Malaria remains one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapies. The need for novel therapeutic targets and drugs is therefore of the utmost importance and detailed knowledge of the biochemistry of the parasite is imperative. This study was directed at the biochemical characterisation of the polyamine metabolic pathway of P. falciparum in order to elucidate differences between the parasite and its human host that can be exploited in the design of novel antimalarials. The thesis focussed on the two rate-limiting enzymes in polyamine biosynthesis, S¬adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), which occur as a unique bifunctional complex in P. falciparum. The genomic structure of the bifunctional gene indicated a single, monocistronic transcript with large untranslated regions that were predicted to be involved in unique translational regulatory mechanisms. This gives rise to a bifunctional protein containing both decarboxylase activities on a single polypeptide forming a heterotetrameric complex. Activity of the decarboxylases decreases dramatically if these proteins are expressed in their monofunctional forms as homodimeric ODC and heterotetrameric AdoMetDC. The deduced amino acid sequence indicated that all the essential residues for catalysis are conserved and highlighted the presence of three parasite-specific insertions. The parasite-specific inserts were shown to be essential for the catalytic activity of the respective domains and also to influence the activity of the neighbouring domain, indicating that intramolecular communication exists in the heterotetrameric complex. The most structured and smallest insert was also shown to mediate protein-protein interactions between the two domains and to stabilise the complex. Further structure- functional characterisations of specifically the ODC domain were deduced from a comparative homology model. The model predicted an overall structure corresponding to those of other homologous proteins. The validity of the model is supported by mutagenesis results. However, certain parasite-specific properties were identified in the active site pocket and dimerisation interface. The former was exploited in the rational design of novel putative ODC inhibitors directed only against the P. falciparumprotein by in silico screening of chemical structure libraries. This study therefore describes the identification of certain parasite-specific properties in a unique bifunctional protein involved in regulation of polyamine metabolism of P. falciparum. Such discoveries are invaluable in strategies aimed at elucidating biochemical and metabolic differences between the parasite and its human host that could be exploited in the design of alternative, parasite-specific chemotherapies. Moreover, the thesis also contributed new knowledge on certain less well-understood biological phenomena characteristic of P. falciparum, the nature and origin of bifunctional proteins and the functional properties of parasite-specific inserts found in some proteins of the parasite. / Thesis (PhD (Biochemistry))--University of Pretoria, 2002. / Biochemistry / unrestricted

Trotz intensiver Forschung und des Global-Eradication-of-Malaria-Programms der WHO in den 1950er Jahren zählt Malaria neben AIDS und Tuberkulose auch heute immer noch zu den bedeutendsten Infektionskrankheiten weltweit. Aufgrund rasch zunehmender Resistenzentwicklung der Erreger gegen gängige Prophylaxe- sowie Therapiepräparate und dem Fehlen eines Impfstoffs sterben jährlich bis zu 3 Mio. Menschen an Malaria. Seit vor etwa 2 Jahrzehnten das wissenschaftliche Interesse an transmissionsblockierenden Vakzinen gegen den Malariaerreger erwachte, rückten sexualstadienspezifische Oberflächenproteine in den Fokus der Impfstoffforschung. Im Zuge der vollständigen Sequenzierung des P.-falciparum-Genoms wurde bei dem Screenen nach Genen, die multiple tier- oder bakterienähnliche, adhäsive, extrazelluläre Domänen kodieren, die PfCCp-Familie identifiziert. Ihre 6 Mitglieder besitzen eine bemerkenswerte Vielfalt an hoch konservierten, adhäsiven Modulen, die eine Beteiligung an Protein-Protein-, -Polysaccharid- oder -Lipid-Interaktionen vermuten lassen. Die Multiadhäsionsdomänenproteine wurden aufgrund des gemeinsamen LCCL-Moduls PfCCp1 bis PfCCp5 benannt. Dem sechsten Mitglied, PfFNPA, fehlt zwar die namensgebende Domäne, doch die ausgeprägte Ähnlichkeit zu PfCCp5 führte zur Integration des Proteins in die PfCCp-Familie. Die Charakterisierung der ersten 3 Mitglieder zeigte, dass PfCCp1, PfCCp2 und PfCCp3 sexualstadienspezifisch exprimiert werden und in der parasitophoren Vakuole reifer Gametozyten lokalisiert sind. Immunfluoreszenzstudien ließen außerdem erkennen, dass die Proteine während der Gametenbildung partiell freigesetzt werden und in einer matrix-ähnlichen Struktur Exflagellationszentren umgeben. In KO-Studien erwiesen sich PfCCp2 und PfCCp3 zusätzlich als essentielle Faktoren für die Migration reifer Sporozoiten aus den Mitteldarmoozysten in die Speicheldrüsen der Mücken. Damit erfüllen sie die 2 grundlegenden Kriterien für Komponenten transmissionsblockierender Vakzine: eine sexual-stadienspezifische Expression und essentielle Funktion während der Parasitenentwicklung in der Mücke. Aufgrund dieser viel versprechenden Daten wurde im Rahmen der vorliegenden Arbeit die Analyse der PfCCp-Familie durch funktionale Charakterisierung von PfCCp4 sowie Interaktionsstudien an den PfCCp-Proteinen fortgesetzt. Die Expressionsanalysen mittels RT-PCR, Western Blot und Immunfluoreszenzstudien ergaben für PfCCp4 ebenfalls eine sexualstadienspezifische, Plasmamembran-assoziierte Expression innerhalb der parasitophoren Vakuole reifer Gametozyten. Die Expression beginnt bereits in Gametozyten des Stadium I und erfolgt hauptsächlich in Makrogametozyten. Im Gegensatz zu PfCCp1, PfCCp2 und PfCCp3 wird PfCCp4 jedoch homogen verteilt exprimiert. Während der Gametogenese wird PfCCp4 nicht freigesetzt, sondern verbleibt an der Oberfläche von Makrogameten. Es ist zudem das einzige Mitglied der PfCCp-Familie, dessen Expression im Zuge der Ookinetenreifung wieder aufgenommen wird. KO-Studien durch Membranfütterungen von Anopheles-Mücken lassen allerdings darauf schließen, dass PfCCp4 keine essentielle Funktion bei der Parasitenentwicklung ausübt. Der Verlust von PfCCp4 beeinträchtigte weder die Fertilisation noch die Bildung, Reifung oder Migration von Ookineten, Oozysten oder Sporozoiten. PfCCp4 kann somit nicht als Kandidat transmissionsblockierender Vakzine betrachtet werden, obwohl es mit den viel versprechenden Kandidaten Pfs230 und Pfs48/45 interagiert, wie funktionelle Analysen nativer PfCCp-Proteine mittels Ko-Immunpräzipitation ergaben. Weitere Ko-Immunpräzipitationsstudien identifizierten Interaktionen innerhalb der PfCCp-Familie, wie bereits die Ko-Lokalisation und ko-abhängige Expression von PfCCp1, PfCCp2 und PfCCp3, ihre Freisetzung bei der Gametogenese und die matrixähnliche Verteilung um Exflagellationszentren vermuten ließen. In Affinitätschromatographiestudien unter Verwendung rekombinanter PfCCp-Proteine konnte gezeigt werden, dass es sich dabei um direkte Interaktionen handelt, an denen besonders die LCCL- und die SR-Domäne beteiligt zu sein scheinen. In Zelladhäsionsstudien konnte außerdem eine Bindungsaffinität ausgewählter rekombinanter PfCCp-Proteine an Makrogameten beobachtet werden. Insgesamt bestätigen diese Daten unsere Hypothese, dass PfCCp-Proteine unter Beteiligung weiterer sexualstadienspezifischer Proteine während der Gametozytenreifung und Gametogenese Proteinkomplexe ausbilden. In zukünftigen Studien gilt es einerseits, ausgewählte PfCCp-Proteine durch transmissionsblockierende Experimente in ihrem Potential als Impfstoffkomponenten zu evaluieren. Andererseits nimmt die funktionale Charakterisierung der Proteinkomplexe während der Gamogonie eine zentrale Rolle ein, um ihre Funktion in der Sexualphase von P. falciparum zu klären und die Beteiligung der PfCCp-Proteine an der Regulation dieses komplexen Lebenszyklus zu verstehen. / Despite intense research and the global eradication of malaria program of the WHO in the 1950’s, malaria is among AIDS and tuberculosis still one of the major infectious diseases worldwide. Rapidly increasing resistance of the pathogen against common treatment, and the persistent lack of a vaccine against malaria lead to a death toll of up to three million people annually. Since the scientific interest in transmission blocking strategies against P. falciparum awoke two decades ago, sexual stage-specific surface proteins became an important focus of antimalarial vaccine research. Especially, proteins containing multiple adhesion domains are regarded as promising candidates for subunits of transmission blocking vaccines due to their possible involvement in parasite-parasite or parasite-host interactions. Following the completion of the P. falciparum genome sequence and its screening for multiple animal- or bacterial-like, extracellular adhesion domains, the PfCCp protein family had been identified. It consists of six members with a striking variety of highly conserved adhesive modules, which were predicted to be involved in protein-protein, protein-polysaccharide or protein-lipid binding. These multidomain adhesion proteins were named PfCCp1 through PfCCp5 due to their common LCCL-domain. Though PfFNPA, the sixth member, lacks this domain, its strong similarity to the PfCCp5 architecture warranted its integration into the PfCCp family. The characterization of the first three family members showed, that PfCCp1, PfCCp2 and PfCCp3 are specifically expressed within the parasitophorous vacuole of mature gameto-cytes. Studies via immunofluorescence assays revealed, that they are partly released during emergence and surround exflagellation centers extracellularly in a matrix-like pattern. Moreover, PfCCp2 and PfCCp3 were shown in knockout experiments to be essential for the transition of sporozoites from the midgut oocysts to the salivary glands of the mosquito. Thus, they fulfil two basic criteria for prospective components of transmission blocking vaccines: sexual stage-specific expression and an essential role for the parasite development within the mosquito. Based upon this promising data the present thesis dealt with further analyses of the PfCCp family via functional characterization of PfCCp4 and interaction studies of the PfCCp proteins. The expression analysis of PfCCp4 using RT-PCR, Western Blot and immunofluorescence assays showed that it is also expressed in association with the plasma membrane within the parasitophorous vacuole of mature gametocytes. Expression starts as early as stage I of gametocytogenesis and is mainly restricted to macrogametocytes. In contrast to PfCCp1, PfCCp2 and PfCCp3, PfCCp4 is not expressed in a punctuated pattern but is distributed homogenously, and instead of being released during emergence it remains on the surface of macrogametes. In addition, PfCCp4 is the only PfCCp member that resumes expression during ookinete maturation. Knockout experiments revealed in membrane feedings of Anopheles mosquitoes that PfCCp4 plays no essential role in the parasite development. The lack of PfCCp4 neither affected fertilization nor formation, maturation or migration of ookinetes, oocysts or sporozoites. Thus, PfCCp4 does not display any potential as a candidate for transmission blocking vaccines, though it interacts with the promising candidates Pfs230 and Pfs48/45, as functional characterizations of native PfCCp proteins showed via co-immunoprecipitation assays. Further co-immunoprecipitation experiments revealed protein-protein interactions within the PfCCp family, as the co-localization data had suggested, supported by the co-dependent expression of PfCCp1, PfCCp2 and PfCCp3, their release during emergence and their matrix-like surrounding of exflagellation centers. Affinitychromatography studies on recombi-nant PfCCp proteins demonstrated, that these interactions are direct interactions, which appear to be mediated predominantly by the LCCL- and SR-domains. Cell adhesion assays revealed in addition a prominent binding affinity of select recombinant PfCCp proteins to macrogametes. Taken together, this data support our hypothesis of protein complex formation mediated by the members of the PfCCp family and other sexual stage-specific proteins during the sexual development of P. falciparum. Future studies aim on one hand at the evaluation of the potential of select PfCCp proteins as vaccine subunits by transmission blocking assays. On the other hand, another focus will be the characterization of the proposed protein complexes to gain deeper insight into their function during sexual development, and to understand the role of the PfCCp proteins in the regulation of the intricate life cycle of the human malaria pathogen P. falciparum.

Malaria is caused by Plasmodium parasites and is the world’s most devastating tropical infectious disease. The need for identifying novel drug targets is fuelled by an increased resistance of these parasites against available drugs. The human host red cell membrane plays an important role during invasion and subsequent development of the parasite within the red cell and undergoes several structural, functional and biochemical changes triggered by various protein-protein interactions between the parasite and the host cells. These interactions form a fundamental part of malaria research, since the parasite spends the pathogenic stage of its life cycle in the human erythrocyte. The Plasmodium kinome is complex and the exact role of protein phosphorylation in malaria parasites is not yet fully understood. This study aims to characterise the kinase domain of Plasmodium falciparum (3D7) Protein Kinase 8 (PfPK8), described as a putative protein on the Plasmodium falciparum database. PfPK8 is encoded by the PfB0150c gene (recently renamed as PF3D7_0203100) situated on chromosome 2 of the parasite genome. A 1 507bp section of the PfB0150c gene, containing a 822bp centrally located kinase domain was cloned into a pTriEx-3 expression vector. A soluble recombinant octa-histidine-tagged PfPK8 was expressed in Escherichia coli Rosetta 2 (DE3) cells, but with relatively low yield and purity.To improve the expression, a recombinant PfB0150c-baculovirus infected Spodoptera frugiperda (Sf9) insect cell system was attempted, but without success. A different tag was employed and glutathione-S-transferase-PfPK8 was successfully expressed in Escherichia coli Rosetta 2 (DE3) cells, with a higher yield and purity. Recombinant GST-PfPK8 was used in non-radioactive coupled spectrophotometric kinase assays in the presence of known kinase substrates casein, MBP and H1 to determine kinetic parameters of the enzyme. It phosphorylated all three substrates at a temperature of 37ºC and pH of 7.4. Recombinant GST-PfPK8 was inactive at a pH below 6 and most active at pH 7.4. The relative activity of the enzyme was highest at a temperature synonymous to a fever spike in a Plasmodium falciparum infected individual. Secondary structural analysis of PfPK8 revealed the position of a conserved substrate binding domain containing an ATP-binding site and binding loop within the kinase domain. The kinase domain of rPfPK8 was modelled using available crystal structures of its identified homologues. The gene is expressed throughout the intraerythrocytic stages of the parasite life cycle, as well as in gametocytes. Protein-protein binding studies revealed that host-parasite protein-protein interactions exist between rPfPK8 and erythrocyte membrane protein, band 3. Plasmodium falciparum PK8 could therefore play a role during invasion of host erythrocytes and during the intraerythrocytic development of the parasite, by phosphorylating red blood cell membrane proteins. This study provides the groundwork for future X-ray crystallographic studies to elucidate the structure of the enzyme, and for additional gene manipulation experiments to ascertain whether it is essential for parasite survival in all the intraerythrocytic stages and therefore a potential new drug target candidate.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg,in fulfillment of the requirements for the degree of Master of Science in Medicine. Johannesburg, South Africa, 2015 / Plasmodium falciparum remains the most virulent cause of malaria. With increasing drug resistance to artemisinin and other antimalarial drugs, combined with an absence of an effective vaccine, there’s a critical need for new agents to complement existing treatment and prophylaxis. Therefore, the aim of the study was to evaluate the in vitro antimalarial activity and potential toxicity to mammalian cells of select synthetic and natural colourants, nucleoside and imidazo[1,2a]pyridine (IP) analogues on the erythrocytic stages of the 3D7 chloroquine-sensitive strain of P. falciparum. The P. falciparum 3D7 strain was maintained in vitro according to standard methods. Quinine and chloroquine were used as positive controls. The tritiated hypoxanthine incorporation assay was used for evaluating the ability of test compounds to inhibit the growth of P. falciparum. Active test compounds were tested in combination studies with quinine. Uninfected human red blood cell (RBC) toxicity was analysed spectrophotometrically. The ability of test compounds to inhibit -haematin formation, a metabolic pathway that sequesters toxic haem within the parasites, was determined. Cytotoxic activity of active compounds was evaluated on two human cell lines (HEK293 and K562) using the [3H]-thymidine incorporation assay. Data was analysed using the one-way ANOVA test and reported as the mean ± standard deviation of at least triplicate experiments and significant difference when p < 0.05. Of the 56 compounds tested, the synthetic colourants showed the most potent antimalarial activity. Methylene blue and safranin O were most potent with IC50 values of 4.19 ± 0.16 nM and 86.50 ± 2.61 nM, respectively, compared to quinine (IC50: 103.90 ± 8.30 nM), and displayed negligible toxicity to uninfected human RBCs. Combination studies with methylene blue and quinine demonstrated a synergistic interaction. Methylene blue also demonstrated the highest selectivity indices (480 and 968) compared to quine (180). Curcumin (diferuloylmethane), a natural extract was active (IC50: 2.29 ± 0.18 μg/ml) against P. falciparum, but significantly (p < 0.05) less potent than quinine. Curcumin was 78-fold more active in inhibiting -haematin formation than quinine, indicating of a possible mechanism of action. The most active nucleoside analogue, JLP118.1 (IC50: 1.79 ± 0.12 μM), demonstrated inhibitory activity against the trophozoite stage of P. falciparum. The imidazopyridine analogue, IP-4, displayed the least potent antimalarial activity (IC50: 15.3 ± 0.41 μM) of the synthetic compounds tested, with low selectivity indices < 1. The study has confirmed the potent antimalarial activity and relative safety of methylene blue as well as its potential as an antimalarial drug. The nucleoside and imidazopyridine analogues showed promising activity and with structural modification their potency and selectivity indices may be enhanced.

Of the four Plasmodium species which cause malaria in humans, P. falciparum is responsible for the majority of the morbidity and mortality associated with this disease. The surface expression of parasite-derived proteins in the middle of the asexual cycle coincides with two important modifications of the host erythrocyte. First, a protective immune response is directed against a family of variant antigens, known as P. falciparum Erythrocyte Membrane Protein-1 (PfEMPl). Second, ligands are detected at the surface which mediate the specific cytoadherence of infected erythrocytes to vascular endothelium, such that infected cells are sequestered away from the peripheral circulation in deep vascular beds. The potentially fatal syndrome known as cerebral malaria can ensue when infected cells sequester at high density in the brain. Indirect studies have shown that the antigenic and adhesive phenotypes at the surface are linked to the expression of PfEMPl. However, there is a paucity of biochemical data which relate to PfEMPl, and this problem is addressed in this thesis. This study has confirmed, at the biochemical level, inferences from serology that clonal antigenic variation occurred rapidly. Variation produced a number of novel antigenic and adhesive phenotypes which were associated with unique forms of PfEMPl. Further insights into the mechanism of sequestration were possible because of the finding that single infected erythrocytes had the capacity to bind to at least three putative endothelial cell receptors; CD36, Intercellular Adhesion Molecule-1 (ICAM1), and Thrombospondin (TSP). It was demonstrated for the first time that PfEMPl was responsible for cytoadherence to CD36 and ICAM1, but was probably not involved in adhesion to TSP. Extensive analysis with sequence-specific proteases proved that adhesive interactions with each receptor were separable properties of the surface, and facilitated the proposal of a domain model for PfEMPl. Detailed analysis of the antigenic and adhesive phenotypes of a series of clonally-derived parasites demonstrated that infected cells expressing all variant antigenic types could adhere to CD36 whereas adhesion to ICAM1 was seen in a restricted subset. This may be clinically relevant if, as current data suggests, adhesion of infected cells to ICAM1 is important in the development of cerebral malaria. Identification of all ICAM1 binding phenotypes could lead to the design of novel therapeutic strategies for this life-threatening condition.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2004. / Includes bibliographical references (leaves 95-103). / Also available by subscription via World Wide Web / xii, 103 leaves, bound ill. (some col.) 29 cm

Hamburg, Universiẗat, Diss., 2007. / Enth. ausserdem 3 Sonderabdr. aus verschiedenen Zeitschr. - Beitr. teilw. dt., teilw. engl.

Tübingen, Univ., Diss., 2007.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2004. / Includes bibliographical references (leaves 95-103).

Thesis (M.Sc.)(Bioinformatics))-University of Pretoria, 2007. / Summary in English. Includes bibliographical references. Available on the Internet via the World Wide Web.