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Pathogenic basis and clinical features of malaria.
The pathogenic process of malaria occurs during the asexual erythrocytic cycle where clinical symptoms including nausea, headache and chills, accompany the characteristic bouts of fever. In the untreated patient, severe complications of P. jalciparum malaria infections manifest as cerebral malaria (with conwlsions and coma), anaemia, hypoglycaemia, renal failure, metabolic acidosis, severe liver failure, respiratory distress, circulatory collapse, raised intracranial pressure and non-cardiac pulmonary oedema often resulting in death (Marsh, 1999; Mendis and Carter, 1995; Ramasamy, 1998; White, 1998).
The abovementioned morbid conditions associated with the disease are ascribed to various host-parasite interactions. Several substances are released by the intraerythrocytic parasite including malarial mitogens, toxic proteins, prostaglandins <D2, ~ and F2a) and polar lipids, particularly glycophosphatidyl inositol (GPI; covalently bound to the merozoite surface antigens, MSAI and MSA2) that has been speculatively named malaria toxins. The proposed main consequence of these bio-active molecules is to direct the systemic release of several pro-inflammatory cytokines, in particular tumour necrosis factor <X (TNFa), interferon y (IFN y), interleukin 1 (ILl), IL6 and ll..10 (Clarke and Schofield, 2000; Hommel, 1997; Miller, et al., 2002; Miller, et al., 1994; Ramasamy, 1998; White, 1998). A major role for the pro-inflammatory cytokines is to generate the inducible form of nitric oxide synthase (iNOS) to produce a continuous release of the nitric oxide (NO) mediator (Clarke and Schofield, 2000). This could be clinically important in some reversible cerebral symptoms, immunosuppression and weight loss seen in malaria (Clarke and Schofield, 2000; Miller, et al., 2002).
One of the most distinctive pathophysiological characteristics, which evolved. for the survival of P.jalciparum, is to effectively modify the ultrastructure of the erythrocyte in which it resides. Several parasite-specific proteins are produced on the surface to allow the unique, parasite-induced ability of infected erythrocytes to adhere to post-capillary microvascular endothelial cells (a process termed sequestration), and the in vitro adherence to uninfected erythrocytes (rosetting), other infected erythrocytes (autoagglutination or clumping) and to platelets, monocytes and lymphocytes (Berendt, et al., 1994; Miller, et al., 2002; White, 1998). Collectively, cytoadherence enables the parasite to avoid destruction by the reticulo-endothelial system in the spleen and has as consequence a decreased peripheral parasitaemia with only ring stage parasites visible in peripheral blood. Sequestration occurs non-homogeneously in various organs including heart, lung, brain, liver, kidney, subcutaneous tissues and placenta resulting in considerable obstruction to tissue perfusion with systemic or local production of cytokines as mentioned above (Miller, et al., 2002).
CHAPTER 1: Literature Overview
1.1Malaria:The disease
1.2The etiologicagents of malaria
1.2.1Life cycle of the humanmalaria parasites
1.2.2Ultrastructureof the erytrocyticstages of P.jalciparum
1.3Pathogenicbasis and clinical features of malaria
1.4Globalcontrol strategiesof malaria
1.4.1Chemotherapyand -prophylaxis
1.4.2Strategiesfor vector control
1.4.3Malariavaccines
1.5Biochemistryand metabolicpathwaysofPlasmodium
1.6Polyaminemetabolism
1.6.1Polyaminemetabolismin the parasiticprotozoa
1.6.2Polyaminemetabolismas an antiprotozoaltarget
1.7 Researchobjectives
CHAPTER 2: Molecular genetic analyses of P. jakiparum 8-adenosylmethionine decarboxylase (Adomettlc), omithine decarboxylase (Ode) and the bifunctional AdometdclOde genes
2.1 Introduction
2.1.1 Geneticanalysesof Plasmodia
2.1.2 Molecularcharacteristicsof the Adometdc and Ode genes
2.1.3 The molecularcharacterisationof genes and their mRNAs
PART I: Identitlcation of Adomettlc and Ode cDNAswith RACE
2.2 Materialsand methods
2.2.1 In vitro cultivationof malaria parasites
2.2.2 Nucleicacid isolationfromP.jalciparum cultures
2.2.3 Nucleicacid quantificatio
2.2.4 Primerdesign
2.2.5 3′-RACEof Ode andAdometdc cDN
2.2.6 5′-RACEof P.jalciparum Ode cDNA
2.2.7 Agarosegel electrophoresisofPCR products
2.2.8 Purificationof agarose-electrophoresedDNAfragments
2.2.9 Cloningprotocols
2.2.10 Aff cloningstrategies
2.2.11 Automatednucleotidesequencing
2.2.12Northernblot analysesof P.jalciparum totalRNAwith Ode-specificprobe
2.3 Results
2.3.1 Primer design
2.3.2 3′-RACE of the P. jalciparum Ode and Adometdc cDNA from the uncloned cDNA library
2.3.3 5′-RACE of Ode cDNA
2.3.4 Northern blot analyses of P. jalciparum total RNA with Ode-specific probe
PART ll: Molecular genetics of the full-length p/AdometdclOtlc
2.4 Materials and methods
2.4.1 Long-distance PCR of the full-length bifunctional PjAdometdc/Ode
2.4.2/n silico nucleotide sequence analyses of the PjAdometdc/Ode gene
2.5 Results
2.5.1 Amplification of the full-length cDNA of the bifunctional PjAdometdc/Ode
2.5.2 Analyses of the nucleotide sequence of the full-length PjAdometdc/Ode gene
2.5 Discussion
2.5.1 Design ofAdometdc and Ode-specific degenerate primers for 3′-RACE
2.5.2 Identification of the Ode and Adometdc cDNAs with 3′-RACE
2.5.3 Analyses of the mRNA transcript of Ode
2.5.4 5′-RACE ofAdometdc and Ode
2.5.5 Amplification of the full-IengthPjAdometdclOde
2.5.6 Genomic structure of PjAdometdc/Ode gene and structure of the single transcript
CHAPTER 3: Recombinant· expression and characterisation of monofunctional AdoMetDC and ODC as well as bifunctional PfAdoMetDC/ODC of P.jalciparum
3.1 Introduction’
3.1.1 Ornithine decarboxylase
3.1.2 S-Adenosylmethionine decarboxylase
3.1.3 AdoMetDC and ODC inP.jalciparum
3.1.4 Recombinant protein expression and analyses
3.2 Materials and methods
3.2.1 Recombinant expression of His-Tag fusion proteins
3.2.2 Recombinant expression ofStrep-Tag fusion proteins
3.2.3 Size-exclusion HPLC of the monofunctional OOC
3.2.4 Size-exclusion FLPC of monofunctional AdoMetDC and bifunctional PfAdoMetDClODC
3.2.5 Quantitation of proteins
3.2.6 SDS-PAGE of proteins
3.2.7 AdoMetDC and OOC enzyme activity assays
3.2.8/n silico analyses of the predicted amino acid sequence ofPfAdoMetDC/ODC
3.3 Results
3.3.1 Directional cloning strategy of individual OOC and AdoMetDC domains
3.3.2 Expression strategy of monofunctional AdoMetDC and OOC as well as bifunctional PfAdoMetDClO
3.3.3 Recombinant expression of monofunctional AdoMetDC and OOC domains
3.3.4 Determination of the oligomeric state of the monofunctional AdoMetDC and ODC
3.3.5 Expression and purification of the bifunctional PfAdoMetDClOOC
3.3.6 Decarboxylase activities of the monofunctional and bifunctional proteins
3.3.7 Analyses of the deduced amino acid sequence of the bifunctional PfAdoMetDC/OC
3.4 Discussion
3.4.1 Heterologous expression of the decarboxylase proteins
3.4.2 Multimeric states of the monofunctional and bifunctional proteins
3.4.3 Decarboxylase activities of the monofunctional and bifunctional proteins
3.4.4 Sequence analyses of the deduced amino acid sequence ofPfAdoMetDC/ODC
CHAPTER 4: FUDdioDai aDd structunl roles of pansite-specitic iDserts iD the birUDdioDai PfAdoMetDCIODC
4.1 Introduction
4.2 Materials and methods
4.2.1 Amino acid sequence and structural analyses
4.2.2 Deletion mutagenesis
4.2.3 Nucleotide sequencing of the various mutants
4.2.4 Recombinant expression and purification of wild-type and mutant prteins
4.2.5 Protein-protein interaction determinations
4.2.6 Enzyme assays
4.3 Results
4.3.1 Explanations for the bifunctional nature ofPfAdoMetDC/ODC
4.3.2 Parasite-specific regions in PfAdoMetDC/ODC
4.3.3 Sequence and structure analyses of the parasite-specific regions
4.3.4 Deletion mutagenesis of parasite-specific regions in PfAdoMetDC/ODC
4.3.5 Effect of deletion mutagenesis on the decarboxylase activities
4.3.6 Deletion mutagenesis in the monofunctional AdoMetDC and ODC
4.3.7 Oligomeric state of deletion mutant forms ofPfAdoMetDC/ODC
4.3.8 Complex forming ability of deletion mutants of monofunctional proteins
4.4 Discussion
4.4.1 Explanations for the bifunctional nature ofPfAdoMetDC/ODC
4.4.2 Defining the parasite-specific inserts in PfAdoMetDC/ODC
4.4.3 Structural properties of the parasite-specific inserts
4.4.4 Involvement of the parasite-specific inserts in the decarboxylase activities
4.4.5 Characterisation of the physical association between the domains
CHAPTER 5: Compantive properties of a homology model of the ODC compoDeDt of
PfAdoMetDC/ODC
CIIAPTER. 6: Stnacture-based ligand binding and discovery of novel inhibiton against 160 PtODC
