Student Number : 9503239E - PhD thesis - Faculty of Science / Malaria is one of the most debilitating pathogenic infections known to man, responsible for approximately three million deaths annually, primarily children in sub-Saharan Africa. The parasite has evaded multiple attempts at eradication, predominantly through the complexity of its life cycle, the ability to elude host immune response, and gametocyte formation to ensure dissemination. The recent completion of the genome sequence has opened up a multitude of avenues for exploration and identification of novel drug and vaccine targets, as well as providing a glimpse into the complex mechanisms that have contributed to the success of this pathogen. The mechanisms of gene regulation, especially those governing gametocytogenesis, have, however, not yet been elucidated. In this research, differential display has been used to identify some of the genes that are differentially expressed between the asexual parasite and gametocyte stages of P. falciparum. Numerous genes involved in diverse aspects of metabolism, protein synthesis and immune evasion were identified. A combination of BLASTN and BLASTX similarity searches was used to categorize and increase the confidence with which a transcript could be identified. Expression data for confidently identified genes were confirmed using reverse slot blot and available microarray data. PfMyb2, a novel transcription factor which may regulate genes involved in gametocytogenesis, was characterized. The DNA binding domains of the protein were cloned and expressed as a histidine fusion protein. Mobility shift assays were used to assess the in vitro binding capability of the recombinant 6xHis-PfMyb2, which bound to oligonucleotides containing the consensus Myb regulatory element. Two of the oligonucleotides represent sequences located within promoters of P. falciparum genes (Pfcrk1 and Pfmap1) known to play a role in regulating the cell cycle, a function ascribed to many members of the vertebrate Myb family. The identification of PfMyb2 as a bona fide transcription factor is a first step into gaining some insight into the many regulatory processes that occur during the life cycle of this complex organism. A better understanding of the molecular mechanisms that govern its survival is essential for the ultimate eradication of this deadly parasite.

La découverte de nouveaux agents antipaludiques est primordiale. A travers le monde, les chercheurs se sont focalisés sur plusieurs stratégies. Les plus développées sont : soit les tests de molécules issues de bibliothèques chimiques dans une recherche phénotypique (comme le test direct d’agents sur des cultures de parasites in vitro), soit la recherche de nouvelles molécules agissant sur l’activité d’une cible ou d’une voie spécifique et essentielle. Cette thèse est centrée sur le second type d’approche. Nous nous sommes intéressés aux transporteurs membranaires de P. falciparum. Pour cela, nous exprimons les protéines d’intérêt dans la levure et nous les purifions. Nous optimisons les tests fonctionnels, dans le but de : a) déterminer l’effet des molécules sur les cibles spécifiques ; b) tester leur effet sur les cultures d’érythrocytes infectés par P. falciparum in vitro ; c) vérifier leur toxicité sur des cellules de mammifères ; et d) réaliser le test des molécules les plus efficaces in vivo dans un modèle de paludisme murin. Notre travail actuel est focalisé sur l’ATPase6 de P. falciparum (PfATP6) et l’adénylate translocase (PfAdT), deux protéines membranaires essentielles localisées respectivement sur le réticulum endoplasmique et la membrane mitochondriale. Nous exprimons de manière hétérologue PfATP6 dans les membranes de levure, nous purifions la protéine et mesurons une activité ATPase spécifique. Nous avons ainsi pu tester une bibliothèque chimique importante et identifier des inhibiteurs spécifiques. Ces derniers ont ensuite été testés pour évaluer leur effet sur les stades érythrocytaires du parasite in vitro et leur cytotoxicité sur des cellules de mammifères. Pour le transporteur PfAdT, nous procédons comme pour PfATP6, mais nous avons choisi un autre type de test fonctionnel dans lequel la protéine est directement exprimée sur la membrane plasmique d’E. coli. Cela devrait permettre de mesurer le transport d’ATP radiomarqué, et l’identification d’inhibiteurs spécifiques dont les effets pourront être évalués sur des cultures de parasites in vitro et dans des essais de cytotoxicité. / New drug discovery for malaria treatment urges, now more than ever. There is no optimal solution to the search for new antimalarials. Worldwide, researchers have focused their energies on several strategies. The most commonly employed are: either by screening molecules issued from chemical libraries in a phenotypic way (i.e., direct testing of drugs on in vitro parasite cultures), or by searching for new molecules acting upon the activity of a specific essential target or pathway. This PhD thesis centers on the second type of approach. We are interested in targeting membrane transporters of P. falciparum. For this, we plan to express proteins of interest in yeast and proceed to their isolation. With optimized functional tests, we aim to: a) Determine the effect of molecules upon specific targets; b) Test their effect on P. falciparum in vitro erythrocytes cultures; c) As well as verify their toxicity on mammalian cells; and d) Perform in vivo testing of the best molecules on a rodent model for malaria. Our actual work is focused on the P. falciparum Ca2+ - ATPase 6 (PfATP6) and adenylate translocase (PfAdT), two essential membrane proteins localized on the endoplasmic-reticulum and the mitochondrial membrane, respectively. We were able to express heterologously PfATP6 in yeast membranes, purify the protein and measure a specific ATPase activity. With this, we have tested a large chemical library and identified specific inhibitors. These were then tested for their effect on in vitro blood stages of P. falciparum and for their cytotoxicity on mammalian cells. For the ATP/ADP carrier PfAdT, we proceeded as previously done with PfATP6 but we have also chosen another type of functional test where we express directly this protein in the plasma membrane of E. coli. This will enable in the future the measurement of radiolabelled ATP uptake, and the identification of specific inhibitors that could then be tested for their effect on P. falciparum in vitro cultures and for their cytotoxicity.

Malaria persists today as a significant burden for a large part of the world. However, over the past few decades, a concerted effort by governments, non-governmental organizations, researchers, and community health workers worldwide has yielded progress in reducing the deadly impact of this disease. Today, some of these gains are threatened by the rise of antimalarial drug resistance, a recurring problem that has impeded global malaria reduction efforts before. Research on Plasmodium falciprum resistance to the numerous antimalarial compounds used today and in the past has made significant progress on determining which specific mutations modulate drug susceptibility and to what degree they do so. To gain a comprehensive understanding of drug resistance, we need to elucidate how and why it arises. Therefore, it is important to elucidate whether some malaria parasites acquire resistance-conferring mutations faster than others and why the native function of the genetic factors involved lend themselves to modulating drug resistance. For instance, resistance to multiple antimalarial therapies has repeatedly emerged in Southeast Asia. We investigated the long-held hypothesis that this was due to the ability of these parasites to mutate significantly faster than non-Southeast Asian strains. Elucidating whether this hypermutability phenotype accurately represents Southeast Asian parasite evolvability is important, as it can inform when resistance would be expected to next arise, particularly in the Greater Mekong Subregion in Southeast Asia. Here, we have adapted a fluctuation assay to Plasmodium falciparum and determined that some contemporaneous Cambodian parasites exhibit a mild mutator, but not a hypermutator, phenotype. We also show that this is likely driven by mutations in DNA repair genes carried predominantly by multidrug resistant Southeast Asian parasites. One of the most common genes in which drug resistance-conferring mutations occurs is the P. falciparum chloroquine resistance transporter (pfcrt). Mutations in pfcrt are associated with parasite susceptibility to many of the antimalarial compounds that have been used in a clinical setting to date. However, beyond its role in drug resistance, little is known about the native function of PfCRT. To facilitate the study of pfcrt, we have designed a zinc-finger nuclease (ZFN)-based gene engineering system that introduces a single double-strand break in intron 1 of pfcrt. Our ZFN strategy enables replacing nearly any endogenous pfcrt locus with a user-defined recombinant pfcrt allele. We show that our method of pfcrt allelic replacement is fast, efficient, and reliable. We used this system to generate a unique mutant parasite encoding a pfcrt-L272F mutation, which enlarges the parasite digestive vacuole, the lysosome-like organelle used to catabolize host-derived hemoglobin for amino acid salvage. Our results provide clear evidence that PfCRT is associated with the terminal steps of hemoglobin degradation, overall parasite fitness, and the balance of osmolytes across the digestive vacuole membrane. Bringing clarity to the native function of PfCRT can reveal how and why this single genetic factor has been and continues to be involved in the resistance to many different antimalarial compounds.

Made available in DSpace on 2015-04-11T13:38:45Z (GMT). No. of bitstreams: 1 Bruna Lovizutto Protti Wohlke.pdf: 1421829 bytes, checksum: 1654b1beeb77d5566e82213f52b50a17 (MD5) Previous issue date: 2012-03-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A malária continua a ser a maior causa de morbidade e mortalidade mundial com até três milhões de mortes anuais. O tratamento da malária, causada por Plasmodium. falciparum, dependeu por décadas do uso da aminoquinolina cloroquina. Contudo a resistência à cloroquina em uma escala global expôs a capacidade com que o parasita pode desenvolver resistência a drogas. É, portanto, necessário o desenvolvimento de estudos que assegurem que drogas mais eficazes sejam descobertas de forma sustentável e que novos alvos moleculares para agentes antimalária sejam revelados. Através de análises de biologia celular e molecular foi possível sequenciar o genoma de P. falciparum e identificou-se novos alvos alvos terapêuticos antimalária. A proteína alvo estudada neste projeto foi a Hipoxantina guanina fosforribosiltransferase do P. falciparum, que está relacionada com a via de recuperação das purinas .O gene da enzima foi identificado no genoma do P. falciparum, e produzido por síntese química com códons preferenciais de Escherichia coli, clonado em um forte promotor de expressão para esta bactéria, expresso e a proteína recombinante purificada mostrou-se ativa. A enzima será utilizada futuramente em estudos de binding e inibição com novos compostos químicos e/ou componentes de extratos de microrganismos e plantas amazônicas

Die Hälfte der Weltbevölkerung lebt mit dem Risiko, an einer schweren Malaria tropica zu erkranken. Zunehmende Resistenzen von Plasmodium falciparum gegen gängige Therapeutika erschweren eine Behandlung, und es existiert keine Möglichkeit frühzeitig die Wirksamkeit der angewandten Medikation festzustellen. Die Bestimmung der Parasitämie als einzig verfügbarer Parameter kann auch bei erfolgreicher Therapie noch über den ersten Tag ansteigen. Das Ziel dieser Studie war, lichtmikroskopische Parameter zu finden, mit denen der Erfolg einer Therapie frühzeitig festgestellt werden kann. So wurden im Rahmen einer Fallstudie die Plasmodien eines an einer schweren Malaria tropica erkrankten Patienten auf morphologische Veränderungen im Verlauf der Chinin-Therapie untersucht. Die Beurteilung der Plasmodien erfolgte durch eine Einteilung nach ihrer Lage im Erythrozyten und der Kern-Plasma-Relation der Ringformen, anschliessend wurden die Ergebnisse durch eine Vermessung der Plasmodien am Computer verifiziert. Es zeigte sich, dass ein Therapieerfolg anhand der Veränderung in der Morphologie der Ringformen bereits in den ersten Stunden nach Therapiebeginn festgestellt werden kann. So lässt sich innerhalb der ersten drei Stunden ein Wechsel von kleinen Ringformen mit dünnem, homogenem Zytoplasmaband zu vergrösserten Ringformen mit einem verbreiterten und inhomogenen Zytoplasma finden. Im weiteren konnten ab der 7. Therapiestunde eine zunehmende Lageveränderungen der Plasmodien im Erythrozyten aufgezeigt werden. So waren ab diesem Zeitpunkt zunehmend Plasmodien, die die Erythrozyten-Membran hervorwölben (Arbeitstitel „Accentué“-Formen), im peripheren Blutausstrich des Patienten zu sehen. Dass die Änderung der Kern-Plasma-Relation der Ringformen ursächlich einer direkten Medikamentenwirkung zuzuschreiben sind, konnte in einem abschliessenden „in vitro“-Studienteil gezeigt werden, in welchem Plasmodien-Kulturen unter Chinin-Einfluss mit Kontrollkulturen ohne Medikamenteneinfluss verglichen wurden. / This case study examines early morphological changes of Plasmodium falciparum under treatment, visible by light microscopy. A transition from small thin ring shapes to thick rings during the early hours of treatment could be demonstrated, as well as an increase in erythrocyte surface distorsion by the plasmodia, beginning after 7 hours of treatment. These findings could help to recognise resistances to medication within hours of beginning treatment and may save crucial time for patients.

Transmission of the malaria parasite from man to the mosquito requires the formation of sexual parasite stages, the gametocytes. The gametocytes are the only parasite stage that is able to survive in the mosquito midgut and to undergo further development – gamete formation and fertilization. Numerous sexual stage-specific proteins have been discovered, some of which play crucial roles for parasite transmission. However, the functions of many sexual stage proteins remain elusive. Amongst the sexual stage-specific proteins are the proteins of the PfCCp proteins family, which exhibit numerous adhesion domains in their protein structures. For four members of the protein family, PfCCp1 to PfCCp4 gene-disruptant parasite lines had been already studied. Amongst these, PfCCp2 and PfCCp3 showed an important role for development of the parasites in the mosquito. In the present work the study of gene-disrupted parasites of the PfCCp Protein family was completed. PfCCp5-KO and PfFNPA-KO parasite lines were characterized to a great extent and many properties were similar to those of other PfCCp proteins. The co-dependent expression previously reported to be a phenomenon of PfCCp proteins was also observed in these two mutants, although to lesser extent. When either PfCCp5 or PfFNPA were absent, all other proteins were detected in reduced abundance only. Co-dependent expression manifests exclusively on the protein level. Transcript levels were not altered as RT-PCR showed. Amongst PfCCp proteins numerous proteinproteins interactions are taking place. The previously described multimeric protein complexes also include further sexual stage-specific proteins like Pfs230, Pfs48/45 and Pfs25. Recently, a new component of PfCCp-based multimeric protein complexes had been identified. The protein was named PfWLP1 (WD repeat protein-like protein 1) due to its possession of several WD40 repeats. In the present study expression of this uncharacterized protein was investigated via indirect IFA. It was expressed in asexual blood stages and gametocytes. Upon gamete formation and fertilization its expression ceased. Another sexual stage protein studied in this work was PfactinII. It was shown to be exclusively expressed in sexual stages. In gametocytes it co-localizes with Pfs230 and correct localization of PfactinII depends on presence of Pfs230. Transcript analysis by means of RT-PCR revealed the expression of several components of the IMC in gametocytes. Furthermore, five or six myosin genes encoded in the P. falciparum genome were detected in gametocytes. Gametocyte egress was studied on the ultrastructural level via transmission electron microscopy and an inside-out type of egress was observed. Firstly, the membrane of the parasitophorous vacuole (PVM) was lysed and only thereafter the membrane of the red blood cell (RBCM) ruptured. Furthermore, a new inductor of gametogenesis was identified: The K+/H+ ionophore nigericin induced gametocytes activation in the absence of xanthurenic acid (XA), which is responsible for gamtetocyte activation in the mosquito midgut. Selective permeabilization of RBCM and PVM by the mild detergent saponin, showed that in the absence of these membranes male gametocytes were still able to perceive both XA and the drop in temperature. Thus, the receptors for both factors signaling the parasite transmission to the mosquito, seem to be of parasitic origin. LC/MS/MS analysis confirmed the ability of RBCs to take up XA. With malaria eradication on the agenda of malaria research targeting the sexual stages becomes a crucial part of intervention strategies. The sexual stages are especially attractive target as they represent a population bottleneck. The here reported findings on P. falciparum gametocytes provide several potential candidate proteins for developing tools to interrupt transmission from man to mosquito. Such tools might include Transmission blocking vaccines and drugs. / Die Übertragung der Malaria vom menschlichen Wirt auf die Überträgermücke erfordert die Bildung von Sexualstadien, der Gametozyten. Dieses Parasitenstadium ist in der Lage im Mitteldarm der Mücke zu überleben und sich zu Gameten zu entwickeln, gefolgt von Befruchtung und ygotenbildung. Eine Vielzahl von in den Sexualstadien exprimierter Proteine wurde bereits entdeckt. Einige von diesen haben essentielle Funktionen für die Transmission der Parasiten auf die Mücke. Die Rolle der meisten dieser spezifisch exprimierter Protein ist jedoch ungeklärt. Zu den sexualstadienspezifischen Proteinen gehören die Proteine der PfCCp-Proteinfamilie. Für vier Proteine diese Proteinfamilie wurden bereits KO-Mutanten untersucht. Zwei Mutanten, PfCCp2-KO und PfCCp3-KO besitzen eine wichtige Funktion während der Entwicklung der Parasiten in der Mücke. In der vorliegenden Arbeit wurde die Studie der PfCCp-Proteine komplettiert. PfCCp5- und PfFNPA-defiziente Parasitenlinien wurden zu einem Großteil charakterisiert. Viele Eigenschaften dieser beiden Parasitenlinien wiesen Ähnlichkeiten zu den bisher untersuchten PfCCp-KO-Mutanten auf. Die ko-abhängige Expression welche in der PfCCp-Proteinfamilie vorkommt, wurde auch in diesen beiden Mutanten beobachtet, wenngleich in geringerem Ausmaß. In den Mutanten, in welchem entweder PfCCp5 oder PfFNPA fehlten, waren alle übrigen PfCCp-Proteine nur in reduzierter Menge nachzuweisen. Diese ko-abhängige Expression ist ausschließlich auf dem Proteinlevel zu beobachten. Die Transkription der jeweiligen Gene hingegen ist unbeeinflusst. Zahlreiche Protein-Protein-Interaktionen finden zwischen den Proteinen der Proteinfamilie statt. The zuvor beschriebenen multimeren Proteinkomplexe schließen auch weitere sexualstadienspezifische Proteine ein, wie Pfs230, Pfs48/45 und Pfs25. Kürzlich wurde eine neue Komponente der PfCCp-basierten Multiproteinkomplexe identifiziert. Dieses Protein wurde PfWLP1 (WD repeat protein-like protein 1) genannt, da es mehrere WD40 repeat Domänen besitzt. In der vorliegenden Arbeit wurde das bisher unbeschriebene Protein mittels indirekter immunfluoreszenzstudien charakterisiert. PfWLP1 ist sowohl in asexuellen Blutstadien als auch in Gametozyten exprimiert. Nach der Gametenbildung und Fertilisation nimmt die Expression des Proteins ab. Ein weiteres Protein der Sexualstadien, welches in dieser Arbeit untersucht wurde, ist PfactinII. Es wurde gezeigt, dass dieses Protein ausschließlich in den Sexualstadien vorliegt. In Gametozyten ko-lokalisiert es mit Pfs230 und die korrekte Lokalisierung ist abhängig von der Anwesenheit von Pfs230. Mittels RT-PCR wurden mehrere Komponenten des inneren Membrankomplexes in Gametozyten nachgewiesen. Weiterhin wurden Transkripte für fünf der sechs Myosin-Gene, welche im Genom von P. falciparum exprimiert sind, nachgewiesen. Der Austritt der Gametozyten aus der Wirtszelle wurde auf ultrastruktureller Ebene mittels Transmissionselektronenmikroskopie untersucht. Hierbei wurde gezeigt, dass die Lyse der den Parasiten umgebenden Membranen von innen nach außen geschieht. Das heißt, dass zunächst die Membran der parasitophoren Vakuole (PVM) lysiert wird, und erst anschließend die Erythrozyten-Plasmamembran (RBCM). Als neuer Induktor der Gametozytenaktivierung wurde Nigericin identifiziert. Nigericin ist ein K+/H+-Ionophor, welcher in Abwesenheit von XA in der Lage ist, die Gametenbildung zu identifizieren. Die selektive Permeabilisierung der beiden den Gametozyten umgebenden Membranen, PVM und RBCM, zeigte, dass männliche Gametozyten nach Entfernung der beiden Membranen, in der Lage sind, den Temperaturabfall und XA zu perzipieren. Somit kann geschlussfolgert werden, dass die Rezeptoren beider Stimuli parasitischen Ursprungs sind. LC/MS/MS-Analysen bestätigten, dass Erythrozyten in der Lage sind, XA aufzunehmen. Die Sexualstadien des Malariaparasiten nehmen mehr und mehr an Bedeutung zu, da langfristig nicht nur eine Eindämmung der Malaria in endemischen Gebieten sondern die Auslöschung der Malaria angestrebt wird. Die Sexualstadien sind ein attraktiver Angriffspunkt aufgrund ihrer Bedeutung für die Transmission der Krankheit. Zum anderen ist die auf den Vektor übertragene Parasitenanzahl vergleichsweise gering. Die Ergebnisse der vorliegenden Arbeit zeigen mehrere potentielle Kandidatenproteine auf, welche für die Entwicklung von Interventionsstrategien von Bedeutung sein könnten. Als Interventionsstrategien wären sowohl transmissionsblockierenden Vakzine als auch transmissionsblockierende Wirkstoffe denkbar.

Besides HIV and tuberculosis, malaria still is one of the most devastating infectious diseases especially in developing countries, with Plasmodium falciparum being responsible for the frequently lethal form of malaria tropica. It is a major cause of mortality as well as morbidity, whereby pregnant women and children under the age of five years are most severely affected. Rapidly emerging drug resistances and the lack of an effective and safe vaccine hamper the combat against malaria by chemical and pharmacological regimens, and moreover the poor socio-economic and healthcare conditions in malaria-endemic countries are compromising the extermination of this deadly tropical disease to a large extent. Malaria research is still questing for druggable targets in the parasitic protozoan which pledge to be refractory against evolving resistance-mediating mutations and yet constitute affordable and compliant antimalarial chemotherapeutics. The parasite kinome consists of members that represent most eukaryotic protein kinase groups, but also contains several groups that can not be assigned to conservative ePK groups. Moreover, given the remarkable divergence of plasmodial kinases in respect to the human host kinome and the fact that several plasmodial kinases have been identified that are essential for the intraerythrocytic developmental cycle, these parasite enzymes represent auspicious targets for antimalarial regimens. Despite elaborate investigations on several other ePK groups, merely scant research has been conducted regarding the four identified members of the cyclin-dependent kinase-like kinase (CLK) family, PfCLK-1-4. In other eukaryotes, CLKs are involved in mRNA processing and splicing by means of phosphorylation of serine/arginine-rich (SR) proteins, which are crucial components of the splicing machinery in the alternative splicing pathway. All four PfCLKs are abundantly expressed in asexual parasites and gametocytes, and stage-specific expression profiles of PfCLK-1 and PfCLK-2 exhibited nucleus-associated localization and an association with phosphorylation activity. In the course of this study, PfCLK-3 and PfCLK-4 were functionally characterized by indirect immunofluorescence, Western blot analysis and kinase activity assays. These data confirm that the two kinases are primarily expressed in the nucleus of trophozoites and both kinases possess in vitro phosphorylation activity on physiological substrates. Likewise PfCLK-1 and PfCLK-2, reverse genetic studies exhibited the indispensability of both PfCLKs on the asexual life cycle of P. falciparum, rendering them as potential candidates for antiplasmodial strategies. Moreover, this study was conducted to identify putative SR proteins as substrates of all four PfCLKs. Previous alignments revealed a significant homology of the parasite CLKs to yeast SR protein kinase Sky1p. Kinase activity assays showed in vitro phosphorylation of the yeast Sky1p substrate and SR protein Npl3p by precipitated PfCLKs. In addition, four homologous plasmodial SR proteins were identified that are phosphorylated by PfCLKs in vitro: PfASF-1, PFSRSF12, PfSFRS4 and PfSR-1. All four parasite SR splicing factors are predominantly expressed in the nuclei of trophozoites. For PfCLK-1, a co-localization with the SR proteins was verified. Finally, a library of human and microbial CLK inhibitors and the antiseptic chlorhexidine (CHX) was screened to determine their inhibitory effect on different parasite life cycle stages and on the PfCLKs specifically. Five inhibitors out of 63 compounds from the investigated library were selected that show a moderate inhibition on asexual life cycle stages with IC50 values ranging between approximately 4 and 8 µM. Noteworthy, these inhibitors belong to the substance classes of aminopyrimidines or oxo-β-carbolines. Actually, the antibiotic compound CHX demonstrated an IC50 in the low nanomolar range. Stage-of-inhibition assays revealed that CHX severely affects the formation of schizonts. All of the selected CLKs inhibitors also affect gametocytogenesis as well as gametogenesis, as scrutinized in gametocyte toxicity assays and exflagellation assays, respectively. Kinase activity assays confirm a specific inhibition of CLK-mediated phosphorylation of all four kinases, when the CLK inhibitors are applied on immunoprecipitated PfCLKs. These findings on PfCLK-inhibiting compounds are initial attempts to determine putative antimalarial compounds targeting the PfCLKs. Moreover, these results provide an effective means to generate chemical kinase KOs in order to phenotypically study the role of the PfCLKs especially in splicing events and mRNA metabolism. This approach of functionally characterizing the CLKs in P. falciparum is of particular interest since the malarial spliceosome is still poorly understood and will gain further insight into the parasite splicing machinery. / Neben HIV und Tuberkulose stellt Malaria vor allem in Entwicklungsländern immer noch eine der verheerendsten Infektionskrankheiten dar, wobei Plasmodium falciparum für die oft tödlich verlaufende Form der Malaria tropica verantwortlich ist. Sie ist eine der Hauptgründe für Mortalität und Morbitität, von der vor allem schwangere Frauen und Kinder unter fünf Jahren am schlimmsten betroffen sind. Das Fehlen eines effektiven und ungefährlichen Impfstoffes und sich schnell ausbreitende Medikamentenresistenzen erschweren die Bekämpfung von Malaria mit Arzneimitteln. Darüber hinaus beeinträchtigen die schlechten sozioökonomischen Bedingungen und der mangelhafte Zustand des Gesundheitssystems in Malaria-endemischen Ländern die Elimination dieser tödlichen Tropenkrankheit in hohem Maße. Die Malariaforschung ist immer noch auf der Suche nach vielversprechenden Angriffspunkten im Parasiten, die widerstandsfähig gegenüber sich entwickelnden resistenz-vermittelnden Mutationen sind und dennoch erschwingliche und verträgliche Chemotherapeutika gegen Malaria darstellen. Das Kinom des Parasiten besteht aus Vertretern der meisten eukaryotischen Proteinkinase-Gruppen und enthält zudem einige Gruppen, die keiner der konventionellen Gruppen zuordenbar sind. Darüber hinaus stellen Kinasen vielversprechende Angriffspunkte für Malariamedikamente dar, da das Parasitenkinom bemerkenswerte Divergenzen gegenüber dem Wirtskinom aufweist und zudem einige Parasitenkinasen identifiziert wurden, die unerlässlich für den Replikationszyklus von asexuellen Parasiten sind. Trotz umfangreicher Untersuchungen anderer Kinasegruppen des Parasiten wurden die vier identifizierten Vertreter der Zyklin-abhängige-Kinase-ähnlichen Kinasen (cyclin-dependent kinase-like kinases, CLKs) bisher kaum untersucht. In anderen Eukaryoten sind CLKs an der mRNA-Prozessierung und am Spleißen durch die Phosphorylierung von Serin/Arginin-reichen (SR-) Proteinen beteiligt, welche wiederum Komponenten der Spleißmaschinerie sind. Alle vier PfCLKs sind abundant exprimiert in asexuellen Parasiten sowie Gametozyten, und stadien-spezifische Expressionsprofile von PfCLK-1 und PfCLK-2 zeigten eine Kern-assoziierte Expression sowie Phosphorylierungsaktivität in in vitro-Aktivitätsstudien. Im Verlauf dieser Studie wurden PfCLK-3 und PfCLK-4 mittels indirekter Immunfluoreszenzstudien, Western Blot-Analysen und Kinaseaktivitätsassays funktionell charakterisiert. Die Ergebnisse bestätigen, dass beide Kinasen vorrangig im Nukleus von P. falciparum-Trophozoiten lokalisiert sind und Phosphorylierungsaktivität gegenüber physiologischen Substraten in vitro aufweisen. Ähnlich wie für PfCLK-1 und PfCLK-2 konnte in Reverse-Genetik-Studien gezeigt werden, dass sowohl PfCLK-3 als auch PfCLK-4 essentiell für den asexuellen Replikationszyklus von P. falciparum sind. Dieser Umstand macht beide Kinasen zu potenziellen Angriffspunkten für antiplasmodiale Bekämpfungsstrategien. Des Weiteren wurde diese Studie ausgeführt, um mögliche Interaktionspartner aller vier PfCLKs zu identifizieren. Vorangegangene Sequenzabgleiche brachten eine bemerkenswerte Homologie der Parasiten-CLKs zur SR-Proteinkinase Sky1p der Bäckerhefe zu Tage. Kinaseaktivitätsassays zeigten Phosphorylierung des Sky1p-Substrates und SR-Proteins Npl3p durch präzipitierte PfCLKs in vitro. Außerdem wurden vier homologe plasmodiale SR-Proteine bzw. mutmaßliche Spleißfaktoren identifiziert, die ebenso von den PfCLKs in vitro phosphoryliert werden: PfASF-1, PFSRSF12, PfSFRS4 und PfSR-1. Alle vier Parasiten-Spleißfaktoren sind vorwiegend in Kernen von Trophozoiten exprimiert. Für PfCLK-1 konnte eine Ko-Lokalisation mit den SR-Proteinen nachgewiesen werden. Abschließend wurden eine Sammlung humaner und mikrobieller CLK-Inhibitoren sowie das Antiseptikum Chlorhexidin (CHX) auf ihren hemmenden Effekt auf verschiedene Lebenszyklusstadien von P. falciparum und gezielt auf die PfCLKs überprüft. Es wurden fünf Inhibitoren aus einer Sammlung von 63 Substanzen auserwählt, die eine moderate Hemmung auf asexuelle Lebenszyklusstadien aufwiesen, mit IC50-Werten zwischen ungefähr 4 und 8 µM. Das Antibiotikum CHX zeigte sogar einen IC50-Wert im niedrigen nanomolaren Bereich. Nachfolgende Stage-of-Inhibition-Assays deckten auf, dass CHX die Entwicklung von Schizonten enorm beeinträchtigt. Wie in Gametozyten-Toxizitätsassays und Exflagellationsassays ermittelt wurde, hemmen alle ausgewählten CLK-Inhibitoren ferner sowohl die Gametozytogenese als auch die Gametogenese. Kinaseaktivitätsassays bestätigen eine spezifische Hemmung der CLK-vermittelten Phosphorylierung aller vier Kinasen, wenn die CLK-Inhibitoren auf immunopräzipitierte PfCLKs angewendet wurden. Diese Erkenntnisse über PfCLK-hemmende Substanzen sind erste Ansätze, um mögliche Wirkstoffe gegen Malaria zu finden, die die PfCLKs als Angriffspunkte haben. Zudem stellen diese Resultate ein wirksames Mittel zur Verfügung, um chemische Kinase-Knockout-Parasiten zu generieren. Diese können dann verwendet werden, um die Rolle der PfCLKs vor allen in Bezug auf Spleißvorgänge und mRNA-Metabolismus phänotypisch zu untersuchen. Der Ansatz, die CLKs des Parasiten funktionell zu charakterisieren, ist von besonderem Interesse, da das Spleißosom des Malariaparasiten immer noch nicht ausreichend erforscht ist. Dadurch können weitere Erkenntnisse über die Spleißmaschinerie des Parasiten gewonnen werden.

<p>Malaria antigen-induced polarization of T cells into effectors Th1 and/or Th2 cells and their subsequent release of cytokines is known to affect antibody production. This thesis includes studies on early innate responses to the parasite, with a focus on γδT cells, and acquired specific responses in African sympatric ethnic tribes. In the last part of this thesis, a method for enrichment for the asexual blood stages of <i>P. falciparum </i>and their use in <i>in vitro</i> T-cell studies is presented.</p><p>To investigate mechanisms involved in parasite growth inhibition by γδT cells, an <i>in vitro</i> system was set up using blood stage parasites co-cultured with differently treated γδT cells. The results showed that Vγ9/δ2⁺ γδT cells inhibited the in vitro growth of <i>P. falciparum</i> parasites whereas CD4⁺and CD8⁺ T cells did not. This inhibition was positively correlated with the expression of cytolytic molecules in the cell lines tested. Anti-granulysin antibodies reversed γδT cell-mediated inhibition, suggesting a role for granulysin in the parasite growth inhibition. Thus, our data suggest that Vγ9/δ2⁺ γδT cells inhibit the parasite growth by a granulysin-exocytosis dependent cytotoxic pathway that needs perforin.</p><p>To study the humoral responses and their relation to Th1/Th2 cytokine profiles, antibody levels, numbers of cytokine-producing cells and spleen rates were measured in two sympatric tribes living in Mali, the Fulani and the Dogon. Our results revealed significantly elevated malaria-specific IgG and IgE antibody levels and spleen rates in the Fulani compared to the Dogon. The Fulani exhibited elevated numbers of both IL-4 and IFN-γ-producing cells, a typical profile seen of CD1-restricted NKT cells. This together with the higher spleen rates and elevated anti-malarial antibodies suggests a role of CD1-restricted cells in the different responses seen between these tribes.</p><p>To investigate whether such responses were specifically confined to malaria or a reflection of a generally activated immune system, total levels of IgG and of IgM as well as IgG antibodies to non-malarial antigens were examined in the Fulani in Burkina Faso and Mali. The results showed that the Fulani consistently mounted stronger malaria-specific IgG, IgG1, IgG3 and IgM responses. Total IgM levels were significantly higher in the Fulani than the non-Fulani, whereas total IgG did not differ between the two tribes. While IgG levels to some non-malarial antigens were significantly higher in the Fulani, no such differences were seen in the responses to several other non-malarial antigens suggesting that the Fulani are not generally hyper-reactive and that other specific factors are of importance for their higher malaria resistance.</p><p>Finally, a new method to enrich for early and late asexual blood stages of <i>P. falciparum</i> parasite from a single parasite culture was developed, using a 3-step centrifugation procedure. Such enriched parasite fractions beside other malaria-parasite antigen preparations were used in an in vitro system to analyse T-cell responses in malaria-exposed and non-exposed donors. Such analysis revealed significant proliferative cell response and CD4⁺ T cell expansion to whole-cell parasite antigens, but not to acellular parasite fractions, in the malaria-exposed as compared to the non-exposed ones. Our data suggest that natural infection preferentially leads to formation of memory cells against certain antigen expressed in live parasites.</p><p>Malaria antigen-induced polarization of T cells into effectors Th1 and/or Th2 cells and their subsequent release of cytokines is known to affect antibody production. This thesis includes studies on early innate responses to the parasite, with a focus on γδT cells, and acquired specific responses in African sympatric ethnic tribes. In the last part of this thesis, a method for enrichment for the asexual blood stages of P. falciparum and their use in in vitro T-cell studies is presented.</p><p>To investigate mechanisms involved in parasite growth inhibition by γδT cells, an in vitro system was set up using blood stage parasites co-cultured with differently treated γδT cells. The results showed that Vγ9/δ2⁺ γδT cells inhibited the in vitro growth of P. falciparum parasites whereas CD4⁺and CD8⁺T cells did not. This inhibition was positively correlated with the expression of cytolytic molecules in the cell lines tested. Anti-granulysin antibodies reversed γδT cell-mediated inhibition, suggesting a role for granulysin in the parasite growth inhibition. Thus, our data suggest that Vγ9/δ2⁺ γδT cells inhibit the parasite growth by a granulysin-exocytosis dependent cytotoxic pathway that needs perforin.</p><p>To study the humoral responses and their relation to Th1/Th2 cytokine profiles, antibody levels, numbers of cytokine-producing cells and spleen rates were measured in two sympatric tribes living in Mali, the Fulani and the Dogon. Our results revealed significantly elevated malaria-specific IgG and IgE antibody levels and spleen rates in the Fulani compared to the Dogon. The Fulani exhibited elevated numbers of both IL-4 and IFN-γ-producing cells, a typical profile seen of CD1-restricted NKT cells. This together with the higher spleen rates and elevated anti-malarial antibodies suggests a role of CD1-restricted cells in the different responses seen between these tribes.</p><p>To investigate whether such responses were specifically confined to malaria or a reflection of a generally activated immune system, total levels of IgG and of IgM as well as IgG antibodies to non-malarial antigens were examined in the Fulani in Burkina Faso and Mali. The results showed that the Fulani consistently mounted stronger malaria-specific IgG, IgG1, IgG3 and IgM responses. Total IgM levels were significantly higher in the Fulani than the non-Fulani, whereas total IgG did not differ between the two tribes. While IgG levels to some non-malarial antigens were significantly higher in the Fulani, no such differences were seen in the responses to several other non-malarial antigens suggesting that the Fulani are not generally hyper-reactive and that other specific factors are of importance for their higher malaria resistance.</p><p>Finally, a new method to enrich for early and late asexual blood stages of <i>P. falciparum</i> parasite from a single parasite culture was developed, using a 3-step centrifugation procedure. Such enriched parasite fractions beside other malaria-parasite antigen preparations were used in an in vitro system to analyse T-cell responses in malaria-exposed and non-exposed donors. Such analysis revealed significant proliferative cell response and CD4⁺ T cell expansion to whole-cell parasite antigens, but not to acellular parasite fractions, in the malaria-exposed as compared to the non-exposed ones. Our data suggest that natural infection preferentially leads to formation of memory cells against certain antigen expressed in live parasites.</p><p>Malaria antigen-induced polarization of T cells into effectors Th1 and/or Th2 cells and their subsequent release of cytokines is known to affect antibody production. This thesis includes studies on early innate responses to the parasite, with a focus on γδT cells, and acquired specific responses in African sympatric ethnic tribes. In the last part of this thesis, a method for enrichment for the asexual blood stages of <i>P. falciparum</i> and their use in <i>in vitro</i> T-cell studies is presented.</p><p>To investigate mechanisms involved in parasite growth inhibition by γδT cells, an <i>in vitro</i> system was set up using blood stage parasites co-cultured with differently treated γδT cells. The results showed that Vγ9/δ2⁺ γδT cells inhibited the in vitro growth of<i> P. falciparum</i> parasites whereas CD4⁺ and CD8⁺ T cells did not. This inhibition was positively correlated with the expression of cytolytic molecules in the cell lines tested. Anti-granulysin antibodies reversed γδT cell-mediated inhibition, suggesting a role for granulysin in the parasite growth inhibition. Thus, our data suggest that Vγ9/δ2⁺ γδT cells inhibit the parasite growth by a granulysin-exocytosis dependent cytotoxic pathway that needs perforin.</p><p>To study the humoral responses and their relation to Th1/Th2 cytokine profiles, antibody levels, numbers of cytokine-producing cells and spleen rates were measured in two sympatric tribes living in Mali, the Fulani and the Dogon. Our results revealed significantly elevated malaria-specific IgG and IgE antibody levels and spleen rates in the Fulani compared to the Dogon. The Fulani exhibited elevated numbers of both IL-4 and IFN-γ-producing cells, a typical profile seen of CD1-restricted NKT cells. This together with the higher spleen rates and elevated anti-malarial antibodies suggests a role of CD1-restricted cells in the different responses seen between these tribes.</p><p>To investigate whether such responses were specifically confined to malaria or a reflection of a generally activated immune system, total levels of IgG and of IgM as well as IgG antibodies to non-malarial antigens were examined in the Fulani in Burkina Faso and Mali. The results showed that the Fulani consistently mounted stronger malaria-specific IgG, IgG1, IgG3 and IgM responses. Total IgM levels were significantly higher in the Fulani than the non-Fulani, whereas total IgG did not differ between the two tribes. While IgG levels to some non-malarial antigens were significantly higher in the Fulani, no such differences were seen in the responses to several other non-malarial antigens suggesting that the Fulani are not generally hyper-reactive and that other specific factors are of importance for their higher malaria resistance.</p><p>Finally, a new method to enrich for early and late asexual blood stages of <i>P. falciparum</i> parasite from a single parasite culture was developed, using a 3-step centrifugation procedure. Such enriched parasite fractions beside other malaria-parasite antigen preparations were used in an <i>in vitro</i> system to analyse T-cell responses in malaria-exposed and non-exposed donors. Such analysis revealed significant proliferative cell response and CD4⁺ T cell expansion to whole-cell parasite antigens, but not to acellular parasite fractions, in the malaria-exposed as compared to the non-exposed ones. Our data suggest that natural infection preferentially leads to formation of memory cells against certain antigen expressed in live parasites.</p>

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Undergraduate honors paper--Mount Holyoke College, 2006. Dept. of Biological Sciences. / Includes bibliographical references (leaves 80-84).