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Assessing the Function of Aspartic Proteinases of the Plasmodium falciparum Digestive Vacuole Using Gene-Knockout Strategies

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Title:
Assessing the Function of Aspartic Proteinases of the Plasmodium falciparum Digestive Vacuole Using Gene-Knockout Strategies
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BONILLA, JORGE ALFREDO ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Antimalarials ( jstor )
Digestion ( jstor )
Enzymes ( jstor )
Erythrocytes ( jstor )
Hemoglobins ( jstor )
Malaria ( jstor )
Parasitemia ( jstor )
Parasites ( jstor )
Plasmids ( jstor )
Vacuoles ( jstor )

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University of Florida
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University of Florida
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Copyright Jorge Alfredo Bonilla. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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5/31/2007

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ASSESSING THE FUNCTION OF THE ASPARTIC PROTEINASES OF THE Plasmodium falciparum DIGESTIVE VACUOLE USING GENE-KNOCKOUT STRATEGIES By JORGE ALFREDO BONILLA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Jorge Alfredo Bonilla

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ACKNOWLEDGMENTS I would like to thank Dr. John B. Dame for serving as my supervisory committee chair a providing valuable guidance during my studies at the University of Florida. I would also like to thank Dr. David Allred, Dr. Anthony Barbet, Dr. Ellis Greiner, Dr. Peter Kima, and Dr. Carol Palmer for serving on my dissertation committee. I learned a great deal from all of them and feel fortunate to have had such an outstanding committee. I thank all the lab members I worked alongside including Mr. Charles Yowell and Mr. Carlos Sulsona. To my wife, Tonya Bonilla, I owe tremendous gratitude for all her assistance with the laboratory experiments, as well as support and encouragement throughout the project. iii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iii LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .........................................................................................................viii ABSTRACT .........................................................................................................................x CHAPTER 1 INTRODUCTION...........................................................................................................1 Malaria: Introduction....................................................................................................1 The Burden of Malaria..................................................................................................2 Antimalarials and Emerging Resistance.......................................................................4 Parasite Life Cycle........................................................................................................6 Hemoglobin Digestion..................................................................................................9 Aspartic Proteinases......................................................................................................9 Plasmepsins.................................................................................................................11 Rationale for Study.....................................................................................................15 Hypothesis..................................................................................................................16 Specific Objectives..............................................................................................16 2 PHENOTYPIC CHARACTERIZATION OF SINGLE-PLASMEPSIN KNOCKOUTS............................................................................................................24 Summary.....................................................................................................................24 Introduction.................................................................................................................25 Material and Methods.................................................................................................27 Culturing of Malaria Parasites.............................................................................27 Comparative Growth Study.................................................................................28 Purification of Hemozoin....................................................................................28 Quantitative RT-PCR..........................................................................................29 Western Blot Analysis.........................................................................................29 Results.........................................................................................................................30 Comparative Growth Study.................................................................................30 Stage-specific Accumulation of Intracellular Hemozoin....................................31 Stage-specific Accumulation of RNA Transcripts by qRT-PCR........................31 iv

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Stage-specific Accumulation of Protein by Western Blot Analysis....................32 Discussion...................................................................................................................32 3 CREATION AND CHARACTERIZATION OF DOUBLE-PLASMEPSIN KNOCKOUTS............................................................................................................39 Summary.....................................................................................................................39 Introduction.................................................................................................................40 Materials and Methods...............................................................................................41 Plasmids...............................................................................................................41 Transfections and Selection.................................................................................41 PCR and Western Blot Analysis..........................................................................42 Parasite Growth Comparison...............................................................................42 Hemozoin Purification for Scanning Electron Microscopy................................43 Results and Discussion...............................................................................................43 4 SIMULTANEOUS DELETION OF THREE AND FOUR DV PLASMEPSINS AND THEIR SUCESPTIBILTY TO HIV PROTEINASE INHIBITORS................53 Summary.....................................................................................................................53 Introduction.................................................................................................................54 Materials and Methods...............................................................................................55 Plasmids...............................................................................................................55 Transfections and Selection.................................................................................56 Southern Blot Analysis of gDNA........................................................................56 PCR and Western Blot Analysis for Plasmepsins...............................................57 Culturing and Growth Characteristics.................................................................57 Transmission Electron Microscopy Analysis......................................................57 Neutral Lipid Staining with Nile Red..................................................................58 Western Blot Analysis of Falcipain 3..................................................................58 In vitro Antimalarial Drug Assays......................................................................58 Results.........................................................................................................................59 Transfections and Selection.................................................................................59 Confirmation of the Creation of Triple and Quadruple Plasmepsin Knockouts.59 Growth of the Parasite in Nutrient-rich Medium................................................61 Intracellular Hemozoin Production.....................................................................61 Transmission Electron Microscopy Analysis of the Mutant Parasites and 3D7.62 Nile Red staining of Neutral Lipids.....................................................................62 Western Blot Analysis of Falcipain 3..................................................................62 Sensitivity to HIV Proteinase Inhibitors, General Proteinase Inhibitors and Antimalarials....................................................................................................63 Discussion...................................................................................................................64 5 THE Plasmodium berghei MODEL..............................................................................77 Summary.....................................................................................................................77 Introduction.................................................................................................................78 v

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Materials and Methods...............................................................................................79 Plasmodium berghei Propagation........................................................................79 Immunofluorescence Staining and Microscopy..................................................79 Plasmid Construction...........................................................................................80 Recombinant PbPM4 Activity Against Hemoglobin Substrates.........................80 Results.........................................................................................................................81 Light Microscopy and Immunofluorescence.......................................................81 Plasmepsin 4 Knockout in P. berghei.................................................................81 Hemoglobin Proteolysis by Recombinant PbPM4..............................................82 Discussion...................................................................................................................83 6 DISCUSSION................................................................................................................91 LIST OF REFERENCES...................................................................................................94 BIOGRAPHICAL SKETCH...........................................................................................110 vi

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LIST OF TABLES Table page 2-1. Parasitemia increase every 48 hours of Dd2 and single-plasmepsin knockout clones.........................................................................................................................30 4-1. Sensitivity of wild-type (3D7), triple-plasmepsin KO (B12) and quadrudple-plasmepsin KO (C10) to HIV and other proteinase inhibitors..................................63 vii

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LIST OF FIGURES Figure page 1-1. Global distribution of malaria, 2004..........................................................................18 1-2. Life cycle of the malaria parasite..............................................................................19 1-3. Asexual stages of the malaria parasite.......................................................................20 1-4. Ultrastructure morphology of the Plasmodium falciparum digestive vacuole..........21 1-5. Proposed hemoglobin digestion pathway in Plasmodium falciparum......................22 1-6. ClustalW alignment of the four digestive vacuole plasmepsins of Plasmodium falciparum..................................................................................................................23 2-1. Flow cytometry analysis for determining parasitemia using SYTO-24....................35 2-2. Intracellular hemozoin accumulation in single-plasmepsin knockouts and the parental line Dd2.......................................................................................................36 2-3. RNA transcript levels of the DV plasmepsins in the single-plasmepsin knockouts and Dd2.....................................................................................................................37 2-4. Protein levels of the DV plasmepsins over 36 hours in Dd2 and the four single-plasmepsin knockouts................................................................................................38 3-1. Schematic of DV plasmepsin locus. The four DV plasmepsins are located in tandem array on chromosome 14..............................................................................46 3-2. Single-crossover plasmids pBSD-CAM-pfpm1 and pBSD-CAM-pfpm2.................47 3-3. PCR and Southern blot confirmation of double-plasmepsin knockouts....................48 3-4. Western blot analysis of clones isolated after limiting dilution................................49 3-5. Growth rate comparison of Dd2 and the double-plasmepsin knockouts...................50 3-6. Light microscopy images of Dd2 and the double-plasmepsin knockouts.................51 3-7. SEM images of hemozoin crystals purified from Dd2 (A), and the double-plasmepsin knockouts 21C3 (B) and 32A7 (C).........................................................52 viii

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4-1. Double-crossover plasmids pHTK-1546 and pHTK-1550 used to create a triple-plasmepsin KO (pfpm1, pfpm2 and pfhap) and a quadruple-plasmepsin KO (pfpm4, pfpm1, pfpm2 and pfhap)..............................................................................67 4-2. Schematic of the expected loci of the wild-type 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10).....................................68 4-3. Southern blot of gDNA from 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10) to confirm targeted disruption of the plasmepsin locus........................................................................................................69 4-4. PCR and western blot analysis confirming the creation of a triple and quadruple-plasmepsin knockout.................................................................................................70 4-5. Growth rate in doubling time of 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10)......................................................................71 4-6. Growth rate of 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10) in rich medium and limited-nutrient medium..............72 4-7. Intracellular hemozoin accumulation in 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10)....................................................73 4-8. TEM images of the 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10).......................................................................................74 4-9. Nile red staining of neutral lipid bodies produced in 3D7 (upper panels) and the quadruple-plasmepsin knockout (C10)......................................................................75 4-10. Western blot analysis of 3D7 and quadruple-plasmepsin knockout (C10) for falcipain 3..................................................................................................................53 5-1. Plasmid construct pL0001-pbpm4 for targeting plasmepsin 4 in P. berghei..........85 5-2. Light microscopy images of P. berghei (ANKA strain) stained with modified Geinsa Stain...............................................................................................................86 5-3. Immunofluorescence microscopy of P. berghei with anti-PbPM4 (green) and Hoescht DNA stain (blue).........................................................................................87 5-4. PCR analysis showing integration of plasmid pL0001-pbpm4 and disruption of the gene......................................................................................................................88 5-5. Hemoglobin hydrolysis of native human hemoglobin and native rat hemoglobin by recombinant PbPM4.............................................................................................89 5-5. Alignment of human and rat -globin.......................................................................90 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ASSESSING THE FUNCTION OF THE ASPARTIC PROTEINASES OF THE Plasmodium falciparum DIGESTIVE VACUOLE USING GENE-KNOCKOUT STRATEGIES By Jorge Alfredo Bonilla May 2006 Chair: John B. Dame Major Department: Veterinary Medical Sciences – Infectious Diseases and Pathology During the intra-erythrocytic stage of infection by Plasmodium falciparum (the causative agent of malaria) the parasite consumes 70 to 80% of the host cell’s hemoglobin by proteolytic digestion with enzymes located in the digestive vacuole (DV). Four DV aspartic proteinases (plasmepsins) in P. falciparum (PfPM1, PfPM2, PfHAP and PfPM4) were targeted for study using gene-knockout approaches. We tested the hypothesis that despite functional redundancy among the DV plasmepsins, at least one DV plasmepsin is necessary for the hemoglobin digestion process. Further, we postulate that PfPM4 (in the absence of PfPM1, PfPM2, and PfHAP) is the plasmepsin most likely to be capable of maintaining fitness of P. falciparum sufficient for survival during the asexual stage growth. Creating a triple-plasmepsin knockout (with only PfPM4 remaining) established that PfPM4 alone is sufficient to fulfill any function of this set of enzymes required to maintain the normal rate of asexual growth of the parasite. However, successful creation of a quadruple-plasmepsin knockout lacking PfPM1, PfPM2, PfHAP, and PfPM4 proved that none of the DV plasmepsins are essential for asexual-stage growth. The slower growth phenotype and the reduced rate of hemozoin formation of the quadruplex

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plasmepsin knockout established that the function of the plasmepsins was required for efficient growth in vitro. The quadruple-plasmepsin knockout showed an increase in susceptibility to cysteine protease inhibitors E-64 and leupeptin, suggestive of an increased dependence on the falcipain class of enzymes. Despite several reports suggesting that one or more of the DV plasmepsins is the target of HIV proteinase inhibitors, our study showed that the target of inhibition of these clinically important compounds is not found among the four DV plasmepsins. The digestive vacuole of the quadruple-plasmepsin knockout contained membranous whorls characteristic of the insufficient breakdown of autophagic vacuoles within a lysosome. Growth of the quadruple-plasmepsin knockout in amino acid-limited medium was severely affected; suggesting lack of the DV plasmepsins increases the susceptibility to starvation-induced autophagy. The DV plasmepsins play a role in the hemoglobin digestion pathway and are involved in the endosome/autophagosome processing of the digestive vacuole. xi

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CHAPTER 1 INTRODUCTION Malaria: Introduction Malaria is one of the earliest known diseases and continues to be an extremely important disease of the developing world. The agent of the disease was first described in 1880 when the French physician Alphonse Laveran, an army doctor working in Algeria, observed the malaria parasite in the blood of an infected patient (Laveran 1880). The mode of transmission was unproven, but evidence that mosquitoes transmitted important tropical diseases (such as filariasis and yellow fever) was mounting around the end of the nineteenth century (Manson 1877, Finlay 1881, Manson 1884, Reed et al. 1900). In 1897, the English physician Ronald Ross demonstrated the entire life-cycle of avian malaria (Ross 1897), and in 1898 the Italian protozoologist Battista Grassi and colleagues showed that the malarial parasite was injected into the human bloodstream by the bite of an infected female mosquito (Grassi et al. 1899). Malarial parasites belong to the protozoan subkingdom in the phylum Apicomplexa, class Sporozoa, order Haemosporida, family Plasmodiidae and genus Plasmodium. Four species are primarily responsible for human malaria: P. falciparum, P. vivax, P. malariae and P. ovale (Coatney et al. 1971). Phylogenetic analysis performed utilizing rRNA and merozoite surface protein (MSP) gene sequences suggest a common ancestry for P. falciparum and avian malarial parasites that diverged from a second lineage leading to monkey and rodent malarial parasites (Escalante & Ayala 1994, Polley et al. 2005). However, investigating the phylogeny using the mitochondrial 1

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2 cytochrome b gene did not support this association (Escalante et al. 1998, Perkins & Schall 2002). The female Anopheles mosquito is the natural vector of the Plasmodium species infecting humans (Coatney et al. 1971). Anopheles gambiae and An. funestus are the primary vectors responsible for the high transmission of malaria in tropical Africa (Coetzee & Fontenille 2004). However, over 60 species of Anopheles are capable of naturally transmitting malaria (Breman 2001). In the Americas, An. darlingi, An. albimanus, An. nuneztovai, and An. aquasalis are the primary vectors (Zimmerman 1992, Laubach et al. 2001). Clinical manifestations of malaria vary among species. A malarial infection can result in asymptomatic parasitemia, clinical malaria (febrile episodes with parasitemia), severe malaria (anemia, neurological syndromes, coma), and mortality. Plasmodium falciparum is the most virulent of the four malaria species that infect humans. Plasmodium falciparum modifies the surface of the infected erythrocyte and renders it adherent to endothelial cells (Udeinya et al. 1981, Gardner et al. 1996) resulting in the sequestration of mature parasites in the deep vasculature or to uninfected red cells leading to the formation of rosettes (Hasler et al. 1990). Sequestration of parasites in the brain may lead to cerebral malaria (Berendt et al. 1994), and the rosetting phenotype has been clearly associated with severe disease (Carlson et al. 1990, Rowe et al. 1995). The Burden of Malaria Studies and estimates on the burden of malaria show that the disease is a significant factor in long-term economic growth and development in regions where it is endemic (Malaney et al. 2004). Global distribution and endemicity of malaria depend mostly on the type of mosquito vector, the parasite species, and the climate of a region (Sachs &

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3 Malaney 2002). The malaria burden is greatest in underdeveloped countries where climate, economic conditions (poverty), inadequate health infrastructure, and lack of political commitment to effective control and prevention all contribute to the problem (Breman 2001). Currently, malaria is considered endemic in about 100 countries, almost half of them in Africa (Figure 1-1), and more than 3.2 billion people are at risk worldwide (WHO 2005). Each year, up to 3 million deaths are due to malaria and there are over 510 million episodes worldwide of clinical illness due to P. falciparum (Snow et al. 2005). About 60% of the malaria cases worldwide, about 70% of the global cases of falciparum malaria and more than 80% of the malaria deaths occur in Africa south of the Sahara (Snow et al. 2005, WHO 2005). Most of the deaths are among African children under 5 years old and these childhood deaths, resulting mainly from cerebral malaria and anemia, constitute nearly 25% of child mortality in Africa (WHO 2005). Fatality rates of 10 to 30% have been reported among children referred to hospitals with severe malaria, although these rates are even higher in rural and remote areas where patients’ access to adequate treatment is restricted (WHO 2005). Why the risk of death after a clinical attack of P. falciparum is much higher in Africa than in Southeast Asia and the Western Pacific is not well understood. People in Southeast Asia and the Western Pacific are believed to have better access to prompt treatment and cross-species protection against severe disease outcomes as a result of multiple Plasmodium species circulating in the region (Snow et al. 2005).

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4 Antimalarials and Emerging Resistance Perhaps the greatest challenge facing malaria control worldwide, and contributing increasingly to its burden, is the spread and intensification of parasite resistance to antimalarial drugs. The limited number of effective antimalarial drugs has contributed greatly to the growing challenge to develop cogent antimalarial drug-use policies and adequately manage the disease (Fidock et al. 2004). For many years, chloroquine and sulfadoxine-pyrimethamine have been the two most widely used antimalarial drugs. However, these drugs are failing at an accelerating rate in most malaria-endemic regions. As a consequence, malaria-related morbidity and mortality is increasing. To combat the malaria burden, new drugs are urgently needed to replace existing drugs for which resistance has become a problem. New drugs will be introduced in combination with other drugs for which parasite resistance is not yet a problem, to delay the development of resistant parasites. Drug resistance in malaria is a critical public-health concern since it has arisen and expanded amid a huge disease burden. Resistance to most antimalarial drugs in current use has been reported. Attempts to elucidate the molecular mechanisms that underlie this phenomenon are expected to provide valuable information for new drug development. The resistance pattern in many geographical regions has also been investigated using molecular epidemiological studies and provides useful treatment guidance. Because there are no bedside methods to assess antimalarial-drug susceptibility, any such developments would also be valuable for controlling malaria in affected areas. Chloroquine was introduced in 1948 and has been the cornerstone of antimalarial treatment for the past 50 years. However, since the first reports of chloroquine-resistant Plasmodium falciparum in Southeast Asia and South America a few years after its

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5 introduction, the burden of drug-resistance has continued to increase. The advent of chloroquine resistance led to the development of other drugs such as sulfadoxine-pyrimethamine (1967), mefloquine (1977), and atovaquone (1996). Proguanil and pyrimethamine were introduced nearly 50 years ago and were powerful additions to the spectrum of antimalarial agents. Both drugs are specific inhibitors of the dihydrofolate reductase (DHFR) enzyme and, in combination with sulfa drugs (pyrimethamine-sulfadoxine and pyrimethamine-dapsone), have been distributed widely for malaria infections. In China, infusions of wormwood (Artemisia annua) were used for treating fever over 1000 years ago. The efficacy was attributed to the sesquiterpene lactone, artemisinin (Rosenthal 2001). Artemisinin and semisynthetic derivatives have become an important antimalarial drug group. By the late 1980s, resistance to sulfadoxine-pyrimethamine and to mefloquine was also prevalent on the Thai-Cambodia and Thai-Burmese border (Hoffman et al. 1985). Chloroquine resistance continued to spread across Africa during the 1980s, and severe resistance is especially prevalent in east Africa. As a result, more than 10 African countries have switched their first-line antimalarial drug to sulfadoxine-pyrimethamine (Kublin et al. 2002). However, this drug is also losing efficacy and efforts are needed to lengthen its lifespan, and to identify effective and affordable alternative antimalarial drugs. In South America and Asia, drug-resistant strains of Plasmodium also pose an increasing therapeutic dilemma. By 1980, all endemic areas of South America were reporting chloroquine resistance in P. falciparum. By 1989, almost all endemic areas in Asia and Oceania had widespread resistance (Wongsrichanalai et al. 2002). Some focal

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6 points in the Amazon Basin are thought to have emerging multi-drug-resistant (MDR) malaria parasites. These areas are classified on the basis of widespread loss of clinical efficacy of chloroquine and sulfadoxine-pyrimethamine, and the potential for emergence of resistance to a third class of drug (Wongsrichanalai et al. 2002). By this definition, MDR-regions include areas of east Africa, the Amazon Basin and Southeast Asia. However, because of the lack of reliable drug-susceptibility surveillance data, precise boundaries of MDR areas are difficult to determine and all areas with signs of progressive loss of sulfadoxine-pyrimethamine efficacy are considered at risk for the emergence of MDR-malaria parasites (Wongsrichanalai et al. 2002). There have been sporadic reports of mefloquine resistance and reduced susceptibility to quinine in the Amazon Basin. As a result, artesunate-mefloquine combination therapy is being adopted as the first-line regimen on the border of Peru and Brazil (Llanos-Cuentas et al. 2001). Currently, the only individual drugs effective against MDR-malaria parasites are artemisinin derivatives such as artesunate. Effective drug-combination therapies have been developed, but they are significantly more expensive than treatment with individual drugs such as chloroquine or sulfadoxine-pyrimethamine. If steps are not taken to address the root causes of drug resistance, these drug combinations will also lose their effectiveness in the near future. These circumstances exacerbate the need for the validation of potential drug targets and the development of new antimalarial compounds. Parasite Life Cycle The biology of the four human species of Plasmodium is generally similar and consists of two discrete phases: sexual and asexual (Figure 1-2). The asexual cycle occurs in the human host, initiating in the liver and continuing in the circulating erythrocytes. The sexual cycle occurs in the female Anopheles mosquito (Ross 1897).

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7 When the infected mosquito takes a blood meal, it injects salivary fluids into the wound. These fluids contain sporozoites, small (10-15 m long), spindle-shaped, motile forms of the parasite that initiate the infection. The sporozoites are cleared from the circulation in an hour and eventually reach parenchymal cells of the liver (Krotoski et al. 1982). Once inside the liver cell, the parasites undergo asexual division: exoerythrocytic schizogony. The length of this exoerythrocytic phase and the number of progeny (merozoites) produced in each infected cell is a characteristic of the individual species of Plasmodium. Plasmodium falciparum typically matures within 5 to 7 days and produces about 40,000 merozoites per infected liver cell. Plasmodium vivax can mature within 8 to14 days, and each of its sporozoites produces about 10,000 daughter parasites. For P. ovale, maturation takes about 11 to 16 days and produces 15,000 merozoites. Plasmodium malariae takes 12 to 16 days, producing about 2000 merozoites (Coatney et al. 1971). These prepatent periods are estimates: factors such as parasite strain and host factors play a role in duration (Coatney et al. 1971). When the merozoites are released from the liver schizonts into the blood stream, they invade erythrocytes and initiate the erythrocytic phase of infection. Invasion of the erythrocytes consists of a complex series of events beginning with contact between a free-floating merozoite and the red blood cell (Dvorak et al. 1975). Attachment of the merozoite to the erythrocyte involves interaction with specific receptors, such as the Duffy blood group antigen for P. vivax (Miller et al. 1976), or sialic acid residues on glycophorin A for P. falciparum (Howard et al. 1982, Pasvol et al. 1982). Parasite proteins involved in the interaction with red cell receptors belong to a family of erythrocyte binding proteins (EBPs). The EBP family includes P. vivax Duffy binding

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8 proteins (PvDBP) (Wertheimer & Barnwell 1989), P. falciparum erythrocyte binding antigen EBA-175, which binds to the sialic acid residues on glycophorin A (Adams et al. 1992), and its paralogues, EBA-140 and EBA-181, which bind receptors other than sialic acid-glycophorin A (Dolan et al. 1994). After apical reorientation, the parasite enters the cell by a localized endocytic invagination of the erythrocyte membrane, using a moving junction between the parasite and the host cell membrane (Aikawa et al. 1978, Miller et al. 1979). Once inside the erythrocyte, the parasite begins to grow, first forming the ring-like early trophozoite and then enlarging to fill the cell (Figure 1-3). The organism then undergoes asexual division and becomes a schizont composed of daughter merozoites (Figure 1-3). The erythrocytic cycle is completed when the erythrocyte ruptures and releases the merozoites, which proceed to invade other erythrocytes (Figure 1-3). The asexual cycle is characteristically synchronous and periodic in the human host. Plasmodium falciparum, P. vivax and P. ovale complete the development from invasion by merozoites to rupture of the erythrocyte within 48 hours, thus exhibiting “tertian” periodicity. Plasmodium malariae, which produces “quartan” malaria, requires 72 hours to complete the cycle (Coatney et al. 1971). Not all merozoites develop asexually. A small proportion differentiate into sexual forms called macrogametocytes (female) and microgametocytes (male) which must complete their development in the gut of an appropriate mosquito vector. On ingestion by the female Anopheles mosquito, the gametocytes in the blood meal shed their protective erythrocyte membrane in the midgut of the vector. Male gametocytes initiate exflagellation, a rapid process that produces up to eight active, sperm-like microgametes,

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9 each of which can fertilize a macrogamete. The resulting zygote elongates into a diploid vermiform ookinete, which penetrates the gut wall and comes to lie under the basal membrane. The parasite then transforms into an oocyst within 24 hours after ingesting the blood meal. Sporozoites develop in the oocysts, leading to the production of more than 1000 of these haploid forms per oocyst. They mature within 10 to14 days, escape from the ruptured oocyst, and travel through the hemolymph to mature in the salivary glands. When the infected mosquito bites the human host again, a new cycle begins. Hemoglobin Digestion The parasite digests up to 80% of the host-cell hemoglobin during the erythrocytic phase of its life cycle (Lew et al. 2004). The metabolism of hemoglobin initiates soon after invasion, but is most pronounced during the trophozoite stage. The mechanism by which the parasite ingests hemoglobin involves the pynocytosis of the parasitophorous vacuole membrane (Rudzinska et al. 1965) resulting in the formation of a cytostome (Figure 1-4). The cytostome transports hemoglobin as a vesicle to a specialized acidic digestive vacuole where hemoglobin digestion occurs (Goldberg 1992). Several enzymes are believed to be involved in the digestion of hemoglobin (Figure 1-5). The digestion of hemoglobin is an essential function and the enzymes involved in this pathway make attractive drug targets. The role of each of the enzymes is the subject of ongoing research. It has been proposed that the parasite evolved redundancy in this pathway to increase its fitness (Omara-Opyene et al. 2004, Sijwali & Rosenthal 2004, Liu et al. 2005a). Aspartic Proteinases Aspartic proteinases are common in eukaryotes and are involved in a number of important biological processes. Inhibitors to these enzymes have been much sought after

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10 as potential therapeutic agents. In P. falciparum, a total of 11 aspartic proteinases have been identified: 10 belong to the clan AA and family A1 of proteinases, and are called plasmepsins (PfPM 1, 2, HAP, 4, 5, 6, 7, 8, 9, 10) (Rawlings et al. 2006), and 1 belongs to clan AD family A22 (the presenilin family). A clan contains proteinases that have arisen from a single evolutionary progenitor. There are 5 clans of aspartic proteinases and each represents one or more families that show evidence of evolutionary relationship as a result of their similar tertiary structures, or when structures are not available, by the order of catalytic-site residues in the polypeptide chain and often by common sequence motifs around the catalytic residues (Rawlings et al. 2006). Clan AA family A1, to which the 10 plasmepsins belong, contains proteinases that were formerly known as “acid proteinases” or “carboxyl proteinases” because most of them are active at acidic pH. The type proteinase of this group of enzymes is pepsin A, which has long been known for its contribution to proteolysis of acid-denatured food proteins in the vertebrate stomach, and was one of the first enzymes crystallized (Northrop 1939). The catalytic active site is formed by two Asp residues that activate a water molecule which mediates a nucleophilic attack on the peptide bond (James 2004). Proteinases in this family have evolved by gene-duplication, and the two catalytic Asp residues, like the lobes in which they are contained, are homologous (Tang 2004). The translated gene sequences encoding these enzymes contain signal peptides and propeptides. The catalytic site is located between the two lobes of the enzyme, and a “flap” structure containing a conserved Tyr residue controls specificity (Davies 1990, Hong & Tang 2004, James 2004). Several of the proteinases are glycosylated, such as cathepsin D, a lysosomal aspartic proteinase in mammalian cells (Tang & Wong 1987,

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11 Fortenberry & Chirgwin 1995); and a few are membrane-bound, such as memapsin-2 studied extensively because it was identified as a -secretase that initiates production of amyloidleading to the pathogenesis of Alzheimer's disease (Selkoe 2003, Selkoe & Schenk 2003). Retroviral aspartic proteinases of human immunodeficiency virus (HIV) have been studied extensively as they are essential for maturation of the virus particle (Navia et al. 1989) and inhibitors to these enzymes have a proven therapeutic record in the treatment of AIDS (Babe et al. 1995). Aspartic proteinases are also virulence factors implicated in the mechanisms of host colonization by the yeast Candida albicans in different types of candidiasis, as such they are targeted in the development of treatment for and immunization against this common fungal infection (Bein et al. 2002, Schaller et al. 2003, Vilanova et al. 2004). Research showing the importance of aspartic proteinases in many biological functions and disease, and the successful development of inhibitors to these enzymes, suggests that the aspartic proteinases of P. falciparum are potential drug targets. Plasmepsins The intraerythrocytic malaria parasite degrades hemoglobin to provide nutrients for its growth and maturation (Rosenthal 1999). This process is thought to occur in the digestive vacuole (DV) (Goldberg et al. 1990), an acidic, organelle unique to the genus Plasmodium (Krugliak et al. 2003). Extensive hemoglobin digestion occurs here during the trophozoite stage of development, and the DV is the apparent site of action of several important antimalarial drugs (Fidock et al. 2000). The catabolism of hemoglobin in the DV is thought to occur in a semi-ordered sequence of proteolytic events involving plasmepsins (PfPM) 1, 2, and 4 (Francis et al. 1994, Gluzman et al. 1994, Egan 2002);

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12 a histo-aspartic proteinase (PfHAP) (Berry et al. 1999); cysteine proteinases (falcipain 2A, 2B, 3) (Shenai et al. 2000); and a metalloproteinase (falcilysin) (Eggleson et al. 1999). The residual heme (which is toxic to the parasite) is sequestered as a crystalline pigment called hemozoin (Sullivan et al. 1996). The specific role of each of these proteolytic enzymes in digesting hemoglobin is not entirely understood. However, using diagnostic inhibitors that block heme release and kill parasites in culture (Francis et al. 1994, Moon et al. 1997), PfPM1 and PfPM2 have been implicated in the early stages of the hemoglobin digestion process. PfPM1 readily cleaves native hemoglobin in vitro, but PfPM2 appears to prefer acid-denatured globin (Gluzman et al. 1994). Both of these closely related enzymes (74% DNA sequence identity; 66% amino acid sequence identity) (Figure 1-6) can cleave the Phe33–Leu34 bond in the hinge region of hemoglobin, considered the crucial first step in the digestion pathway (Goldberg et al. 1991). After this initial hydrolysis, the partially degraded hemoglobin molecule is thought to unravel, thus becoming susceptible to further proteolysis by the other proteinases in the DV. Several proteinases are capable of acting on the denatured or fragmented globin, including two cysteine proteinases, falcipain 2 and 3 (Salas et al. 1995, Francis et al. 1996, Sijwali et al. 2001); and two aspartic proteinases, PfHAP and PfPM4 (Berry et al. 1999, Banerjee et al. 2002). The proteinases PfHAP and PfPM4 are approximately 60% identical to PfPM1 and PfPM2 at the amino acid level (Figure 1-6) (Dame et al. 2003). PfHAP is an active enzyme despite having an unusual active site with a histidine in place of one of the two catalytic aspartic acid residues present in all other plasmepsins.

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13 A metalloproteinase, falcilysin, has been shown to act on 15 amino acid globin fragments to generate small peptides (Eggleson et al. 1999). A dipeptidyl aminopeptidase 1 (DPAP1) located in the DV of P. falciparum has recently been characterized and shown to possess hydrolytic activity against fluorogenic peptide substrates (Klemba et al. 2004b). The resulting dipeptides may be exported out of the DV for terminal degradation to amino acids in the parasite cytosol catalyzed by neutral aminopeptidases (Klemba et al. 2004b). It is also possible that a yet-to-be discovered acidic aminopeptidase exists and is functional in the DV. The biosynthesis of the four DV plasmepsins is similar (Francis et al. 1997, Banerjee et al. 2003). Each proenzyme is synthesized as a 51 kD type II integral membrane protein containing a transmembrane domain within its prosegment. Following proteolytic cleavage of the prosegment, a 37 kD active, soluble enzyme is formed. In vivo pulse-chase studies using specific PfPM1, PfPM2, and PfHAP antibodies revealed that the proenzymes are processed rapidly, with a t 1/2 of 20 min (Francis et al. 1997, Banerjee et al. 2003). In culture, this processing has been shown to be blocked by tripeptide aldehydes, N-acetyl-Leu-Leu-norleucinal (ALLN) and N-acetyl-Leu-Leu-methioninal (ALLM). These compounds, known as calpain inhibitors, block the calpain family of cysteine proteinases as well as other cysteine proteinases and proteasomes. Importantly, proplasmepsin processing was not blocked by EGTA, or general inhibitors of cysteine proteinases (E64, leupeptin) and proteasomes (lactacystin) (Banerjee et al. 2003). It has therefore been proposed that a novel convertase is responsible for the removal of the proregion but it is not clear where proplasmepsin activation occurs or what protein may be responsible for this process.

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14 Studies suggest that newly synthesized DV plasmepsins reach the digestive vacuole via the ‘classical’ secretory pathway. Maturation of PfPM 1, 2, and HAP is blocked by treatment of parasites with brefeldin A (BFA), an inhibitor of anterograde protein traffic from the ER (Francis et al. 1997, Banerjee et al. 2003). PfPM1 has been detected at the parasite plasma membrane and in a specialized organelle called a cytostome (Francis et al. 1994). The cytostome is a double membrane invagination of the parasite plasma membrane (PPM) and parasitophorous vacuole membrane (PVM) which pinches off to form a hemoglobin-containing vesicle that traverses to and fuses with the DV where the hemoglobin is released and digested. The presence of PfPM1 in the cytostome suggests that the enzyme is transported to the digestive vacuole via the cytostome (Francis et al. 1994). Histidine-rich protein 2 is an example of a protein that is transported to the erythrocyte cytosol in a BFA-sensitive manner and a direct transport route from the ER to the digestive vacuole has been proposed (Akompong et al. 2002). However, it has also been proposed that small amounts may be transported to the digestive vacuole via the cytostome (Sullivan et al. 1996). The trafficking of PfPM2 has recently been shown to involve insertion into the ER, followed by transport to the cytostome where it accumulates and is brought into the DV with hemoglobin (Klemba et al. 2004a). PfPM4 has been shown to localize with BiP in fixed BFA treated cells also suggesting transport via the ER (Banerjee et al. 2003). Taken together, the data suggest that the plasmepsins are secreted via the ER to the plasma membrane where the cytostome forms by pinching off and brings the proteins to the DV. An analysis of the sequenced genomes of other Plasmodium spp. indicates that among the DV plasmepsins only the ortholog of P. falciparum plasmepsin 4 is present

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15 (Dame et al. 2003). It has been suggested that PfPM4 has an active site that is similar to, but yet distinct from those of PfPM1 and PfPM2 so that the three enzymes might well act in concert in the food vacuole of P. falciparum (Brinkworth et al. 2001, Wyatt & Berry 2002). Moreover, PfPM4 has been shown to share highly similar subsite specificities to the orthologous plasmepsins in P. malariae, P. ovale and P. vivax (Li et al. 2004, Beyer et al. 2005). The roles of four closely related plasmepsins in the P. falciparum DV are open to speculation. Has P. falciparum evolved a more complex pathway of catabolizing hemoglobin? Do the paralogs simply provide redundant functions to assure the survival of the parasite? Do they contain little or no specialized functions? These issues are important ones to investigate when analyzing the hemoglobin digestion pathway for potential targets for developing new antimalarial chemotherapies. A fundamental assumption has been that one or more of the DV plasmepsins performs an essential role in metabolism of hemoglobin. Inhibition of individual enzymes, however, might be insufficient to kill the parasite (Omara-Opyene et al. 2004, Liu et al. 2005a). The broad similarities between the DV plasmepsins may permit the development of compounds able to inhibit all four enzymes to produce a drug capable of imposing a multiple blockade on the pathway of hemoglobin digestion. An inhibitor that inhibits the DV plasmepsins of P. falciparum as well as the closely related homologs of PfPM4 in other species could prove to be a drug for general use. Rationale for Study The complete sequencing of the P. falciparum genome and advances in malaria genetics are providing new approaches for developing vaccines and drugs to combat the growing malaria pandemic. The sequencing of the malaria parasite has revealed over 5400 genes in the genome (Gardner et al. 2002), the majority for which there is no known

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16 function. Drug design approaches based on targeting a specific gene or family of genes must first involve validating them as potential targets. To this end, genetic manipulations including gene knockout strategies provide useful methods of evaluating the suitability of a potential drug target. Hypothesis I hypothesize that one or more of the DV plasmepsins is required to provide sufficient aspartic proteinase activity in the DV for the multiplication and survival of the parasite during the asexual blood stage. Since PfPM4 is the ortholog of the single DV plasmepsin found in other Plasmodium species, I hypothesize that the function of this one enzyme alone will be sufficient for parasite survival. Several specific questions include: Is PfPM4 capable of supporting the asexual growth of the parasite in the absence of the other DV plasmepsins? Can the parasite survive with all of the DV plasmepsins deleted? Is the plasmepsin 4 ortholog in P. berghei truly an orthologous protein in function? Is there an increased dependence on other mechanistic classes of DV proteinases when one or more of the plasmepsins is deleted? Are other DV proteinases transcriptionally upregulated in the absence of one or more plasmepsin genes? Specific Objectives Objective 1: Create knockout mutants in P. falciparum where multiple (two, three or four) DV plasmepsin genes have been ablated. These studies will help define which plasmepsin genes are sufficient for parasite survival. Successful ablation of all four genes will establish that none of the DV plasmepsins are required for growth in the asexual blood stage. Failure to successfully ablate all four genes will suggest that one or

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17 more are required. Phenotypes of these KO mutants will be characterized to better understand any unique yet non-essential functions of these ablated genes. Objective 2: Phenotypes of mutants obtained in these studies will be defined by characterizing: growth rates, accumulation of hemozoin, accumulation of remaining plasmepsins, amount of plasmepsin and/or falcipain transcripts, and EC 50 values for proteinase inhibitors of all major classes including E64 (cysteine proteinase inhibitor), leupeptin (serine proteinase inhibitor), pepstatin (aspartic proteinase inhibitor) and HIV proteinase inhibitors. Antimalarials such as chloroquine will also be included as controls. Objective 3: Create a knockout mutant in P. berghei of PbPM4, the ortholog of PfPM4. This objective will be pursued if we fail to successfully knockout all four DV plasmepsin genes in P. falciparum. Use of this animal model (P. berghei–rodents) for the preparation of gene knockouts has the advantage of ablating a single gene in a system where the efficiency of targeted gene deletion is so high that unsuccessful attempts provide compelling evidence for the essential function of the gene. Since the subcellular location of PbPM4 has not been established, immuno-fluorescence and immunoEM will be performed to localize the protein in the asexual blood stage parasite. Active recombinant PbPM4 is available in the laboratory and will be assayed for its ability to digest hemoglobin.

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18 Figure 1-1. Global distribution of malaria, 2004. Reproduced with permission from the World Health Organization.

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19 Figure 1-2. Life cycle of the malaria parasite. Reproduced with permission from the Center for Disease Control and Prevention, USA.

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20 Figure 1-3. Asexual stages of the malaria parasite: A) ring-form parasite, B) trophozoite. C) schizont, D) rupturing schizont

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21 transport vesiclecytostomedigestive vacuoleneutral lipid body hemozoin transport vesiclecytostomedigestive vacuoleneutral lipid body hemozoin Figure 1-4. Ultrastructure morphology of the Plasmodium falciparum digestive vacuole.

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22 HEMOGLOBIN SMALL PEPTIDESSMALLER PEPTIDES Plasmepsins I, II , IV, HAP?Falcipains II and IIIFalcilysinDPAP1 AMINO ACIDSAminopeptidases(cytoplasm)HEME HEMATINHEMOZOIN OxidationPolymerizationHEMOGLOBIN SMALL PEPTIDESSMALLER PEPTIDES Plasmepsins I, II , IV, HAP?Falcipains II and IIIFalcilysinDPAP1 AMINO ACIDSAminopeptidases(cytoplasm)HEME HEMATINHEMOZOIN OxidationPolymerization Figure 1-5. Proposed hemoglobin digestion pathway in Plasmodium falciparum.

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23 PfPM11 M A L S I KE D F S SA FA K N E S AV N SSTF N N NMKT W K I Q K R F Q I L Y VFF F L L IPfPM21 M D I T V R EH D FK HG F I K SN S T F DG LN I D N S KNKK K I Q KG F Q I L Y V L L F C S VPfHAP1 M N L T I K E E D F T N T F M K N E E S F N TF R VTK V KRWNA K RL F K I L F VT V F I V LPfPM41 M A L T V K E E E F S N TL I K NA S A F DR L K LG N L KN-L K I Q K KL QF L Y L I L F V L IClustal Consensus1* :::: .:*. : *. .:.: . : . : :: :::*:: .* :PfPM150 T GA L F Y Y L I D NV L FP K N K K I N E I MNT S K H VI I G FS I E N S H D R I M K T V K Q HPfPM251M C G L F Y Y V Y E NV WL Q R DN E M N E I L KN S E H L T I G FK V E N A H D R I L K T I KT HPfHAP50A G G FS Y Y I F E NF V F Q K N R K I NH I I KT S K Y S T V G FN I E N S Y D R L M K T I K E HPfPM450 T GV F F F F L IG NF YSH R-K LY Q V I KN T K H T T I G FK I DRP H D K V LS S VL K NClustal Consensus : ::: *. : :: .:::.::: :**.::..:*:::.:: :PfPM1100 R L K N Y I K E S L K F F K T G L T Q KP H L G N AG D S VT L N D V A N V M Y Y G E A Q I G D N KPfPM2101 K L K N Y I K E S VN FL N S G L T KT N Y L G S S N D N I E LV DFQ N I M F Y G D A E V G D N QPfHAP100 K L K N Y I K E S V KL F NK G L T K K S Y L G S EF D N V E LK D L A N V LS F G E A K L G D NGPfPM498 K LST Y V K E SF K F F K S GYA Q KG Y L G S E N D S I E L D D V A N L M F Y G EG Q I GT N KClustal Consensus51:*..*:***.::::.* ::. :**. *.: * *. *:: :*:.::* * PfPM1150 Q K FA F I F D T G S A N L W V P SA Q C N T I G C K T K N L Y D S N K S K T Y E K D G T K V E M NPfPM2151 Q P FT F IL D T G S A N L W V P S V K C T TA G CL T K H L Y D S S K S R T Y E K D G T K V E M NPfHAP150 Q K FN F L FH TA S S N V W V P S I K C T SS E C E S K NH Y D S S K S K T Y E K D D TP V K LTPfPM4148 Q P FM F I F D T G S A N L W V P S VN C D S I G CS T K H L Y D A SA S K S Y E K D G T K V E I SClustal Consensus82* * *::.*.*:*:**** :* : .* :*: **:. *::****.* *::.PfPM1200 Y V S G T V S G F F S K D I V T IA N L SF P Y K F I E V T D T N G F E PA Y TLG Q F D G I V G LPfPM2201 Y V S G T V S G F F S K D L V T V G N L S L P Y K F I E VI D T N G F E PT Y TA ST F D G I L G LPfHAP200SK A G T I S GI F S K D L V T I GK L S V P Y K F I E M T EIV G F E PF Y S E S DV D G VF G LPfPM4198 YG S G T VR G Y F S K D V I S L G D L S L P Y K F I E V T DA DL D E PI Y SG S E F D G I L G LClustal Consensus :**: * ****::::..**.******: : .:** *: . .**:.**PfPM1250 G W K D L S I G S V D P V V V E L KN Q N K I E Q A V F T F Y L PF D D K H K G Y L T I G G I E D RPfPM2251 G W K D L S I G S V D P I V V E L KN Q N K I E N A L F T F Y L P VH D K HT G F L T I G G I E E RPfHAP250 G W K D L S I G S I D PY I V E L KT Q N K I E Q A V Y SI Y L PP E N K N K G Y L T I G G I E E RPfPM4248 G W K D L S I G S I D P V V V E L KK Q N K I D N A L F T F Y L P VH D K HV G Y L T I G G I ESClustal Consensus150*********:** :****.****::*::::*** .:*: *:*******. PfPM1300 D F Y E GQ L T Y E K L N H D L Y W Q V D L D L H F G N L T V E K AT A I V D S G T S S I TA P T E FPfPM2301 F Y E G P L T Y E K L N H D L Y W Q IT L DA HV G N I M L E K A N C I V D S G T S A I TV P T D FPfHAP300 F F D G P LN Y E K L N H D LM W Q V D L D V H F G N V SS K K A NV I L D SA T SV I TV P T E FPfPM4298 F Y E G P L T Y E K L N H D L Y W Q I D L D I H F GKY V M Q K A N A V V D S G T S T I TA P TS FClustal Consensus193*::* *.******** **: ** *.*: :**. ::**.** **.**.*PfPM1350 L N K FF EG L D V V K I P F L P L Y I T T C N NP K L P T L E F R SAT N V Y T L E P E Y Y L QQPfPM2351 L N KML QN L D V I K V P F L PF Y V TL C N N S K L P TF E FT S E NG K Y T L E P E Y Y L QHPfHAP350F N Q FV ESAS VF K V P F LS L Y V T T CG N T K L P T L E Y R SP NK V Y T L E P KQ Y L E PPfPM4348 L N K FFR D M N V I K V P F L P L Y V T T C D N DD L P T L E FH SR N N K Y T L E P E F Y M D PClustal Consensus228:*::... .*.*:***.:*:* *.* .***:*: * . *****: *:: PfPM1400 IF DF GI S L C M V S I I P V D L N K N T F I L G D P F M R K Y F T V F D Y D NH T V G F A L A KPfPM2401 I E D V G PG L C M L N I IG L DFPVP T F I L G D P F M R K Y F T V F D Y D NQ S V GI A L A KPfHAP400 L E N IFS A L C M L N I V P I D L E K N T F V L G D P F M R K Y F T V Y D Y D NH T V G F A L A KPfPM4398 LS D I D P A L C M LY I L P V D I D D N T F I L G D P F M R K Y F T V F D Y EK E S V G F A V A KClustal Consensus260: :. .***: *: :*: **:************:**::.:**:*:**PfPM1450 KK LPfPM2451 KN LPfHAP450 NL-PfPM4448 NLClustal Consensus299: Figure 1-6. ClustalW alignment of the four digestive vacuole plasmepsins of Plasmodium falciparum.

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CHAPTER 2 PHENOTYPIC CHARACTERIZATION OF SINGLE-PLASMEPSIN KNOCKOUTS Summary Four of the ten plasmepsins encoded by the Plasmodium falciparum genome (PfPM1, PfPM2, PfPM4 and PfHAP) are localized in the digestive vacuole (DV) of the asexual blood stage parasite and each had been previously targeted for gene disruption (knockout, KO) in our laboratory (Omara-Opyene et al. 2004). This chapter describes phenotypic characterizations performed on these single-plasmepsin KO mutants. Growth-rate analysis showed that KO mutants of P. falciparum strain Dd2, with either the gene pfpm4 or pfpm1 disrupted, had slowe r growth rates in vitro. However, those with pfpm2 or pfhap disrupted had no apparent reduction in growth rate during the asexual erythrocytic stages of development. To measure the effect of these knockout mutations on hemoglobin digestion, the amount of c rystalline hemozoin produced per parasite during the asexual cycle was measured. The pfpm4 knockout mutant (Tx4) produced about two-thirds the amount of hemozoin compared with the parental line Dd2 36-40 hr after synchronization of rings, whereas the other three knockout mutants produced 80-100%. The steady state levels of mRNA and protein products of the remaining intact DV plasmepsin genes were measured to discern whether disruption of a DV plasmepsin gene elicits a compensatory increase in RNA and/or protein accumulation. There were no compensatory increases in RNA or protein accumulation observed in knockout mutatnts pfpm1 (Tx1), pfpm2 (Tx2) and pfhap (Tx3). The 24

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25 pfpm4 mutant had an increase in pfpm1 mRNA accumulation at the schizont stage, but no increase in PfPM1 protein expression was evident by western blot. Introduction Asexual development of P. falciparum occurs within the human erythrocyte where the parasite consumes most of the host cell hemoglobin for protein biosynthesis (Krugliak et al. 2002), osmotic stability (Lew et al. 2003, Lew et al. 2004) and room for growth (Ginsburg 1996). Since the digestion of hemoglobin is a unique, presumably essential catabolic function performed by the blood stage parasites, the proteinases participating in this pathway have been proposed as targets for the development of novel antimalarial drugs (Olliaro & Goldberg 1995, Rosenthal 1998, Rosenthal et al. 2002). Proteinases of several mechanistic classes are found within the digestive vacuole including plasmepsins PfPM1, PfPM2, PfHAP and PfPM4 (Berry et al. 1999, Banerjee et al. 2002, Egan 2002), three falcipains (PfFP2, PfFP2, PfFP3) (Rosenthal et al. 1988, Salas et al. 1995, Francis et al. 1996, Sijwali et al. 2001, Singh et al. 2005) and a metalloproteinase, falcilysin (Eggleson et al. 1999, Murata & Goldberg 2003). The specific roles of the various proteinases in this catabolic pathway are only partially understood, but evidence supports the potential involvement of PfPM1 and PfPM2 in initiating digestion by cleaving the hinge region of the hemoglobin molecule allowing the protein to unfold and expose additional peptide bonds to cleavage by the other peptidases (Goldberg et al. 1991, Gluzman et al. 1994, Olliaro & Goldberg 1995). Additionally, cysteine proteinase inhibitors have been shown to block hemoglobin hydrolysis suggesting an essential role of one or more falcipains early in the hemoglobin hydrolysis pathway (Rosenthal et al. 1988, Rosenthal et al. 1991). Moreover, targeted disruption of the falcipain-2 gene caused trophozoites to accumulate undegraded hemoglobin in the parasite food vacuole

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26 and became more sensitive to aspartic proteinase inhibitors (Sijwali & Rosenthal 2004). As a result of these genetic studies, it has been suggested that redundancy likely exists among both classes of proteinases (Omara-Opyene et al. 2004, Sijwali & Rosenthal 2004, Liu et al. 2005a), however, any specific function of each of these proteinases has not been elucidated. The role of PfPM4 in hemoglobin digestion is less well studied but it has been demonstrated in silico that pfpm4 is the only DV plasmepsin gene present in six other species of Plasmodium (Dame et al. 2003). Orthologs of pfpm1, pfpm2 and pfhap were not found in the other species of Plasmodium. It has been suggested the three paralogs pfpm1, pfpm2 and pfhap arose as a result of gene duplication (Dame et al. 2003). More recent analysis suggests that this duplication occurred prior to the divergence of P. falciparum and P. reichenowi since P. reichenowi also encodes orthologs of all four DV plasmepsins found in P. falciparum. The successful creation of knockout mutants each having a single DV plasmepsin disrupted was previously reported (Omara-Opyene et al. 2004). The single-plasmepsin KOs had no obvious deleterious phenotype in the asexual erythrocytic stages of development leading to the conclusion that there is likely redundancy among the DV plasmepsins with regards to digesting hemoglobin. In this study, we attempted to address whether the apparent functional redundancy observed in the single-plasmepsin knockouts is marked by a compensatory response from the parasite. The RNA transcript levels of the remaining DV plasmepsins were analyzed, as well as the accumulation of the protein products. Studies to determine the comparative growth rates of the single-plasmepsin KOs relative to the wild-type Dd2 were performed. Experiments were also performed to

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27 determine whether the mutant parasites demonstrated any decrease in the polymerization of heme into hemozoin, perhaps as a consequence of digesting a smaller percentage of the host cell hemoglobin prior to rupture. Material and Methods Culturing of Malaria Parasites Erythrocytic stages of P. falciparum strain Dd2 and mutant cell lines were cultured at 4-5% hematocrit in RPMI 1640 medium supplemented with 0.5% Albumax (Invitrogen), 0.225% sodium bicarbonate, and 0.01 mg/ml gentamycin (Trager & Jensen 1976, Jensen 2002). Parasite growth-chambers were flushed with a gas mixture (1% O 2 , 5% CO 2 , 94% N 2 ) for 1 min. Parasite cultures were synchronized for stage-specific sampling by treating with 5% sorbitol for 10 min (Lambros & Vanderberg 1979). The sorbitol treatment is deleterious to the late stage parasites, and only the early trophozoites (ring-forms) survive. The window of synchrony from one sorbitol treatment, however, was too large. Therefore, 3 sorbitol treatments, approximately 44 to 48 hours apart, were performed for all stage-specific sampling experiments to increase synchrony. Two methods were used throughout the study to determine the parasitemia of the culture. Primarily, parasitemia was determined from thin smears prepared on microscope slides and stained with Hema Wright-Giemsa Stain (Fisher). In an effort to facilitate and optimize for accuracy, a method of determining parasitemia using flow cytometry was also employed. Parasite culture (0.5-1.0 mL) was transferred to a microcentrifuge tube and stained with 500 nM SYTO-24 (Molecular Probes). The sample was diluted >100 fold in PBS prior to flow cytometry analysis. A minimum of 50,000 cells was analyzed for each sample. A sample of uninfected erythrocytes treated identically with SYTO-24

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28 was used as a control (Figure 2.1). The hematocrit (RBCs/mL) was determined using a Beckman Coulter Counter (Beckman). Comparative Growth Study For comparative growth studies, parasites were removed from drug-containing medium and cultured in 1 ml volumes in a 24 well culture dish. Culture medium was changed every 24 hours and parasitemia was determined every 48 hours for a period of 21 days. Cultures were split 1:10 to 1:2 every 48 hours to maintain a <1% parasitemia. Thin smears were stained as described above and the mean fold-increase was calculated based on the parasitemia and the split ratios employed over time. The mean as a percentage of Dd2 was determined and the two-tailed Student’s t-test was computed using SigmaPlot. Purification of Hemozoin For hemozoin isolation and purification, culture medium was removed and the cells were resuspended in fresh RPMI 1640 medium. One ml of culture was treated with 0.05% saponin for 5 min, added to 1 ml of DNAzol BD (Invitrogen) and vortexed vigorously for >30 sec several times. All samples were frozen at -80C. After thawing samples, the hemozoin was pelleted at 215,000 g for 30 min at 20C. The supernatant was removed and the pellet was washed with dH 2 O and centrifuged at 84,000 g. Hemozoin was dissolved with 0.2 N NaOH for 1 h at 50C and periodic mixing of the sample. The Abs at 400 nm was measured and the amount of heme was calculated from an extinction coefficient of 91,000 cm -1 M -1 (Asakura et al. 1977, Pandey et al. 1999, Tripathi et al. 2002).

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29 Quantitative RT-PCR For RNA isolation and purification, culture medium was removed and the cells were resuspended in fresh medium. One ml of culture was treated with 0.05% saponin for 10 min at room temperature and the parasites were pelleted at 10,000 g for 10 min. Parasites were washed with PBS followed by centrifugation and 1 mL of Trizol (Invitrogen) was added to the pellet and vortexed vigorously. All samples were frozen at -80C in Trizol. Total RNA was purified per manufacturer’s instructions. Samples were treated with DNase I to remove traces of genomic DNA. cDNA synthesis was performed with the First-Strand cDNA Synthesis Kit (Invitrogen). Gene-specific primers and probes for qPCR were designed using Primer Express Software (ABI), the fluoregenic probes were from IDT (Coralville, IA) and the QuantiTect Probe Master Mix (Qiagen) was used. The qRT-PCR reactions were run on an MJ Research Opticon DNA Engine thermocycler or an ABI 7000 thermocycler. A single-strand oligonucleotide, identical in sequence and length to each target template, was synthesized (Sigma-Genosys) and used as a control for generating a standard curve. Using genomic DNA, the primer efficiency of all sets of primer and probes used in the experiment were evaluated for their comparability. The genes analyzed (pfpm1, pfpm2, pfhap, pfpm4 and pfasl) are all single copy genes and the qPCR analysis of gDNA resulted in comparable Ct (cutoff threshold) values (data not shown). The value obtained for RNA transcript levels of each plasmepsin, interpolated from the standard curve, was normalized to the value of pfasl, a constitutively expressed gene (Ben Mamoun et al. 2001). Western Blot Analysis Saponin-treated parasites were prepared as for RNA extraction except the washed parasite pellet from 1 mL of culture was resuspended in 50 L SDS buffer and boiled for

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30 5 min. All samples were frozen at -80C in SDS Buffer. For each cell line, total protein from an equal number of cells was collected at each time point, electrophoresed on a 10% or 12% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were blocked for 1 hour with TTBS (0.2% casein, 0.1% Tween-20 in TBS pH 7.5) and probed with 1:10,000 dilutions of anti-PfPM1, anti-PfPM2, anti-PfHAP or anti-PfPM4 (Banerjee et al. 2002). After 5-6 washes with TTBS for 10 min each, secondary antibodies were added for 1 hour and enhanced chemiluminescence was used to identify the protein (Pierce). Results Comparative Growth Study The mean fold-increase in parasitemia was calculated every 48 hours over a 3 week period (Table 2-1). The parasitemia of Dd2 increased 9.90 fold every 48 hours. Tx1 increased 5.80 fold, Tx2 increased 9.44 fold, Tx3 increased 9.11 fold and Tx4 increased 5.77 fold. These values are 58.6 %, 95.4%, 92.0% and 58.3% of the fold increase of Dd2, respectively. For comparing the fold increase, a statistical analysis of the mean parasitemia was performed for each of the single-plasmepsin KOs to Dd2. The Student t-test for unpaired samples was utilized with the SigmaPlot statistics program. The P value represents the probability that the two means are not different. Data on the growth rates Table 2-1. Parasitemia increase every 48 hours of Dd2 and single-plasmepsin knockout clones Dd2 Tx 1 Tx 2 Tx 3 Tx 4 Mean 9.90 5.80 9.44 9.11 5.77 SEM 1 1.20 0.85 2.20 0.54 0.53 No. samples 8 7 7 7 7 Mean as % Dd2 100.0% 58.6% 95.4% 92.0% 58.3% P value 2 0.007 0.948 0.313 0.002 1 SEM, standard error of the mean. 2 Unpaired two-tailed Student t-tests showed a statistically significant difference in the growth rates of Tx1 and Tx4 when compared to Dd2 (p< 0.01 in both cases).

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31 of Tx1 and Tx4 compared to Dd2 gave P values of 0.007 and 0.002, respectively, indicating that their lower growth rates are statistically significant. It is important to note, the decrease in growth rate is under in vitro growth conditions using complete RPMI 1640 medium supplemented with AlbuMax (Invitrogen). Stage-specific Accumulation of Intracellular Hemozoin The amount of intracellular hemozoin was calculated from triplicate 1 ml aliquots of culture (Figure 2-2). The mean values (and the standard error of the means) of 2 to 4 experiments, depending on the cell line, demonstrate that the amount of hemozoin produced during the ring stage (10-12 hr post-synchronization) was not affected in any of the knockout parasites. At the trophozoite stage (24-26 hr), pfpm1 had more hemozoin per parasite than Dd2 while pfhap had less than Dd2. At the schizont stage (36-40 hr), pfpm1 and pfpm2 had about the same amount of hemozoin as Dd2, pfhap had slightly less and pfpm4 had the least amount of hemozoin per parasite. Stage-specific Accumulation of RNA Transcripts by qRT-PCR The RNA transcript of each DV plasmepsin was normalized to a constitutively expressed gene, pfasl (Ben Mamoun et al. 2001) (Figure 2-3). In the ring and trophozoite stage, the normalized level of each transcript in all KO parasites was generally (considering error bars) similar to that of Dd2. In the schizont stage, the amount of pfpm2 and pfhap transcripts in the pfpm1 mutant was lower than in Dd2 but there was a comparable level of pfpm4 transcripts. The pfpm4 mutant had an increase in pfpm1 transcript levels compared to Dd2 in both rings and schizonts, while the levels of pfpm2 and pfhap transcripts remained unchanged.

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32 Stage-specific Accumulation of Protein by Western Blot Analysis Cultures of Dd2 and the four KO parasite lines were tightly synchronized and the amount of each plasmepsin expressed was analyzed at 12, 24, and 36 hrs post-synchronization. The experiment was performed 3-5 times and a representative image of the data is presented. The patterns of protein accumulation in the parasite culture over time were relatively comparable to each other (Figure 2-4). Each time point contains total protein from an equal number of cells harvested at the previous time point. However, not all cultures had identical parasitemias and hematocrits so the accumulation of the plasmepsins over time should be compared between the cell lines. The protein expression patterns over the time course analyzed did not demonstrate any dramatic increase or decrease in accumulation of any of the remaining DV plasmepsins when one is ablated. Discussion Plasmodium falciparum has four DV plasmepsins that presumably evolved both complementary and unique roles in hemoglobin hydrolysis and other functions (Dame et al. 2003, Omara-Opyene et al. 2004, Liu et al. 2005a). Our studies of single-plasmepsin KO mutants show that mutants lacking either PfPM4 or PfPM1 grow more slowly than the parental line. Moreover, only PfPM4 has a function (albeit, not entirely identified) that when absent, the parasite fails to digest hemoglobin and polymerize free heme into hemozoin as efficiently as the wild-type Dd2. It is not certain whether the deletion of the gene is directly affecting the hydrolysis of hemoglobin and therefore the formation of hemozoin and its rate of growth. Deletion of gene pfpm4 may have an effect on another biological pathway that affects the rate of growth and, in turn, affects hemozoin formation in the parasite.

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33 In the schizont samples, the pfpm4 KO had an increase in the accumulation of transcripts of pfpm1, and the pfpm1 and pfhap KOs had lower levels of pfpm2 transcription. Microarray analysis has shown that the timing of transcription of pfpm1 and pfpm4 is similar, and pfpm2 and pfhap is similar (Bozdech et al. 2003). If the parasite has the ability to compensate for the loss of a particular plasmepsin by increasing the transcription of another gene in the family, a detectable increase in transcription of pfpm1 rather than the pfpm2 or pfhap, at a particular time in the cycle, might be expected in the pfpm4 KO. Compensation by redundant expression of other members of this group of enzymes was not observed in the other single-plasmepsin KOs or in the other stages of pfpm4. While an induction of transcription appears to be a mechanism of survival detectable in the parasite when exposed to drug, these parasite lines were targeted for gene disruption and selected for over many weeks. We investigated the transcript levels of the remaining plasmepsins because induction of transcription has been shown to occur in other cell types as a result of a compensatory reaction. For example, the transcription of multiple drug resistance (mdr) genes is affected when rat liver cells are treated with antimicrobial agents (Fardel et al. 1997). In HIV infections, treatment with certain proteinase inhibitors induces transcription of host cell mdr, leading to a decrease in the uptake of the drug (Perloff et al. 2000, Fellay et al. 2002, Khoo et al. 2002). In C. albicans, treatment with certain antifungal drugs has also shown a marked increase in transcription levels of ABC transporters cdr1p and cdr2p (Henry et al. 1999). In P. falciparum, a 2.5 to 3.3 fold induction in mRNA levels of pfmdr1 after treatment with chloroquine has been demonstarted (Myrick et al. 2003). On the other hand, other reports have described P.

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34 falciparum as having a hard-wired gene transcription profile, making it difficult to observe changes in the levels of mRNA after exposure to stresses such as drug treatment (Ganesan et al. 2002). To observe whether compensatory effects were occurring at the protein level, we chose to analyze the protein expression patterns of the three remaining DV plasmepsins in all 4 KOs at specific time points after synchronization. The kinetics of expression over the time course analyzed were not dramatically different for the single-plasmepsin knockouts and Dd2 to suggest there was a compensatory upregulation in protein expression when one plasmepsin is ablated. The lack of evidence for an induction of protein expression of any of the 3 remaining plasmepsins in all 4 KOs was intriguing. If these enzymes are redundant in function, the data suggest they are expressed in excess and/or modified post-translationally dependending on their necessity in the cell. Alternatively, the enzymes may be redundant in digesting hemoglobin in the DV but this is a secondary or non-specific function.

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35 Figure 2-1. Flow cytometry analysis for determining parasitemia using SYTO-24 and 50,000 cell counts. The top panel represents the control uninfected-RBCs followed to the right by rings, early trophozoites, late trophozoites, schizonts and rings (second generation).

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36 Time post-synchronization 12 hr24 hr36 hr Hemozoin [fmole/parasite] 0.01.02.03.04.0 Dd2 Tx1 Tx2 Tx3 Tx4 Figure 2-2. Intracellular hemozoin accumulation in single-plasmepsin knockouts and the parental line Dd2.

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37 Early RingsCell Line Dd2Tx1Tx2Tx3Tx4 Plasmepsin/ASL 010203040 Early TrophsCell Line Dd2Tx1Tx2Tx3Tx4 Plasmepsins/ASL 010203040 Late TrophsCell Line Dd2Tx1Tx2Tx3Tx4 Plasmepsin/ASL 010203040 SchizontsCell Line Dd2Tx1Tx2Tx3Tx4 Plasmepsin/ASL 010203040 Pm1 Pm2 Pm3 Pm4 Inactive Transcript Early RingsCell Line Dd2Tx1Tx2Tx3Tx4 Plasmepsin/ASL 010203040 Early TrophsCell Line Dd2Tx1Tx2Tx3Tx4 Plasmepsins/ASL 010203040 Late TrophsCell Line Dd2Tx1Tx2Tx3Tx4 Plasmepsin/ASL 010203040 SchizontsCell Line Dd2Tx1Tx2Tx3Tx4 Plasmepsin/ASL 010203040 Pm1 Pm2 Pm3 Pm4 Inactive Transcript Figure 2-3. RNA transcript levels of the DV plasmepsins in the single-plasmepsin knockouts and Dd2.

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38 Pm2 HAP Pm4Tx1Dd2Tx2Tx3Tx4Pm1 122436122436122436122436 hr Pm2 HAP Pm4Tx1Dd2Tx2Tx3Tx4Tx1Dd2Tx2Tx3Tx4Pm1 122436122436122436122436122436122436122436122436 hr Figure 2-4. Protein levels of the DV plasmepsins over 36 hours in Dd2 and the four single-plasmepsin knockouts. Total protein from an equal number of parasites were collected at each time point and analyzed by SDS-PAGE. Cell lines did not begin with identical parasitemias.

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CHAPTER 3 CREATION AND CHARACTERIZATION OF DOUBLE-PLASMEPSIN KNOCKOUTS Summary Characterization of the single-plasmepsin knockouts demonstrated that none of the known DV plasmepsins is essential for the asexual growth of the parasite. If the DV plasmepsins are valid targets for the development of antimalarial compounds, more than one may need to be targeted by a drug. It appears feasible to develop a single inhibitor that can target both PfPM1 and PfPM2 (Noteberg et al. 2003, Muthas et al. 2005). Therefore, attempts were made to knockout both of these enzymes in the same P. falciparum clone to determine whether their combined loss is compatible with parasite survival. A double-plasmepsin knockout mutant lacking complete copies of genes pfpm2 and pfpm1, and another lacking complete copies of pfhap and pfpm2, were created by sequential integration of plasmid constructs designed to provide resistance to two different selective agents, WR99210 and blasticidin. PCR and Southern blot analysis confirmed integration of each construct into the targeted gene via single-crossover integration. Western blot analysis utilizing specific antibodies confirmed the absence of PfPM2 and PfPM1 expression in the clone where pfpm1 and pfpm2 were disrupted. Likewise, PfHAP and PfPM2 were absent in the clone where pfhap and pfpm2 were disrupted. The asexual growth of both of the double-plasmepsin KOs was comparable to the wild-type parasite, Dd2. Some morphological abnormalities were evident in both double-plasmepsin KOs in the first 3 months post-cloning but later the morphology 39

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40 became normal, possibly reflecting an adaptation phase of the parasite to the lack of two genes or an adaptation to the presence of the drug used for selection. Introduction Genetic manipulation of the malaria parasite has provided a valuable tool in the study of malaria. Early transfection success with Plasmodium species causing rodent malaria (Goonewardene et al. 1993, Vandijk et al. 1995) was followed by a breakthrough in transient transfection of P. falciparum (Wu et al. 1995). Subsequently, electroporation-based transfection of ring-stage P. falciparum was used for stable expression and disruption of malaria genes (Crabb & Cowman 1996, Wu et al. 1996, Crabb et al. 1997, Fidock & Wellems 1997). When early intraerythrocytic parasites (rings) are electroporated in the presence of plasmid DNA, the DNA is able to cross the erythrocytic membrane, the parasitophorous vacuole membrane (PVM) surrounding the parasite, and the nuclear membrane to gain entry into the nucleus. The parasite can maintain episomal plasmids indefinitely under selective pressure. After some time, and at a low frequency, homologous integration occurs and can be exploited for allelic exchange or gene disruption at a specific locus. Selection for the desired event is achieved by cloning and screening clonal populations. Transfection of the malaria parasite initially relied on a single marker, the human or Toxoplasma gondii dihydrofolate reductase genes that confer resistance to methotrexate or pyrimethamine (Wu et al. 1995). Later, the use of bsd (encoding blasticidin S deaminase of Aspergillus terreus) and neo (encoding neomycin phosphotransferase II from transposon Tn 5) genes as positive selectable markers for P. falciparum transfection was demonstrated to confer resistance to the compounds blasticidin S and G418, respectively (Mamoun et al. 1999).

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41 A plasmid containing the bsd gene (Mamoun et al. 1999) was used to target a second plasmepsin in the previously generated mutants. Gene pfpm1 was targeted in the pfpm2 mutant (Tx2) and pfpm2 was targeted in the pfhap mutant (Tx3). In both instances, the second targeted gene lies immediately 5 of the gene already disrupted (Figure 3-1). Materials and Methods Plasmids The plasmid pBSD-CAM expresses BSD under the control of the P. falciparum calmodulin (CAM) gene 5 UTR and pfhrp2 3 UTR. It was used to create plasmids that can integrate into the genome via single-crossover homologous recombination (Mamoun et al. 1999). The BSD-CAM knockout constructs were prepared by PCR amplifying a genomic DNA fragment and ligating it directionally following restriction enzyme digestion into the plasmid vector in a position flanking the BSD selection marker. For creating plasmid pBSD-CAM-pfpm1, a 715 bp fragment of pfpm1 (nucleotides 481 to 1196) was cloned into pBSD-CAM (Figure 3-2). For plasmid pBSD-CAM-pfpm2, a 716 bp fragment of pfpm2 (nucleotides 484 to 1200) was cloned (Figure 3-2). Transfections and Selection Plasmodium falciparum asexual stages were maintained (Trager & Jensen 1976) and sorbitol-synchronized (Lambros & Vanderberg 1979) by standard procedures as described in Chapter 2. Predominantly ring-stage parasites were transfected with 100 g of purified (Qiagen) plasmid DNA (Fidock & Wellems 1997). After 1 complete asexual cycle, the transfected culture was treated with 5 g/mL blasticidin to select for parasites that had received the plasmid and were expressing BSD. There was an immediate

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42 decrease in the number of viable cells. Transformants were obtained between 17 and 21 days following transfection. Cloning was performed by limiting dilution method (Francois et al. 1994). The parasitemia and hematocrit of the culture were calculated and an equivalent of 35 infected erythrocytes was added to 20 mL of complete culture medium plus uninfected erythrocytes (5% hematocrit). This suspension was aliquoted into a 96-well tissue culture plate (200 L/well) and the chamber was flushed with gas as described in Chapter 2. The medium was changed every 1-2 days and a MalStat assay was performed at days 12, 16, and 20 to detect growth. The Malstat assay is a sensitive colorometric assay which detects P. falciparum lactate dehydrogenase (Makler & Hinrichs 1993). PCR and Western Blot Analysis Parasites were prepared free of excess erythrocytes by treatment with 0.05% saponin and washed with phosphate-buffered saline. Genomic DNA was extracted using TELT lysis buffer [50 mM Tris (pH 8.0), 62.5 mM EDTA (pH 8.0), 2.5 M LiCl, 4% Triton X-100], purified by phenol/chloroform/isoamyl alcohol (25:24:1), chloroform/isoamyl (24:1) and isopropyl alcohol precipitation. PCR was performed on the DNA using primers designed to amplify the entire plasmepsin gene from start codon to stop codon. A control pair of primers (Pf1/Pf2) that amplifies the coxI gene was used to confirm the presence of DNA. The remaining plasmepsin genes also served as a control as amplification efficiency was consistent for all plasmepsin primer pairs. The western blot analysis was performed as described in Chapter 2. Parasite Growth Comparison For comparative growth studies, parasites were removed from drug-containing medium and cultured in 1 ml volumes in a 24 well culture dish. Culture medium was

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43 changed every 24 hours and parasitemia was determined every 48 hours for a period of 14 days. Cultures were split between 1:10 to 1:2 every 48 hours to maintain approximately a 1% parasitemia. Thin smears were stained as described above and the projected parasitemia was calculated taking into account all the dilutions made over the course of the experiment. Hemozoin Purification for Scanning Electron Microscopy The hemozoin of the wild-type (Dd2) and the double-plasmepsin KOs (21C3 and 32A7) was purified as described in Chapter 2 with modifications. Additional washes with 0.25% SDS and Proteinase K (50g/ml) were repeated to achieve free-floating crystals. The washing was originally tried for the hemozoin quantitation assay, however, a loss of sample quantity was observed. Samples were sent to the EM core facility at the University of Florida for SEM analysis. Results and Discussion Parasites having only two functional plasmepsin genes were successfully created and cloned for analysis. PCR was used to confirm the disruption of the gene (Figures 3-3) and western blot analysis to confirm that only two of the plasmepsins were being expressed (Figure 3-4). The growth rates of the double-knockout parasites showed that the deletion of two plasmepsin genes, either pfpm2 and pfpm1 or pfhap and pfpm2, had no deleterious effect on the asexual growth of the parasite (Figure 3-5). The double-plasmepsin KOs grew nearly identically to Dd2 over the two week period of the experiment. Morphological differences were observed within the first 3 months in the double-plasmepsin knockouts compared to Dd2 (Figure 3-6). The trophozoites, in particular, had abnormal morphology. Within the first three months, gametocytes were also observed with regular frequency. It is possible that during the first three months the

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44 parasites were still adjusting to the loss of the plasmepsins, but after this period, the morphology became normal. The structure and size of the hemozoin crystals being produced by the double-plasmepsin knockouts and Dd2 did not differ (Figure 3-7). Since these enzymes are active in the DV where hemoglobin is digested and hemozoin is produced, it was felt that an analysis of the crystal structure might show a difference in the mutants. However, like the growth and morphology, the double-plasmepsin knockouts did not differ in hemozoin structure or size. The hypothesis that two plasmepsins could be deleted in the same parasite was formulated after the successful ablation of each of the plasmepsins singly in Dd2. The fact that the pfpm2 and pfhap knockout mutants demonstrated minimal effects on the fitness of the parasite suggested that a knockout mutant with these two genes disrupted was likely to be successful. In fact, the pfhap/pfpm2 mutant grew out relatively quickly after only 15 days of selection. Clones also grew out rapidly after the limiting dilution experiment for cloning. Previous studies implicated PfPM2 in the egress of the parasite from the erythrocyte by cleaving actin and protein 4.1 at a neutral pH (Le Bonniec et al. 1999). However, no effect on this process was observed. Successful creation of the pfpm2/pfpm1 KO mutant was a little more surprising because the pfpm1 mutant did not grow as well as the wild-type parasite (Omara-Opyene et al. 2004). When the growth rate of this double-plasmepsin KO was analyzed, the slow-growth phenotype was not observed. One important difference is that pfpm2 was the first disrupted gene (with normal growth) and pfpm1 was subsequently targeted. The parasite line lacking only PfPM2 may have established a compensatory mechanism

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45 for dealing with the lack of this protein for a period of time that allowed PfPM1 to be disrupted without significant effects on growth. Perhaps epigenetic factors such as the number of plasmid copies each transfection introduced and the resultant architecture of the chromosome played a role on whether the growth rate was affected. Further studies would be necessary to address these issues. The deletion of two plasmepsins in the same parasite shows that these plasmepsins have a non-essential function and supports the notion that their function may be performed by other proteases. The creation of a double-plasmepsin KO that included PfPM4 was not successful in this study but others have shown that it is attainable and the parasites continue to grow in in vitro culture (Liu et al. 2005a). A slow growth phenotype of 60% fold decrease over 7 days compared to 3D7, and a 13% longer doubling time compared to 3D7 was observed for a double-plasmepsin KO of PfPM1/PfPM4 (Liu et al. 2005a). The data support our hypothesis of a higher reliance on PfPM4 by the parasite than any of the other 3 DV plasmepsins. Therefore, future experiments were focused on disrupting 3 and 4 plasmepsins simultaneously.

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46 pfpm4 pfpm1 pfpm2 pfhap NsiI (20425) NsiI (22) NsiI (4945) P. falciparum Chromosome 14(pfpm4 to pfhaplocus = 15373 bp) NsiI (20425) pfpm4 pfpm1 pfpm2 pfhap NsiI (22) NsiI (4945) P. falciparum Chromosome 14(pfpm4 to pfhaplocus = 15373 bp) Figure 3-1. Schematic of DV plasmepsin locus. The four DV plasmepsins are located in tandem array on chromosome 14.

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47 pBSD-CAM-pfpm15211 bp pfcam-5' BSD amp pfpm1 481-1196 pfhrp2-3' AvaI (2260) BamHI (2266) HindIII (1688) NcoI (660) PstI (2258) SmaI (2262) XmaI (2260) BglII (2751) NsiI (964) SacI (3010) SacII (3001) SpeI (2809) ApaLI (3718) ApaLI (4964) BssHII (620) BssHII (3043) EcoRI (666) EcoRI (2833) EcoRI (2932) NciI (2261) NciI (2262) NciI (3784) NciI (4480) NciI (4831) pBSD-CAM-pfpm25212 bp pfcam-5' BSD amp pfpm2 484-1200 pfhrp2-3' AvaI (2260) BamHI (2266) EcoRI (666) HindIII (1688) NcoI (660) SmaI (2262) XmaI (2260) NsiI (964) SacI (3011) SacII (3002) SpeI (2809) NotI (2990) ApaLI (3719) ApaLI (4965) PstI (2258) PstI (2310) BssHII (620) BssHII (3044) NciI (2261) NciI (2262) NciI (3785) NciI (4481) NciI (4832)a.b. pBSD-CAM-pfpm15211 bp pfcam-5' BSD amp pfpm1 481-1196 pfhrp2-3' AvaI (2260) BamHI (2266) HindIII (1688) NcoI (660) PstI (2258) SmaI (2262) XmaI (2260) BglII (2751) NsiI (964) SacI (3010) SacII (3001) SpeI (2809) ApaLI (3718) ApaLI (4964) BssHII (620) BssHII (3043) EcoRI (666) EcoRI (2833) EcoRI (2932) NciI (2261) NciI (2262) NciI (3784) NciI (4480) NciI (4831) pBSD-CAM-pfpm25212 bp pfcam-5' BSD amp pfpm2 484-1200 pfhrp2-3' AvaI (2260) BamHI (2266) EcoRI (666) HindIII (1688) NcoI (660) SmaI (2262) XmaI (2260) NsiI (964) SacI (3011) SacII (3002) SpeI (2809) NotI (2990) ApaLI (3719) ApaLI (4965) PstI (2258) PstI (2310) BssHII (620) BssHII (3044) NciI (2261) NciI (2262) NciI (3785) NciI (4481) NciI (4832)a.b. Figure 3-2. Single-crossover plasmids pBSD-CAM-pfpm1 and pBSD-CAM-pfpm2. One fragment of the plasmepsin gene targeted was targeted for homologous recombination and single-crossover integration.

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48 PM1PM2PMHAPPM4PM1PM2PMHAPPM4 a b c32A721C3WT-Dd2 1 kb 1 kbPM1PM2PMHAPPM4PM1PM2PMHAPPM4 a b c32A721C3WT-Dd2 1 kb 1 kb 1 kb 1 kbA. PM3 5.761.5 Dd221C3PM1 20 5.74Dd232A7PM2 Dd232A7 PM3 5.761.5 Dd221C3PM1 20 5.74Dd232A7PM2 Dd232A7 B.PM1PM2PMHAPPM4PM1PM2PMHAPPM4 a b c32A721C3WT-Dd2 1 kb 1 kbPM1PM2PMHAPPM4PM1PM2PMHAPPM4 a b c32A721C3WT-Dd2 1 kb 1 kb 1 kb 1 kbA.PM1PM2PMHAPPM4PM1PM2PMHAPPM4 a b c32A721C3WT-Dd2 1 kb 1 kbPM1PM2PMHAPPM4PM1PM2PMHAPPM4 a b c32A721C3WT-Dd2 1 kb 1 kb 1 kb 1 kbA. PM3 5.761.5 Dd221C3PM1 20 5.74Dd232A7PM2 Dd232A7 PM3 5.761.5 Dd221C3PM1 20 5.74Dd232A7PM2 Dd232A7 B. PM3 5.761.5 Dd221C3PM1 20 5.74Dd232A7PM2 Dd232A7 PM3 5.761.5 Dd221C3PM1 20 5.74Dd232A7PM2 Dd232A7 B. Figure 3-3. PCR and Southern blot confirmation of double-plasmepsin knockouts. A. PCR for the full-length plasmepsin genes in clones 21C3, 32A7 and Dd2. B. Southern blot of gDNA probed with pfpm1, pfpm2 and pfhap sequences.

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49 Dd221C3-PfPM1-PfPM2-PfHAP-PfPM432A7 kDa5037255037373725Dd221C3-PfPM1-PfPM2-PfHAP-PfPM4-PfPM1-PfPM2-PfHAP-PfPM432A7 kDa5037255037373725 kDa5037255037373725 Figure 3-4. Western blot analysis of clones isolated after limiting dilution. The first number in the clone name represents the plasmepsin previously disrupted from the single-plasmepsin KO group (Tx2 and Tx3). Clones 21C3 was a PfPM2 knockout and now lacks a functional copy of PfPM1. Clones 32A7 was a PfHAP knockout and now lacks a functional copy of PfPM2.

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50 Day 0246810121416 Growth 10-310-210-1100101102103104105 Dd2 21C3 32A7 Figure 3-5. Growth rate comparison of Dd2 and the double-plasmepsin knockouts. The growth rates were determined concurrently by calculating parasitemias every 48 hours from Geimsa-stained smears.

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51 Figure 3-6. Light microscopy images of Dd2 and the double-plasmepsin knockouts.

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52 Figure 3-7. SEM images of hemozoin crystals purified from Dd2 (A), and the double-plasmepsin knockouts 21C3 (B) and 32A7 (C).

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CHAPTER 4 SIMULTANEOUS DELETION OF THREE AND FOUR DV PLASMEPSINS AND THEIR SUCESPTIBILTY TO HIV PROTEINASE INHIBITORS Summary The locus containing the four DV plasmepsins in tandem array was targeted for disruption using a plasmid construct containing a negative-selection marker to promote double-crossover integration. A clone of a triple-plasmepsin knockout (KO) mutant lacking functional copies of pfpm1, pfpm2 and pfhap was created and named clone B12. A clone of a quadruple-plasmepsin KO mutant lacking functional copies of all four DV plasmepsins (pfpm4, pfpm1, pfpm2 and pfhap), called C10, was also created. Disruption of the locus via integration of the plasmid was confirmed by PCR and Southern blot analysis. The lack of protein expression was confirmed by western blot analysis using specific antibodies. The quadruple-KO (C10) showed a slower rate of growth. The retarded growth was accompanied with a delay in the formation of hemozoin compared to the parental 3D7 cell line. Clone C10 was also slightly more sensitive to E64 and leupeptin, but there was no upregulation in the expression of the cysteine proteinases falcipain 2 and 3. Interestingly, the sensitivity of both the triple and quadruple knockout mutants to the HIV aspartic proteinase inhibitors lopinavir, ritonavir, indinavir, atazanavir, and saquinavir remained comparable to the wild-type 3D7, however, nelfinavir showed an increased potency to both mutants. The data indicate that the primary targets of inhibition of these clinically important compounds are not found among the four DV plasmepsins. The data also indicate that the DV plasmepsins play a 53

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54 role in hemoglobin digestion and contribute to the fitness of the parasite but that they are not essential for the survival of the asexual stage parasite. Introduction The creation and characterization of the double-plasmepsin KOs further supported the hypothesis that there was redundant hemoglobinase activity among the DV plasmepsins. It should also be considered that while this family of proteinases is capable of digesting hemoglobin in the DV, there are additional functions to each plasmepsin that may not be redundant but are yet uncharacterized. PfPM2 has been proposed to be involved in cytoskeleton cleavage of infected erythrocytes as well as hemoglobin digestion (Le Bonniec et al. 1999). No other function has been attributed to PfPM1, PfPM4 and PfHAP. However, the data demonstrate that the DV plasmepsins are not individually essential. The hypothesis formulated for this study stated that at least one DV plasmepsin would need to remain undisrupted in the parasite’s genome for the parasite to survive. Based on our previous data, including the molecular phylogeny and the single-plasmepsin KO growth characteristics and morphology (Dame et al. 2003, Omara-Opyene et al. 2004), PfPM4 would be the single paralog most likely to be able to fulfill this requirement. To address this hypothesis the wild-type parasite (3D7) was targeted for disruption at the DV plasmepsin locus where the genes lie in tandem. In this study, new vectors containing a negative-selection marker were used that allow for double-crossover homologous recombination. The negative selection of cells expressing transformed genes has been developed in mammalian cells to increase the efficiency of gene targeting, and has proved useful for the selective killing of P. falciparum (Duraisingh et al. 2002).

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55 The thymidine kinase gene of Herpes simplex virus (Moolten 1986) converts a normally innocuous metabolite into a toxic one. The viral thymidine kinase is a promiscuous enzyme that activates the nucleoside analogue ganciclovir into toxic metabolite inhibiting the de novo pyrimidine biosynthesis pathway and nucleic acid synthesis directly (Rogulski et al. 1997). With the transfection of one double-crossover, negative-selection plasmid, three of the four DV plasmepsins were targeted for simultaneous disruption. With another plasmid, all four plasmepsin genes were targeted for simultaneous disruption. Materials and Methods Plasmids The plasmid pHHT-TK was engineered to integrate via double crossover into the genome and disrupt either 3 or 4 tandemly arranged plasmepsin genes at once. Two genomic fragments (600-900 bp each) were PCR-amplified and inserted into the plasmid vector flanking the gene expressing human dihydrofolate reductase, the positive selection marker. For creating plasmid pHHTK-1546, a 619 bp fragment of pfpm1 (nucleotides 1 to 619 where ” represents the first nucleotide of the start codon) and a 865 bp fragment of pfhap (nucleotides 816 to +325 where “+” represents the 3 UTR) were cloned into pHHT-TK flanking the human dhfr gene which provides the positive-selection marker when the parasites are cultured in the presence of WR99210 (Figure 4-1) (Fidock & Wellems 1997). For plasmid pHHTK-1550, a 794 bp fragment of pfpm4 (nucleotides -348 to 446 where “-“ represents the 5 UTR) and the identical 865 bp fragment of pfhap used in pHHTK-1546 was cloned into pHHT-TK (Figure 4-1).

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56 Transfections and Selection Plasmodium falciparum asexual stage parasites were cultured in vtro (Trager & Jensen 1976) and synchronized using sorbitol as described (Lambros & Vanderberg 1979). Two and a half milliliters of predominantly ring-stage parasites (5-8% parasitemia; 4% hematocrit) were transfected with 100 g of plasmid DNA by electroporation in 2 mm cuvettes with low voltage, high capacitance (0.31 Kv, 960 F) conditions (Fidock & Wellems 1997). Transformants were selected with the antifolate drug WR99210 (10 nM). Resistant parasites were obtained between 19 and 23 days following transfection. When the parasitemia reached 4-5%, gancyclovir was added to the culture medium for 9 days. Gancyclovir was removed from the medium for 9 days. This cyclic treatment with gancyclovir was repeated 2 more times. PCR and Southern blot analysis was used to confirm whether the expected knockout was present among the parasites in the culture (Figure 4-2). Upon confirmation, the parasites were cloned by limiting dilution (Francois et al. 1994). Southern Blot Analysis of gDNA Genomic DNA was digested with NsiI and electrophoresed in 0.75% agarose. The DNA was nicked for 5 min under UV light, denatured with 1.5 M NaCl, 0.5 N NaOH, neutralized with 0.15M Tris-HCl (pH 7.5), 0.5 M NaCl, and transferred to positively-charged nylon membranes. Membranes were treated with UV light to crosslink the DNA and dried at room temp. PCR products (450 to 740 bp in length) were purified and labeled using gene-specific primers, DIG-labeled dUTP (Roche), and exo(-) Klenow DNA polymerase (Stratagene). Membranes were blocked for 1-2 hr at 65C and the heat-denatured probe was added and hybridized overnight at 65C. High stringency washes (2SSC/0.1% SDS twice for 5 min at room temp followed by two 15 min washes

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57 at 60C with 0.1SSC/0.1%SDS) were used prior to the addition of a secondary antibody (1:15000) recognizing the DIG molecule. After washing, the chemiluminescent substrate CDP-Star (Roche) was added and the membrane was exposed to film. PCR and Western Blot Analysis for Plasmepsins The PCR and western blot analysis was performed as described in chapter 3. Culturing and Growth Characteristics All parasite lines were cultured at 37C in sealed chambers gassed with 1% O 2 ; 5% CO 2 ; 94% N 2 . The 3 parasite lines were followed simultaneously over a 2 week period. The culture medium was changed daily, and the parasitemia was calculated every 48 h by counting thin smears stained with Hema Wright-Giemsa Stain (Fisher). The doubling time (in hours) was calculated for each cell line. Growth in limited-nutrient medium was measured over a period of 120 h. Cultures of each clone were prepared identically in duplicate, one in RPMI-1640 (standard medium) and one in Jensen basal medium (JBM) (Divo et al. 1985). Culture medium was changed daily, thin smears were prepared and the parasites were counted. Transmission Electron Microscopy Analysis Parasites were pelleted and fixed in 2.5% glutaraldehyde, 0.05M phosphate buffer (pH 7.4) and 4% sucrose, first at room temperature for 30 min and then at 4C for 90 min. The fixative solution to sample volume ratio was approximately 10:1. The cells were washed (3 changes over 15-30 minutes elapsed time) in 0.1M phosphate buffer (pH 7.4) at 4C and left in 0.1M phosphate buffer prior to shipment. Dr. Hisashi Fujioka at Case Western University kindly performed the TEM analysis on the parasite lines.

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58 Neutral Lipid Staining with Nile Red Parasites were grown under standard conditions. After washing twice with PBS, Nile Red was added (1 g/ml) to cells in fresh medium and incubated at room temperature for 1 hour. After 1 hour, cells were washed with PBS and viewed. Western Blot Analysis of Falcipain 3 Saponin-treated parasites were prepared from stage-specific samples as described above. All samples were frozen at -80C in SDS Buffer. Parasitemia and hematocrit values were calculated and used to determine the volume of extracted protein equivalent to 4 million and 2 million parasites for each stage; ring, early trophozoite, late trophozoite, and schizont. Protein samples were electrophoresed on a 10% or 12% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were blocked for 1 hour with TTBS (0.2% casein, 0.1% Tween-20 in TBS pH 7.5) and probed with 1:10,000 dilutions of anti-PfFP3 (Sijwali et al. 2001). After 5-6 washes with TTBS for 10 min each, a secondary antibody was added for 1 hour and enhanced chemiluminescence was used to identify the protein (Pierce). In vitro Antimalarial Drug Assays Antimalarial activity of various proteinase inhibitors was measured in vitro using [ 3 H]-hypoxanthine uptake assays (Desjardins et al. 1979, Fidock et al. 1998). Dilutions of drug were added to P. falciparum 3D7 cultures and the KO-mutants (0.5-1.0% parasitemia, 1.5-2.0% hematocrit) in triplicate in 96-well plates at a final volume of 200 l/well. The parasites were cultured in low-hypoxanthine containing medium for 48 h in gas (94% N 2 , 5% CO 2 , 1% O 2 ) at 37C. After 48 h, 100 l of medium was removed and replaced with 100 l of ‘low-hypoxanthine’ medium containing [ 3 H]-hypoxanthine at a concentration of 0.5 Ci/mL. After an additional 24 h, cells were harvested onto glass

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59 fiber filters and washed thoroughly with distilled water. Dried filters were placed in sample bags and immersed in scintillation fluid (PerkinElmer), and radioactive emissions were counted in a Model 1450 MicroBeta reader (PerkinElmer). Percentage reduction in hypoxanthine uptake (a marker of growth inhibition) was calculated as follows: reduction = 100 [(geometric mean cpm of no-drug samples)-(mean cpm of test samples)]/(geometric mean cpm of no-drug samples). The percentage reductions were plotted as a function of drug concentration. Fifty percent effective concentrations (EC 50 ) were determined using the variable-slope sigmoidal dose-response nonlinear regression equation (Systat Software Inc.). Results Transfections and Selection Parasites electroporated with plasmids pHTK-1546 and pHTK-1550 were selected with WR99210 and resistant parasites were found in 14-18 days. After 3 cycles of ganciclovir treatment for 9 days on and 9 days off, the parasites showed no further susceptibility to ganciclovir. Knockout parasites were cloned by limiting dilution (Francois et al. 1994) into 96 well plates. Growth of the clones was detected by day 12. PCR and qPCR was performed on gDNA to confirm absence of the full-length genes. Clone B12 of the pHTK-1546 transfection was negative by qPCR for the 3 plasmepsin genes targeted for disruption. Clone C10 of the pHTK-1550 transfection was negative by qPCR for all 4 DV plasmepsins (data not shown). These 2 clones were selected for further characterization. Confirmation of the Creation of Triple and Quadruple Plasmepsin Knockouts The expected double-crossover integration of pHTK-1546 and pHTK-1550 is outlined in Figure 4-2, panels A and B, respectively. If integrated as expected, the triple

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60 plasmepsin KO, clone B12, would have a full-length pfpm4 gene located upstream of the integration site, a truncated fragment of pfpm1, followed by the drug resistance cassette, and a truncated fragment of the pfhap coding region. The quadruple-plasmepsin KO, clone C10, would have a truncated fragment of pfpm4, followed by the integrated drug resistance cassette and a truncated fragment of the pfhap coding region (Figure 4-2). Southern blot analysis of gDNA from both KO clones B12 and C10 confirmed the expected integration of the plasmid and disruption of the target locus (Figure 4-3). Probe prepared from nucleotides 1-530 of pfpm4 hybridized to NsiI digestion fragments of 4.9 kb in both the wild-type 3D7 and the mutant clone B12 (Figure 4-3). The same probe, pfpm4 (1-530), hybridized to a fragment of 5.4 kb in the mutant clone C10. A probe prepared from pfpm1 (1-619) hybridized to a fragment of approximately 15.9 kb in the 3D7 digest and 5.9 kb in clone B12. The probe failed to hybridize to gDNA from clone C10. A probe prepared from the coding region of hdhfr (1-725) from the drug resistance cassette hybridized equivalently to a 1.9 kb fragment in digests of gDNA from clones B12 and C10 (Figure 4-3). The data from the hybridization of all 3 probes confirmed the expected integration of pHTK-1456 in clone B12, and pHTK-1550 in clone C10. The clones were also analyzed by PCR and western blot analysis to confirm the genes of interest had been disrupted. PCR assays using primers designed to amplify full-length copies of pfpm1, pfpm2 and pfhap were only positive with gDNA from 3D7 while that of clones B12 and C10 did not produce any signal (Figure 4-4). A PCR assay for the full-length pfpm4 gene was positive with gDNA from 3D7 and B12 but negative from C10. Total parasite protein from 3D7, B12 and C10 was assayed by western blot analysis with polyclonal antibodies monospecific to PfPM1, PfPM2, PfHAP and PfPM4 (Banerjee

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61 et al. 2002). Expression of all 4 DV plasmepsins was detected in 3D7 (Figure 4-4). In clone B12 only PfPM4 was detected, and in clone C10 none of the 4 DV plasmepsins was detected (Figure 4-4). Growth of the Parasite in Nutrient-rich Medium All three parasite lines grew in the nutrient-rich RPMI 1640-based medium in which we routinely culture the parasites. 3D7 and B12 grew normally and indistinguishably from each other while C10 grew more slowly. Over a two week period, 3D7 and B12 demonstrated a calculated doubling time of 17.4 h and 17.2 h, respectively, whereas clone C10 had a doubling time of 21.3 h (Figure 4-5). This represents a 25% increase in doubling time for the quadruple-plasmepsin KO. When grown in amino acid-limited medium (JBM) (Divo et al. 1985), B12 and C10 grew more slowly than 3D7 but growth of C10 was essentially halted. After two 48-hour cycles, growth of B12 began to decrease significantly, having a 38-fold increase at 120 hours compared to 119-fold increase in its growth with the nutrient-rich RPC medium (Figure 4-6). Growth of B12 in JBM was still better than growth of C10 in RPC. C10 in JBM grew very poorly maintaining a parasitemia below 1%. Intracellular Hemozoin Production The intracellular hemozoin produced by each cell line was measured over the first 34 hours post-synchronization and expressed as the amount of heme per parasite. The two mutant cell lines, B12 and C10, and 3D7 had similar amounts of hemozoin isolated from the purified parasites at 8 hours post-synchronization (Figure 4-7). After 16 hours, the amount of hemozoin in the three cell lines was comparable although C10 had the lowest mean. At 24 hours, the amount of hemozoin isolated from C10 was significantly less than that from either 3D7 (~60%) or B12 (~75%). After 34 hours, C10 had produced

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62 less hemozoin than 3D7 (~65%) and the amount in B12 was equal to that in 3D7 (Figure 4-7). Transmission Electron Microscopy Analysis of the Mutant Parasites and 3D7 To assess whether there were any morphological changes in the KO clones, 3D7 and the KO clones B12 and C10 were prepared for TEM analysis at the late-trophozoite/schizont stage. No morphological differences were noted between B12 and 3D7, but there was a striking difference in the morphology of the C10 digestive vacuole. Multilaminate structures, that appeared to be compossed of lipid bilayer of membranes, were found within the digestive vacuoles of ~40% of the C10 parasites (Figure 4-8). Nile Red staining of Neutral Lipids Having found membranous vesicles within the DV of the quadruple-KO, and since membrane vesicles were also observed in the DV of the single-KO clone lacking PfPM4 (Omara-Opyene et al. 2004), we decided to investigate lipid trafficking and storage in mutants lacking PfPM4. The number of neutral lipid bodies in the quadruple-KO and 3D7 was similar (Figure 4-9). Both parasite lines had a higher number in the schizont stage with typically 2 to 4 lipid bodies closely associated with the DV (Coppens & Vielemeyer 2005). There was no staining of neutral lipid bodies within the DV of either parasite line suggesting that the metabolism of triacylglycerol lipids was normal. Western Blot Analysis of Falcipain 3 Western blot analysis of the expression of falcipain 3 (PfFP3) did not show a significant alteration of PfFP3 expression in the quadruple-KO C10 versus 3D7 (Figure 4-10). Total protein from 2 million and 4 million parasites were used to avoid saturation of signal when too much protein is loaded. Both had equivalent amounts of protein and

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63 were processed at about the same time (Figure 4-10). The hemoglobinase PfFP3 was not upregulated in the absence all 4 DV plasmepsins. Sensitivity to HIV Proteinase Inhibitors, General Proteinase Inhibitors and Antimalarials The sensitivity of the triple-KO (B12), the quadruple-KO (C10) and 3D7 to various proteinase inhibitors was determined by calculating the effective concentration for 50% inhibition of growth (EC 50 ) (Table 4-1). Parasite lines 3D7, B12 and C10 had comparable EC 50 concentrations for the HIV proteinase inhibitors atazanavir, lopinavir, indinavir, ritonavir and saquinavir. The EC 50 concentration of nelfinavir, also an HIV proteinase inhibitor, was less than half (~44%) of the value for 3D7, and B12 was ~65% of that for 3D7 (Table 4.1). The EC 50 concentration of E64, a cysteine proteinase inhibitor, for C10 was ~50% of that for 3D7, and for B12 it was ~75% of the value for 3D7. Leupeptin, also showed a lower (~44%) EC 50 concentration for C10 compared to Table 4-1. Sensitivity of wild-type (3D7), triple-plasmepsin KO (B12) and quadrudple-plasmepsin KO (C10) to HIV and other proteinase inhibitors Protease Inhibitor 3D7 B12 C10 Atazanavir [M] 12.4 5.6 14.4 1.7 12.2 0.1 Lopinavir [M] 3.4 0.1 3.6 0.4 3.1 0.1 Indinavir [M] 18.8 3.6 17.6 6.2 14.3 2.5 Ritonavir [M] 17.1 1.8 20.8 3.6 18.1 3.0 Nelfinavir [M] 15.6 1.1 10.0 1.1 7.0 1.3 Saquinavir [M] 15.9 1.4 16.3 1.2 13.5 1.2 E64 [M] 5.6 0.5 4.3 0.2 2.9 0.5 Leupeptin [M] 8.2 1.1 7.6 1.1 3.6 1.1 Chloroquine [nM] 44.1 3.7 36.4 4.0 32.4 4.2

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64 3D7, but the EC 50 values for B12 and 3D7 were similar (Table 4-1). There was not a significant difference in EC 50 concentrations for chloroquine between the 2 KO lines and 3D7. Discussion The simultaneous deletion of all 4 DV plasmepsins is an important achievement in the understanding of this family of proteases. Creating a triple-KO with only pfpm4 functional is also a valuable attainment for studying the role of plasmepsin 4 in the parasite. These two successful KOs signify the first time more than two genes have been deleted simultaneously with one plasmid construct. Furthermore, the removal of a genomic fragment >14 kb in length is an impressive feat that may suggest even larger deletions are possible. These mutants add to the repertoire of parasite lines created in our laboratory to further dissect the role and importance of this class of enzyme in Plasmodium species. Our hypothesis that at least one DV plasmepsin is required in the asexual stage of P. falciparum was not proven, and instead a null hypothesis that none of the known DV plasmepsins is required in this stage must be accepted. Beginning with the single-plasmepsin KOs created in our laboratory (Omara-Opyene et al. 2004) and by others (Liu et al. 2005a), the notion that redundancy in hemoglobin digestion exists within this family of proteinases was being realized. The deletion of members of the falcipain family of proteinases, and the effects this had on the parasite, suggested that redundancy might extend across proteinase classes (Sijwali & Rosenthal 2004). However, the issue of redundancy was always based on the presumption that hemoglobin hydrolysis was the primary function of all these enzymes.

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65 We investigated whether the quadruple-KO had an increase in expression of falcipain 3, a hemoglobinase in the DV believed to be essential (Sabnis et al. 2003). No up-regulation was observed in the expression of falcipain 3 (Figure4-10). Compensation by this proteinase may still be occurring despite the lack of an obvious increase in expression. When we investigated the single-plasmepsin KOs for up-regulation of other plasmepsins, none was observed (Figure 2-4). The proteinases may be present in abundance and performing multiple functions but readily able to adjust substrate specificity. One observation still suggests that these proteinases may be involved in the hemoglobin digestion pathway. The triple-KO (B12) grew comparably to the wild-type (3D7) when given nutrient-rich medium, but when given amino-acid limited medium it grew significantly slower than 3D7 (Figure 4-6). Wild-type 3D7 was capable of utilizing an alternative source of amino acids that B12 could not revert to. The most likely source of these amino acids is the hydrolysis of hemoglobin. In mammalian cells, the lysosomal compartment, and its membrane, have various functions including the turnover of cellular proteins, down-regulation and recycling of cell-surface receptors and lipid components, release of recycled nutrients, transport of degradation products from the lysosomal lumen to the cytoplasm and acidification of the interior (Andrejewski et al. 1999, Eskelinen et al. 2003). Cathepsin D, a lysosomal aspartic proteinase, has been demonstrated to be involved in the fusion of a vesicle to an autophagic vacuole (Eskelinen et al. 2002). Cathepsin D deficiency has been associated with lysosomal storage disorders and accumulation of undigested lipoproteins (Shacka & Roth 2005) and vacuolar structures (Koike et al. 2005). All eukaryotic cells are known

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66 to use autophagy as a central mechanism in cellular metabolism to degrade parts of their cytoplasm and organelles (Klionsky & Emr 2000). In Plasmodium, the process of autophagy may be analogous to the formation of a transport vesicle and fusion with the digestive vacuole. The vacuolar structures accumulated in the DV of the quadruple-plasmepsin KO (C10) had a morphology very similar to the structures observed in the neuronal cells of cathepsin D-deficient mice (Koike et al. 2005). PfPM4 appears to have a role in the recycling of the lipid components of the transport vesicles (synonymous with autophagic vesicles) after they reach the digestive vacuole. The accumulation of these vacuolar structures is believed to be result of a retardation in their degradation as opposed to an increase in their formation. Very few 3D7 and B12 cell contained these vesicles while over 40% of the C10 parasites contained multiple vesicles. Nile Red was used to stain neutral lipid bodies. Neutral Lipid bodies have been associated with the DV and are believed to be valuable storage deposits for cholesterol and triacylglycerol (TAG) (Jackson et al. 2004, Palacpac et al. 2004, Vielemeyer et al. 2004). A transport vesicle fuses its outer membrane with the DV membrane and the vesicles inner membrane is thought to be processed by a lysophopholipase (Jackson et al. 2004). When we investigated the morphology of neutral lipid bodies, no difference was apperent between the wild-type (3D7) and the quadruple-KO (C10) (Figure 4-9)

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67 pHHTK-15509284 bp hDHFR hsp3-5' CAM-5' thymidine kinase amp ^PfHAP 816-1681 ^pfPM4 -348-446 3' hrp2 pHHTK-15469108 bp hDHFR hsp3-5' CAM-5' thymidine kinase amp ^PfPM1 1-619 ^PfHAP 816-1681 3' hrp2 pHHTK-15509284 bp hDHFR hsp3-5' CAM-5' thymidine kinase amp ^PfHAP 816-1681 ^pfPM4 -348-446 3' hrp2 pHHTK-15469108 bp hDHFR hsp3-5' CAM-5' thymidine kinase amp ^PfPM1 1-619 ^PfHAP 816-1681 3' hrp2 Figure 4-1. Double-crossover plasmids pHTK-1546 and pHTK-1550. Plasmid pHTK-1546 was used to create a triple-plasmepsin KO (pfpm1, pfpm2 and pfhap), and pHTK-1550 was used to create a quadruple-plasmepsin KO (pfpm4, pfpm1, pfpm2 and pfhap).

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68 NsiI (7865) NsiI (9834) NsiI (1571) PfPM4 5' CAM hrp2-3' hDHFR ^pfpm1 ^pfhap NsiI (7522)Nsi I NsiI (5841) 5' CAM hrp2-3' hDHFR ^pfhap ^pfpm4 NsiI (7) NsiI (5498) NsiI (7810) pfpm4 pfpm1 pfpm2 pfhap NsiI (22) NsiI (4945) NsiI (20425) WTTripleQuadruple NsiI (7865) NsiI (9834) NsiI (1571) PfPM4 5' CAM hrp2-3' hDHFR ^pfpm1 ^pfhap NsiI (7522)Nsi I NsiI (7865) NsiI (9834) NsiI (1571) PfPM4 5' CAM hrp2-3' hDHFR ^pfpm1 ^pfhap NsiI (7522)Nsi I NsiI (5841) 5' CAM hrp2-3' hDHFR ^pfhap ^pfpm4 NsiI (7) NsiI (5498) NsiI (7810) pfpm4 pfpm1 pfpm2 pfhap NsiI (22) NsiI (4945) WTTripleQuadruple NsiI (20425) Figure 4-2. Schematic of the expected loci of the wild-type 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10). NsiI was used to digest gDNA for Southern blot analysis.

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69 Figure 4-3. Southern blot of gDNA from 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10) to confirm targeted disruption of the plasmepsin locus. Genomic DNA was digested with NsiI and probed with DNA probes specific to the proregion of PfPM4 (proPfPM4), the proregion of PfPM1 (proPfPM1) and the coding region of the human dihydrofolate reductase gene (hDHFR).

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70 3D7B12C10 3D7B12C10 375075kDaanti-PfPM1anti-PfPM2 3D7B12C10 3D7B12C10 375075kDaanti-PfHAPanti-PfPM4 3D7B12C10 3D7B12C10 375075kDaanti-PfPM1anti-PfPM2 3D7B12C10 3D7B12C103D7B12C10 3D7B12C10 3D7B12C103D7B12C10 375075kDa 375075kDaanti-PfPM1anti-PfPM2 3D7B12C10 3D7B12C10 375075kDaanti-PfHAPanti-PfPM4 3D7B12C10 3D7B12C103D7B12C10 3D7B12C10 3D7B12C103D7B12C10 375075kDa 375075kDaanti-PfHAPanti-PfPM4 3D7B12C10Neg3D7B12C10Neg pfpm1pfpm2 3D7B12C10Neg3D7B12C10Neg pfhappfpm4 3D7B12C10Neg3D7B12C10Neg pfpm1pfpm2 3D7B12C10Neg3D7B12C10Neg 3D7B12C10Neg3D7B12C10Neg3D7B12C10Neg3D7B12C10Neg pfpm1pfpm2 3D7B12C10Neg3D7B12C10Neg pfhappfpm4 3D7B12C10Neg3D7B12C10Neg 3D7B12C10Neg3D7B12C10Neg3D7B12C10Neg3D7B12C10Neg pfhappfpm4a.b. 3D7B12C10 3D7B12C10 375075kDaanti-PfPM1anti-PfPM2 3D7B12C10 3D7B12C10 375075kDaanti-PfHAPanti-PfPM4 3D7B12C10 3D7B12C10 375075kDaanti-PfPM1anti-PfPM2 3D7B12C10 3D7B12C103D7B12C10 3D7B12C10 3D7B12C103D7B12C10 375075kDa 375075kDaanti-PfPM1anti-PfPM2 3D7B12C10 3D7B12C10 375075kDaanti-PfHAPanti-PfPM4 3D7B12C10 3D7B12C103D7B12C10 3D7B12C10 3D7B12C103D7B12C10 375075kDa 375075kDaanti-PfHAPanti-PfPM4 3D7B12C10Neg3D7B12C10Neg pfpm1pfpm2 3D7B12C10Neg3D7B12C10Neg pfhappfpm4 3D7B12C10Neg3D7B12C10Neg pfpm1pfpm2 3D7B12C10Neg3D7B12C10Neg 3D7B12C10Neg3D7B12C10Neg3D7B12C10Neg3D7B12C10Neg pfpm1pfpm2 3D7B12C10Neg3D7B12C10Neg pfhappfpm4 3D7B12C10Neg3D7B12C10Neg 3D7B12C10Neg3D7B12C10Neg3D7B12C10Neg3D7B12C10Neg pfhappfpm4a.b. Figure 4-4. PCR (a) and western blot (b) analysis for confirming the creation of a triple and quadruple-plasmepsin knockout. The PCR was performed with primers designed to amplify the entire gene. The western blot was performed on asynchronous parasites with monospecific, polyclonal antibodies.

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71 Parasite Doubling Time Doubling Time (hr) 0510152025 3D7 B12 C10 Figure 4-5. Growth rate in doubling time of 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10).

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72 Growth in Standard and Limited-Nutrient MediumHours 024487296120Fold Increase 020406080100120 3D7 Standard medium 3D7 Limited medium B12 Standard medium B12 Limited medium C10 Stanard medium C10 Limited medium Figure 4-6. Growth rate of 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10) in rich medium and limited-nutrient medium. Fold-increase was calculated from parasitemias determined every 48 hours.

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73 Hemozoin FormationHours (post-synchronization) 8162434 Heme [fmol/parasite] 0.01.02.03.04.05.0 3D7 B12 C10 Figure 4-7. Intracellular hemozoin accumulation in 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10). The mean SD of three experiments, each run in triplicate, is plotted.

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74 Figure 4-8. TEM images of the 3D7, the triple-plasmepsin knockout (B12) and quadruple-plasmepsin knockout (C10). The digestive vacuoles of 3D7 and B12 show abundant hemozoin crystals. The digestive vacuole of C10 has membranous whorls characteristic of deficient autophagosome/endosome processing.

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75 Figure 4-9. Nile red staining of neutral lipid bodies produced in 3D7 (upper panels) and the quadruple-plasmepsin knockout (C10).

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76 anti-PfFP3 50-25-37-75-8 h16 h24 h36 h424242423D7 50-25-37-75-8 h16 h24 h36 h42424242C104 = 4 x 106parasites2 = 2 x 106parasiteskDakDa anti-PfFP3 50-25-37-75-50-25-37-75-8 h16 h24 h36 h42424242424242423D7 50-25-37-75-50-25-37-75-8 h16 h24 h36 h4242424242424242C104 = 4 x 106parasites2 = 2 x 106parasiteskDakDa Figure 4-10. Western blot analysis of 3D7 and quadruple-plasmepsin knockout (C10) for falcipain 3. Total protein from equal number of parasites (2 million and 4 million) collected every 8 hours for 36 hours was analyzed by western blot.

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CHAPTER 5 THE Plasmodium berghei MODEL Summary Plasmodium berghei is one of over 200 species of malaria parasites that infect mammals other than humans (Landau & Boulard 1978, Qari et al. 1996, Valkiunas 2005). The basic biology of this rodent parasite is similar to that of human malarial parasites to provide an excellent model for functional genomic studies in Plasmodium. Because P. berghei appears to have the ortholog of pfpm4 only, and not the other three plasmepsins associated with the digestive vacuole (DV) in P. falciparum, we chose to target pbpm4 in P. berghei as an alternative approach to deleting the aspartic proteinase activity in the DV. Knockout parasites with PbPM4 disrupted were created in collaboration with Dr. Andrew P. Waters at Leiden University, The Netherlands. Immunofluorescence microscopy using antibodies generated to a synthetic peptide sequence of PbPM4 localized the protein to hemozoin-containing vesicles throughout the parasite. Recombinant PbPM4 was expressed and its activity against rat and human hemoglobin was assessed. Visual examination of SDS-PAGE images suggests that the rodent recombinant PbPM4 enzyme was more effective at digesting human hemoglobin than rat hemoglobin. The data of the study demonstrate that PbPM4 is not essential for the survival of the parasite in the asexual stage and suggest that digestion of hemoglobin by other classes of proteases is sufficient for asexual growth. 77

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78 Introduction New technological advances in cell biology manipulation and post-genomics analysis are making members of the genus Plasmodium model organisms for intracellular eukaryotic pathogens (Fraunholz 2005). The rodent malaria parasites, in particular, are recognized as valuable model parasites providing useful opportunities to study the biology of the parasite, host–parasite interactions, vaccine development and drug testing. Methods for the genetic modification of the parasite, technologies for in vitro cultivation, and knowledge of its genome sequence and organization make the P. berghei–rodent model a very useful complement to research on P. falciparum. P. berghei infects laboratory hamsters, rats and mice. The life cycle and the developmental stages of P. berghei and P. falciparum are conserved as both are only infectious to Anopheline mosquitoes; have haploid sporozoites then invade and develop in hepatocytes; produce merozoites that invade and multiply in red blood cells; have relatively small percentage of parasites develop into gametocytes; fertilize and develop a diploid zygote into a ookinete in the midgut of the mosquito; and develop oocysts on the outside of the midgut after ookinete penetration. The genome organization between P. berghei and P. falciparum is highly conserved and both genomes are organized into 14 linear chromosomes ranging in size from 0.5 to 3.8 MB (Kooij et al. 2005). Despite the overall similarity between P. berghei and P. falciparum, there are some differences. For instance, the time and size of the developmental stages vary between the species. In addition, synteny maps show that each species has species-specific genes and gene families, some of which are rapidly evolving in P. falciparum (Kooij et al. 2005). We chose to exploit the difference in the number of DV plasmepsins between P. falciparum and P. berghei and collaborate with the laboratory of Dr. Andrew P. Waters

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79 in The Netherlands on targeting pbpm4, the plasmepsin 4 ortholog in P. berghei, for disruption. Modifications of the P. berghei genome, including allelic exchange and gene-knockouts, are more feasible due in part to a higher efficiency of transfection (Carvalho & Menard 2005, Janse et al. 2006). Intracellular localization of PbPM4 had not been reported in the literature so we chose to localize the protein by immunofluorescence staining, and to characterize the presumed proteinase activity of recombinant PbPM4 with human and rat hemoglobin as substrates. Materials and Methods Plasmodium berghei Propagation Plasmodium berghei (ANKA) parasites were propagated by intraperitoneal injection into 5-7 week-old C57BL/6 mice. The parasitemia was monitored by analyzing stained slides of blood drawn from the tail-vein. When the parasitemia reached 20-40% infected erythrocytes, the animal was euthanized and blood was collected by cardiac puncture for analysis. All protocols were in accordance with prior approval obtained from the University of Florida Institutional Animal Care and Use Committee (IACUC). Immunofluorescence Staining and Microscopy Parasitized blood samples collected from an infected mouse were washed twice with incomplete malaria culture medium, lacking Albumax, as defined in chapter 2. Thin smears of P. berghei-infected blood were prepared on coverslips and allowed to air dry. Cells were fixed for 10 min with ice-cold acetone/methanol (1:1), rinsed with phosphate-buffered saline (PBS) pH 7.2, and permeabilized with 0.1% Triton X-100/PBS for 10 min. Cells were washed twice in PBS and blocked with 3% bovine serum albumin (BSA)/PBS for 1 hour. Anti-PbPM4 antibody or pre-immune serum (diluted 1:1000) was added and allowed to bind overnight at 4C in 3%BSA/PBS. Cells were washed 3 times

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80 with PBS for 10 min each to remove excess primary antibody. Alexa-Fluor 488 goat anti-rabbit secondary antibody (1:1000 dilution in 3%BSA/PBS) was added and allowed to bind for 1 hour. Cells were washed 3 times with PBS for 10 min each, counter-stained with Hoescht 33258 nuclear stain and inverted onto a glass microscope slide with AntiFade (Molecular Probes). Plasmid Construction Plasmid pL0001 was kindly provided by A. P. Waters at Leiden University, The Netherlands. A 680 bp fragment of the pbpm4 gene (including 310 bp pf 5 UTR sequence) was PCR-amplified and ligated into pL0001 using the restriction enzymes KpnI and ClaI, and a 755 bp fragment of the 3 end of the gene (including 134 bp of 3 UTR sequence) was ligated into pL0001 using restriction enzymes EcoRI and SacII (Figure 5-1). The plasmid was subsequently linearized by digestion with restriction enzymes SapI, NaeI, and ScaI (Figure 5-1). For transfection of the parasites, the linearized plasmid was sent to Dr. Waters’ laboratory. Recombinant PbPM4 Activity Against Hemoglobin Substrates Recombinant proPbPM4 was over-expressed in E. coli, refolded in vitro, and the zymogen activated by preincubation under acidic conditions (Liu et al. 2005b). The enzyme was incubated at 37C for 5 min in 0.1 M sodium citrate buffer (pH 5.0). Human or rat hemoglobin (25 g) was added and the reactions were analyzed at time 0, 5, 30, 60, 120, and 240 min. Final enzyme concentration was 120 nM. Digestion was stopped at each respective time point by adding SDS sample buffer, boiling for 5min and placing at -80C until all samples were collected. For controls, hemoglobin was incubated at the same pH in the absence of the enzyme and for all time points. The samples were

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81 analyzed by SDS-PAGE on a 15% polyacrylamide gel and densitometric measurements were calculated using ImageJ software. Results Light Microscopy and Immunofluorescence Observations of stained blood smears taken from infected mice revealed that the P. berghei (ANKA) strain we were using produced multiple small vesicles containing hemozoin crystals. In P. falciparum, the digestive vacuole (DV) is easily recognized by light microscopy because of the amount of golden/brown hemozoin it contains. In the P. berghei slides, the hemozoin crystals were distributed throughout the cytoplasm of the cell in several vesicles and not in any one compartment (Figures 5-2). All cells displayed this characteristic. Immunolocalization of PbPM4showed that the protein is also scattered throughout the cytoplasm and not in any one compartment (Figure 5-3). Using differential interference contrast (DIC) and phase contrast when analyzing the sample, PbPM4 appeared to be localized to the many light-reflecting particles in the cytoplasm characteristic of hemozoin crystals (data not shown). Plasmepsin 4 Knockout in P. berghei The data on the targeted disruption of PbPM4 suggest the gene was successfully disrupted. The parasites are growing in mice treated with pyrimethamine for selection. Blots of P. berghei chromosomes separated by pulsed field gel electrophoresis were probed by the Waters’ laboratory, and the site of plasmid integration was shown to be chromosome 10 (data not shown). This is the location of the PbPM4 (Kooij et al. 2005). We designed primers to amplify the integration locus based on the expected locus architecture. Figure 5-4 shows the results of PCR amplification of gDNA extracted from

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82 the transfected parasites after selection. The locations of primers (Pr) 1-4 are marked on the schematic of the expected locus (Figure 5-4). For confirming integration at either the 5 or 3 end of the locus, one primer is in the undisrupted region not included in the plasmid construct and upstream of the integration (Pr-1 or Pr-4), and the other primer is in the drug cassette (Pr-2 or Pr-3). Only positive-integrants should produce the expected amplicon size. PCR analysis for the endogenous gene (Pr-5 and Pr-6) showed no amplification of the full length gene. Primers Pr-7 and Pr-8 were designed to anneal to the plasmid construct (in the backbone region) and were used to confirm that primers Pr-2 and Pr-3 can amplify DNA. They also indicate that unincorporated plasmid remains in the cell. The annealing temperature was 43C, below optimal to be conservative in the first analysis, and some presumably non-specific bands in several of the lanes may be removed upon further optimization of the PCR conditions. Hemoglobin Proteolysis by Recombinant PbPM4 The activity of recombinant PbPM4 against native human and rat hemoglobin (Hb) was investigated. At pH 5.0, human Hb was digested in the presence of enzyme beginning at about 30 min and continuing through the 4 h incubation period (Figure 5-5). The human Hb treated identically except for the addition of recombinant PbPM4 showed little degradation over the 4 h experiment. Densitometric measurements showed that after 2 h of incubation the protein band intensity was 22 % of the no-enzyme control, and after 4 h of incubation the protein band intensity was 12 % of the control. Interestingly, the rat hemoglobin was not digested as efficiently by recombinant PbPM4 over the 4 h experiment (Figure 5-5). After 2 h of incubation, the protein band intensity was 37% of the no-enzyme control, and after 4 h of incubation it was 30% of the control.

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83 Discussion The number of hemozoin-containing vesicles in the cytoplasm of the parasite has been described as varying between 2 to 7 depending on the strain of P. berghei (Sinden 1978, Saxena et al. 1989) and the erythrocyte type that it enters (Slomianny 1990, Slomianny & Prensier 1990). The initial or early-stage replication of P. berghei in rats and mice occurs in normal erythrocyte, but later there is a predilection for reticulocytes (immature erythrocytes) in the circulation (Landau & Boulard 1978). In this study, P. berghei also demonstrated a predilection for reticulocytes. The parasite morphology by light microscopy showed a large number of small golden/brownish crystals believed to be hemozoin (Figure 5-2). Our objective was to localize PbPM4 and determine whether it is localized to the DV as has been described for P. falciparum (Banerjee et al. 2002). By light microscopy no primary DV was evident and by immunofluorescence the anti-PbPM4 antibody bound throughout the cytoplasm suggesting it was associated with the hemozoin crystals but the hemozoin crystals and the enzyme are not in a centralized DV. The hemoglobin digestion experiment shows that the rodent PbPM4 can digest native human hemoglobin more effectively than native rat hemoglobin. These data agree with the hypothesis that the DV plasmepsins are not the primary hemoglobinases in the parasite, but are, perhaps, digestive vacuole proteinases capable of digesting a broad spectrum of substrates. The affinity for a particular substrate may vary depending the type of substrate or whether the proteinases are involved in other cellular processes. The human and rat globin-A sequence share a high level of homology (Figure 5-6) yet differ at the Phe33-Leu34 site where it has been described that the action of the DV plasmepsins involved in Hb digestion is initiated, causing an unraveling of the molecule and making it susceptible to further digestion (Gluzman et al. 1994). We have not

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84 determined whether PbPM4 is digesting native human hemoglobin at this particular site. However, it could be that this particular peptide bond is susceptible to hydrolysis by the plasmepsins in general, leading to an increase in digestion of this substrate by the recombinant PbPM4. One would expect the PbPM4 enzyme to digest rat hemoglobin as efficiently as human hemoglobin if this molecule was the primary substrate of this family of enzymes. The quadruple-plasmepsin KO in P. falciparum demonstrates that they are not essential for hemoglobin digestion. The creation of a plasmepsin 4 KO in P. berghei further proves that they are not essential for survival of the asexual stages and suggest there is a lot of redundancy in the hemoglobin digestion pathway. However, since PbPM4 does not digest native rat hemoglobin, this supports the hypothesis that this family of enzymes has an entirely different function and the proteolysis of hemoglobin is not their primary function in the cell. Further work is needed on the P. berghei plasmepsin 4 KO. It remains to be cloned and characterized. The P. berghei–rodent model of this particular KO together with the quadruple-KO in P. falciparum should be valuable in assessing the role of these enzymes. Transmission of the sexual stages and analysis of the sexual stages in the mosquito vector need to be investigated and compared between both mutant parasite lines to determine whether this family of enzymes have important roles in the sexual cycle of the parasite.

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85 pL0001-^Pbpm48908 bp 3' Pb-dhfr/ts 3' ^Pb-pm4 5' Pbdhfr/ts Tg-dhfr/ts 5' ^Pb-pm4 ClaI (683) EcoRI (5279) KpnI (8908) NaeI (6373) SacII (6044) SapI (8624) ScaI (7138) AgeI (2971) NheI (4809) A.B. 1 kb ladderDigested plasmid[SapI, NaeI,ScaI]Undigested plasmidSupercoiledladder 1 kb3 kb4 kb2 kb 2 kb6 kb4 kb3 kb8 kb 1 kb ladderDigested plasmid[SapI, NaeI,ScaI]Undigested plasmidSupercoiledladder 1 kb3 kb4 kb2 kb 2 kb6 kb4 kb3 kb8 kb pL0001-^Pbpm48908 bp 3' Pb-dhfr/ts 3' ^Pb-pm4 5' Pbdhfr/ts Tg-dhfr/ts 5' ^Pb-pm4 ClaI (683) EcoRI (5279) KpnI (8908) NaeI (6373) SacII (6044) SapI (8624) ScaI (7138) AgeI (2971) NheI (4809) A.B. 1 kb ladderDigested plasmid[SapI, NaeI,ScaI]Undigested plasmidSupercoiledladder 1 kb3 kb4 kb2 kb 2 kb6 kb4 kb3 kb8 kb 1 kb ladderDigested plasmid[SapI, NaeI,ScaI]Undigested plasmidSupercoiledladder 1 kb3 kb4 kb2 kb 2 kb6 kb4 kb3 kb8 kb Figure 5-1. Plasmid construct pL0001-pbpm4 for targeting plasmepsin 4 in P. berghei. A. Plasmid map with features identified. B. Linearized and unlinearized plasmid confirms full digestion and plasmid map.

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86 Figure 5-2. Light microscopy images of P. berghei (ANKA strain) stained with modified Geimsa Stain.

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87 Figure 5-3. Immunofluorescence microscopy of P. berghei with anti-PbPM4 (green) and Hoescht DNA stain (blue). The PbPM4 localized to the entire cytoplasm. Pre-immune sera were non-reactive with the cells (data not shown).

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88 Pr-1/Pr-2(5’int)Pr-3/Pr-4(3’int)Pr-5/Pr-6(whole gene)Pr-2/Pr-7(plasmid)Pr-3/Pr-8(plasmid)DNA LadderPr-1/Pr-2(5’int)Pr-3/Pr-4(3’int)Pr-5/Pr-6(whole gene)Pr-2/Pr-7(plasmid)Pr-3/Pr-8(plasmid)^pbpm4-KO unclonedP. berghei(ANKA)wild-type Pr-1Pr-2Pr-3Pr-4A.B. Pr-1/Pr-2(5’int)Pr-3/Pr-4(3’int)Pr-5/Pr-6(whole gene)Pr-2/Pr-7(plasmid)Pr-3/Pr-8(plasmid)DNA LadderPr-1/Pr-2(5’int)Pr-3/Pr-4(3’int)Pr-5/Pr-6(whole gene)Pr-2/Pr-7(plasmid)Pr-3/Pr-8(plasmid)^pbpm4-KO unclonedP. berghei(ANKA)wild-type Pr-1Pr-2Pr-3Pr-4 Pr-1Pr-2Pr-3Pr-4A.B. Figure 5-4. PCR analysis showing integration of plasmid pL0001-pbpm4 and disruption of the gene. A. Agarose gel electrophoresis of amplicons from the presumptive KO (left side) and the parental P. berghei strain (right side). B. Schematic of integration with wildtype locus (straight line), sequence fragments used for homologous recombination (pink) and the selectable-marker cassette of the plasmid in the middle (blue).

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89 Human Hb(no enzyme)Human Hb% Undigested 98% 91% 63% 38% 22% 12% Human Hb(no enzyme)Human Hb% Undigested 103% 88% 59% 57% 37% 30%053060120240Digestion time (min)Human Hb(no enzyme)Human Hb% Undigested 98% 91% 63% 38% 22% 12%Human Hb(no enzyme)Human Hb% Undigested 98% 91% 63% 38% 22% 12% Human Hb(no enzyme)Human Hb% Undigested 103% 88% 59% 57% 37% 30% Human Hb(no enzyme)Human Hb% Undigested 103% 88% 59% 57% 37% 30%053060120240Digestion time (min) Figure 5-5. Hemoglobin hydrolysis of native human hemoglobin and native rat hemoglobin by recombinant PbPM4. Equal amounts of hemoglobin were used to begin the experiment. The % of hemoglobin undigested was calculated by densitometric measurement of the treated and no-enzyme control at each time point.

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90 10 20 30 40 50 MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLS :::: ::::.: :::.: : :::: :::.::: .:::::::: : :.: MVLSADDKTNIKNCWGKIGGHGGEYGEEALQRMFAAFPTTKTYFSHIDVS 10 20 30 40 50 60 70 80 90 100 HGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFK :::::: ::::::::: : ::.:.: ::: ::::::::::::::::: PGSAQVKAHGKKVADALAKAADHVEDLPGALSTLSDLHAHKLRVDPVNFK 60 70 80 90 100 110 120 130 140 LLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR :::::::::: : : .::::.:::::::::::::::::::: FLSHCLLVTLACHHPGDFTPAMHASLDKFLASVSTVLTSKYR 110 120 130 140 Human HbRat HbHuman HbRat HbHuman HbRat Hb Identity = 78.2% Similarity = 84.5% 10 20 30 40 50 MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLS :::: ::::.: :::.: : :::: :::.::: .:::::::: : :.: MVLSADDKTNIKNCWGKIGGHGGEYGEEALQRMFAAFPTTKTYFSHIDVS 10 20 30 40 50 60 70 80 90 100 HGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFK :::::: ::::::::: : ::.:.: ::: ::::::::::::::::: PGSAQVKAHGKKVADALAKAADHVEDLPGALSTLSDLHAHKLRVDPVNFK 60 70 80 90 100 110 120 130 140 LLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR :::::::::: : : .::::.:::::::::::::::::::: FLSHCLLVTLACHHPGDFTPAMHASLDKFLASVSTVLTSKYR 110 120 130 140 Human HbRat HbHuman HbRat HbHuman HbRat Hb 10 20 30 40 50 MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLS :::: ::::.: :::.: : :::: :::.::: .:::::::: : :.: MVLSADDKTNIKNCWGKIGGHGGEYGEEALQRMFAAFPTTKTYFSHIDVS 10 20 30 40 50 60 70 80 90 100 HGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFK :::::: ::::::::: : ::.:.: ::: ::::::::::::::::: PGSAQVKAHGKKVADALAKAADHVEDLPGALSTLSDLHAHKLRVDPVNFK 60 70 80 90 100 110 120 130 140 LLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR :::::::::: : : .::::.:::::::::::::::::::: FLSHCLLVTLACHHPGDFTPAMHASLDKFLASVSTVLTSKYR 110 120 130 140 Human HbRat HbHuman HbRat HbHuman HbRat Hb Identity = 78.2% Similarity = 84.5% Figure 5-5. Alignment of human and rat -globin. Circled is the Phe-Leu bond in human Hb described as being the site of initial plasmepsin hydrolysis. Rat Hb contains an Phe-Ala bond at this site.

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CHAPTER 6 DISCUSSION The hypothesis of the study postulated that deleting the function of PfPM1, PfPM2 and PfHAP, and leaving only one of the DV plasmepsins, PfPM4, undisrupted would be sufficient for the parasite to complete the asexual cycle. Genetic knockouts of the DV plasmepsins in which 2 plasmepsins were deleted, and, subsequently, 3 plasmepsins deleted proved that the function of PfPM4 is sufficient to maintain the asexual growth of the parasite. The data are consistent with the hypothesis that the 4 DV plasmepsins are paralogs which evolved in P. falciparum to increase the fitness of the parasite and that they have a level of redundancy in the ability to digest hemoglobin in the DV. The DV plasmepsins, however, are not essential for the parasite to digest hemoglobin and survive the asexual cycle. The creation and characteristics of the quadruple-plasmepsin KO strongly suggest that this family of enzymes does not contribute significantly to the digestion of hemoglobin in the DV. The function of PfPM4 appears to be important as evidenced by the growth rate and morphology, and additional experiments are underway to determine whether they have functions outside of the food vacuole, perhaps in parasite egress and/or in lipid metabolism. More speculatively, the plasmepsins may be involved in the digestion of hemoglobin in individuals with hemoglobinopathies. The plasmepsins are actively being pursued as therapeutic drug targets (Johansson et al. 2005, Ersmark et al. 2006). The digestion of hemoglobin is viewed as an essential metabolic pathway and a target for new antimalarials. As such, drug targets should be 91

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92 validated and this study aims at addressing the validity of the plasmepsins as therapeutic drug targets. Our data suggest that they are not ideal drug targets as the parasite can grow asexually when they are all deleted. However, it should be noted that the in vitro culture of the Plasmodium parasite represents only one stage of growth and in the absence of immune pressure by the host. Therefore, demonstrating that the deletion of the DV plasmepsins has deleterious effects on the multiplication of the parasite in another stage (mosquito stage or liver stage) of the life cycle would be extremely important. The antimalarial activity of the HIV proteinase inhibitors and the interaction and treatment of these diseases where they are co-endemic is an important area of research (Nathoo et al. 2003, Kublin et al. 2005, Whitworth & Hewitt 2005, Kublin & Steketee 2006). Nearly all studies describing the antimalarial activity of clinically-available HIV proteinase inhibitors propose that the DV plasmepsins are the likely targets of these inhibitors (Skinner-Adams et al. 2004, Parikh et al. 2005, Andrews et al. 2006). If the DV plasmepsins were the targets of these inhibitors, an increase in resistance to the compounds would be observed. Except for nelfinavir, the triple and quadruple-plasmepsin KOs demonstrated no difference in susceptibility to the HIV proteinase inhibitors. Interestingly, both the triple and quadruple-plasmepsin KOs were more sensitive to nelfinavir, perhaps signifying an increase dependence on the function of the target of nelfinavir. This target may be an aspartic proteinase essential to the parasite and should be identified. One important question we asked was whether the deletion of the DV plasmepsins can be achieved in more than one Plasmodium species. To address this, we targeted plasmepsin 4 in P. berghei under the notion that because the genome of P. berghei

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93 encodes only the ortholog to pfpm4, and not the other 3 plasmepsin genes, deleting it would be equivalent to deleting all four paralogs in P. falciparum. It might be argued that this premise relies heavily on the in silico analysis of the sequences. However, there is no doubt these genes are paralogous and the general life cycle of the parasites are similar. In fact, other Plasmodium species that infect humans and have similar life cycles also lack the paralogs present in P. falciparum. Therefore, we feel the P. berghei–rodent model for studying the role of the DV plasmepsins will be useful when investigating other stages of the life cycle. Plasmodium falciparum has over 90 proteinases (Wu et al. 2003) and many of them represent potential targets for rational drug design (Wang & Wu 2004). Among them, are the aspartic proteinases of Plasmodium species (Coombs et al. 2001). Validation of a potential drug target is a difficult yet important step to the development of novel compounds. Functional genomic studies, immunological studies and many other types of analyses are need in the course of development. The gene-knockout studies presented in this work address the functional genomic analysis of 4 plasmepsins regarded as drug targets for malaria. Parasite lines lacking various combinations of plasmepsins, and a KO parasite for use in an animal model of malaria, have been created to continue studying many important aspect of this important class of enzymes.

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LIST OF REFERENCES Adams JH, Sim BKL, Dolan SA, Fang XD, Kaslow DC, Miller LH. 1992. A Family of Erythrocyte Binding-Proteins of Malaria Parasites. Proceedings of the National Academy of Sciences of the United States of America 89: 7085-7089 Aikawa M, Miller LH, Johnson J, Rabbege J. 1978. Erythrocyte entry by malarial parasites: A moving junction between erythrocyte and parasite. Journal of Cell Biology 77: 72-82 Akompong T, Kadekoppala M, Harrison T, Oksman A, Goldberg DE, Fujioka H, Samuel BU, Sullivan D, Haldar K. 2002. Trans expression of a Plasmodium falciparum histidine-rich protein II (HRPII) reveals sorting of soluble proteins in the periphery of the host erythrocyte and disrupts transport to the malarial food vacuole. Journal of Biological Chemistry 277: 28923-28933 Andrejewski N, Punnonen E-L, Guhde G, Tanaka Y, Lullmann-Rauch R, Hartmann D, von Figura K, Saftig P. 1999. Normal Lysosomal Morphology and Function in LAMP-1-deficient Mice. Journal of Biological Chemistry 274: 12692-12701 Andrews KT, Fairlie DP, Madala PK, Ray J, Wyatt DM, Hilton PM, Melville LA, Beattie L, Gardiner DL, Reid RC, Stoermer MJ, Skinner-Adams T, Berry C, McCarthy JS. 2006. Potencies of Human Immunodeficiency Virus Protease Inhibitors In Vitro against Plasmodium falciparum and In Vivo against Murine Malaria. Antimicrobial Agents and Chemotherapy 50: 639-648 Asakura T, Minakata K, Adachi K, Russell MO, Schwartz E. 1977. Denatured hemoglobin in sickle erythrocytes. The Journal of Clinical Investigation 59: 633-640 Babe LM, Rose J, Craik CS. 1995. Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation. Proceedings of the National Academy of Sciences of the United States of America 92: 10069-10073 Banerjee R, Francis SE, Goldberg DE. 2003. Food vacuole plasmepsins are processed at a conserved site by an acidic convertase activity in Plasmodium falciparum. Molecular and Biochemical Parasitology 129: 157-165 Banerjee R, Liu J, Beatty W, Pelosof L, Klemba M, Goldberg DE. 2002. Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine. Proceedings of the National Academy of Sciences of the United States of America 99: 990-995 Bein M, Schaller M, Korting HC. 2002. The secreted aspartic proteinases as a new target in the therapy of candidiasis. Current Drug Targets 3: 351-357 94

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BIOGRAPHICAL SKETCH I received my Bachelor of Science degree in Biological Sciences from the University of California at Irvine. After working for several years at the water utility in Orange County and a biotechnology company in Los Angeles, I decided to pursue graduate work at the University of Hawaii. I received a Master of Science degree in Microbiology for studying the microbiology of pollution in coastal waters and streams. I moved to Florida and conducted field studies on malaria, and other tropical diseases, spending many weeks in malaria endemic countries. My Ph.D. dissertation work at the University of Florida focused on studying the molecular mechanisms of pathogenesis of Plasmodium falciparum, the causative agent of malaria. My time at the University of Florida’s Department of Infectious Diseases and Pathology has been a rewarding experience and I am indebted to many people with whom I’ve had the opportunity to work alongside. 110