sensitivity of the different parasite stages. Again testing for the reducing enzymes in the two populations, sensitive and insensitive, may give an indication as to the likelyhood of this as would testing for the O 2 carrying capacity of the infected blood cells.
3) Finally, mitomycin C may be inhibiting parasite growth via some, as yet, unknown pathway. It is possible that it is an effect of the general toxicity of the drug or that it acts in a manner that has not been described in other systems. This however still remains to be discovered.
3.4.1. Conclusion
The work described in this chapter details a simple method for the successful production of high yields of Plasmodium berghei gametocytes and ookinetes.
87 Chapter 4
The Effect of Metabolic Inhibitors on the Development of Plasmodium berghei Gametocytes and Ookinetes
88 4.1 Introduction
Despite the unexpected failure of mitomycin C to act against DNA synthesis in P. berghei, the effects of other drugs on the development of P. berghei were studied. The use of metabolic inhibitors with alleged specific effects can be a useful method to study metabolic process underlying a morphological change. Toy6 et al (1977) described the use of inhibitors to study the requirement for DNA, RNA and protein synthesis in gametogenesis (exflagellation). To extend this further here, a range of drugs are tested not only against microgametogenesis but also ookinete development.
The thinking behind this study was that by the use of specific metabolic inhibitors affecting transcription, translation and protein synthesis it should be possible to determine particular metabolic process underlying the visible events exflagellation and ookinete formation. Since DNA synthesis, RNA synthesis and protein synthesis are closely linked ("DNA makes RNA makes protein" (Hunt et al, 1983)) it was hoped that the effects of inhibitors to any of these processes could be seen by studying the incorporation of radiolabel into proteins during synthesis. Thus, If the addition of DNA synthesis inhibitors to the culture brought about changes in the spectrum of proteins synthesised then they would be expected to include proteins that are intimately associated with DNA or its replication, eg the histones. In the case of RNA synthesis inhibitors the synthesis of the proteins would be expected to continue for all those proteins for which the coding mRNA was present at the time of radiolabel addition. However, one would anticipate that for protein species where translation takes place immediately following transcription, or where the mRNA has a short half life, synthesis would show reduced or zero incorporation of the label. Following the identification of protein synthesis during gametogenesis and ookinete development (Kaushal et at, 1983) and the inhibition of exflagellation by protein synthesis inhibitors (Toy 6 et al, 1977) a single protein synthesis inhibitor was included as a form of positive control. It was expected that protein synthesis would be dramatically affected by cycloheximide.
While certain effects were expected from the drugs tested, based on their reported action in mammalian/bacterial systems, ie inhibition of DNA, RNA and protein synthesis and the inhibition of microtubule assembly, some intracellular organelles have been shown not to be affected by metabolic
89 inhibitors that are active against the cell in general, eg cycloheximide does not inhibit mitochondrial protein synthesis (Alberts et al, 1983). Reasons for this differential activity could be that 1) many organelles are bounded by their own membranes which show different permeability properties to the cell/nuclear membranes, thus preventing entry of the drug or 2) the metabolic processes in the organelles follow alternate routes, ie, mitochondrial DNA is more akin to bacterial DNA than eukaryotic DNA and is therefore not affected by certain DNA synthesis inhibitors that are active against eukaryotic DNA and vice versa..
It was also hoped to identify proteins which may be involved with particular events in the life cycle by comparing changes in protein synthesis in the presence of inhibitors with alterations in the morphology/ development of the parasite. By observing the effects of the various metabolic inhibitors on the development of the parasite at the morphological level it was hoped to be able to determine the underlying biochemical events. Evaluation of the effects of these inhibitors may identify stages in the parasite life cycle where a particular metabolic activity or protein appear critical for further development and are worthy of further investigation.
The drugs tested fall into four categories: (1) DNA synthesis inhibitors, (2) RNA synthesis inhibitors, (3) Protein synthesis inhibitors and (4) anti microtubule agents. For both the DNA and RNA synthesis inhibitors a range of drugs was used, thus within a group all act against the same process but through a range of different mechanisms, which guards against the wrong conclusions being reached through a single drug acting in a non-attributable manner.
The DNA synthesis inhibitors tested were, mitomycin C, aphidicolin and hydroxyurea. Mitomycin C, has been described as a rapidly acting, selective inhibitor of DNA synthesis, that acts by covalently cross-linking the two DNA strands due to its action as a bifunctional alkylating agent (Gale et al, 1981), although the incorporation of 32p into P. berghei DNA was not affected by the addition of mitomycin C (See Chapter 2 and Janse et al, 1986). Aphidicolin is an inhibitor of DNA synthesis which occurs naturally. It is reported to act by blocking DNA polymerase a by mimicking the purine nucleotides as a substrate for DNA synthesis and forming non-Watson-Crick bonds between the inhibitor and template pyrimidine at a site that bears a template-primer
90 structure. The result is the formation of a tight but reversible complex consisting of inhibitor, template and enzyme, thus inactivating the sequestered enzyme (Gale et al, 1981). Hydroxyurea has been demonstrated, in numerous systems, to be a potent inhibitor of DNA synthesis, but in addition has various effects on protein synthesis, particularly that of lysine- rich histones, while having little or no effect on RNA synthesis (Yarbro, 1967). Hydroxyurea acts by inhibiting ribonucleotide reductase (Young & Hodas, 1964). This inhibits the reduction of ribonucleoside diphosphate to deoxyribonucleoside diphosphate at the level of the ribonucleotide, thus preventing DNA synthesis.
The RNA synthesis inhibitors tested were cordycepin (3' deoxyadenosine), a- amanitin and actinomycin D. Cordycepin is an analogue of the nucleotide adenosine, and works by blocking the elongation of RNA chains. After intracellular phosphorylation to the triphosphate cordycepin becomes incorporated into the growing RNA chains, however cordycepin lacks a 3' hydroxyl group, thus preventing any further phosphodiester bonds from forming (Penman et al, 1970; Gale et al, 1981). Cordycepin has been shown to affect only the synthesis of rRNA precursors. Messenger RNA and hnRNA synthesis remains unaffected by the drug while it has little or no effect on DNA or protein synthesis. oc-amanitin is a potent toxic peptide produced by the mushroom Amanita phalloides. Its toxicity to animal cells is related to inhibition of RNA synthesis due to its action on nuclear DNA-dependent RNA polymerase II (pol II) while having no effect on pol I and only affecting pol III at high concentrations (Gale et al , 1981). Actinomycin D inhibits both nuclear and the cytoplasmic RNA synthesis by binding to DNA, preventing the movement of RNA polymerase, thus preventing chain elongation. At high concentrations it also inhibits DNA and phosphate metabolism.
The only protein synthesis inhibitor tested was cycloheximide, but the effects of other protein synthesis inhibitors were described by Toy 6 et al (1977) and were found to inhibit exflagellation. Cycloheximide acts by inhibiting the enzyme peptidyl transferase, thus preventing polypeptide chain elongation on cytoplasmic ribosomes, while not affecting that which occurs on the mitochondrial ribosomes (Alberts et al, 1983).
91 Colchicine is an alkaloid derived from the autumn crocus, Colchicum autumnale, and has been shown to interfere with many cellular functions involving microtubules. Colchicine acts by interfering with the tubulin equilibrium by binding to free tubulin subunits in the cytoplasmic pool at a concentration of 1 mole of colchicine to 1 mole of tubulin (Wilson & Meza, 1973). This brings about a change in the delicate equilibrium established between free unpolymerised tubulin and the polymerised form in the microtubules, thus causing depolymerisation of susceptible microtubules. The difference in the stability of the microtubules also has an effect on the activity of colchicine. For example microtubules found in cytoplasmic organelles, such as spindle microtubules, are highly sensitive to depolymerisation under the influence of colchicine, while those found in cilia and flagella do not readily depolymerise.
The effects of the drugs were studied at two levels. The morphological events of exflagellation and ookinete development were examined by light and electron microscopy, and the biochemical level by looking at the effect of the drugs on protein synthesis during ookinete development.
92 4.2 Materials and Methods
4.2.1 Micro Drug Culture Technique (fig 4.1)
In order to allow for the rapid assessment of metabolic inhibitors on exflagellation and ookinete development a new system of microcultures was established. The microcultures were carried out as follows:
Serial dilutions of the drugs to be studied were set up in 20|il complete RPMI 1640 (2.2.5.1) in a microtitre plate (plate 1) and warmed to 37°C. Two similar plates, containing the drug solutions to be tested, were also set up and equilibrated at 19°C (plates 2 and 3). Parasitised blood (2.2.1) obtained by cardiac puncture from phenylhydrazine treated mice as described in sections 2.2.1. and 2.2.2, was carefully maintained at 37°C and added to plate 1 (20|il/well). The plate was then returned to the 37°C incubator for lh. At the end of this incubation the contents of plate 2 (160jil drug treated RPMI 1640/well at 19°C) were transferred to plate 1, bringing about a rapid drop in temperature to 19°C, thus inducing exflagellation some 10-15 min later. The final preparation shows a blood dilution of 1:10 in RPMI 1640 in a total volume of 200}il. Before incubating the plate at 19°C for 24h 5jil aliquots of each sample (now in plate 1) were transferred to plate 3 (which contained 195|il drug treated ookinete medium/well), resulting in a blood dilution of 1:400 in a total volume of 200^.1. After centrifugation of plate 3 at 150g for 10 min at 19°C each well was scored directly for exflagellation using phase contrast optics in an inverted microscope at 320x magnification. Owing to the limitations of time, exflagellation being a short lived event, it was only possible to score each well as positive (+) or negative (-). To allow for the comparison of three runs these were subsequently awarded the values of (+) = 2 and (-) = 0 (table 4.2). These values of (0) and (2) were chosen to coincide with the total score values given for ookinete production, where a more thorough examination was possible.
At the end of the 24h incubation the excess medium was removed from the cultures in plate 1 by inclining the plate and careful removal of the spent medium by pipette, and blood films were made. These were then stained with Giemsa (section 2.2.3) and then examined for ookinete production. The slides were scored (+), good ookinete production; (+/-), low numbers of 'sickly' ookinetes; (-) no ookinete production. As before each was awarded a
93 DRUG CULTURE TECHNIQUE
Parasitized blood
Add blood______^ Plate 1 y Incubate at 37t) for 1h
Add drug solns Plate 2 to plate 1
Plate 1 Add 5u.l of cultures ------► Plate 3 l from plate 1 Incubate at 19°C for 24h
Centrifuge at 1 150 g for Remove excess 10 min medium 1 Make & Giemsa stain blood film s i Examine for Examine for ookinete exflagellation production
j
Score (+), (-) Score (+) or (+/-) or (-)
Fig 4.1 score; (+) = 2, (+/-) = 1 and (-) = 0. Again the accumulated total was recorded for three runs (table 4.3).
The effect of the drugs on ookinete formation specifically, was tested by the addition of drug treated medium to the culture after the gametocyte infected blood had had 2h incubation at 19°C, ie following the normal completion of exflagellation and fertilization (Sinden, 1983). The cultures were then left at 19°C for a further 24h before examination, which followed the scheme described immediately above. The accumulated total for three runs is given in table 4.4.
The approximateED sqs were then calculated for each drug by taking the mid point drug dilution between full ookinete production and total inhibition of ookinete production.
4.2.2 Electron Microscopy
Ookinete cultures were set up in the presence of the lowest concentration of each drug that would inhibit their formation. Parasitised blood was diluted 1:2 in complete ookinete medium (2.2.5.1) with the added drug and incubated at 37°C for lh. Ookinete medium, equilibrated at 19°C was then added to give a final blood dilution of 1:10 and resulted in a rapid drop in temperature to 19°C, thereby stimulating exflagellation 10 -15 min later. The cultures were then incubated at 19°C for 24h, after which each sample was processed for electron microscopy, as described by Sinden et al (1985a).
Samples were fixed in 1.25% (v/v) glutaraldehyde in 0.1 M cacodylate containing 0.15M sucrose, pH7.4 for 30 min at 4°C. After fixing they were then washed three times in 0.1M cacodylate/0.15M sucrose buffer at 4°C and post-fixed in 1% osmium tetroxide in the same buffer. Following dehydration in acetone the samples were embedded in araldite, sectioned and stained with Reynold's lead citrate and 8% methanolic uranyl acetate, then examined in a Philips EM 300 (Sinden et al, 1985a).
95 4.2.3 35s-Methionine Labelling of Ookinete Cultures
'Ookinete 1 cultures, using blood from the mixed infection (2.34L) and the asexual infection (2.33L), were set up as in (2.2.4) with the notable exception that methionine-free RPMI 1640 (2.2.5.4) was used. It was found that to prevent initiation of gamete formation the Whatmann CF11 column had to be equilibrated at 37°C instead of 19°C, and the eluate diluted 1:5 to allow for a further dilution of the blood after incubation. The culture was then incubated for 2h at 37°C in the presence of the drug to be tested. After the incubation the blood was further diluted to 1:10 with methionine-free RPMI 1640 to bring about a rapid drop in temperature to 19°C, thus inducing exflagellation 10 -15 min later. 5jiCi/ml of 35s-methionine (Amersham International - SJ235) were added to the culture at this time.
At the end of the 24h labelling period the cells were pelleted, washed once in serum-free methionine-free RPMI 1640 and then lysed with NH 4 CI, to remove the bulk of unwanted red blood cells, giving a cell population consisting mainly of extracellular parasites (fig 4.2). For NH 4 CI lysis the cells were pelleted at 160g and the resulting supernatant removed with a pasteur pipette. The cells were then resuspended in ice-cold NH4 CI (0.17 M) (10ml NH4 CI per 1 ml of packed blood) and incubated on ice for 10 min. At the end of this incubation an equal volume of RPMI 1640 was added to halt the lysis reaction and the cells pelleted by spinning at 160gfor 10 min at 4°C. The resulting pellet was then washed twice in PBS (Winger et al, 1988).
The parasites were then solubilised in lysis buffer (150mM NaCl, 10 mM Tris, 1% Triton X-100, 2mM PMSF) for 10 min at 4°C and clarified by centrifugation at 14,000g for 10 min. The supernatant was collected and stored frozen at -20°C until needed.
4.2.4 Analysis of Protein Samples
After LSC of TCA precipitated protein samples (2.2.7) to determine the incorporation of 35s-methionine into the total protein samples (precipitable counts), aliquots of protein solution containing 50,000 cpm were ethanol precipitated by the addition of 3 volumes of ethanol at -20°C. The samples were left to stand for at least 2h at -20°C, to obtain maximum protein
96 FIG 4.2 Giemsa stained blood film made from 24 hour P. berghei ookinete cultures after lysis with ammonium chloride. * precipitation. The resulting precipitate was pelleted by centrifugation at 14,000# for 5 min and vacuum dried. The precipitate was resuspended in 150|il of Laemmli sample buffer (LSB - 0.01 M Tris, 0.001 M EDTA, 5% SDS, 5% 2-mercaptoethanol, 10% glycerol, 2m M PMSF). The samples were then boiled for 5 min in LSB, which also served to denature the proteins and provide an even negative charge on the molecules, thus allowing separation due to size rather than charge. Separation was achieved by 15% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) based on the method of Laemmli (1970).
4.2.5 SDS-PAGE
Stock acrylamide was prepared by dissolving acrylamide (Sigma) (30% w/v), bis-acrylamide (Sigma) (0.9% w/v) in distilled water in the presence of MBI ion exchange resin (Sigma) (0.9%w/v). This was then filtered through Whatman filter paper number 1 and stored in a dark bottle at 4°C until needed.
To prepare 15% polyacrylamide gels 50 ml of stock acrylamide, 25ml separating buffer (1.5mM Tris-HCl, pH8.8) and 24ml distilled water were mixed in a Buchner flask and then degassed under vacuum.
Immediately prior to pouring O.lg SDS, 0.1ml TEMED and 1ml ammonium persulphate solution (AMPS - 10%w/v) were added to the acrylamide solution and gently mixed. This was then poured into 3 mm cassettes constructed using the Biorad protein I system, to within 3 cm of the top of the plates, overlaid with distilled water to give and even surface and left to set. All glassware used in this system had previously been cleaned in detergent solution followed by absolute ethanol before pouring.
5% stacking gels were prepared as above with the following modifications: 8.3ml stock acrylamide, 12.5 ml stacking buffer (0.5mM Tris-HCl pH6.8) and 28.7ml distilled H 2O. SDS (0.05g), TEMED (0.05ml) and AMPS (0.5ml) were added. Just prior to running the stacking gel was poured onto the top of the set separating gel, and a comb inserted to form the wells. The gel was then left to set.
99 The comb was carefully removed and the samples loaded into the wells using a Gilson pipette. Separation was carried out overnight at 50V and 8mA, with cooling (tap water 4 - 10°C).
After separation the gels were carefully removed from the cassettes and the protein bands visualised by Coomassie blue staining (4.2.6).and the gels were then prepared for autoradiography (4.2.7).
4.2.6 Coomassie Blue Staining of Gels
Coomassie Brilliant Blue (R250) (0.2g) was dissolved in 250ml staining solution (50% methanol (v/v), 10% glacial acetic acid (v/v) made up to 250ml with distilled water). Undissolved Coomassie was removed by filtering.
Gels were stained by incubating in the Coomassie blue stain for 2h at room temperature with gentle rocking agitation. The staining solution was then removed and the gels rinsed in 5-10ml of the first destain solution (50% methanol (v/v), 10% glacial acetic acid (v/v) in distilled water) and then destained for a further 2 times in this solution, first for 2h and then overnight, both with gentle rocking. The gels were then transferred to a second destain solution (5% methanol, 10% glacial acetic acid in distilled water) for a minimum of 24h before drying.
4.2.7 Drying and Autoradiography of Gels
The Coomassie blue stained gels were soaked in preservative (30% methanol (v/v), 10% glacial acetic acid (v/v), 10% glycerol in distilled water) for lh to prevent the gel from cracking on drying followed by 30min in Amplify (Amersham International), to enhance the effect of the emitted p-radiation on later exposure to X-ray film, before being dried using a slab-gel-dryer according to the manufacturers technical information.
Detection of incorporated label was carried out in cassettes using Fuji X-ray film, that had been presensitised by flashing (Laskey & Mills, 1977), at a temperature of -80°C. An image on film is formed when two adjacent silver atoms are raised to a higher energetic state and form a stable complex. This
100 reaction is however reversible, but the speed with which the silver atoms return to the lower energy level can be reduced by incubating at -80°C, thus at this temperature there is an increased chance of obtaining an image at low levels of isotope incorporation. The film was subsequently developed using universal developer (1:4) at 20°C for 5min. The film was washed briefly before being fixed in Hypam fixer for 2 min, then washed in cold running water for 20 min and dried.
4.2.8 Densitometry of Autoradiographs
The autoradiographs were scanned and analysed using a Zeineth Soft Laser scanning densitometer, model SL-2D and the supplied software, before being printed.
101 4.3 Results
4.3.1 Total Protein Synthesis
Scanning densitometry of autoradiographs from ookinete cultures labelled with 35s-methionine clearly shows, where examination of the autographs by eye does not (figs 4.3 & 4.4), that the profile of proteins synthesised by the asexual (fig 4.5a) and mixed (4.5b) lines of infection are different. The molecular weights and origins of the identified proteins are summarised in table 4.1. Proteins found exclusively in the mixed culture during gametogenesis and formation, as identified by molecular weight are: 105 kD, 89 kD, 87 kD, 75 kD, 71 kD, 52 kD, 43 kD, 25 kD, 21 kD and 16kD. Of these proteins the 71kD (Section 4.3.4.1) and the 21kD (See Chapter 5) proved of note.
4.3.2 Exflagellation
Mitomycin C failed to inhibit exflagellation, even at a concentration of 5.98 x 10"4m . Hydroxyurea was similarly ineffectual giving an approximate ED 50 of 1.67 x lO'^M. Contrasting to this aphidicolin inhibited exflagellation showing an ED50 of 5.76 x 10'^M (at this concentration of aphidicolin the DMSO solvent was 32x more dilute than its minimum inhibitory concentration). Therefore a specific inhibitory effect of the drug is shown. Both cordycepin and actinomycin D were consistently effective in preventing exflagellation giving approximate EDsos of 4.72 x 1(H m and 4.95 x 10'^M respectively. On the other hand a-amanitin showed no inhibition of exflagellation at concentrations of 3.98 x 1 (H m . The only protein synthesis inhibitor tested was cycloheximide, which invariably inhibited exflagellation with and ED 50 of 4.4 x 10_^M (table 4.2). Colchicine was a most effective inhibitor of exflagellation giving an ED50 of 4.88 x 10"^M, which is in close agreement with that found by Toye et al (1977) and Sinden et al (1985).
102 Table 4.1 Computer identified proteins from densitometer scans of 35s- methionine labelled proteins from ookinete cultures of mixed and asexual infections. Localization of the proteins determined by 125I surface labelling of parasites (see chapter 5)
Protein M.wt (kD) Parasite stage Location in cell 112 Asexual Internal 105 Gametocyte / ookinete Internal 98 Unknown Surface 95 Asexual Internal 89 Gametocyte / ookinete Surface 87 Gametocyte/ookinete Surface 83 Asexual Internal 75 Gametocyte / ookinete Surface 71 Gametocyte / ookinete Internal 66 (see section 5.3.1.3) Asexual (trophozoite) Surface 60 Asexual Surface 53 Asexual Surface 52 Gametocyte/ ookinete Surface 45 (see section 5.3.1.2) Asexual / sexual/host Internal 43 Gametocyte / ookinete Surface 40 Asexual Internal 39 Asexual Surface 36 (see section 5.3.1.2) Asexual/host Internal 34 (see section 5.3.1.1) Asexual/host Surface 31 Asexual Surface 27 Asexual/host Internal 25 Gametocyte/ookinete Surface 23 Asexual Surface 21 (Section 5.3.2) Ookinete Surface 16 Gametocyte/ookinete Internal
103 Table 4.2. The effect of metabolic inhibitors on exflagellation of Plasmodium berghei, as assessed by the rapid assay method. A doubling dilution series for each drug was set up in wells numbered 1-12 and each well was scored for exflagellation and awarded a value of (2) for positive exflagellation and (0) for no exflagellation. The total for three runs was the obtained to give readings between 0 and 6 (6 = normal development; 0 = no development). An approximate ED50 was then established by taking the mid-point between normal development and no development.
Well Number Drug Initial 1 2 3 4 5 6 7 8 9 10 11 12 Cone
Control - 6 6 6 6 6 6 6 6 6 6 6 6 Mitomycin C 334.3jiM 6 6 6 6 6 6 6 6 6 6 6 6 Hydroxyurea 76.1mM 0 0 4 6 6 6 6 6 6 6 6 6 Aphidicolin 338.5 jiM 0 0 0 0 0 0 0 4 6 6 6 6 DMSO 78.1mM 0 0 0 6 6 6 6 6 6 6 6 6 Cordycepin 251.2jiM 0 0 4 6 6 6 6 6 6 6 6 6 a-Amanitin 108.0|iM 6 6 6 6 6 6 6 6 6 6 6 6 Actinomycin D 199.0{iM 0 0 0 2 6 6 6 6 6 6 6 6 Cycloheximide 281.3jiM 0 0 2 6 6 6 6 6 6 6 6 6 Colchicine 399.4JJ.M 0 0 0 0 0 0 0 2 4 6 6 6
104 Table 4.3. The effect of metabolic inhibitors on ookinete production in Plasmodium berghei, as assessed by the rapid assay method. A doubling dilution series for each drug was set up in wells numbered 1-12 and ookinete cultures run for 24h (section 4.2.1). After the preparation of Giemsa stained smears from each well the slides were scored for ookinete production and awarded values of (2) for good ookinete production, (1) for low numbers of sickly ookinetes and (0) for no ookinete production. The total for three runs was obtained to give readings between 0 and 6 (6 = normal development; 0 = no development). An approximate ED50 was then established by taking the mid-point between normal development and no development.
Well Number Drug Initial 1 2 3 4 5 6 7 8 9 10 11 12 Cone
Control - 6 6 6 6 6 6 6 6 6 6 6 6 Mitomycin C 334.3|iM 0 0 1 4 6 6 6 6 6 6 6 6 Hydroxyurea 76.1mM 0 0 0 0 0 2 0 3 4 5 5 6 Aphidicolin 338.5|iM 0 0 0 0 0 0 1 2 3 4 4 6 DMSO 78.1mM 0 0 0 0 5 6 6 6 6 6 6 6 Cordycepin 251.2nM 0 0 0 0 0 0 0 2 3 3 3 4 oc-Amanatin 108.0jiM 0 0 0 1 3 3 3 2 2 3 6 6 Actinomycin D 199.0JJ.M 0 0 0 0 0 0 0 0 1 2 3 3 Cycloheximide 281.3|iM 0 0 0 0 0 0 0 0 0 1 1 1 Colchicine 399.4jiM 0 0 0 0 0 0 0 1 2 4 4 6
105 Table 4.4. The effect of metabolic inhibitors on ookinete production subsequent to exflagellation in Plasmodium berghei, as assessed by the rapid assay method. A doubling dilution series for each drug was set up in wells numbered 1-12 and ookinete cultures run for 24h (section 4.2.1) however, in this case the drug solutions were only added after the first two hours of ookinete formation, in order to avoid affecting the critical periods associated with exflagellation and fertilization. After the preparation of Giemsa stained smears from each well the slides were scored for ookinete production and awarded values of (2) for good ookinete production, (1) for low numbers of sickly ookinetes and (0) for no ookinete production. The total for three runs was the obtained to give readings between 0 and 6 (6 = normal development; 0 = no development). An approximate ED50 was then established by taking the mid-point between normal development and no development.
Well Number Drug Initial 1 2 3 4 5 6 7 8 9 10 11 12 Cone
Control - 6 6 6 6 6 6 6 6 6 6 6 6 Mitomycin C 334.3|xM 3 5 6 6 6 6 6 6 6 6 6 6 Hydroxyurea 76.1mM 5 5 6 6 6 6 6 6 6 6 6 6 Aphidicolin 338.5{iM 0 0 0 0 0 5 5 5 6 6 6 6 DMSO 78.1mM 0 0 0 0 0 6 6 6 6 6 6 6 Cordycepin 251.2|iM 0 0 0 0 0 0 0 2 3 3 3 4 a-Amanatin 1Q8.0HM 0 0 0 1 1 3 4 5 6 6 6 6 Actinomycin D 199.0|iM 0 0 0 0 0 0 0 0 1 2 3 3 Cycloheximide 281.3JJ.M 0 0 0 0 0 0 0 0 0 0 0 1 Colchicine 399.4|iM 0 0 0 0 0 1 4 6 6 6 6 6
106 Fig 4.3 Autoradiograph of an asexual P. berghei infection labelled with 35S-methionine during ookinete culture in the presence of selected metabolic inhibitors.
Lane 1 Control Lane 2 Aphidicolin Lane 3 Hydroxyurea Lane 4 Cordycepin Lane 5 Actinomycin D Lane 6 a-amanatin Lane 7 Cycloheximide Lane 8 Colchicine H- FIG 4.4 Autoradiographs of a mixed P. berghei infection labelled with 35S methionine during ookinete culture in the presence of selected metabolic inhibitors. Lane 1A Control Lane 2A Aphidicolin Lane 3A Hydroxyurea Lane 4A Cordycepin Lane 5A Cycloheximide Lane 6A Colchicine
Lane IB Control Lane 2B Cordycepin Lane 3B Actinomycin D Lane 4B a-amanatin A 5 r—i A —L.i--’”i-..•*'
112 .98 95 1 _ 6 6 -66 45 45: 43
36: 36 34 29- 24- = .23
— ^ 21
Fig 4.5 Densitometer traces of 35s-methionine labelled ookinete cultures of an asexual infection (A) and a mixed infection (B). Labelled markers indicate the relative positions of the molecular weight markers. The unlabelled tabs show the positions of the computer recognised peaks which are numbered. 4.3.3 Ookinete Development
4.3.3.1 Electron Microscopy of Untreated Control Cultures. The development of the control preparations was consistent with the previous studies of Sinden et al (1985a). The preparations showed normal apical complex assembly (fig 4.6B & C) development of the crystalloid (fig 4.6A), associated vesicle structures and meiosis (fig 4.6D). An additional observation, made possible by the presence of unexflagellated, but extracellular microgametocytes was that these cells will also bind to the 'macrogamete' rafts that form in culture, and will equally bind to each other (Sinden et alt 1987). Thus all extracellular gametocytes (or their products) appear to have specific and mutual binding affinity. All the drug treated cultures were compared with this untreated control.
4.3.3.2 DNA Synthesis Inhibitors 4.3.3.2.1 Light Microscopy Examination of the Giemsa stained blood films taken at the end of the 24h culture period showed that all the inhibitors tested blocked the development of the ookinete from the fertilized zygote more effectively than they block exflagellation (see section 4.3.2). The three drugs tested here again showed very varied abilities in inhibiting ookinete production giving approximate ED5QS of:- mitomycin C, 1.12.x 10"^M; hydroxyurea, 3.8 x 10“7M; and aphidicolin 9.25 x lO'^M.
4.3.322 Electron microscopy Mitomycin C (1.5 x 10'^M). Despite being used at a concentration that inhibited full ookinete morphogenesis, the sole consistent departure from normal morphology was frequent 'granulation' of the nuclear matrix (fig 4.7A). This phenomenon however did not prevent normal morphological condensation to the chromatids at meiosis (Sinden & Hartley, 1985).
Hydroxyurea (5.26 x 10"5 m ). Development was largely normal, with only two detectable effects seen; an apparent increase in the overall density of the cytoplasm and the induction of surface 'blebs' in the zygote plasmalemma (fig 4.7B). Neither of these observed morphological changes can be directly attributed to the recorded activity of hydroxyurea against DNA or protein synthesis but may be an indirect consequence of general metabolic blockade of both DNA and protein synthesis. Although used at a concentration that
112 inhibited mature ookinete formation, well advanced retort form ookinetes were found in which all development appeared normal, including meiotic division.
Aphidicolin (1.18 x 10~5m ). At the ultrastructural level this drug was found to induce no major lesions under the conditions of this experiment. In particular normal single chromatids were seen to be condensing prior to meiotic division and meiotic spindle poles were seen (fig 4.7C). These observations do not however indicate the successful completion of division, only its onset. An electron dense intranuclear structure was seen in this and other experimental groups, which was not in the controls of this study, and is similar to a structure seen in other in vivo radiolabelling studies to incorporate ^H-adenosine readily (Sinden, 1978) and is therefore simply inteipreted as the nucleolus (fig 4.7D).
4.3.3.3 RNA Synthesis Inhibitors 4.3.3.3.1 Light Microscopy As with the DNA synthesis inhibitors, examination of the blood films showed that ookinete development was more effectively blocked than exflagellation (Section 4.3.2). The estimated EDsos were:- cordycepin 5.9 x 10"5M; actinomycin D, 5.86 x 10_^M; and a-amanitin, 4.25 x 10‘5 m (table 4.3).
4.33.3.2 Electron Microscopy Cordycepin (2.59 x 10'^M). Treated cultures showed that following an apparently normal initial development, morphogenesis ceased at the time of apical complex expansion. In addition the mitochondria were found to have a matrix, the electron density of which contrasted more consistently with the cytoplasm when compared to the control (fig 4.8A). The endoplasmic reticulum was less well developed, with a possible decrease in the attachment of ribosomes to this membrane 'domain'. Polyribosomes were however still found within the cytoplasm. Chromatid condensation, which precedes meiosis was seen ( Sinden & Hartley, 1985).
Actinomycin D (3.2 x 10"^M). The drug had the major effect of causing extensive vesiculation of those cytoplasmic organelles limited by a single unit membrane (fig 4.8B). One additional observation made on a single parasite in this group was that the inner membranes of the apical complex showed a
113 FIG 4.6 Electron micrographs of P. berghei ookinete cultures. A) Shows a group of four parasites; one mature ookinete and three macrogametes of which one is degenerate, c - indicates the crystalloid (magnification 17,000x) B) Shows normal apical complex formation (magnification 32,000x) C) Shows a zygote showing early apical complex formation (magnification 17,000x). D) Meiotic cell showing MTOC and condensed chromatids aligned on the spindle microtubules (magnification 75,000x)
FIG 4.7 Electron micrographs of P. berghei in ookinete culture in the presence of DNA synthesis inhibitors A) Shows the granulation of nuclear matrix in mitomycin C treated cultures (magnification 17,000x) B) Shows the induction of surface blebs in the zygote plasmalemma under the influence of hydroxyurea (magnification 17,000x) C) Shows the events leading up to meiosis with normal spindle pole (sp) formation in the presence of aphidicolin (magnification 27,000x) D) Shows an electron dense structure interpreted as the nucleolus (n) in the presence of aphidicolin (magnification 17,00 Ox)
FIG 4.8 Electron micrograph of P. berghei in ookinete culture in the presence of RNA synthesis inhibitors. A) Shows the increased electron density of the mitochondrial matrix (m) in the presence of cordycepin (magnification 26,000x) B) Shows the extensive vesiculation of the cell in the presence of actinomycin D (magnification 21,000x) C) Shows the normal development of sexual stage parasites in the presence of oc-amanatin (magnification 70;000x) D) Shows an unusual periodicity in the electron density of the inner membranes of apical complex in the presence of actinomycin D (magnification 37,000x) E) Shows vesiculation of the cells and blebbing of the membranes in the presence of oc-amanatin
FIG 4.9 Electron micrographs of P. berghei in ookinete culture in the presence of metabolic inhibitors A) Shows the enlarged endoplasmic reticulum (er), vesiculation of the cytoplasm and early apical complex formation in cycloheximide treated cultures (magnification 21,000x) B) Shows spindle pole development in a zygote treated with colchicine (magnification 17,000x) C) Shows a magnification of the spindle pole (sp) seen in (B) (magnification 45,000x) D) Shows the occurrence of microtubules (mt) in the cytoplasm of a parasite cell despite treatment with colchicine (magnification 45,000x) E) Shows subpellicular microtubules in the developing apical complex in the presence of colchicine (magnification 56,000x) B remarkable 70nm periodicity in electron density (fig 4.8D). Other than this unexplained phenomenon ookinete development was apparently normal. a-Amanitin (8.7 x 10"^M). The treated sexual stage parasites could be divided into two distinct classes. The first group showed no apparent effects following a-amanitin treatment, with the result that normal ookinete development followed (fig 4.8C). The second group was formed by highly vesiculated zygotes, in which the internal and surface membranes were heavily blebbed or formed whorls (fig 4.8E).
4.3.3.4 Protein Synthesis Inhibitors 4.3.3.4.1 Light Microscopy Cycloheximide proved to be an extremely effective inhibitor of ookinete formation showing and ED50 of 1.7 X lO'^M.
4.3.3.4.2 Electron Microscopy Cycloheximide (1.42 x 10"^M). Treated preparations were markedly different from any of the others examined. There were many more undifferentiated or partly differentiated male and female gametocytes. The cells were very condensed and the cytoplasm electron dense; osmiophilic bodies were still present, often forming stacked arrays of flattened discs; the parasites were still intracellular. Male gametocytes had sometimes initiated exflagellation and axoneme assembly had occurred normally. Some early retort form ookinetes were found, and these cells showed all the relevant micromorphology (apical complex, nuclear structure etc) associated with this transformation. However the endoplasmic reticulum was greatly enlarged (fig 4.9A), the cisternal space forming a swollen electron luscent network (fig 4.9A), and the surrounding cytoplasm was unusually electron dense; numerous cytoplasmic vesicles with a typical peripheral array of small membrane blebs were found; and some nuclei contained large electron dense masses.
4.3.35 Inhibitors of Microtubule Assemble 4.3.35.1 Light Microscopy The only drug tested in this category was colchicine, which effectively inhibited ookinete formation giving an approximate ED50 of 3.7 x 10"^M.
122 4.33.52 Electron Microscopy Colchicine used at a concentration of 1 x 10‘^M totally inhibited ookinete formation. However, microtubule assembly was found not to be completely inhibited by the drug at this concentration following the 24h incubation period (fig 4.9D). Again parasites were inhibited from transforming from the fertilized zygote into the retort form ookinete. It is at this time that the expansion of the newly initiated microtubule based subpellicular apical complex occurs. The major observed subpellicular effect of colchicine was a reduction or elimination of microtubules in those organelles where they are normally found (fig 4.9B, C & E) and frequently small vacuoles were associated with the developing complex, this association was not noted in the control preparations. Meiotic figures were found both with and without microtubules, other structures of the meiotic apparatus, including condensed chromatids, kinetochores, spindle poles (MTOC) were all present. Colchicine was different from the other drugs tested in that microgametocytes were found in the 24h preparation, indicating that microgametogenesis had failed to go to completion in all cells. In all these cells microtubule assembly in axoneme formation had occurred on the kinetosomes and long microtubules had polymerised, nonetheless these parasites failed to exflagellate and escape the host erythrocyte. No other significant variation from the control cultures could be detected.
4.3.4 The Inhibition of Protein Synthesis
Ookinete cultures were set up in the presence of 5|iCi ^s-methionine, as described earlier (4.2.3). As with the electron microscopy study, the lowest concentration of each drug that totally blocked ookinete formation was used. In all cases exflagellation was seen to occur, but not ookinete formation.
4.3.4.1 DNA Synthesis Inhibitors The DNA synthesis inhibitors tested were aphidicolin (1.15 x 10'^M) and hydroxyurea (5.26 x 10"^M). In the presence of these two drugs protein synthesis continued as shown by the sustained incorporation of 35s- methionine (fig 4.4, lanes 1 - 4). However one protein band with a molecular weight of 71kD was synthesised in reduced amounts in the mixed cultures (fig 4.4). The reduction of the peak of this density band from the drug treated cultures was confirmed by densitometry of the autoradiograph (fig 4.10).
123 A L__ — ■«.: 98 95 P71_| •75 66 H ------66 ~j i 1..= *.60 —i 53 45d s i T ” -4 H o 36 36 34
29- 31 2 4 i 25 4~
21 - =— 21 16 14J A
B -— ------.. "■*“ “ ......
__ '9—S7 P71_: — 75 66~ = = — 66 =..53 45- — ------^ 40 B6- ■^=--•--—36 = c 29^j H 3 1 2 4 - j-1- —T'L 21- 21 S'16f 14------f - . _
Fig 4.10 Densitometer traces of aphidicolin (A) and hydroxyurea (B) treated ookinete cultures. Labelled markers indicate the relative positions of the molecular weight markers. The unlabelled tabs show the positions of the computer recognised peaks which are numbered. Comparison of densitometiy traces from the control labellings of both the asexual and mixed cultures would suggest that this is a sexual stage protein (fig 4.5).
43.4, ? RN.A Synthesis Inhibitors The radiolabelling and autoradiography of the electorphoretically separated proteins of drug treated asexual and mixed cultures (figs 4.3 & 4.4) showed that none of the drugs tested block protein synthesis in P. berghei parasites in ookinete culture. No inhibitory effect can be detected by eye and this was confirmed by a densitometer scan (not shown).
4.3.4.3 Protein Synthesis Inhibitors Consistent with the proposed mode of action cycloheximide at 1.42 x 10'^M had a devastating effect on the incorporation of 35s-methionine during ookinete culture. The autoradiograph shows that cycloheximide inhibits protein synthesis in both asexual (fig 4.3 ) and mixed cultures (fig 4.4), with the densitometer showing that the effect is more pronounced in the asexual cultures (fig 4.11).
4.3.4.4. Inhibitors of Microtubule Assembly Colchicine, as expected, had no effect on the incorporation of 35s-methionine into protein during ookinete culture for both the asexual (fig 4.3) and mixed cultures (fig 4.4), with the densitometer scans confirming this (not shown).
125 A
r
- —r“ 66-
45- — 36= _ ---
29- —"i,._? _ 24
21- — 14- £ r‘„ . .
Fig 4.11 Densitometer traces of ^^S-methionine labelled parasites showing the different sensitivities of an asexual (A) and a mixed (B) infection in ookinete culture to cycloheximide. Labelled markers indicate the relative positions of the molecular weight markers. The unlabelled tabs show the positions of the computer recognised peaks. 4.4 Discussion
The object lesson learnt in this section of the study, in conjunction with the data on mitomycin C in chapter 2, is that whilst metabolic inhibitors provide useful tools for the study of specific events it should be stressed that the results must be treated with a considerable degree of caution, since they assume 1) susceptibility of the target cell, 2) the specificity and 3) action of the drug in question. Similarly, certain antimalarial drugs have been described as being effective due to their specific action against metabolic events. For example, the sulphonamides and antifolates are thought to have their effect by interfering with the synthesis of folate co-factors by the parasite enzymes. Of the other major anti-malarials, chloroquine and other 4-aminoquinolines interact with the DNA of the parasite, but some allege this is not their primary mode of action (Ginsburg, 1988). Primaquine, one of the 8- aminoquinolines is described as acting through interference with the mitochondrial respiratory processes. While these are the reported effects of the drugs, the present understanding of plasmodial metabolism is not fully understood and such interpretation should be treated with considerable caution. Clearly, therefore, any increase in our understanding of the metabolic processes of plasmodia may offer guidance in the evaluation of candidate anti-malarials (Bruce-Chwatt, 1986). In the meantime however, care in the interpretation of such results must be taken. It must be emphasised that the apparent lack of inhibition is not indicative of the absence of a particular process. In the case of mitomycin C, Toy6 et al (1977) showed an absence of effect and assumed the absence of DNA synthesis, however it has now been shown that DNA synthesis occurs during microgametogenesis (Janse et al, 1986). Finally it should be mentioned that some drugs when used at higher concentrations may bring about an effect due to general toxicity rather than through a specific effect. One way of trying to avoid such misinterpretations is to use several drugs that act on the same event but allegedly via different routes, on the basis that different drugs act in different manners and will therefore be unlikely to have identical non- attributable side effects.
127 4.4.1 Exflagellation
With this in mind examination of the data from cultures treated with DNA synthesis inhibitors is contradictory. While mitomycin C failed to inhibit exflagellation completely and hydroxyurea only did so at high concentrations, aphidicolin proved very effective. Therefore since both aphidicolin and hydroxyurea do have an effect it would appear that the earlier interpretation, that DNA synthesis has been completed by the time the gametocyte reaches maturity (Sinden, 1983) is incorrect and that rapid DNA synthesis does occur during gametogenesis. Since these studies were made it has been shown exquisitely by Janse et al (1986) that this is indeed the case with the DNA content of the microgametocyte increasing 8-fold between activation and exflagellation. Since the absence of DNA synthesis as an explanation for the inactivity has now been ruled out, the remaining possible explanations for the inactivity of mitomycin C are: (1) the mitomycin C does not enter the cell or (2) extraneous conditions prevent the drug from working. The first explanation that mitomycin C does not enter, or does not have an effect within the cell was discounted on evidence from the electron microscope study that reveals an intranuclear effect, implying entry of the drug into the cell. This leaves only the explanation of extraneous conditions. One possibility that fits with this theoiy is the observation that mitomycin C is activated through a reductive pathway (Moore, 1977) which involves the conversion of a fully reduced hydroquinone to a semiquinone, ultimately yielding a biologically active bifunctional alkylating agent (see Chapter 3). If for some reason the culture system or some property of the plasmodial cell allows the drug to persist only in its non-reduced form then the apparent discrepancy in the activity of the drugs may be explained.
The results from the parasites treated with RNA and protein synthesis inhibitors before exflagellation confirm the finding of Toy6 et al (1977) that both transcription and translation are necessary if exflagellation is to occur and that they take place in the 10 minutes between induction and exflagellation. The RNA and protein synthesis inhibitor data from the rapid assay method, although far less quantitative than the methods described by Toy6 et al (1977) gives inhibitory values for the drugs of approximately the same order as those found by these workers, thus supporting the validity and usefulness of this rapid and simple method in establishing the concentrations at which to use the drugs in later experiments.
128 4.4.2 Ookinete Development
The effect of the metabolic inhibitors of DNA, RNA and protein synthesis on ookinete development if added before exflagellation were similar to those found with exflagellation, except that a lower concentration of the drug was required for inhibitory effect to be detected. The effects of the drugs however, are veiy different if they are added after exflagellation with an apparent decrease in the "sensitivity" to the drugs (table 4.4), supporting the view above, that de novo RNA and protein synthesis are involved in exflagellation and fertilization and are clearly critical to ookinete development and that these processes are complete within two hours of the initiation of gametogenesis. Certainly DNA synthesis in P. berghei has been shown to occur within 2-3 hours of fertilization resulting in an approximately tetraploid DNA value in the young zygote, followed by at least 21 hours during which no DNA synthesis has been seen to occur (Janse et al, 1986b). Also within 2.5 hours of fertilization the completion of normal eukaryotic meiosis has been identified (Sinden & Hartley, 1985; Sinden et al, 1985a) and Janse et al (1986b) have associated the early DNA synthesis with the genome replication that precedes the formation of synaptonemal complexes during normal two-step meiosis. The pattern of reported DNA synthesis in the gametocyte and young zygote (up to -3 hours) also fits with the observed sensitivities of the parasite to the drugs aphidicolin and hydroxyurea reported here, which decrease when added at 2 hours after exflagellation has been initiated, ie at about the time that DNA synthesis and meiosis have been reported as being complete.
The electron microscope revealed very little concerning the molecular mechanisms through which the drugs act, although it does show that in all cases inhibition commonly occurs just after the apical complex has been laid down, implying that this is a very sensitive phase of ookinete development. It is tempting to draw the analogy to the very sensitive period of gastrulation in amphibian development, which is the time at which many zygote genes are switched on for the first time (Davidson, 1976). The one surprising finding though was the fact that apical complex formation, which is thought to be under the guidance of microtubules nonetheless occurred in the presence of colchicine. It was also seen that inhibition occurred mid exflagellation in a large number of cells even though the drug concentration was not selected specifically to block exflagellation. It may be that during
129 exflagellation and ookinete development, colchicine interrupts the flow of tubulin monomers at a time when their utilization is greatest.
Another cellular effect that is common to all the drug treated cultures, but which is not seen in the untreated controls, is the inhibition of crystalloid formation. Since this structure is linked to lipoprotein mobilization (Trefiak & Desser, 1973) it would suggest that all the drugs are having a Trnock on' effect.
At the molecular level one process that is certainly occurring de novo throughout the transition of zygotes to ookinetes is protein synthesis (Kaushal et al, 1983) and this was studied by radiolabelling.
Cycloheximide had a devastating inhibitory effect on protein synthesis, although in the mixed culture protein synthesis was still seen (Fig. 4.5b). One possible explanation considered for this is contamination of the culture by bacterial cells, but this was discounted because the underlying pattern of synthesis is similar to that of the control. It is therefore likely that the concentration of the drug used was not high enough to achieve total blockade, possibly due to a variation in the drug/parasite ratio with the result that the drug concentration per cell in not at an effective level. Repeating the experiments having established the parasite concentration before adding the drugs could determine whether this were so.
The only visible effect on protein synthesis of any of the other drugs was seen with aphidicolin and hydroxyurea, on a 71kD protein which was synthesised at reduced levels in the presence of these two drugs. The fact that RNA synthesis inhibitors failed to block synthesis of this protein suggests that it may be coded for by a long lived messenger RNA. Since the production 71kD is inhibited by the presence of DNA synthesis inhibitors it follows that it may be either a DNA packaging molecule like the histones or a regulatory factor or enzyme that is involved in gene expression, which is being translated from the mRNA as it is required by the cell.
130 4.4.3 Comments on Methodology and Future Directions of Analysis
Densitometry proved a useful method of confirming differences visible to the naked eye. It also showed that there are definite differences between the mixed and asexual cultures highlighting the different spectrum of proteins synthesised by each population of parasites. While the quantitative potential of the densitometer was not fully exploited in these studies as the gels were designed to give a qualitative assessment of protein synthesis there is no reason why in the future the scanning densitometer should not be used to great effect to determine both qualitatively and quantitatively changes in protein synthesis under experimental condition, eg in the presence of inhibitory drugs.
The use of drugs to study metabolic events in ookinete development did not reveal a great deal of information and did to a certain degree highlight areas of controversy with contradictory results being found (eg the results from the study of mitomycin C vs aphidicolin). It may be possible to clarify the situation by repeating the study using further metabolic inhibitors, however considering the results obtained in this study it may be more productive to investigate the effects of the drugs in in vitro test systems against the isolated plasmodial cellular components to establish drug sensitivities. It may also be useful to determine the intracellular concentrations of the drugs. This may involve the use of expensive labelled drugs but it may also show whether a particular metabolic process is susceptible or whether the drug is actually achieving entry to the parasite cell.
Further investigation of the results obtained here, using the parasite system could include (1) to study the events occurring at the time of apical complex formation and (2) the characterization of the 71kD protein described in this section. 1
1) The first route suggests itself as apical complex formation is the time at which all the drugs tested so far block ookinete formation. It may therefore be that this is a highly sensitive time, during which some 'special' events are occurring. It is possible that the biological mechanisms that have evolved "protect" the zygote early in its development in the hostile environment of the mosquito midgut serve in this instance to protect it against the action of drug attack. Another
131 possibility is that it is during apical complex formation the drug sensitive biosynthetic machinery responsible for the synthesis of critical, regulatory proteins is switched on for the first time in the zygote. It is therefore the first opportunity that the drugs have to act and that prior to this prior to this all protein synthesis has been of a general non-essential nature. This argument does not hold for protein synthesis inhibitors with the demonstration of de novo protein synthesis of zygote/ookinete specific proteins occurring as early as two hours postfertilization (Kumar & Carter, 1984; Kaushal et at, 1983; Winger et a)' 1988; Ch 5). Likewise DNA synthesis has been demonstrated as taking place during exflagellation and ookinete development (Janse et at, 1986b). By comparison RNA synthesis has not been so precisely timed and it is therefore possible that presynthesis of the necessary RNA molecules by the macrogametocyte furnishes the macrogamete with all the necessary equipment to complete development up to the point of apical complex formation. If such were the case a presynthesised pool of mRNA would explain why protein synthesis could occur in the presence of RNA synthesis inhibitors. A comparable situation is seen during embryo development, which in the presence of actinomycin D continues to develop normally up until gastrulation. Prior to this the embryo develops from presynthesised messengers, but at the point at which gastrulation takes place the biosynthetic machineiy is switched on (Davidson, 1976). It is interesting to note that it is at this stage of the life cycle that the parasite switches from a 'high temperature1 rRNA to a ‘low-temperature’ rRNA (Waters et al, 1989) and that this low temperature rRNA might be presynthesised and stored in the prominent nucleolus found so readily in the macrogametocyte.
2) The second path of study would be the characterization of the 71kD protein, whose synthesis appears to be altered by the addition of DNA synthesis inhibitors and this inhibition suggests that this protein may offer an interesting area of study. The fact that it is affected by the DNA synthesis inhibitors suggests that it may be a DNA-associated protein. Naturally the first step to characterizing the protein would be to raise an antibody against it, in order to facilitate the preparation of the protein. This could then be used to determine the protein's location within the cell, whether or not it was DNA-associated, whether it showed any stage specificity and its patterns of synthesis. The fact that this protein does
132 show sensitivity to the DNA inhibitors also suggests that this may be an area within the parasite metabolism through which it would be possible to block parasite development, and thereby lead to a site against which drug development could be aimed.
One predominant feature of the ookinete stage gels was the presence and synthesis of a 21kD protein as a major species. With the availability of a monoclonal antibody that recognised a protein of this size and that illicited a transmission blocking effect it was decided to concentrate study on this protein rather than parasite metabolism in general which had not yielded as much interesting data as had been hoped for.
4.4.4 Conclusions
The work in this chapter has shown:
1. A micro culture technique that allows for the rapid assessment of metabolic inhibitors on exflagellation and ookinete development.
2. DNA, RNA and protein synthesis are all necessary events for the normal development of the zygote and ookinete stages of ?. bergheias was demonstrated by the ability of metabolic inhibitors against these processes to block development.
3. Apical complex formation appears to be a highly sensitive time in zygote/ookinete development.
133 Chapter 5
Studies on the 21kD, Sexual Stage Transmission Blocking Protein of Easmodium berghei
Paper in preparation.
134 5.1 Introduction
The transmission blocking or altruistic vaccine relies upon preventing the transmission of the parasite from the vertebrate host to the mosquito vector. Targets for such vaccines have been identified on the gametocyte, gamete, zygote and ookinete stages of Plasmodium (reviewed Carter et al, 1988) and in view of the the identification of naturally occurring antibodies against such targets (Mendis et al, 1987; Ranwaka et al, 1988) have taken on an even greater significance in attempts to control malaria.
Of direct relevance to the work reported in this chapter are those identified on the zygote/ookinete stages of P. gallinaceum and P. falciparum. In P. gallinaceum a protein with a molecular weight of 26kD (now known as Pgs 25) has be identified as such a target (Carter & Kaushal, 1984) while in P. falciparum a 25kD protein (now known as Pfs 25) has been found (Kumar & Carter, 1984). Both of these proteins have been shown to be recognised by monoclonal antibodies that block transmission of the parasite to the mosquito (Carter & Kaushal, 1984; Vermeulen et al, 1985; 1986). Likewise in P. berghei a protein with a molecular weight of 21kD (which on occasions shows and apparent Mr of 23kD) has been found and this protein is now called Pbs 21. Pbs 21 is synthesised by the zygote and ookinete stages of P. berghei as will be demonstrated in this chapter (Section 5.3.2) and is detectable on the surface of the zygote within 2h of the initiation of ookinete production using immunogold electron microscopy (Sinden et al, 1987) and iodobead labelling (section 5.3.4). Pbs 21 is recognised by a monoclonal antibody designated 13.1 and this antibody elicits a transmission blocking response during mosquito feeds (Winger et al, 1988), suggesting that Pbs 21 plays an important part in the transmission ofP. berghei and is therefore a good target for the development of transmission blocking studies, and that it may also be an 'equivalent' protein to the Pfs 25 and Pgs 25 seen in the P. gallinaceum and P. falciparum. It was therefore deemed important to characterise this protein. Furthermore in the light of the differences identified in the times of the reported synthesis of the P. falciparum and P. gallinaceum proteins: the P. gallinaceum protein is only synthesised in the zygote and ookinete stages, (Carter & Kaushal, 1984), while the P. falciparum equivalent is in addition synthesised in the immature gametocyte (Vermeulen et al, 1986). As a result of this difference between the P. falciparum and P. gallinaceum species it became even more important to characterise the Pbs 21 protein to see how Pbs
135 21 fits into the picture and also to try to determine the degree to which these three proteins may be related to one another based on the synthesis patterns and their basic biochemical characteristics. This in turn could provide a basis for more detailed studies.
Monoclonal antibody 13.1 (mAb 13.1), a mouse monoclonal, class IgG 2a was prepared by immunising one Balb/C mouse via the i.p. route with ~107 zygotes /ookinetes in complete Freund's adjuvant (Winger et al, 1988). A month later the same dose (~107 zygotes/ookinetes) without adjuvant was given as a booster and this was followed after a further interval of one month by an i.v. dose of 1-2 x 106 enriched freeze-thawed zygotes/ookinetes in PBS. Four days after this final boost the mouse was killed, the spleen cells removed and the cells fused with Ag 8 myeloma cell fusion partner (Winger et al, 1988). Two weeks after fusion the clones were screened and subcloned. Selected subclones were then expanded for freezing and ascites production (Winger et al, 1988).
In this chapter mAb 13.1 is used as a tool in the study of Pbs 21. The synthesis of the 21kD protein of P. berghei is described. In addition to this, the protein has been examined for other characteristics typical of membrane proteins, as has been done for both Pgs 25 and Pfs 25 (see section 5.4) and a method for extraction of the protein tested, with a view to the further purification necessary for the development and analysis of both antibodies and vaccines.
136 5.2 Materials and Methods
5.2.1 35S-Methionine Labelling of Asexual and Gametocyte Stages of P. b erg h ei (fig 5.1)
P. berghei parasites synchronised in vivo, were kindly prepared by Dr B Mons and his colleagues (Mons, 1986). To prepare the synchronised cultures four Wistar rats (8-10 weeks old) were injected with 0.5-lxl05 P. berghei parasites. When the parasitaemia had reached 1-3%, around day 3-4 post inoculation, the infected blood was collected by cardiac puncture from animals under deep ether anaethesia and the cells prepared for culture (Mons, 1986). After centrifugation at 200g for lOmin, the packed cells were resuspended in RPMI 1640 supplemented with HEPES (5.94g/l), NaHC03 (38ml of a 5% solution), FCS (200ml/l) and neomycin (5,000 iu/1) to give a 2- 4% cell suspension (v/v, packed cells/medium) and incubated in 500ml Erlenmeyer flasks, which were continually gassed with a mixture of 10% O 2, 5% CO2 and 85% N 2 (- 10cm3/min) and placed on an orbital shaker (ca 60 rev/min) at 37°C. After -20 hours, when the parasites had developed into early schizonts the shaker was switched off thus allowing the cells to sediment. In this static culture morphologically mature schizonts persist for extended periods without rupture and the associated release of merozoites (Mons et alt 1985). To assess the maturity of the schizonts in culture, samples were taken at hourly intervals, Giemsa stained and examined microscopically. When 80-90% of the schizonts were found to be mature (containing 12-16 merozoites) the schizonts were purified in the following manner:
Schizont purification was achieved on a one-step density gradient prepared by mixing 58% (v/v) Nycodenz sterile isotonic solution (Nyegaard & Co) with 42% (v/v) sterile PBS. 10 ml aliquots of the Nycodenz/PBS were dispensed into 50 ml tubes (Greiner) and then 40 ml of the culture material was layered on to this Nycodenz cushion. The tubes were then centrifuged at 200gfor 5 min followed by 450gfor 20 min, both runs being at 37°C. The resulting interface, which contained >90% of the schizonts was then collected and centrifuged at 450gfor 7 min to pellet the cells. The pellet was resuspended in RPMI 1640 to give -2 x 108 schizonts/ml or -2 x 109 merozoites.
137 METHOD FOR THE SYNCHRONIZATION OF ASEXUAL AND GAMETOCYTE STAGE PARASITES OF P. BERGHE/
1%-3% parasitaemia
Cardiac puncture. One wash step.
10%02.5%C02.85%N
s
Culture at 37°C in RPMI 1640 pH7.3 for 20h followed by 16h static culture * /
Purified schizonts
Nycodenz centrifugation
v
i.v. inoculation into phenylhycfrazine treated mice.
in v iv o development v Cardiac puncture. Wash in RPM11640
Laemmli sample Culture at 37°C for 3h buffer added in presence of 35S-methionine I . i Pellet cells and wash in Freeze at -20°C serum-free medium \ v Lyse at 4^C for 1h Western blotting I Centrifuge at 10,000rpm for 10 min \ Fig 5.1 Immunoprecipitations To establish synchronised cultures TO mice were inoculated, via the intravenous route, with 0.2ml of the schizont suspension. After ~7 h post inoculation bloodfilms were made from tail blood and after Giemsa staining assessed for infection. Those assessed as having the "best" infections, ie those with the greatest numbers of young parasites visible in Giemsa stained smears, were then used to supply parasite samples for radiolabelling with 35S- methionine and for the collection of material for Western blotting.
At various times throughout the highly synchronised infection (9 - 30h post infection) blood was collected by cardiac puncture from an infected mouse. On each occasion a different animal was used to collect the parasites. The collected blood was divided into two, half was frozen in Laemmli sample buffer (LSB) for Western blotting experiments and half was labelled in vitro in gametocyte culture medium, (methionine-free RPMI 1640 pH7.2)(2.2.5.4) in the presence of 5jiCi/ml of 35S-methionine (Amersham International). For the labelling the parasitised red blood cells were diluted 1:10 in gametocyte culture medium at 37°C with added radiolabel and cultured at this temperature for 3h. The cells were then pelleted by centrifugation at 14,000g for 5 min. The pellet was resuspended, washed in gametocyte culture medium and repelleted. The pellet was then resuspended a second time and washed in serum-free gametocyte culture medium (2.2.5.5). After a further 3 washes in the serum-free medium the cells were lysed in lysis buffer (see section 4.2.3) for lh at 4°C, to solubilise the proteins. The resulting solution was clarified by centrifugation in a microcentrifuge at 14,000g for 10 min. The supernatant was collected and stored frozen at -20°C until needed. The incorporation of labelled methionine was then ascertained by TCA precipitation (3.2.7) and liquid scintillation counting (3.2.7).
5.2.2 35S-Methionine Labelling of Ookinete Cultures (fig 5.2)
Ookinete cultures were set up in methionine-free RPMI 1640 (2.2.5.2) following the method described in Section 3.2.4. Radiolabelling was carried out by the addition of 5jiCi/ml 35S-methionine (Amersham International - SJ235) to the culture at various time throughout the culture period (0-3h, 3- 6h, 6-12h, 12-18h & 18-24h post exflagellation). The cells were purified by Nycodenz gradient centrifugation (3.2.5), followed by the washing and lysis procedure
139 METHOD FOR THE CULTURE OF P. BERGHE! OOKINETES.
Mitomycin C treatment of infected mice (25p.g/g body wt)
24h later
Cardac puncture
v
r
Whatmann CF11 cellulose column
Culture at 19°C in Unlabelled ookinete RPM11640 pH8.0in culture presence of 35S-methionine
Nycodenz centrifugation. \ Wash purified parasites in serum-free medium
Centrifuge at 10.OOCXpm for Frozen at -20°C 10 min .1 Immunoprecipitations Western blotting described in 5.2.1, with the sole difference being the use of methionine-free RPMI (2.2.5.2) and serum-free ookinete medium for the washes (22.5.3).
Similar ookinete cultures were run in the absence of radiolabelled methionine in order to supply similar untreated parasite material for Western blotting experiments.
5.2.3 Immunoprecipitation
IOjj.1 of monoclonal antibody 13.1 (either ascites or purified IgG), was added to 20jil aliquots of the solubilised protein, and left overnight at 4°C. To this, lOOjil of a 50% suspension of protein A-sepharose (Sigma) was added and incubated at room temperature for 2h with rotary agitation, to bind the IgG. The sepharose beads were then pelleted by centrifugation at 14,000g for 2 min. The supernatant was removed, and the pellet resuspended in PBS +1% Triton X-100 and repelleted. This washing procedure was repeated two further times. LSB was then added to the samples, which were stored frozen at -20°C before protein separation by SDS-PAGE on 15% gels as described in section 4.2.5.
5.2.4 Western Blotting
Total protein samples were separated by SDS-PAGE and transferred to nitrocellulose paper (Biorad) following the method of Burnette (1981). The gels were overlaid with nitrocellulose paper that had been wetted in transfer buffer (20mM Tris, 150mM glycine, 20% methanol (v/v) in distilled water), and sandwiched between two Scotchbrite pads. The sandwich was then placed in the perspex holder of the Biorad Transblot cell and immersed in the transfer tank with the nitrocellulose lying between the gel and the anode. The proteins were then transferred at a voltage of 60V and a current of 0.2A for 6h, with cooling by running tap water.
After removing the marker strip, which was stained in Amido black, the nitrocellulose sheets were incubated in blocking solution (0.016M Na2HP042H20, 0.004M NaH2PO42H20, 0.14M NaCl, 5% (w/v) skimmed milk powder, 5% (v/v) normal goat serum, pH7.4) at 37°C for 2h, to prevent the
141 non-specific binding of antibodies to the nitrocellulose or the transferred proteins. This was then followed by incubation of the nitrocellulose in the monoclonal antibody 13.1, diluted 1:100 in blocking solution, for 2h at 37°C. The nitrocellulose was then washed (4 x 15 min) in milk wash (0.016M Na2HP04.2H20, 0.004M NaH2PO42H20, 0.14M NaCl, 5% (w/v) skimmed milk powder), followed by incubation in l25I-protein A (Amersham International) (lH-Ci/ml) diluted in milk wash for Ih at room temperature. Following this the l25I-protein A solution was discarded and the nitrocellulose washed as before (4 x 15 min in the milk wash), followed by 2 x 15 min washes in PBS + 1% Triton X-100 +1% Tween 20, and 2 x 15 min high salt washes (0.4M NaCl). The filters were then blotted dry before being set up for autoradiography.
In order to establish the background binding patterns associated with the monoclonal antibody, blots were also carried out where the first antibody (mAb 13.1) was replaced by an incubation with normal mouse serum (NMS) or was omitted completely, thus giving the backgrounds due to the non specific binding of the radiolabelled antibody.
5.2.5 Autoradiography of Western Blots
The filters were blotted dry, mounted on exposed X-ray film (Fuji), covered in cling film and then placed in cassettes containing DuPont Cronex intensifying screens to enhance the detection of isotope (Lasky & Mills, 1977; Lasky, 1980) and were then incubated overnight at -80°C with Fuji X-ray film. The film was developed as described in section 4.2.7.
5.2.6 35S-Methionine Labelling in the Presence of Microtubule Assembly Inhibiting Drugs
In order to try to prevent exflagellation and fertilization occuring, ookinete cultures were set up in methionine-free ookinete culture medium (2.2.5.2) at 37°C in the presence of either 50jig/ml colchicine (Sigma) or lOOjig/ml vinblastine (Sigma) and incubated at this temperature for lh. Ookinete production was then initiated by placing the cultures in a 19°C incubator. At this time 5}iCi/ml 35S-methionine was added to each culture. At the end of the 24h culture period the labelled proteins from the parasites were processed
142 for immunoprecipitation (section 5.2.3), electrophoresis (section 4.2.5) and autoradiography (section 4.2.7).
5.2.7 Immunofluorescence Assay (IFA) of Ookinetes Cultured in the Presence and Absence of Tunicamycin
Ookinetes were cultured as described earlier (section 3.2.4) in the presence (50p.g/ml) and absence of tunicamycin (Sigma), which is a drug that is described as inhibiting dolichol diphosphate dependent protein glycosylation (Duskin & Mahoney, 1982). At the end of the culture period 100p.l of the culture was added to each well of a microtitre plate containing a doubling dilution of the anti-ookinete monoclonal antibody 13.1. The plate was then incubated at 4°C for l-2h. The cells were then pelleted by spinning the plate at 150g for 10 min and were resuspended and washed twice in complete RPMI 1640 (2.2.5.1). This was followed by the addition of goat anti-mouse serum conjugated with fluorescein-isothiocyanate diluted 1:25 in RPMI 1640, and incubated for lh at 4°C. The cells were then pelleted and washed twice in RPMI 1640. Citifluor was added to each sample before an aliquot was taken and examined for fluorescence under ultra violet light and photographed using a Leitz orthoplan Epi-illuminator, fitted with an Orthomat camera and using Kodak Technical pan film.
5.2.8 Glucosamine/Mannose Labelling of Ookinete Cultures
Ookinete cultures were set up (3.2.4) with and without the addition of tunicamycin (50jj,g/ml). After 2h incubation in the presence of the drug 3H- glucosamine and 3H-mannose were added. For each of three untreated and three treated cultures either 3H-glucosamine (20p.Ci/ml), or 3H-mannose (20p.Ci/ml) or 3H-glucosamine and 3H-mannose (lOjiCi/ml of each) was added. The cultures were then incubated for 24h at 19°C. The ookinetes were purified by Nycodenz centrifugation as described in section 3.2.5, lysed (section 3.2.3) and the resulting protein solution used for immunoprecipitation (5.2.3). The proteins were separated by SDS-PAGE (section 4.2.5) and the incorporation of the radiolabel visualised by autoradiography (section 4.2.7).
143 5.2.9 Surface Labelling of Ookinete Cultures
Ookinete cultures were set up as described earlier (section 3.2) and using iodobeads (Pierce) the cells were surface labelled at various times throughout the culture period (Pierce Technical Bulletin; Markwell, 1982). Before surface labelling the beads were washed in Dulbecco’s PBS (136mM NaCl, 2.6mM KC1, 7.9mM Na 2HP04 .2H20,1.4mM KH2PO4) pH7.4. The beads were then loaded by incubating them at room temperature for 5 min in 125I solution (50jiCi NaI/2 iodobeads). The ookinete culture was then pelleted by centrifugation at 150g, the parasites resuspended in PBS, added to the beads and left at room temperature for 15 min, after which the cells were pipetted off, thereby terminating the labelling reaction. The parasites were pelleted at 150g, the supernatant removed and the cell pellet washed in complete RPMI1640 (2.2.5.1), followed by serum-free ookinete medium (2.2.5.2). RPMI 1640 was used in the washing procedure in the hope that it would better preserve the parasite and therefore improve yields as compared to PBS washes. Giemsa stained smears showed that the technique gave morphologically intact parasites.
Following labelling the parasites were lysed (section 4.2.3) and the incoiporation of 125I established by gamma counting 10p.l samples to determine the specific activity of each preparation. Samples containing equal counts were then immunoprecipitated (section 5.2.3) and separated by SDS- PAGE (section 4.2.5) for drying and autoradiography (section 4.2.7).
5.2.10 Triton X-114 Extraction of 35S-Methionine Labelled Proteins (fig 5.3)
Parasites that had been 35S-labelled as described earlier (Section 4.2.3) were solubilised in 1% Triton X-114 in tris-buffered saline stock solution(TBS - lOmM Tris, 150mM NaCl, pH7.5) at 4°C for Ih and centrifuged at 40,000g for 30 min at 4°C (Bordier, 1981). The supernatant was collected on ice using pre chilled pipettes to prevent phase separation, and stored in lOOjil aliquots at -80°G
144 Triton X-114 extraction of protein samples radiolabelled with 35 S-methionine during ookinete culture
To labelled parasites add 1ml 1% Triton X-114 in TBS ♦ Incubate at 4 C for 1h I Centrifuge at 40000g for 30 min at 4 C I Collect supernatant and store at -80 C
Thaw samples i Add 0.001 %BPB to samples ♦ Warm to 37 C for 5 min
Microcentrifuge for 1 min at room temp
Separate the Aqueous phase from the Detergent phase
UP1 LP1 I I Add triton X-114 to Add TBS to give 0.5% and BPB to final volume of 0.001% 10Oul ♦ ♦ Incubate at 4 C for 10 min i i Warm to 37 C for 5 min for phase separation I I Microcentrifuge for 1 min at room temp
Separate the aqueous and detergent phases Aqueous Detergent Aqueous Detergent i ♦ i i UP2 Discard Discard LP2
Fig 5.3 For detergent extraction the samples were thawed and then prior to phase separation 0.001% bromophenol blue (BPB) was added to the samples (Kumar, 1985). They were then warmed to 37°C for 5 min followed by microcentrifugation at room temperature for 1 min. The aqueous phase was separated from the detergent phase (the blue pellet). The detergent phase (LP1) was then redissolved in TBS equivalent to the original volume by incubating at 4°C for 10 min. More stock Triton X-114 and BPB were added to the aqueous phase (UP1) to give final concentrations of 0.5% and 0.001% respectively. The phase separations were then repeated by rewarming the samples to 30° C for 5 min, followed by 1 min micro centrifugation at room temperature. The pellet from the second separation of LP1 was collected and labelled LP2 and the supernatant discarded. Conversely the pellet from the repeat separation of UP1 was discarded and the supernatant (aqueous phase) saved and labelled UP2. The samples were then analysed by SDS-PAGE (section 3.2.5) and autoradiography (section 3.2.7) to identify the separation pattern of the proteins, particularly the 21kD protein.
146 5.3 Results
5.3.1 Characterization of the Monoclonal Antibody 13.1
The monoclonal antibody has been shown to bind to a 21kD protein (Pbs 21) (Winger et al, 1988) of Plasmodium berghei. To characterise the proteins recognised by m Ab 13.1 and to look for the presence of Pbs 21 three sets of blots were carried out against synchronised gametocyte and ookinete preparations. These blots were:
1. Blotting using NMS as the first antibody followed by detection with protein A to look for non-specific binding to molecules present in serum, eg, Ig molecules or their component light and heavy chains.
2. Blotting where the first antibody step was omitted completely to try to identity proteins that were "sticky" to protein A and would therefore show as recognised bands although this might have come about via a nonspecific reaction.
3. Blotting using mAb 13.1 in order to look for the presence of Pbs 21 in different parasite stages.
The result of these blots, was the identification, in the synchronised infections of P. berghei, of bands with Mr of 45, 36 and 27kD which were recognised by NMS; of 27kD which was recognised by protein A (fig 5.4), and at 66kD which was recognised by mAb 13.1. In the ookinete preps two bands were recognised one at 33kD by NMS and one, as expected, at 21kD by mAb 13.1 (fig 5.5)
By comparing these results with those obtained from the biosynthetic labelling of the cultures with 35S-methionine and iodobead labelling of surface proteins the following characterisation of those bands other than Pbs 21 has been formed.
5.3.1.1 The 36/27/33 kD Proteins The proteins found at 36 and 27kD in the synchronised cultures and at 33kD in the ookinete cultures (figure 5.4a & b) would appear to be contamination of the blots by proteins of unknown origin. As they are recognised by NMS they
147 FIG 5.4 Western blots on material taken at different times during synchronised gametocyte production and ookinete culture of P.berghei in which the monoclonal antibody has been replaced by normal mouse serum (A) and omitted (B) to indicate nonspecific binding by mouse serum globulin and binding of protein A to proteins found in certain parasites. Lane 1 9 hours post infection Lane 2 12 hours post infection Lane 3 15 hours post infection Lane 4 18 hours post infection Lane 5 21 hours post infection Lane 6 24 hours post infection Lane 7 27 hours post infection Lane 8 30 hours post infection Lane 9 3 hours ookinete culture Lane 10 6 hours ookinete culture Lane 11 12 hours ookinete culture Lane 12 18 hours ookinete culture Lane 13 24 hours ookinete culture I A 1 2 3 4 5 6 7 8 9 10 11 12 13 > A
66-
45- *
36-
29- 2 4 -
21-
B- 1 2 3 4 5 6 7 8 9 10 11 12 13
66-
45-
36 - ^ A ** 29- 24"
21- FIG 5.5 Western blots of material collected from synchronised gametocyte infections (lanes 1 - 8) and ookinete culture (lanes 9-13) after varying time durations. Samples were blotted against monoclonal antibody 13.1, which recognises the 21kD P. berghei sexual stage protein Pbs 21. Lane 1 9 hours post infection Lane 2 12 hours post infection Lane 3 15 hours post infection Lane 4 18 hours post infection Lane 5 21 hours post infection Lane 6 24 hours post infection Lane 7 27 hours post infection Lane 8 30 hours post infection Lane 9 3 hours ookinete culture Lane 10 6 hours ookinete culture Lane 11 12 hours ookinete culture Lane 12 18 hours ookinete culture Lane 13 24 hours ookinete culture n~~
0 4
ill II I VO LTlvo Ov 5.3.1.2 The 45kD Protein Initial blotting experiments with NMS or with the ommission of the first antibody (fig 5.4) identified a 45kD band in the synchronised cultures that was recognised by NMS. Recognition by NMS suggests that this protein is not host in origin. It also suggests that it is not parasite. However blotting against Babesia bovis antigens has shown niave sera to recognise parasite antigens (Goodger et al, 1984). Therefore the possiblitiy of this being a parasite antigen cannot be eliminated. In addition to the 45kD protein recognised by NMS in sychronised cultures a band at 45kD was found to be synthesised in ookinete cultures (fig 5.6B; lanes 3 & 5). This protein was not recognised by NMS. In view of the different recognition patterns obtained in blots with NMS it is possible that these two proteins are not one and the same. The use of a technique such as isoelectric focusing could might determine whether these bands are in fact two different proteins. 5.3.1.3 The 66kD Protein The last of the bands identified in the synchronised infection is that seen at a Mr of 66kD. This protein was not identified by NMS and therefore may be host in origin, possibly the heavy chains of Ig that were bound to the samples when they were taken. This could have been investigated by blotting using anti-mouse or anti-Ig antibodies. This view is supported by the fact that (1) they correspond to the molecular weight of heavy chain Ig (50-77kD) (Roitt et a], 1985) and (2) they are recognised by protein A (fig 5.4B). Additionally, it has been suggested that malarial infections will induce a general inflammatory response (Maegraith, 1977) with the result that all levels of Ig molecules are present at raised levels. Furthermore non-specific immune responses to P.yoelii have been identified with all classes of Ig being present at raised levels (Cohen et al, 1961; Rosenberg, 1978; Freeman & Parish, 1978; Langhome et al 1985; Taylor et al 1988). Thus the chances of finding Ig in the samples are increased. Alternatively, since this protein can be detected using mAb 13.1 in the synchronised cultures between 15 and 21 hours post infection (fig 5.5, lanes 3-5), which corresponds to the time at which trophozoites appear in the infection (Mons, 1986) and again at 30h post infection, the time at which the synchronisation breaks down and further rounds of schizogony take place, with trophozoites again being seen in the 152 FIG 5.6 Autoradiograph of ^S-methionine labelled, material immunoprecipitated with monoclonal antibody 13.1 and collected at different times during synchronised, in vivo gametocyte and ookinete culture of P. berghei. Lane 1A 9-12 hours post infection Lane 2A 12 -15 hours post infection Lane 3A 15-18 hours post infection Lane 4A 18-21 hours post infection Lane 5A 21 - 24 hours post infection Lane 6A 24 - 27 hours post infection Lane 7 A 27 - 30 hours post infection Lane IB 0-3 hours ookinete culture Lane 2B 3 - 6 hours ookinete culture Lane 3B 6-12 hours ookinete culture Lane 4B 12-18 hours ookinete culture Lane 5B 18-24 hours ookinete culture > NO |N O h O LU -F> O n CD •P* v£> O n U 1 O n I r 1 1 - 1 1 I I NO I UJ I I LT1 infection. It is therefore possible that the 66kD protein is in fact a trophozoiteprotein. This view is further strengthened by the incorporation of 35S-methionine into this protein during labelling of synchronised infections between 12 and 15 hours post infection. Finally, this protein is labelled by iodobead labelling of ookinete cultures (figs 5.11 & 5.12) and therefore suggests that this is a surface protein (table 4.1). 5.3.2 Synthesis of the 21kD Protein - Pbs 21 The bands detected in blotting e>q3eriments have been described above (Section 5.3.1). Additionally, in lane 8 (30 hour gametocytes) a much heavier band is seen at 27kD than in the other lanes. This may be due to a technical error where a wash was omitted in error before the samples were lysed and frozen for later analysis, leading to higher levels of this contaminant proteinthan in other samples. Whilst the source of this protein is unknown it was considered unlikely to be parasite in origin as no evidence of synthesis was found (Fig 5.6). The two next most likely sources might be a component of the culture system or a mouse protein. This could be checked using anti mouse antibodies. This protein is possibly a molecule present in serum such as the Ig or its component light chain. Unfortunately it was not possible to repeat the work as the samples were prepared with the kind help of Dr Mons and his colleagues. Unfortunately, the facilities were not available to obtain a second batch. However, this banding pattern does not coincide with Pbs 21 or any previously recorded bands and so is suspected of being a heavy artefact. However the underlying fact is that the autradiographs in figs 5.4 and 5.5 clearly show that the 21kD protein band seen in figs 5.5 and 5.6 is the only consistent label. To see in which parasite stages Pbs 21 was present unlabelled protein samples taken at the same intervals as the radiolabelled material, were separated electrophoretically and blotted against monoclonal antibody 13.1. The results of this blot, shown in fig 5.4 (lanes 1-8), also suggest that the 21kD protein is not found in either the asexual stages or the immature gametocytes or the mature gametocyte in the vertebrate host. Western blotting of unlabelled material (fig 5.4, lanes 9 -13) shows the presence of Pbs 21 in ookinete samples as early as 3 hours post ookinete culture. 155 The synthesis of the 21kD protein was examined by the biosynthetic labelling of parasites in culture with 35S-methionine and immunoprecipitation using mAb 13.1 at different stages of development, starting with the young trophozoite at 9h post invasion (Mons, 1986), through gametocyte development, and then throughout ookinete formation in vitro. Bands at 36kD and 45kD (Fig 5.4) were seen in some of the samples as is described above (Section 4.3.1.2). At each sampling point blood films were made and Giemsa stained to check for the presence and stages of parasites present at each time point. Immunoprecipitation and autoradiography of 35S-labelled proteins from synchronised gametocyte cultures (fig 5.6a) shows that the synthesis of the 21kD protein cannot be detected in any of the blood stage parasites including the gametocytes. Compared to this, the results obtained from the radiolabelling of parasites in ookinete culture (fig 5.6.b) show that the synthesis of the 21kD protein starts within the first three hours of ookinete culture. Further, more detailed, analysis of the first three hour period of ookinete formation, obtained by labelling for the shorter time period of Ih shows that the synthesis of the 21kD protein does in fact start within the first hour of ookinete culture (fig 5.7, lanes 2 & 3), but is not seen in a similar culture radiolabelled for lh at 37°C (fig 5.7, lane 1). In samples from both the synchronised gametocyte cultures and the ookinete cultures high molecular weight proteins are coprecipitated and these include the 66kD protein described above. In some gametocyte samples low molecular weight proteins are also found. Whether this is due to a specific reaction of mAb 13.1 against the proteins is unknown. If it were however, it might be indicative of a common sequence shared between these proteins and 21kD suggesting the possibility of a precursor. It may also be indicative of a product precursor relationship, but this would need to be further investigated by pulse chase labelling experiments. Alternatively, these proteins may come down as an artefact of the preparation of the samples, and are possibly complete Ig molecules or the separated heavy and light chain subunits that show similar molecular weights to the bands that are being detected (Roitt, 1986). In hindsight precipitation using NMS against these samples would have shown whether these bands constituted a background spectrum of 156 FIG 5.7 Autoradiograph of total protein samples labelled with ^ S - methionine during early ookinete culture. Lane 1 -1-0 hours ookinete culture Lane 2 0-1 hours ookinete culture Lane 3 1 -2 hours ookinete culture 1 2 3 f5 proteins or whether they appeared due to a specific reaction with 13.1. Despite the presence of these bands it still stands that the 21kD protein is not synthesised by the erythrocytic stages but is synthesised by the zygote and ookinete stages. 5.3.3 Trigger of 21kD Protein Synthesis Ookinete cultures treated with colchicine, vinblastine and the untreated control culture were examined for exflagellation at 15 min intervals for the first 4h of ookinete culture. This was achieved by removing a small sample from each culture and sealing it under a vaselined cover slip and examining it under the microscope for exflagellation Exflagellation was seen only to occur in the untreated control culture, but not in those treated with the anti microtubule assembly drugs colchicine and vinblastine. Radiolabelling of these cultures showed that 35S-methionine was incorporated into a 21kD protein which was immunoprecipitated by mAb 13.1 for both the control and colchicine treated parasites (fig 5.8). Synthesis is therefore not triggered by fertilisation. This labelling was not seen for the vinblastine treated cultures, however inspection of Giemsa stained smears showed extensive cell lysis with only a few intact cells remaining in this culture. Therefore the results from the vinblastine treated cultures were not considered meaningful. Unfortunately time did not allow for a repeat of the vinblastine treated cultures using lower concentrations. 5.3.4 125I Surface Labelling of Ookinete Cultures The autoradiograph of electrophoretically separated proteins from iodobead labellings shows that the ookinete dominant 21kD protein is first found on the surface of the cell within 30-45 min post exflagellation and from then on is detected at all times up until 24 hours (Fig. 5.11). This finding is in agreement with the data from immunogold labelling of the ookinete surface (Sinden et al, 1986). Further more the 125I surface labelling in conjuction with densitometry shows that there are 6 major and 12 minor peaks labelled by the iodobead method (Fig 5.12) and these are described in table 4.1. 159 FIG 5.8 Autoradiograph of SDS-PAGE gel of total 35s radiolabelled protein from ookinete cultures treated with anti microtubule agents prior to the start of ookinete culture Lane 1 Vinblastine Lane 2 Colchicine Lane 3 Control F " D 5.3.5 IFA of Ookinetes Cultured in the Presence and Absence of Tunicamycin IFA using the anti-21kD monoclonal antibody showed fluorescence (fig 5.9) even in the cultures treated with tunicamycin throughout the ookinete culture, suggesting that although the 21kD protein is an extracellular membrane protein and therefore likely to be glycosylated, the expression of the 13.1 epitope on the cell surface is not inhibited by tunicamycin. This therefore suggested one of several possibilities: 1) that 13.1 is not recognising a glycosylated epitope; 2) that the 21kD protein is not glycosylated or 3) that tunicamycin is not a active in Plasmodium. In view of finding that recognition of Pbs 21 by mAb 13.1 is not affected by periodate treatment (Tirawanchai, 1989) indicating that it is not a glycosylated epitope that is being recognised it was decided to use glucosamine/mannose labelling techniques to determine whether or not Pbs 21 was glycosylated. 5.3.6 Glucosamine/ Mannose Labelling of Ookinete Cultures To test further whether or not the 21kD protein is glycosylated, ookinetes were cultured in the presence and absence of tunicamycin with mannose, or glucosamine, or mannose and glucosamine. The results of this labelling are shown in fig 5.10. In all cases the label was incorporated into the 21kD protein, even in the presence of tunicamycin and as is clearly visible the intensity of labelling when both tritiated glucosamine and mannose are incorporated is greater than when just the single label is used, as would be expected. The inactivity of tunicamycin against the plasmodial cell is not novel to the ookinete stage and has also been seen for the 195kD protein of P. falciparum (GNewbold, Personal Communication). 5.3.7 Triton X-114 Extraction o f35S-methionine Labelled Proteins The result of the two rounds of triton X-114 extraction are shown in figure 5.13. As expected the 21kD protein separated into the detergent phase. An associated 66kD protein, possibly the same protein that is frequently detected in immunoprecipitations and Western blotting, separated into the aqueous phase, offering a future method of separating the two proteins. 162 FIG 5.9 Fluoresence micrograph showing the typical reaction of monoclonal antibody 13.1 with a retort form P. berghei ookinete in an indirect immunofluoresence assay FIG 5.10 Autoradiograph of the immunoprecipitated 21kD protein (running at 23kD) radiolabelled in the absence (lanes 1-3) and presence (lanes 4 - 6) of tunicamycin Lane 1 ^H-glucosamine Lane 2 ^H-mannose Lane 3 3fi-glucosamine and ^H-mannose Lane 4 ^H-glucosamine Lane 5 ^H-mannose Lane 6 ^H-glucosamine and 3H-mannose 1 2 3 4 5 6 6 6 " 45- 36- 29- 24- 21- 14- P FIG 5.11 Autoradiograph of total protein material collected at different times during in vitro ookinete formation and surface labelled with 125i using the iodobead technique. Lane 1 30 min ookinete culture Lane 2 1 hour ookinete culture Lane 3 1 hour 30 min ookinete culture Lane 4 2 hours ookinete culture Lane 5 3 hours ookinete culture Lane 6 6 hours ookinete culture Lane 7 12 hours ookinete culture Lane 8 18 hours ookinete culture Lane 9 24 hours ookinete culture 29- 24“ ..... FIG 5.12 Densitometer trace of a 24h ookinete culture surface labelled with 125I using the Iodobead method. Labelled markers indicate the relative positions of the molecular weight markers. The unlabelled tabs show the positions of the computer recognised peaks. FIG 5.13 Autoradiograph of proteins labelled with 35s-methionine during in vitro ookinete formation, extracted with Triton X-114 and then immunoprecipitated with mAb 13.1 Lane 1 Total protein Lane 2 Lower phase, first extraction Lane 3 Upper phase, first extraction Lane 4 Lower phase, second extraction Lane 5 Upper phase, second extraction 1 2 3 4 5 5.4 Discussion The development of monoclonal antibodies has provided useful tools for the study of specific proteins. The monoclonal antibody used in this study has been shown to recognise a protein with a molecular weight of 21kD (sometimes seen as 23kD) (Winger eta], 1988) that is expressed on the surface of the zygote and ookinete stages of P. berghei (Sinden et al, 1987), as early as two hours post fertilization. The 13.1, anti-21kD, monoclonal antibody has also been shown to block the transmission of the parasite to the mosquito, therefore making it of great importance as a model for development of potential vaccines. While the monoclonal antibody has much improved the detection of specific proteins caution should be taken in the interpretation of the results. It has been found here (figs 5.4 & 5.5) that using antibody based techniques bands in addition to the target 21kD protein, which was sought, were identified. In section 5.3.1 these proteins were described and suggestions as to their origin put forward. Further work is now required to investigate these ideas. The 21kD P.berghei protein is a zygote/ookinete stage protein as has been demonstrated by the data presented here and that of Sinden et al, (1987). The data obtained for the 21kD protein shows marked similarities to those obtained for a surface protein in both P. falciparum and P. gallinaceum. In P. falciparum Vermeulen et al (1985; 1986) has described a 25kD protein named Pfs 25, which is recognised by monoclonal antibodies that have a transmission blocking effect. The biosynthesis of this protein has been demonstrated in zygote/ookinete stages and also in the young gametocyte (Vermeulen et al, 1986) (Table 5.1). In P.gallinaceum however, two surface determinants have been identified with the molecular weights of 28kD and 26kD and the 26kD protein which will from now on be referred to as Pgs25 after Kaslow et al (1989) has been compared to Pfs25. It has been shown here that the P. berghei protein like the P. gallinaceum 26kD protein is synthesised in the zygote/ookinete stages but not in the immature gametocyte. The finding that Pfs25 is synthesised in the immature gametocyte may be due to the culture method applied to P. falciparum and could have come about through the contamination of the culture with mature gametocytes, that have become accidentally activated, from the previous subculture, thereby 172 Table 5.1 Comparison of the biosynthesis of transmission blocking proteins of zygotes/ookinetes of P. falciparum, P. gallinaceum and P. berghei. P. falciparum P. gallinaceum P.berghei Molecular weight 25kD 26kD 21kD Immature gametocyte + - - Mature gametocyte - - - Activated gametocyte ? ? + Zygote + + + Ookinete + + + 173 indicating the ease with which a contaminated culture could lead to this error. On the other hand this difference in P. falciparum may be due to variation between the species and in this instance would provide a natural boost to the production of transmission blocking antibodies. However, no actual anti-25kD antibodies have been detected in the infected host (Carter et al, 1989). The biosynthesis data for these proteins is summarised in table 5.1. At the molecular level the 21kD protein is much less well characterised than either it P. falciparum or its P. gallinaceum counterpart. The P. falciparum, Pfs25, is a protein in which the sequence determination of the gene encoding it has shown that the most marked feature is the presence of four tandem repeated epidermal growth factor-like domains that constitute the majority of the protein (Kaslow et al, 1989). The domains contain six cysteine residues in a core consensus sequence (Y/F-X-X-C-X-C-X-X-G-Y/F), with the second and fourth EGF-like domains fitting this consensus most closely. The fourth domain also shows a preceding cysteine as is seen in the human LDL receptor (Kaslow et al, 1989). Analysis of the P.gallinaceum gene sequence (Pgs25) has led to the prediction of a sequence containing a putative signal sequence at the amino-terminus, a short-hydrophobic anchor at the C-terminus, four potential glycosylation sites, 22 cysteine residues and a potential polyadenylation signal (Kaslow et al, 1989). Kaslow et al (1989) have also shown a close similarity between the structural organisation of Pfs25 and Pgs25. Like its P.falciparum equivalent Pgs25 shows the four EGF-like domains, a highly conserved spacing of cysteine residues and their surrounding amino acid residues. Also conserved are amino acid residues at the carboxyl-terminus of the putative signal sequence and the N-terminus of the anchor sequence, also two of the four glycosylation sites of Pfs25 have been conserved in Pgs25. While the P. berghei (Pbs21) protein is not so well characterised comparison of the properties of these three proteins may permit speculation on what some of the structural properties of Pbs21 may be. While all the parasite stages show different spectrums of the proteins synthesised some show the synthesis of stage specific antigens. This is seen with the zygote/ookinete stage as it is with the other invasive stages, the merozoite and sporozoite, all of which are involved at a time in the life cycle when the parasite is attempting to evade the host immune responses. Further insights into the 174 21kD protein might be gained by comparison with the surface antigens ofother parasitic organisms, like the variable surface antigens of Trypanosomes (Borst & Cross, 1982; Ferguson et al, 1987) and the immobilization antigens of Paramecium (Capedeville et al, 1987). Like these proteins the Pfs25 protein can be cleaved by phospholipase C without loss of immunogenicity (Vermeulen, unpublished observations) although this has not been confirmed by Freis et al (1989). It may therefore be expected that the P. berghei 21kD protein also shows a similar anchoring tail which may facilitate a method for the sheding of the protein as seen in other parasite systems. Pfs 25 has also recently been shown by Kaslow et al (1988) to have a hydrophobic anchoring tail in the protein sequence. In spite of the fact that Pbs 21 is easily shed it is possible that, depending of the juxta positioning of these putative anchoring mechanisms, the structural conformation of Pbs 21 is held far more rigidly than would be possible with a single anchor. Release of one of these tails could lead to a change in the tertiary structure of the protein and therefore antigenicity. Interestingly, the conformation of the 21kD protein is not critical in the recognition by mAb 13.1 (Winger et al, 1988) but the epitope recognised by another monoclonal (17.9) is conformation dependent. From an evolutionary point of view this may have provided a mechanism by which the ookinete was able to evade an immune response, once it has passed through the midgut wall and becomes open to attack from the humoral components of the insect hosts immune system. One property of membrane proteins is the ease with which they can be extracted using detergents. The triton X-114 method of Bordier (1981) was tested against radiolabelled proteins of P. berghei following the reported extraction of surface proteins of P. gallinaceum by Kumar (1985). The 21kD protein like its P.gallinaceum counterpart was extracted into the detergent phase. As these proteins have shown the transmission blocking effect the use of detergents to easily purify them could provide another step on the path of using such proteins for the all important development of a vaccine against malaria. Another interesting point of note is the labelling properties of theP. falciparum 25kD protein, which shows a poor uptake of 35S-methionine and yet 35S-cysteine gives a good level of labelling (Kaslow et al, 1988). This observation would however fit with the high cysteine and low methionine content predicted and also explains the described reduction-sensitivity of the 175 protein. By comparison the 21kD protein which labels well with radiolabelled methionine and is reduction insensitive with 2-mercaptoethanol (Winger et al, 1988) may be anticipated to have a high methionine content and low levels of cysteine, thereby explaining the differences in the biochemistry of these two proteins. This is consistent with preliminaiy amino acid composition data (Tiraiwanchai, Morris & Sinden. Personal Communication). The trigger for synthesis of the transmission blocking determinants in P. gallinaceum has been shown to be linked with events occurring at exflagellation. Carter & Kaushal (1984) have identified fertilization as the trigger in P. gallinaceum while Sinden et al (1987) suggest that macrogamete activation is responsible The suggestion that macrogamete activation is responsible for the triggering of Pbs 21 synthesis is confirmed by the labelling of gametocytes in which fertilization has been prevented by the addition of anti-microtubule agents. Although fertilization did not take place the 21kD was found to be synthesised in these cultures as determined by 35S- methionine labelling and SDS-PAGE (section 5.3.3). While the actual event responsible for triggering the synthesis remains unknown, the view that it is associated with macrogamete activation is supported by this labelling and the tentative identification of 21kD synthesis and its expression by unfertilized macrogametes (Sinden et al, 1987). The expression of the 21kD protein on the surface of the ookinete has been shown by Sinden et al (1987) using immunogold labelling and combined here with the iodine labelling gives initial expression as occurring between 45 and 110 min after exflagellation has been triggered. Although the earliest time of expression varies slightly between the two methods, both methods agree that it follows soon after ookinete culture is initiated. The discrepancy in absolute timing is almost certainly due to a difference in the sensitivities of the methods with the iodobead labelling being able to detect much smaller amounts of the protein than the immunogold method. Alternatively however, the 21kD protein could have been released by lysis of the cells during the iodobead labelling giving the erroneous indication of surface expression at a time when synthesis has occurred but before the protein could be transported to the cell surface. However, were this the case the whole range of parasite proteins would be expected to be labelled and this was not found. 176 The synthesis of the 21kD protein in P. berghei runs concurrently with the event of exflagellation and ookinete development. Certainly following exflagellation three rounds of DNA synthesis occur in approximately 10 min (Janse et al, 1986) as does RNA synthesis (Toy 6 et al, 1977) and protein synthesis. One way of speeding up these events would be if the precursors were all presynthesised by the gametocyte, only awaiting for the biosynthetic machinery to be switched on and in Chapter 3 it was shown that that the incorporation of 35S-methionine was not affected by the DNA and RNA synthesis inhibitors tested, suggesting that the messenger RNA coding for this protein is stored. While the timing of this does not prohibit the de novo synthesis of 21kD starting at this time a possibility is that, as for the other macromolecules (RNA and DNA), precursors are being presynthesised by the macro gametocyte. Presynthesis of the actual protein by the gametocyte is ruled out by the inability to detect the protein in the gametocyte in P. berghei (see earlier)and P. gallinaceum (Kumar & Carter, 1985), however, the possibility of this happening has not been completely ruled out for P. falciparum. Alternatively, the 21kD may be synthesised in a form whereby it could be stored, as part of a larger protein not recognised by the mAb 13.1, and only requires processing to allow its rapid initial expression. Radiolabelling experiments have shown that Pbs 21 is rapidly labelled following the induction of exflagellation and that this synthesis is cycloheximide sensitive. However, they do not show whether synthesis occurs directly or via the synthesis and processing of a higher molecular weight precursor as is the case for the circumsporozoite and merozoite proteins. If this proves to be the case then the 21kD protein would show marked similarities to the major surface proteins of the other invasive stages, the merozoite and the sporozoite. The sporozoite surface protein, or CS protein inP. berghei, is synthesised as a 54kD protein that undergoes a series of post-synthetic modifications that reduce it in size to firstly a 52kD protein and finally to a protein with a molecular weight of 44kD (Yoshida et al, 1981). Likewise the merozoite protein is synthesised as a high molecular weight protein throughout schizogony and is deposited on the surface of the intracellular parasite (Holder & Freeman, 1984). This protein is then processed either in late schizogony or at merozoite release to produce specific fragments on the surface of the merozoite (Holder et al, 1986; Holder, 1988). To investigate these possibilities gametocyte cultures could be run with the inclusion of radiolabelled amino acids, followed by washing and flushing with an excess of unlabelled amino acids. The presence of radiolabel in Pbs 21 could then be looked for. 177 Based on the evidence from the sporozoite and merozoite systems it might be expected for the 21kD protein to be synthesised in the form of a high molecular weight precursor and then processed later, assuming that a common mechanism is at work. Since the major surface proteins of the merozoite and sporozoite stages undergo modifications after synthesis extending the parallel further it may also be expected of the third. While no evidence was found in this study of such a relationship, it may also be the case that Pbs 21 coprecipitates as a complex as has been found for the 230/48/45kD proteins of P. falciparum (Kumar, 1987). In blotting and immunoprecipitation experiments coprecipitation of high molecular weight proteins was seen as well as those showing relative molecular weights (Mr) of 66kD and 45kD (Sections 5.3.1.2 & 5.3.1.3). Whether there is any relationship between any of these proteins remains to be determined, but possible ways in which they could relate to one another. 1) Protein polymerisation of the 21kD protein to give higher molecular weight products. Dimerisation of the 21kD protein would be expected to lead to a protein of approximately 42kD. A surface protein with an Mr of 45kD was also detected by surface iodination and densitometry of ookinete cultures. Within the accuracy of molecular weight determination on gels it is possible that this protein could be a dimer of Pbs 21. This would however need to be shown using pulse chase labelling techniques. Although, if polymerisation is the case, it would be expected that the polymer appears as the major protein present, and this does not happen with Pbs 21. It may be possible to prove whether or not 21kD spontaneously dimerises by preparing a concentrated solution of the protein, incubating under favourable conditions for dimerisation and then assaying for polymer formation. 2) A precursor/product relationship, where the 21kD protein is synthesised as a high molecular weight precursor and is later modified to yield the final product, Pbs 21. Whether any of the proteins that were coprecipitated do fill the role of precursor is unknown. Immunoprecipitation with mAbs 12.1 and 17.9, which recognise two further epitopes on Pbs 21 (Tirawanchai, 1989) and comparison of the banding patterns seen with these mAbs with those found for 13.1 might identify proteins that are common to all three mAbs strengthen the implication of the 178 product/precursor relationship. Also, the use of pulse chase labelling techniques may further clarify the situation. 3) The third possibility is that there is, in fact, no relationship between any of the proteins that are coprecipitated and that it has come about as an artefact. The association of the 13.1 mAb with the 66kD protein and a 45kD proteins needs clarification as to whether this came about due to the antibody binding to a common sequence in two unrelated proteins, or due to non specific binding. It may also be possible that coprecipitation occurred as a result of the two proteins being located close to one another on the cell surface, eg as part of a complex, but are in fact two totally unrelated proteins. To try to ascertain whether of not the proteins exist as a 'complex' the immunoprecipitation when followed by electrophoresis under non-reducing condition, or molecular sieve column chromatography should provide evidence of possible high molecular weight complexes. Non-specific binding, ie due to "sticky proteins" could be studied by substituting mAb 13.1 with other mAbs to see whether the 66kD and 45kD proteins are also regularly bound. Further biochemical modifications of Pbs21 might also be expected. It has been shown here that Pbs 21 is a glycosylated protein incorporating both tritiated mannose and glucosamine, as was found to be the case for Pgs25 in P. gallinaceum (Kumar & Carter, 1984). As with the labelling of Pgs25 not all the proteins incorporated the radiolabels and so a direct glycosylation event is indicated. In the same study on Pgs25 Kumar & Carter (1984) showed that as well as being glycosylated it was acylated incoiporating 3H-palmitic acid. It would now be logical to perform labellings to identify whether or not Pbs21 is also acylated, further strengthening the evidence that these two proteins share a common role, but in two different species of Plasmodium.. The function(s) of Pbs 21 are unknown, however by extending the the parallel between the merozoite, sporozoite and ookinete stages further it may be possible to speculate. As stated earlier, all are invasive and all show a predominant surface protein. It is therefore possible that these proteins serve as an aid to invasion. Therefore the function of the 21kD protein could be to mediate the ookinete penetration of the midgut of the mosquito, and this has certainly been suggested for Pfs25 (Amerongen et alt 1989). An alternative function of these proteins could be in the evasion of the host immune 179 system. While the value of this latter suggestion in the merozoite and sporozoite is apparent, this role is not as clear in the case of the ookinete. It may be that this protein serves a protective role. Such a function has been associated with the synthesis of the 28kD and 26kD proteins in P. gallinaceum, (Grotendorst et al, 1986; Grotendorst & Carter, 1987), as it has with the P. gallinaceum 55kD protein. The form of protection could be against immune components taken up in the blood meal, for example C3 complement (Grotendorst et al, 1986; Grotendorst & Carter, 1987) or from attack by the digestive enzymes (Gass & Yeates, 1979). However, antibodies against Pfs25 have not been found in the host blood (Carter et al, 1989) and therefore any protection would need to be against a general response. Alternatively in light of the identification of an immune response by insects in response to parasitic attack (Ratcliffe & Rowley, 1987) the 21kD protein may actually have an evasive role once the parasite enters the insect haemoceol. The exact relationship between the Pfs 25, Pgs 25 and Pbs21 proteins still remains to be determined. Differences between the species have been found, for example the different synthesis patterns seen for the 25kD P. falciparum protein (Vermeulen et al, 1986) and the 21kD P. berghei protein and the 25kD P. gallinaceum protein may indicate that the P. falciparum protein is in fact not an equivalent protein to the other two. If such is the case then it reduces the value of each protein as a model for the other two species, although information about each individual still retains its value for that system, ie, data on Pbs 21 still remains valuable as regards the P. berghei situation, but not in terms of defining the P. falciparum or P. gallinaceum events, and vice versa. However, at present the evidence suggests that these proteins are comparable, all being zygote/ookinete surface proteins that exhibit a transmission blocking effect. 5.4.1 Conclusions The work described in this chapter has shown: 1. Pbs 21 is synthesised by the zygote and ookinete stages of P. berghei starting within one hour of exflagellation. However, it is not synthesised by the gametocytes as was suggested for Pfs 25. 180 2. Pbs 21 is expressed on the surface of the zygote within one hour of exflagellation. 3. Synthesis is triggered by an event associated with exflagellation. This trigger is not fertilization. 4. Pbs 21 is glycosylated, incorporating both glucosamine and mannose. 5. Pbs 21 is readily extracted into the detergent phase with Triton X-114. 181 Chapter 6 General Discussion 182 6.1 Background to this Study As stated at the at the beginning of this thesis malaria is one of the biggest problems faced by the Third World and is therefore a major obstacle that must be overcome before those countries can achieve their full potential and reach western standards hygiene and health care (WHO, 1985). The more traditional methods that have been used to control malaria; spraying with insecticides and the use of anti-malarial drugs, began to loose their effectiveness during the 1960s as resistance by the mosquito to insecticides arose (Bruce-Chwatt, 1986) and resistance of the human malarias to the anti- malarial drugs available at that time became commonplace (Moore & Lanier, 1961; WHO, 1978; Peters, 1970; Rieckman, 1983; Reacher et al, 1981; McNamara et al, 1967). However, the development of new anti-malarial drugs for the treatment of the disease (Peters, 1989) and the introduction of complemtary and integrated pest management programmes, which in its simplest form involves the impregnation of bed nets with insecticide (Curtis, 1989) but can include any combination of antimalarial measures (treatment, insecticides, vaccines etc), have now once again made these approaches to malaria control highly effective. In addition to these programmes another aspect of current thinking towards the control of malaria is the development of antimalarial vaccines as a route to either eliminating or at least controlling the incidence of malaria. Three major vaccine strategies have been proposed: the anti sporozoite vaccine (prophylactic), the anti-merozoite vaccine (therapeutic) and the anti-gametocyte/zygote vaccine (transmission blocking or altruistic). These have been described in detail earlier in this thesis (Section 1.4). In recent years a number of optimistic and possibly premature reports have appeared in the quality newspapers based on the initial and promising results of early vaccine trials using the CS protein of P. falciparum . Initial studies in mice using irradiated sporozoite resulted in protection against infection and a resistance that could be maintained by periodically subjecting the animal to the bite of P. berghei infected mosquitoes (Nussenzweig & Nussenzweig, 1989). Following the identification of an immunodominant B cell epitope in the central region of the P. falciparum CS protein with the sequence NANP two synthetic vaccines were constructed. The first consisted of cysteine- (NANP)3 conjugated to tetanus toxoid (Herrington et al, 1987) and the second was constructed in E. coliand was in the form of a polypeptide consisting of 32 NANP repeats with 32 non-relevant amino acids from part of the 183 tetracyclin resistance gene, read out of frame (Ballou et a], 1987). While in each of these trials prevention of malaria was achieved in some individuals and in others the prepatent period was increased, the protection afforded was not as high as had been expected from the mouse studies and the limited success could be said to have resulted from the lack of basic information available concerning the immunological interaction between the parasite and its host. The aim of these trials was to induce protection through high antibody titres (Nussenzweig & Nussenzweig, 1989a). This objective was compromised possibly because (1) NANP is a poor T cell epitope, therefore limiting presentation of the antigen to the antibody producing cells, and (2) immune restiction within the host population, such that a single antigen is not recognised by all members of the population (Good et al, 1988). The low success rate of these trials resulted from a basic lack of information concerning the machanisms through which immunity to malaria is mediated. In recent years significant progress has been made into the indetification and characterisation of relevant malaria antigens, particularly those from the sporozoite, and in the understanding of protective immunity (Nussenzweig & Nussenzweig, 1989a). Evidence now exists to show that protection against infection is the result of anitbody mediated, cell mediated and non-specific responses to the sporozoite and cell mediated responses by CD4 and CD8 against the infected hepatocytes (Nussenzweig & Nussenzweig, 1989,1989a). It has therefore been suggested that further studies are necessary to determine the most effective way in which to incorporate protective antigens into vaccines. Therefore, for all the vaccine studies it becomes necessary to describe the parasite in terms of its mechanism of immunogenicity, the targets available to immune attack and the mechanisms active during the immune responses to parasite infection before the more complex task of vaccine development can be tackled effectively. 6.2 Pbs 21, A Transmission Blocking Protein of P. b e r g h e i The work in this project was conceived to provide some of the basic data on elements of the life cycle and biochemistiy of P. berghei. Extrapolation of such information may provide useful and easily manipulated models for the human malarias. 184 To study the parasite P. berghei and to concentrate on the gametocyte/zygote/ookinete stages it was necessary to characterise the course of the infection of the 2.34L strain used (Chapter 2). The intention of the study was describe the course of the infection and to try to identify some of the characteristics with an aim of being able to obtain a reliable supply of the parasite stages of interest. The study of the parasite in vivo gave new data that indicated clearly that the development of the parasite and its transmission are dependent on a complex set of factors, including the host immune response (Carter etal, 1988), blood factors (Eyles, 1951; 1952; 1952), serum factors (Rosenberg & Koontz, 1984), nutrient factors (Rosenberg & Koontz, 1984; Rosenberg et al, 1984) and behavioural responses to the parasite infection (Rossignol et al, 1984; 1985; 1986). Subsequent to this, routine methods were established to harvest these stages in a 'purified' form (Chapter 3) and then apply immune techniques as tools to study the Pbs 21 protein (Chapter 5). Investigation of the P. berghei transmission blocking protein Pbs 21 identified it as a major protein that is synthesised by and expressed on the surface of the zygote and ookinete stages. As expected of a membrane protein, Pbs 21 was found to be glycosylated, incorporating both labelled mannose and labelled glucosamine. Further chemical characterisation, such as identification of acylation and palmitoylation, still needs to be carried out and this would be the next obvious step in the study of this protein. Based on the findings of Kumar and Carter (1984) for P. gallinaceum it may well be expected that Pbs 21 is palmitoylated. While there are better systems in which to study post- translational modifications of proteins, such information is necessary in order to allow further studies on its physical and antigenic structure, its spatial distribution, and thus its interaction with its surrounding environment which may include those with transmission blocking antibodies. Pbs 21 is recognised by mAb 13.1 (Winger et al, 1988) which elicits a transmission blocking effect when used in passive transfer or in vitro membrane feeds to mosquitoes. Similarly mAbs against the Pfs 25 and Pgs 25 proteins have also been shown to block transmission. This raises questions as to the homology and function of this group of proteins. Possibilities as to the function include (1) acting as a barrier between the host immune components, such as antibodies, cytotoxic cells or cytokines, that are taken up 185 with the blood meal and sensitive targets on the parasite surface and (2) as a mechanism by which the ookinete penetrates the midgut wall. 1 It has not been possible to show synthesis of either Pbs 21 or Pgs 25 in the parasite in the vertebrate host, however this is not the case with Pfs 25 where the protein is found in the immature (2-7 day gametocyte) (Vermeulen et al, 1986). Therefore in the first two species at least it would not be expected to find antibodies against these proteins in the vertebrate host and in the case of Pfs 25 Carter et al (1989) have been unable to find any naturally occurring antibodies in the blood of infected patients. It is therefore possible that the rapid synthesis and expression of Pbs 21, Pgs 25 and Pfs 25 on the surface of the ookinete serves to block the recognition of gametocyte antigens present in the early zygote to which there is a natural immune response, (eg epitopes shared with Pfs 230 or Pfs48/45), from antibodies present in the bloodmeal by masking the expression and the recognition of these proteins, and thus protecting the developing zygote from destruction by host immune factors that would be aquired as part of a natural infection. A comparable mechanism has been suggested for P gallinaceum trypsin- sensitive components on the zygote surface which have been shown to protect the developing zygote from the alternative pathway of complement (APC), components of which are present in the bloodmeal (Grotendorst et al, 1986; Grotendorst & Carter, 1987). This protective function was shown to last for ~8 hours after exflagellation, up until the time at which digestive enzymes had removed these components from the bloodmeal (Grotendorst etal, 1986). After this time zygotes were shown to be sensitive to complement (Grotendorst et al, 1986). Coincidentally and on a speculative note, this timing of complement insensitivity corresponds with the time of synthesis of a 26kD protein (designated PgO-1) in P. gallinaceum which is synthesised for the first 8-10 hours of ookinete development (Kumar & Carter, 1985). It may be that this protein is responsible for conferring resistance to APC upon the P. gallinaceum zygote. 2 The second suggestion regarding the function of Pbs 21 is that it is involved with the escape of the ookinete midgut and therefore penetration of the midgut wall as has been suggested for Pfs 25 186 (Amerongen et al, 1989). This suggestion also fits with the pattern seen for the other invasive stages of the malarial parasite, each of which synthesise a 'major' surface protein: the CS protein in the case of the sporozoite (Yoshida et al, 1981) and PMMSA in the case of the merozoite (Holder & Freeman, 1984; Holder et al, 1986). In both these cases their respective surface proteins or their products have been linked to the invasion of host cell (Aikawa & Kilijian, 1979). It is possible that all these proteins interact with receptor molecules on the surface of the host cell in a receptor-ligand manner, and that this facilitates the passage of the parasite through the cell wall. This phenomenon of ligand-receptor interaction is by no means unique to the plasmodial parasite. The organisation of animal cells in differentiated organs and tissues has long been postulated to depend on cell-surface interactions both with molecules on the surface of other cells and with the extracellular matrix (Springer, 1990). Adhesion receptors participate in the control of cell-cell interactions and the regulation of cell migration. The distinctive antigen specific interactions studied by immunologists, and the cell and tissue-selective adhesive interactions studied by cell biologists, have recently found some common ground with the identificaiton of antigen-specific receptors and cell adhesion molecules on T lymphocytes. Cell-cell adhesion requires accessible cognat sites of the receptor, involves multivalent interactions and may be facilitated by receptor redistribution to the site of adhesion (Springer, 1990). Viruses have also been shown to use adhesion receptors in their interactions with cells. Similarly, the ookinete may use such receptor-ligand interactions to aid its escape from the hostile environment of the midgut. To return to the P. gallinaceum situation and the synthesis of PgO-1 there is associated with the synthesis of this protein a second protein with a molecular weight of 28kD (designated PgO-2) whose synthesis occurs during the latter part of ookinete development and at 22 hours post exflagellation is the major protein species synthesised (Kumar & Carter, 1985). Perhaps PgO-1 serves to protect the young developing zygote from immune components in the bloodmeal while PgO-2 aids the escape from the midgut. This speculation however would require substantial investigation. A question that now follows is how are the transmission blocking antibodies functioning. What makes this question even more interesting are the observations that such antibodies, both m Ab and naturally occurring antibodies to the sexual stages, will both block transmission (at high 187 concentrations) and enhance transmission (at low concentrations) (Mendis et al 1987; Peiris et al, 1988; Tirawanchai, 1989). The mechanism through which a single antibody can have these two opposing effects remains unknown. Does it, when present in high concentrations, aid the action of immune components while low concentrations form some sort of shield that prevent the action of these molecules? If the function of these proteins is receptor- ligand mediated invasion of the cells lining the midgut wall by the parasite do high concentrations of the mAb binding to the parasite prevent the interaction responsible? Conversely, do low concentrations of the mAb binding to the parasite, bring about conformational changes that increase the affinity between the receptor and the ligand as is seen with some enzyme complexes, for example aspartate transcarbamylase (Monod et al, 1963). Or do they act in yet some other manner? This remains to be seen and a full understanding of such mechanisms can only aid in the task of vaccine development. 6.3 Strategies for Vaccine Design In designing an anti-malarial vaccine the natural situation where the vaccine is to be most widely used must be taken into account. Since malaria is a problem that is predominantly faced by communities living in areas of the world that have poor health care and hygiene and in many cases are not easily reached, vaccines must be able to be administered in a single injection and to give long lasting protection to all or at least the majority of the population. Based on studies on the anti-sporozoite vaccine some of the factors that stimulate an immue response, that must be included in the design of a vaccine have been described: it needs to contain both B and T cell epitopes which are covalently linked and the T cell epitopes must be parasite derived to allow effective boosting (Good et al, 1986; Nussenzweig & Nussenzweig 1989; 1989a). In some cases it may be necessary to include some other form of T cell help in order to increase levels of antibody production (Good et al, 1988). Good et al (1986) also showed that immunity requires the induction of high levels of specific antibody and the involvement of non antibody dependent cellular immunity. Turning specifically to the sexual stage vaccines, study is at present focused on three P. falciparum proteins, Pfs 230, Pfs 48/45 and Pfs 25 (the presumed 188 'equivalent' protein to Pbs 21) (Targett et al, 1990; Kaslow, 1990). These proteins have been described as falling into two distinct groups based on their antigenic properties (Kaslow, 1990). The first group (Group I antigens) includes proteins that show a limited immunogenicity and a paucity of T cell epitopes, being unable to produce a cellular response in all individuals (Riley et al, 1990). It has been suggested that this lack of response is a result of MHC restriction (Carter et al, 1989; Good et al 1986), however Targett et al (1990) have suggested that it is a result of the down regulation of the immune system as a result of prolonged exposure to infection. Whatever the cause, with these antigens, it becomes necessary to include a potent adjuvant or a heterologous helper T cell (Kaslow, 1990) as was suggested with the sporozoite antigens (Good et al, 1986) in order to overcome widespread non responsiveness. In view of this non-responsiveness, it is unlikely that boosting of such vaccines would occur (Kaslow, 1990) and thus the maintenance of the high antibody titres essential for an effective immunity to be raised in the population (Good et al, 1986). The use of single group I antigens as candidates for the development of a universial vaccine may offer limited protection as a result of the combination of immune restiction and presence of low numbers of T and B cell epitopes in these antigens. One possible approach to this is to combine several of these group I antigens into a single multivalent vaccine, each of which may be recognised by a proportion of the population. This could then lead to "the universal vaccine", with boosting in individuals occurring to one or more epitopes recognised by the immune system (Carter et al, 1989). Therefore it is important to identify further antigenic proteins of the Group I type, in addition to the 230kD and 48/45kD proteins. The second group or Group II antigens (Kaslow, 1990) are proteins that are highly immunogenic, lack naturally occurring antibodies (Carter et al, 1989) and show a lack of genetic restriction in mice, implying that they have not come under immune pressure in their evolution (Kaslow, 1990) The Pfs 25 protein falls into this category and therefore by analogy so do Pgs 25 and Pbs 21. Whether of not boosting of vaccines would occur remains uncertain but depends upon (1) whether Pfs 25 is expressed in the human host, (2) whether it is expressed at low levels that are insufficient to initiate a primary immune response but at levels that might stimulate a secondary response or (3) whether it is expressed and evades the immune system in some unknown 189 manner (Kaslow, 1990). In the first and third cases boosting would be unlikely to occur. However, two pieces of data available today suggests that the second situation is the most likely. Firstly, the synthesis of Pfs 25 in the immature gametocyte has been demonstrated (Vermeulen et al, 1986). This however may be artefact due to the handling of the parasite, but the conclusion is supported by the findings of Kumar & Carter (1984) for P. gallinaceum and my own observations on P. berghei (Chapter 5). In the latter studies expression has not been detected in any of the parasite stages prior to those found in the mosquito. Whilst it may be argued that is the result of such low levels of the protein being present that detection was not possible at the levels of sensitivity used the availability of the antigen in the model systems used is greater than in P. falciparum~ Secondly, supporting the view that Pfs 25 is synthesised in the gametocyte is the finding of Kaslow et al (1988) that the transcript for this protein is found in the young gametocyte. A problem that must be confronted in the development of the transmission blocking vaccines is that if boosting fails to occur then the titres of antibodies in the blood could fall to levels where instead of blocking transmisssion it is actually enhanced, thereby increasing the spread of the parasite. It is therefore important to ensure that this will not happen before any vaccine is introduced. 6.4 Further Studies on Fbs 21 The study of the P. berghei is of interest both in terms of being able to define a complete parasite system and as a model for the human malaria P. falciparum. In view of the restricted recognition by the human immune system of P. falciparum antigens (Carter et al, 1989; Good et al, 1986; Targett et al, 1990) it would now be wise to determine whether or not native Pbs 21 is recognised by the immune systems of at least the majority of individuals within a host population. This could be achieved through studies on strains of inbred mice that are genetically identical except at the MHC, as has been done for P. falciparum 230-kDa, 48/45-kDa and 25-kDa proteins (Good et al, 1988). It is also necessary to determine the degree of transmission blockade offered by such a scheme. It has been shown that that inoculation with purified Pbs 21, 190 without adjuvant via the intramuscular route, will induced a potent transmission blocking effect that is still 95% effective 34 weeks post immunisation (Tirawanchai, 1989). Were Pfs 25 to show such a long-lived protection, and then used in conjunction with anti-malarial drugs it would allow the disease to be treated without the risk of transmission. It might also be used in conjunction with the "anti-disease" vaccines proposed by Taveme et al (1990a) to allow the development of natural immunity without the risk of transmission. As has already been stated further studies of Pbs 21 must include the full characterisation of this protein, including identification of the post- translational modifications and determination of its primary structure (amino acid sequence), which would provide the basis for further analysis. Logically once the biochemical nature of the protein has been defined it would then be possible to determine the structure by the use of biomolecular modelling techniques involving the application of computer programs (reviewed Blundell et al, 1987). The use of such techniques would allow the identification of regions of hydrophobicity within the protein, secondary structures such as a helical regions, p sheets and looped domains, and sequence alignments between Pbs 21 and other parasite proteins similarly investigated. Obvious candidates for such comparison are the proteins Pfs 25 and Pgs 25. It would also be of help in determining the degree to which these three proteins are related, particularly in conjuction with data obtained from the cloning and sequencing of the DNA coding for these proteins. Computer modelling of the protein could also be used to study the interaction of this protein with its surroundings, possibly giving insight into the way in which it functions. For instance, whether Pbs 21 could be acting in a receptor/co receptor (ligand) interaction with the cell lining the midgut wall of the mosquito to facilitate invasion by the ookinete. Similar studies on the three- dimensional structure and interactions of the adhesion receptors are currently being carried out and promise to provide exciting insights into the mechanisms by which they function (Springer, 1990). It may be possible to determine how Pbs 21 interacts with the transmission blocking antibodies and why they are able to both block and enhance development. Yet another possibility that results from protein modelling is the identification of potential new sites that may provide likely target against which vaccines and novel drugs could be aimed (Blundell et al, 1987). 191 Identification of the gene coding for the protein, would permit studies into the control of this protein's synthesis at the level of gene and RNA expression. It would also allow the insertion of the DNA coding for such proteins into bacterial plasmids and thus the bulk production of the protein. This could be used to test the efficacy of transmission blocking proteins in animal models (Tirawanchai, 1989) as a prelude to the use of the human transmission blocking proteins (eg, Pfs 25). If this proved effective, and once the safety of the native protein had been demonstrated in animal test systems, the way would then be open for the use of Pfs 25 in the form of the fusion protein, as a temporary measure against the spread of malaria, while more sophisticated preparations of the Pfs 25 protein are being developed. 6.5 Treatment of Malaria So far in this discussion I have centred on the prevention of malaria through the development of vaccines but an alternative approach to controlling the disease is through prophylatic treatment. In this section several suggestions for the ways in which drugs may be aimed specifically at the parasite as a result of its characteristics and metabolism will be presented. 1. In Chapter 4 it was shown that the levels of metabolic inhibitors required to block the development of the different parasite stages varied, for example the concentrations of metabolic inhibitors needed to block ookinete development if added prior to gamete formation were, in general, lower than those required if added after gamete formation. This may be due to (1) differences in the intracellular concentration of the drug that arise as a result of variations in the permeability of the membrane systems associated with the parasite or (2) differing sensitivities of the individual stages to the drug in question. One major difference between the gametocytes and zygotes studied in Chapter 4 is the membrane systems that the drug has to pass en route to its site of action: in the gametocyte, there is not only the triple parasite membranes to be traversed but also those surrounding the parasitoporous vacuole and infected erythrocyte membrane, while to enter the zygote the drug only has to pass through the zygote plasmalemma. However, the zygote plasmalemma is the only form of protection that the parasite has against the hostile environment of the mosquito midgut and it may therefore be particulary impermeable. In 192 respect of this drugs need to be developed that are able to cope not only with the differing natures of the parasite membranes (Sinden, 1978) but also the specialised nature of the host cell membranes. Sherman (1988) and Ginsburg (1990) have suggested that parasite invasion of the erythrocyte alters the permeability or the red blood cell membrane. This therefore offers the opportunity for the development of targeted drugs which will only be taken up by the parasitised cells. Thus treatment with such drugs should only affect those cells that parasitised (Ginsburg, 1990). 2. It was shown in Chapter 4 that many of the conventional metabolic inhibitors failed to act against the plasmodial cell, eg mitomycin C while in Chapter 5 tunicamycin was found not to block the glycosylation of Pbs 21. The inactivity of mitomycin C has also been demonstrated by Janse et al, (1986) as has the apparent inactivity of tunicamycin against the 195kD protein of P. falciparum (C. Newbold, Personal Communication). An explanation to those given above for the apparent inactivity of the drugs, the variation of intracellular drug concentration and stage specific sensitivities to the drug, is that atypical metabolic pathways are to be found in Plasmodium. This suggestion needs detailed investigation. However, were it to be found that Plasmodium does not use the usual metabolic pathways then it would open up the possibility for the development of drugs that are targeted at the parasite metabolism and would hopefully have no effect on the host. 3 The drugs could are targeted specifically against the parasite cells by binding therapeutic agents to mAbs against parasite proteins, as has been done with the treatment of cancer patients (Buri & Gumma, 1985). Development of such drugs linked to m Ab against parasite proteins, eg the gametocyte surface proteins or parasite antigens seen in the membrane of the infected erythrocyte (Perlmann et al, 1984), would allow the drug to be delivered directly to the parasite cell. This method of treatment allows the use of drugs with potentially serious side effects, eg ricin, to be used at toxic/effective levels with minimal risk to the patient. 193 The malarial parasite provides an interesting and rewarding system of study. 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WHO A31/19 2. World Health Organization. (1990). Rapp, trimest. Statist, sanit. mond. 43. Yeates, R.A. and Steiger, S. (1981). Ultrastructural damage of in vitro cultured ookinetes of Plasmodium gallinaceum (Brumpt) by purified proteinases of susceptible Aedes aegypti (L.). Zeitschrift fur Para sit enkunde 66, 93-97. 214 Yarbro, J.W. (1967). The effect of deoxyribnucleoside and ribonucleoside addition on the inhibitory effect of hydroxyurea on deoxyribonucleic acid synthesis. Biochemical Journal, 104, 52c-54c. Yoshida, N.; Potocnjak, PI, Nussenzweig V. and Nussenzweig R.S. (1981). Biosynthesis of Pb44, the protective antigen of sporozoites of Plasmodium berghei. Journal of experimental Medicine, 154,1225-1236. Young C.W. and Hodas, S. (1964). Hydroxyurea: inhibitory effect on DNA metabolism. Science, 146,1172-1174. 215 Appendix I Data from Plasmodium berghei in vivo and in vitro (Chapter 2) 216 Table Al.3.1. Parasite counts for a P3 infection in an individual nouse in 10** Tbc. DAY t bc/ml xlO* R T S Ma Mi Total El OP 0 436.6 1 424.8 3 21 0 0 0 24 0 n/d 2 382 2 34 0 3 0 39 0 n/d 3 380.8 8 59 0 15 0 82 4 0 4 334 59 307 0 16 4 386 8 0 5 364 90 447 4 26 4 573 16 0 6 398 102 558 20 24 10 714 32 0 7 353.6 80 743 22 10 47 902 110 1 8 316 63 771 51 38 17 940 62 0 9 254.8 88 718 80 60 94 1040 136 15 10 262.8 51 915 39 36 63 1104 114 17 11 245.6 124 562 46 32 36 800 103 33 12 257.2 62 1478 29 16 28 1613 198 20 13 189.6 131 981 51 17 43 1223 148 14 14 175.2 98 782 97 59 52 1088 176 14 15 160 196 792 30 26 32 1076 174 23 16 105.2 178 824 57 19 33 1111 90 13 17 93.2 278 1173 54 57 30 1531 90 0 18 83.6 274 984 90 24 40 1412 70 0 19 67.2 242 1054 66 28 16 1406 50 0 20 61.2 66 1037 44 7 21 1175 26 1 21 55.2 98 1338 74 26 34 1572 34 7 22 48 171 1224 45 12 9 1461 31 1 23 50.8 112 1260 60 16 17 1465 34 0 24 46.8 103 1484 68 15 43 1713 93 0 25 28 194 1868 66 20 60 2208 24 0 26 40 80 1829 112 21 48 2090 68 0 27 38.4 248 1624 84 44 48 2048 8 0 28 29.6 4 1823 35 26 24 1914 5 0 29 30.4 32 1697 149 16 11 1865 0 0 30 DEAD 217 TABLE A1.3.2 Parasite counts for a P3 infection in an individual mouse in 10** rbc. Mouse 2 DAY rbc/ml xl0e R T S Ma Mi Total El OP 0 420 _, _ IM 1 418.4 0 33 0 2 0 35 0 n/d 2 370 36 87 3 14 8 148 6 3 3 263 78 267 2 35 10 393 23 1 4 324.4 228 544 20 43 10 845 8 1 5 298.4 270 666 39 28 22 1025 43 8 6 402.4 252 962 30 40 28 1314 48 8 7 235.6 537 905 75 120 63 1700 278 10 8 206.4 438 876 75 98 93 1580 212 12 9 210 64 1626 52 52 50 1844 347 20 10 195.2 363 906 92 129 93 1583 256 15 11 158.4 56 1644 103 23 51 1876 379 40 12 161.2 201 1755 93 45 111 2205 378 25 13 149.6 106 956 114 34 36 1246 409 39 14 132 122 808 108 52 132 1222 278 50 15 132 729 1755 345 90 96 3015 218 47 16 73.2 276 1254 218 52 68 1868 171 19 17 59.2 194 1288 132 26 44 1684 90 0 18 49.2 158 1304 150 24 50 1668 72 0 19 49.6 322 1332 26 34 44 1758 39 0 20 38.0 36 1384 66 6 6 1500 33 3 21 36.8 110 1060 120 32 18 1340 32 1 22 34.8 480 1368 108 42 24 2022 22 2 23 38.8 105 1755 131 18 21 2030 95 1 24 42.4 33 1836 90 69 72 2100 96 0 25 41.6 54 1548 87 21 57 1803 40 3 26 38.0 52 1372 104 8 23 1559 76 0 27 29.2 96 1493 144 61 80 1874 1 0 28 29.2 24 1695 78 27 48 1872 0 0 29 DEAD 218 TABLE Al.3.3 Parasite counts f or a P3 infection in an individual mouse in 10* rbc. Mouse 3 DAY rbc/ml xlOe R T S Ma Mi Total El OP 0 432 - - - 1 434.4 1 6 0 0 0 7 0 n/d 2 381.2 1 4 0 0 0 5 0 n/d 3 356.4 0 8 0 0 0 8 0 n/d 4 356.4 1 17 0 1 0 19 0 n/d 5 344.0 9 39 4 3 0 55 1 1 6 412 81 354 6 82 19 542 23 6 7 309.6 432 1250 114 122 44 1966 218 10 8 251.6 56 1428 20 58 48 1610 230 14 9 266.8 42 982 12 54 54 1144 190 10 10 243.2 24 785 47 32 42 930 195 20 11 256.8 198 666 301 54 64 1283 158 10 12 221.2 81 896 102 47 65 1191 350 21 13 200.8 141 1005 57 18 51 1272 164 10 14 183.2 214 862 360 6 82 1324 192 41 15 176 206 786 54 24 56 1126 167 21 16 114.4 42 959 102 13 52 1168 99 25 17 96.4 96 922 144 14 20 1196 83 0 18 94.0 241 1470 127 16 38 1892 52 0 19 69.2 200 1410 121 6 21 1758 16 3 20 53.2 251 1408 50 21 13 1743 12 0 21 48.2 66 896 172 10 16 1160 18 0 22 38.8 198 1414 74 26 14 1726 14 0 23 37.2 98 1269 94 12 5 1478 13 0 24 45.2 42 1419 63 6 18 1548 41 0 25 42.4 132 1785 103 13 32 2065 24 0 26 46.8 101 1676 115 6 27 1925 12 0 27 38.2 78 1914 87 3 24 2106 0 0 28 32.4 81 1692 87 99 11 1995 0 0 29 DEAD 219 TABLE Al.8.1 Parasite counts for a P8 infection in an individual mouse in 10* rbc. Mouse 1 DAY rbc/ml xl0e R T S Ma Mi Total El OP 0 407.2 1 355.6 2 20 0 0 0 22 0 n/d 2 376 19 136 0 12 0 167 0 n/d 3 324 85 322 5 27 7 445 14 0 4 285.6 92 389 17 21 18 538 26 0 5 253.6 58 574 12 30 22 696 28 0 6 285.6 68 532 8 22 20 650 27 1 7 250 104 662 28 8 12 814 54 3 8 212 138 668 24 8 12 860 60 0 9 195.6 114 982 46 10 34 1186 71 14 10 177.6 66 784 16 10 18 894 91 14 11 133.2 134 212 2 30 14 392 75 25 12 145.6 102 862 62 14 12 1052 98 8 13 124 85 1491 69 12 22 1679 7 3 14 98 128 1273 48 10 11 1470 0 0 15 94.8 20 1055 50 2 3 1130 5 1 16 76.4 236 1148 88 0 2 1478 0 0 17 74.8 67 1447 60 12 7 1593 0 0 18 56.8 246 1554 78 4 8 1890 0 0 19 56.4 384 1636 32 30 6 2088 0 0 20 56 224 1912 52 13 5 2206 5 1 21 DEAD 220 TABLE Al.8.2 Parasite counts for a P8 infection in an individual mouse in 10* rbc. Mouse 2 DAY rbc/ml xl0e R T S Ma Mi Total El OP 0 400.4 _ 1 358 0 14 0 0 0 14 0 n/d 2 342.4 0 10 0 0 0 10 0 n/d 3 317.2 112 256 5 16 11 400 0 n/d 4 306.4 5 10 0 0 0 15 0 n/d 5 306.8 4 26 0 2 0 32 0 n/d 6 355.2 2 10 0 0 0 12 2 0 7 298 3 11 0 0 0 14 1 0 8 355.6 3 7 0 0 0 10 0 0 9 348 4 9 0 0 0 13 2 1 10 376 2 14 0 0 0 16 6 0 11 326.4 42 232 10 26 6 316 38 0 12 330 28 272 24 46 34 404 141 20 13 306 12 372 2 30 32 448 79 0 14 259.6 4 566 12 36 36 654 80 6 15 290.4 42 692 10 20 36 800 59 38 16 279.2 34 950 24 34 30 1072 77 52 17 230.2 76 1196 60 24 76 1432 154 5 18 193.2 56 1232 32 57 52 1429 115 54 19 153.2 154 1372 40 20 98 1584 116 19 20 152 21 1209 57 21 27 1335 164 7 21 131.6 60 1503 128 28 58 1777 140 31 22 80 242 1694 46 24 74 2086 127 8 23 47.6 114 1011 303 30 102 1560 11 0 24 32.8 64 2040 245 16 45 2410 0 0 25 DEAD 221 TABLE Al.8.3 Parasite counts for a P8 infection in an individual mouse in 10* rbc. Mouse 3 DAY rbc/ml xl0s R T S Ma Mi .Total El OP 0 413.2 1 432.4 4 16 0 0 0 20 0 n/d 2 399.6 2 7 0 0 0 9 0 n/d 3 344.4 4 10 0 0 0 14 0 n/d 4 296 10 7 0 0 0 17 0 n/d 5 285.6 4 3 0 0 0 7 0 n/d 6 324.8 5 5 0 0 0 10 0 n/d 7 329.6 2 8 0 0 0 10 0 n/d 8 320.4 2 9 0 0 0 11 0 n/d 9 307.2 2 5 0 0 1 8 5 0 10 311.2 5 15 0 0 1 21 13 0 11 262.8 8 48 0 2 5 83 34 0 12 295.2 4 290 12 36 24 376 131 12 13 279.2 18 480 8 32 48 586 42 10 14 215.6 16 454 12 42 32 556 40 0 15 211.6 52 714 12 22 18 818 37 4 16 175.6 80 1048 94 18 44 1284 45 2 17 131.2 24 1947 36 36 36 2079 86 7 18 136.4 264 892 66 18 40 1280 65 2 19 144 206 1744 58 8 30 2046 31 10 20 70.4 88 1640 96 8 45 1877 44 22 21 71.2 200 1662 60 27 27 1976 21 14 22 65.6 157 1558 59 19 18 1811 8 0 23 34.4 175 1830 160 30 12 2107 1 0 24 DEAD 222 TABLE Al.14.1 Parasite counts for a P14 infection in an individual mouse in 10** rbc Mouse 1 DAY rbc/ml xlOe R T S Ma Mi Total El OP 0 449.6 _ , 1 398 1 28 0 1 0 30 0 n/d 2 412.4 6 70 0 2 0 78 0 n/d 3 419.7 58 430 4 29 13 534 12 o o o o o o o 4 333.7 78 574 28 36 4 804 25 5 210 20 261 12 32 5 1330 14 6 200.4 54 1072 98 32 122 1398 9 7 141.7 106 1506 30 50 80 1772 6 8 63.2 232 1026 42 8 8 1318 1 9 58.8 149 1768 109 0 0 2067 0 10 DEAD 223 TABLE Al.14.2 Parasite counts fora P14 infection in an individual mouse in 10* rbc. Mouse 2 DAY rbc/ml xlOe R T S Ma Mi Total El OP 0 438 . 1 348.8 3 21 0 0 0 24 0 n/d 2 307.2 4 56 0 2 0 62 0 n/d 3 345.6 16 166 4 42 5 233 1 n/d 4 337.2 0 806 20 36 28 960 15 1 5 248 54 1280 37 32 24 1437 127 15 6 197.6 90 1601 25 30 31 1777 44 2 7 136 103 2004 65 13 23 2208 55 1 8 86 448 1572 120 8 32 2130 30 1 9 64 210 1792 44 16 10 2072 1 1 10 71.2 42 1788 114 6 0 1950 0 0 11 DEAD 224 TABLE Al.14.3 Parasite counts for a P14 infection in an individual mouse in lO* rbc. Mouse 3 DAY r b c /m l xlO e R T S Ma Mi T o ta l El OP 0 4 1 0 .8 1 3 5 8 .8 5 22 0 2 0 29 0 n /d 2 2 9 8 .8 8 84 0 10 0 102 0 n /d 3 3 0 3 .6 18 464 6 43 6 537 22 0 4 2 7 9 .6 50 1070 72 32 14 1238 30 2 5 1 5 4 .4 160 1274 44 136 32 1546 62 3 6 168 112 1984 149 13 45 2303 2 0 7 DEAD 225 TABLE Al.AS.l Parasite counts for a asexual infection in an individual mouse in 10* rbc Mouse 1 DAY R T S Ma Mi T o ta l 1 22 49 1 0 0 72 2 69 244 0 0 0 313 3 27 672 27 0 0 726 4 90 2457 67 0 0 2614 5 76 2335 121 0 0 2532 6 DEAD 226 ue 2 ouse M AL A.S2 aaie ons o a sxa ifcin n an in infection asexual a for counts Parasite A1.AS.2 TABLE (-*'X»C»'JCn Ma 0 0 0 0 0 0 0 0 0 227 Mi 0 0 0 0 0 0 0 0 0 al ta o T 1858 1258 1114 1192 1785 1037 1867 676 75 TABLE A1.AS.3 Parasite counts for a asexual infection in an individual mouse in 10* rbc Mouse 3 DAY R T S Ma Mi T o ta l 1 16 38 0 0 0 54 2 147 335 0 0 0 482 3 72 1260 94 0 0 1426 4 58 1602 99 0 0 1759 5 36 1390 67 0 0 1493 6 27 1782 27 0 0 1836 7 90 1810 94 0 0 1994 8 18 1876 64 0 0 1958 9 DEAD 228 Appendix II Data from the Purificaiton of Plasmodium berghei gametocytes in vivo and ookinetes in vitro (Chapter 3) 229 Table A2.1.1 The course of a P . b e r g h e i infection after 15/tg mitomycin C g-1 body weight treatment for an individual mouse M ouse 1 No. p er 10 * rb c Tim e R T S Ma Mi T o ta l E l S/P (h ) 0 58 404 7 17 11 497 21 2 /2 6 20 436 11 28 24 519 28 2 /2 12 36 199 7 73 29 344 63 2 /2 18 36 155 4 76 40 311 82 2 /2 24 106 50 6 52 44 258 187 2 /2 30 38 272 9 32 43 394 208 2 /2 36 38 427 1 28 28 522 290 2 /2 42 94 362 5 36 20 517 352 2 /2 48 2 96 0 53 27 178 697 2 /2 54 7 51 5 51 28 142 451 2 /2 60 4 31 3 39 29 106 476 2 /2 66 1 27 0 30 25 83 217 2 /2 72 8 85 1 19 26 139 101 2 /2 78 4 79 0 31 12 126 82 2 /2 T a b le A 2 .1 .2 The c o u r s e o f a P . b e r g h e i i n f e c t i o n a f t e r 1 mitomycin C g-1 body weight treatment for an individual mouse Mouse 2 No. p er 10-* rb c Time R T S Ma Mi T o ta l El S/P (h ) 0 7 291 10 20 7 335 47 2 /2 6 3 227 3 14 11 258 55 2 /2 12 106 93 2 19 34 254 50 2 /2 18 15 40 0 71 28 154 117 2 /2 24 7 31 5 51 58 157 66 2 /2 30 179 259 1 23 21 483 118 2 /2 36 76 283 2 12 20 393 61 2 /2 42 152 91 0 8 23 274 29 2 /2 48 8 304 3 11 13 339 37 2 /2 54 20 316 0 12 14 362 30 2 /2 60 27 308 7 0 0 342 13 2 /2 66 35 616 12 0 0 663 3 2 /2 72 dead 230 A2.2.1 The course of a parallel P . b e r g h e i infection to A 2.1 .1 without mitomycinC treatment fo r an individual mouse 1 No. p e r 10 r b c Tim' R T S Ma Mi T o ta l El S/P (h) 0 44 301 12 13 12 382 25 2 /2 6 8 420 11 24 23 486 33 2 /2 12 92 356 12 23 20 503 63 2 /2 18 51 364 8 75 28 526 61 2 /2 24 20 420 14 34 37 525 137 2 /2 30 31 500 33 44 36 644 158 2 /2 36 32 523 5 31 31 622 247 2 /2 42 14 529 18 41 41 643 357 2 /2 48 27 576 0 34 54 691 991 2 /2 54 37 612 53 41 46 789 706 2 /2 60 40 664 23 40 33 798 772 2 /2 66 95 605 36 28 40 804 695 2 /2 72 45 802 50 79 110 1086 909 2 /2 78 20 868 55 91 91 1125 974 2 /2 Tab A 2 .2 .2 The c o u r s e o f a P . b e r g h e i infection after 15Hg m ito m y cin C g -1 body weight treatment fo r an in d iv id u a l m ouse Mou 2 No. per 10^ rbc Tim R T S Ma Mi T o ta l El S/P (h) 0 20 332 14 14 9 389 18 2 /2 6 16 290 17 21 15 359 36 2 /2 12 33 347 8 22 36 425 45 2 /2 18 45 400 12 18 51 526 79 2 /2 24 12 385 21 45 39 502 98 2 /2 30 33 438 35 60 45 611 123 2 /2 36 40 497 11 55 66 669 179 2 /2 42 27 576 19 31 71 724 251 2 /2 48 21 622 27 37 78 785 387 2 /2 54 35 686 18 46 64 849 380 2 /2 60 41 745 8 50 73 917 532 2 /2 66 34 728 19 68 86 935 498 2 /2 72 27 813 14 71 67 992 579 2 /2 78 26 926 10 69 63 1094 586 2 /2 231