Listeria monocytogenes (L. monocytogenes) are Gram-positive, facultatively anaerobic and intracellular bacilli, occupying a wide range of ecological niches and are responsible for a number of serious infections in man. Primarily transmitted to humans through contaminated food stocks, L. monocytogenes invade mammalian cells in a phagosome, escaping and growing in the cell cytoplasm. Currently, there is a great deal of information about pathogenesis of L. monocytogenes, however, much less is known about the physiology of the bacteria. In particular, very little is known about the physiology during intracellular growth and even less about host cell physiology and changes in response to infection. The focus of this research was to address these issues using a multidisciplinary approach, utilising multiple biological techniques. The catabolic metabolism of L.monocytogenes was elucidated using mutagenesis and protein purification studies. The results are not completely conclusive; however, it was shown that unlike in Escherichia coli, L.moncytogenes may not be dependent on fermentation enzymes Ldh and Pflb during anaerobic growth. Instead anaerobic respiration is hypothesised, utilising a putative fumarate reductase with fumarate as a terminal electron acceptor. The putative fumarate reductase gene was purified and confirmed to have enzymatic activity.External and internal metabolism of HeLa cells, and the effect of L.monocytogenes infection was elucidated by mass spectrometry. The external metabolomic studies proved inconclusive. The internal metabolomic studies show that a number of key amino acids are being sequestered by L.monocytogenes during the course of an infection. Also, the studies show that a large number of carbon compounds are being sequestered by L.monocytogenes, pointing to a complex carbon metabolism for L.monocytogenes during intracellular growth. A targeted analysis of the nitrogen metabolism of L.monocytogenes has shown that L.monocytogenes may utilise a number of nitrogen compounds with glutamine and glutamate being particularly important. The ability to synthesise glutamine de novo is shown to be essential for normal intracellular growth.

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

Magister Scientiae (Biodiversity and Conservation Biology) / Parasites pose a major constraint on livestock farming. Its impacts are represented as transmission of disease to humans and economic costs for prevention and treatment as well as loss in terms of; livestock mortality and, lowered productivity. Although the impacts of parasitic loads can be decreased through proper management, extensive management systems are depended on external factors such as rainfall and forage availability, which, in its absence, promote malnutrition and therefore parasite susceptibility.

Blood flukes of the genus Schistosoma are parasitic trematodes that cause schistosomiasis, a serious disease afflicting more than 240 million people. The proteolytic system of schistosomes is essential for their viability: it participates in important processes during host-parasite interactions such as food digestion, invasion and tissue migration. Thus, schistosomal proteases are promising molecular targets for therapeutic intervention in schistosomiasis treatment. The thesis focuses on the protease cathepsin B2 from S. mansoni (SmCB2) which has not been studied in detail so far in terms of biochemical properties and biological function. Recombinant SmCB2 was prepared using yeast and bacterial expression systems and was chromatographically purified. Using an in vitro activity assay, the first effective inhibitors of SmCB2 were identified which inhibited its proteolytic activity in submicromolar concentrations. Specific polyclonal antibodies against SmCB2 were prepared and used for immunomicroscopic localization of this protease on the surface of the parasite. ELISA analysis demonstrated that SmCB2 is a parasite antigen recognized by the host immune system in the mouse model of schistosomiasis. The thesis provides valuable information about SmCB2 as a potential target molecule for synthetic...

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

6 Abstract Leishmaniasis is a neglected tropical disease, which belongs to the top health problems because it is endemic in 98 countries in Asia, Africa, the Americas and the Mediterranean region, and is gradually expanding to new areas, including Central Europe and USA. Clinical manifestations of leishmaniasis include a diverse range of forms, ranging from non-lethal cutaneous leishmaniasis to potentially lethal visceral leishmaniasis. Asymptomatic cases are known to exist in endemic areas. Different species of Leishmania induce distinct symptoms, but even the patients infected by the same species develop different symptoms and may respond differently to the treatment. Thus, one of the challenges is to explain the observed variability of leishmaniasis that cannot be attributed to the currently known factors. To find novel regulatory factors of the disease we tested molecules that were shown to play role in other infections and mapped loci controlling parasite load after L. major infection. We also determined genetic control of survival after infection with tick-borne encephalitis virus (TBEV) in order to establish whether there are common elements in response to L. major and TBEV. Interferon-induced GTPases (guanylate-binding proteins, GBPs) play an important role in inflammasome activation and mediate...

For many years hexamitids, Hexamita spp. and Spironucleus spp., have frequently been reported in vertebrates, particularly in fish. This suggests a potentially important role of these parasites in the fish culture industry. Though the majority of hexamitids are not known to cause disease in their vertebrate host, those that have been documented as associated with disease are still in need of further investigation into their geographical distribution, host range, life cycle, host-parasite relationship, pathogenicity, diagnosis, prevention, treatment, and control. Spironucleus vortens is a hexamitid recently described from angelfish (Pterophyllum scalare). Although the structure of this parasite has been investigated using the electron microscope (Poynton et al., 1995), other information on this organism is poorly understood. Thus, the purpose of this research was to study the nature of S. vortens in TYI-S-33 culture medium and in the angelfish host. The optimal environmental conditions for S. vortens growth were investigated using variations of temperature, pH, and bile concentrations. This study is useful in helping to understand the locations and environmental conditions in the host that are suitable for the growth of S. vortens. Treatment of S. vortens, using seven chemotherapeutic agents; dimetridazole, metronidazole, pyrimethamine, albendazole, fenbendazole, mebendazole, and magnesium sulfate was evaluated. The pathogenicity of S. vortens in angelfish was investigated in fish experimentally inoculated with trophozoites. This study provided information to help understand the pathogenesis of the parasites in the host. Finally, to examine the protective defense mechanisms, the presence of anti-S. vortens antibodies in angelfish serum were evaluated along with the presence of immune cells (lymphocytes, macrophages, eosinophilic granular cells, neutrophils, and plasma cells) at invaded sites of the intestine and other internal organs in response to an experimental Spironucleus vortens infection. The results of this research provide information on this parasite's effect on the fish host which may be useful in understanding the nature of other hexamitids. A few published reports have suggested the in vitro growth requirement of fish Spironucleus (Poynton et al., 1995; Sterud, 1998), but none have examined the optimal conditions required for growth and the pathogenicity of S. vortens. The first study was to examine the optimal requirements for the in vitro growth of the parasite. The organisms were cultivated in either an artificial medium (TYI-S-33) at different temperatures or various pH conditions, or in medium supplemented with different bile concentrations at 25°C. Criteria used to justify the optimal conditions were average cell number ml-1, growth rate, survival time, and cell conditions (motility and morphology). The organisms survived longest at 22°C, and had the highest average cell number ml-1 at 25°, 28° and 31°C. At 25°C the parasites were highly active and survived up to 6 days. The organisms cultivated at pH 6.5, 7.0 and 7.5 yielded the highest average cell number ml-1 with survival periods up to 13-14 days. Most of the organisms cultivated at a pH lower than 6.0 or a pH higher than 7.5 were suppressed and killed within 5-6 days of cultivation. All cultures supplemented with bovine or fish bile yielded lower maximal numbers of parasites than cultures with no bile. These results indicate that the optimal condition for the in vitro cultivation of S. vortens is 25°C and pH 6.5 to 7.5 without supplementation with bile. In order to treat spironucleosis, the efficacy of various chemotherapeutic agents on the growth of S. vortens was examined in vitro. In this study nitroimidazoles and benzimidazoles, formerly reported as drugs of choice for the treatment of diplomonads, pyrimethamine and magnesium sulfate (Epsom salt) were evaluated at different concentrations on the growth of S. vortens. Dimetridazole and metronidazole were effective in inhibiting the parasite's growth at concentrations of 1 μg ml-1 or higher. Albendazole and fenbendazole suppressed the growth of parasites at concentrations of 1.0 μg ml-1 or higher after 24 h exposure. Mebendazole was the most effective agent of the benzimidazole group; and inhibited the parasite's growth at concentrations of 0.5 μg ml-1 or higher. Pyrimethamine at concentrations of 1-10 μg ml-1 failed to inhibit the parasite's growth. Magnesium sulfate inhibited the growth of the parasites only at high concentrations (70 mg ml-1 or higher) . This study indicates that dimetridazole, metronidazole and mebendazole are the most effective chemotherapeutic agents in vitro at inhibiting the growth of S. vortens. To investigate the pathogenesis of spironucleosis, angelfish were orally (PO) or intraperitoneally (IP) inoculated with S. vortens. Control angelfish which were orally gavaged or intraperitoneally injected with PBS were in normal body condition and had no morbidity or mortality. Compared to the control angelfish, PO-infected angelfish were inappetent with no other clinical signs, while IP-infected angelfish showed clinical signs of inappetite, weakness, respiratory distress, and laying on their sides. Twenty percent of the IP-infected angelfish died within the first three weeks after infection. In PO-infected angelfish, the organisms were located only in the intestinal lumen. In IP-infected angelfish, S. vortens were found in the blood, stomach, intestine, and other internal organs (spleen, gall bladder, and ovary). However, no parasites were observed within the intestinal mucosa of either PO- or IP-infected fish. Histopathologic examination of the intestines revealed mild to moderate multifocal enteritis in both PO- and IP-infected angelfish. The mucosal epithelium appeared undamaged although the parasite was closely located and appeared attached to the intestinal mucosa. The results suggest that S. vortens normally causes mild to moderate multifocal enteritis with no morbidity. However, the parasites can cause granulomatous inflammation in a wide variety of host tissues, and may be lethal if they enter the abdominal cavity and disseminate to other organs via the blood circulation. Immunity, both cell mediated and humoral, against S. vortens was investigated in this study. Histopathologic examination revealed a response from inflammatory cells infiltrated and localized in the affected tissues. Macrophages, lymphocytes, and plasma cells were the most common cell types found in the internal organs. Macrophages were active in the affected tissues where the parasites lived in situ. However, in vitro studies indicated that there were no differences in a production of H₂O₂ or in phagocytosis between macrophages of control and infected angelfish regardless of inoculum dosage and administration route. A preliminary study of humoral antibody indicated that angelfish did not develop anti-S. vortens antibody after they were orally or intraperitoneally infected with either a low or a high number of the organisms. It is suggested that localized leucocyte response may be an important mechanism against Spironucleus vortens infection in angelfish. This research has indicated some of the important environmental factors affecting the parasite's growth, and has provided some initial information on the pathogenicity of S. vortens. In addition, preliminary information on the host's protective immune systems, humoral and cell-mediated immunity, against the parasite have been documented. The results from this research will be useful for aquaculture, particularly of tropical freshwater angelfish, and may help to provide an understanding of the biological roles of other hexamitids. / Ph. D.

Indiana University-Purdue University Indianapolis (IUPUI) / Toxoplasma gondii is an obligate intracellular eukaryotic pathogen that causes severe neurologic disease in immunocompromised adults and congenitally infected neonates. Events critical to the propagation of T. gondii, such as invasion and egress, are regulated by calcium-dependent signaling. In order to identify unique components of the parasite’s calcium signaling networks, members of the Arrizabalaga laboratory have used a forward genetics approach to isolate mutants with altered sensitivity to the calcium ionophore A23187. Exposing extracellular parasites to A23187 induces protein secretion, motility and cytoskeletal rearrangements and prolonged treatment causes exhaustion of factors required for invasion, which results in what is referred to as ionophore induced death (iiDeath). Mutants capable of surviving this treatment were isolated from a chemically mutagenized population. Whole genome sequencing of one such mutant, MBD2.1, identified a nonsense mutation in a protein of unknown function (TGGT1_069070, ToxoDBv7.2) Complementation of MBD 2.1 with a wild-type copy of TGGT1_069070 restored sensitivity to iiDeath treatment. Endogenous tagging of this locus revealed that the encoded protein is secreted from a unique parasite secretory organelle known as the dense granule into the parasitophorous vacuole, leading to its designation as TgGRA41. Complete knockout of TgGRA41 recapitulates the resistance to iiDeath observed in MBD2.1 but also exhibits a dramatic decrease in propagation in tissue culture not seen in the original mutant. The knockout shows defects in multiple steps of the lytic including compromised invasion efficiency and premature egress of parasites from host cells. Cytosolic calcium measurements of extracellular parasites show enhanced uptake of calcium in the knockout strain as compared to parental and complemented, suggesting that the loss of TgGra41 results in calcium dysregulation. Together, these results provide a novel insight into the role that the parasitophorous vacuole of T. gondii plays in calcium homeostasis and calcium-dependent signaling processes.

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