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There is an abundance of information available on the molecular mechanisms of antigenic variation in Plasmodium falciparum. The variant antigen PfEMP1, which mediates antigenic variation as well as cytoadherence and rosetting, has been extensively characterised. Genes coding for the antigen belong to the gene family var, and several var genes have been cloned and characterised. The rodent malaria parasite P. chabaudi is a widely studied in vivo model for P. falciparum. The P. c. chabaudi AS parasite strain has been shown to exhibit antigenic variation and the variant antigen has been detected by surface fluorescence. As with P. falciparum, there is a link between antigenic variation and cytoadherence, however genes coding for the variant antigen in P. chabaudi have not been cloned to date. Therefore, potentially useful in vivo experiments on antigenic variation are restricted. In this thesis it is shown for the first time that the P. c. adami DS parasite strain also exhibits antigenic variation. / Chapter 3 describes efforts to locate genes coding for variant antigens in P. c. adami DS. The main strategy involved a genome survey, by sequencing and analysing randomly selected clones from a P. c. adami DS genomic library. DNA sequences were compared to Plasmodium spp. sequence databases to look for similarity to var genes or other genes encoding variant antigens. Of the 297 clones analysed none had significant sequence similarity to genes coding for variant antigens. However, in a small proportion of sequences some similarity to var genes was noted. Several genes of potential interest were identified, most importantly the gene coding for the vaccine candidate rhoptry associated protein 1 (RAP1), which was subsequently cloned and characterised. Further attempts to locate var gene homologues in P. c. adami involved amplification of P. c. adami genomic DNA using degenerate oligonucleotide primers corresponding to conserved regions of var genes. This strategy proved to be unsuccessful, most likely due to lack of sequence similarity between P. falciparum and P. c. adami genes. In several vaccination studies with the apical membrane antigen 1 (AMA1) of P. c. adami DS, mice were significantly protected against homologous parasite challenge. However, some mice developed late, low-level breakthrough parasitaemias. In Chapter 4, the characterisation of two such breakthrough parasitaemias is described. The ama1 genes of the breakthrough parasites were found to be identical to the ama1 gene of the parental parasites. Similarly, no alteration in AMA1 expression was observed. However, the breakthrough parasites were found to be more resistant than the parental parasites to the effects of passive immunisation with rabbit antisera to AMA1, RAP1 and possibly also MSP119. P. chabaudi infections in mice have been previously shown to consist of a primary parasitaemia followed by a short period of subpatency, and a recrudescent parasitaemia. In surface immunofluorescence studies with P. c. chabaudi, parasites of the recrudescence were shown to be distinct from parasites of the primary parasitaemia, with respect to antigens expressed on the surface of late trophozoite- and schizont-infected erythrocytes. / Chapter 4 describes similar surface immunofluorescence assays carried out with P. c. adami infected erythrocytes, and quantitation of fluorescence by flow cytometry. As with P. c. chabaudi, the recrudescent parasites were found to be antigenically distinct from the primary parasitaemia, indicating that antigenic variation had taken place. Because breakthrough parasites from the AMA1 vaccination trial were similar to recrudescences in peak and duration, we hypothesised that breakthrough parasitaemias, like recrudescent parasitaemias, occur as a result of antigenic variation. In Chapter 4 it was shown by surface immunofluorescence and flow cytometry using hyperimmune sera raised against different parasite populations, that breakthrough parasites express antigens on the surface of late trophozoite- and schizont infected erythrocytes that differ from those expressed by the parental and recrudescent parasites. These results support the hypothesis that switching of the variant antigen on the infected erythrocyte surface enables parasites to evade protective antibody responses directed against merozoite antigens. / Chapter 5 describes the cloning and characterisation of P. c. adami RAP1 which was identified in the process of the genomic survey described in Chapter 3, as well as P. berghei RAP1. Both rodent parasite orthologues of RAP1 were found to have 30% sequence similarity to P. falciparum RAP1, and 6 of 8 cysteines were conserved in the rodent parasite orthologues. However the three polypeptides vary significantly in size. P. c. adami RAP1 and P. berghei RAP1 consist of 691 aa and 604 aa respectively, whereas P. falciparum RAP1 consists of 783 aa residues. These size differences reflect very different N-terminal sequences prior to the first cysteine, whereas the cysteine-rich C-terminal regions are more conserved. Both P. falciparum RAP1 and P. c. adami RAP1 contain N-terminal repeats, however they bear no sequence similarity to each other. P. berghei RAP1 lacks N-terminal sequence repeats that are characteristic of P. falciparum and P. c. adami RAP1. The large cysteine-rich C-terminal region P. c. adami RAP1 (PcRAP1 C3) was expressed in E. coli as a hexa-his fusion protein. Rabbit antiserum to recombinant PcRAP1 C3 was used to characterise the expression and sub-cellular localisation of the RAP1 antigen. P. c. adami RAP1 was found to have a Mr of approximately 80,000 and was shown by immunofluorescence to localise to the merozoite rhoptries. Passive immunisation of mice with rabbit anti-RAP1 serum was shown to protect against fulminant parasitaemia and mortality. In a mouse vaccination trial using the recombinant PcRAP1 C3 polypeptide partial protection was conferred against homologous parasite challenge.

The primary aim of this study was to use a computational structure-based ligand design strategy in finding novel ligands that could act as inhibitors of PfGST as basis for future antimalarial drug development. Since there is only one PfGST isoenzyme present in the parasite and the architecture of the binding site differs significantly from its human counter part, PfGST is considered a highly attractive drug target. Inhibition of PfGST is expected to interfere at more than one metabolic site in synergy: it is likely to disrupt the glutathione-dependent detoxification process, which will lead to an increase in the cytotoxic peroxide concentration and most likely lead to an increase in the levels of ferriprotoporphyrin IX and hemin as well. S-hexyl glutathione was co-crystallized with PfGST (Harwaldt et al., 2004), consequently it was seen as one of the most important lead compounds in the development of PfGST inhibitors. The first step in the rational drug design strategy was to modify GTX, concentrating on its ability to bind competitively to the G site and the hydrocarbon chain protrudes into the H site as well. Considering the 3D structure of the enzyme, modifications to GTX were made by LUDI and NEWLEAD, resulting in a library of active site binding ligands ranked by AutoDock according to their ability to optimally bind to PfGST. Additionally, the ligands were ranked according to their affinity for binding to PfGST produced by AutoDock, LUDI and XScore. Once all the compounds were ranked by these in silico methods they were screened for acquisition or synthetic accessibility and those available were experimentally screened for activity against recombinantly expressed PfGST. Based on in silico predictions NDA was the best inhibitor followed by LAP and EDP. From the biological assay and Lineweaver-Burk analysis the order of inhibition was NDA as the best inhibitor tested, followed by LAP and EDP. EDP and LAP showed competitive inhibition but the inhibition constant values were signi_cantly lower than GTX. With respect to GSH and CDNB, NDA was found to be a non-competitive inhibitor. It was suggested therefore that NDA binds to a non-substrate Summary 93 binding site that may lead to conformational change of the enzyme and hence lead to a loss in enzyme activity. This data leads to the conclusion that the H site should be better exploited in order to find more potent inhibitors or non-substrate binding sites. It was concluded that the experimental results add confidence to the discriminative power of the structure-based ligand design strategy and that these inhibitors could form scaffolds for future antimalarial drug development. / Dissertation (MSc (Bioinformatics))--University of Pretoria, 2008. / Biochemistry / unrestricted

Polyamines are ubiquitous components of all living cells and their depletion usually causes growth arrest or cytostasis, a strategy employed for treatment of West-African trypanosomiasis. In the malaria parasite, Plasmodium falciparum, polyamine biosynthesis is regulated by the uniquely bifunctional protein, Sadenosylmethionine decarboxylase/ornithine decarboxylase (PfAdoMetDC/ODC). The unique nature of this protein could provide a selective mechanism for antimalarial treatment. To validate polyamine depletion and specifically PfAdoMetDC/ODC, as drug target for antimalarial therapeutic intervention, polyamine biosynthesis was completely restrained via the inhibition of both catalytic sites of PfAdoMetDC/ODC with DFMO and MDL73811. The physiological effects during the resulting cytostasis were studied with a comprehensive functional genomics approach. The study was preceded by various assays to determine the treatment dosage that would result in complete cytostasis, without non-specific chemical cytotoxicity. The results obtained revealed that the cytostatic mechanism with growth arrest of the treated parasites and normal progression of the untreated controls require special consideration for basic comparisons of response in terms of the assay methodology used and data analysis. This is particularly important when studying a multistage organism such as P. falciparum, which constantly develops and change during the intraerythrocytic developmental cycle, such that growth arrest compared to normal progression would result in significant differences merely due to stage. This critical principle was kept in mind throughout the investigation and was applied to the relative quantification of RNA, proteins and metabolites via a relative time zero approach as opposed to the standard parallel time point comparison. Three independent functional genomics investigations, namely transcriptomics, proteomics and metabolomics were conducted, in which highly synchronised 3D7 parasite cultures were treated during the schizont stage and parasites were sampled during a time course at three time points (just before and during cytostasis). Transcriptome analysis revealed the occurrence of a generalised transcriptional arrest just prior to the growth arrest. To our knowledge this is the first time that transcriptional arrest as the preceding mechanism of cytostasis due to polyamine depletion, was demonstrated. However, despite the transcriptional arrest, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase (OAT) and the decreased abundance of that for S-adenosylmethionine synthetase (AdoMet synthetase). Pearson correlations indicated more subtle effects of the perturbation on the proteome and even more so on the metabolome where homeostasis was generally maintained, except downstream to the enzymatic blockade at PfAdoMetDC/ODC. The perturbation-specific compensatory roles of OAT in the regulation of ornithine and AdoMet synthetase in the regulation of AdoMet were confirmed on both the protein and metabolite levels, confirming their biological relevance. The results provide evidence that P. falciparum respond to alleviate the detrimental effects of polyamine depletion via the regulation of its transcriptome and subsequently the proteome and metabolome, which supports a role for transcriptional control in the regulation of polyamine and methionine metabolism within the parasite. The study concludes that polyamines are essential molecules for parasite survival and that PfAdoMetDC/ODC is a valid target for antimalarial drug development. / Thesis (PhD)--University of Pretoria, 2008. / Biochemistry / unrestricted

This thesis reports the results of a large number of experiments which were designed to elucidate the mechanisms whereby Gambian children, suffering from acute Plasmodium falciparum malaria may eventually control their infections. These experiments were carried out in vitro and success or failure of the various test systems was judged by their effect on parasite multiplication. Early in the course of these investiqations it was demonstrated that mononuclear cells from these children could cooperate with antibodies present in their serum to bring about a marked reduction in parasite growth. The efficiency of this antibody-dependent cellular cytotoxicity (ADCC) mechanism was related to levels of parasitaemia in the children, being greater in convalescent children than in those with acute malaria. Attempts were now made to identify the effector cells in this ADCC. Purified T and B cells were ineffective and although purified adherent cells (A) had an effect, it was much less than that mediated by the undepleted mononuclear cell population. Adherent cells were, however, fully effective in ADCC if they were exposed to the supernatant from T cells non-specifically activated by PHA. Thus cell cooperation leading to activation appears to play an important role in this system. Finally, experiments were set up to determine whether activated mononuclear cells could exert an inhibitory effect on parasite multiplication which was independent of anti-malarial antibody. It was shown that depression of parasite growth could be achieved by mononuclear cells, either from the children or from Europeans, if these cells were exposed to supernatants of previously stimulated mononuclear cells. These findings can be assembled to provide a tentative model of the development of protective responses in vivo. Perhaps following phagocytosis of parasite antigens and their presentation on the cell surface, T cells become activated: they may cooperate with B cells to produce parasite specific antibodies; they may also activate other mononuclear cells (non T, non B) to become effector cells. These cells, either alone, or perhaps more efficiently in cooperation with antibody, are able to kill parasites by the release of toxic factors, and the infection is brought under control. Finally, large amounts of specific antibodies of appropriate isotypes are synthesized. Acting as opsonins or by activating complement, they may serve to destroy remaining parasites. Their continued presence, by preventing merozoite penetration, may provide at least a temporary defense against reinfection. It is assumed that Gambian adults who have suffered repeated malaria infections and are now immune are defended by their possession of circulating IgG antibodies and B memory cells of all appropriate specificities.

Plasmodium falciparum causes the most severe form of human malaria, and the continual development of resistance of this parasite to current anti-malarial drugs underpins a pressing need for the discovery of novel chemotherapeutic approaches. Polyamines and their biosynthetic enzymes are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite stage, but does not cure infections in vivo. This may be due to the salvage of exogenous polyamines from the host, replenishing the intracellular polyamine pool; however the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study the uptake of the polyamines putrescine and spermidine into P. falciparum-infected erythrocytes (iRBC) well as into P. falciparum parasites functionally isolated from their host cell by saponin-permeabilisation of the erythrocyte membrane was investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected erythrocytes, spermidine entered iRBC in part via the ‘new permeation pathways’ induced by the parasite in the erythrocyte membrane. Both putrescine and spermidine were taken up across the plasma membrane of isolated parasites via a saturable, temperature-dependent process that showed competition between different polyamines as well as the polyamine precursor ornithine and basic amino acids. Inhibition of polyamine biosynthesis led to increased total uptake of both putrescine and spermidine. The influx of putrescine and spermidine into isolated parasites was independent of Na+ but increased with increasing pH and showed a marked dependence on the membrane potential, decreasing with membrane depolarisation and increasing with membrane hyperpolarisation. Both anthracene and polyamine derivatives have been shown to have anti-malarial activity. Anthracene-polyamine conjugates have been developed with the aim of utilising the polyamine uptake mechanisms of cancer cells to deliver the cytotoxic anthracene moieties to these cells. Here, several anthracene-polyamine conjugates showed promising anti-malarial activity. These compounds inhibited parasite proliferation with IC50 values in the nM range, and caused an arrest in the cell cycle, as well as a decrease in the mitochondrial membrane potential. Cytotoxicity could not be reversed by the addition of exogenous polyamines, nor did the conjugates have an effect on intracellular polyamine levels. This doctoral study showed that P. falciparum parasites not only synthesise polyamines, but can also acquire putrescine and spermidine from the extracellular environment and paves the way for interfering with polyamine metabolism as an anti-parasitic strategy. / Thesis (PhD)--University of Pretoria, 2012. / Biochemistry / unrestricted

Malaria, a disease transmitted by female mosquitoes, has plagued the world for many centuries. The disease is associated with high mortality rates, severe poverty, and economic burden. These are factors which hamper effective eradication of the disease. Drug resistant forms of the parasite have caused increasing concerns and questioned the longevity of current effective antimalarials. Efforts are therefore aimed at the identification and exploitation of essential parasite proteins as potential drug targets. The polyamine pathway of Plasmodium falciparum is an exploitable pathway which contains two distinct, chemically validated drug targets; a bifunctional PfAdoMetDC-ODC protein and PfSpdSyn. These enzymes ensure intricate regulation of polyamine production and the pathway contains various distinctive features which could be selectively targetable from the mammalian counterpart pathways. However, inhibition of polyamine production through the use of specific enzyme inhibitors has revealed various compensatory responses that negate the efficacy of these inhibitors. An account is given of the metabolomic fluctuations in the parasite during inhibition of polyamine biosynthesis. From co-inhibited P. falciparum extracts, it could be demonstrated that the characteristic growth-arrest coincided with the depletion in spermidine, the metabolic product of PfSpdSyn. The co-inhibition strategy therefore emphasised the importance of spermidine biosynthesis by PfSpdSyn. Moreover, adenosyl-related metabolite levels were not disrupted during polyamine depletion, supporting the notion that these metabolites are intricately recycled within the parasites. The identified metabolic compensatory mechanisms have further potential for exploitation, and can strategically be combined with polyamine biosynthesis inhibition to ensure parasitic attenuation. In addition, several novel inhibitors were previously computationally identified, based on a dynamic receptor-based pharmacophore model of PfSpdSyn. The in vitro inhibiting activity of these compounds was determined against PfSpdSyn. Results from the in vitro experiments supported the in silico predictions, and emphasized the supportive role of pharmacophore modelling has for the identification of novel inhibitors. The research contributed in understanding parasitic polyamine metabolite regulation, and will aid in the future optimization of therapeutic strategies, aimed at exploitation of the polyamine pathway as a potential antimalarial drug target. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Biochemistry / unrestricted