Three genes (Imcpa, Imcpb and Imcpc) that encode cysteme proteinases (CPs) in L. mexicana have previously been cloned and sequenced. The enzymes encoded differ significantly from each other and host CPs. In an attempt to elucidate the parts played by each CP in the parasite's life history, mutant lines of the parasite which lack Imcpa, Imcpb and Imcpc have been examined in order to determine their phenotypes. Null mutants which have been further modified by transfecting the cells with genes encoding L. mexicana CPs and CPs of other trypanosomatids have also been studied. These lines have been analysed to determine how they differ from the parent line in terms of proteinase and protein content, growth and differentiation axenically in vitro and in peritoneal macrophage cells, infectivity to mice and susceptibility to CP inhibitors. Null mutants for the Imcpa CP gene were found to have a cryptic phenotype, since differences between the Imcpa null mutants and wild type L. mexicana were not detectable. This suggested that either the enzyme has a role which was not detected with the phenotypic tests used or that its activity was being compensated for by one of the other CPs present in L. mexicana. In contrast to previously published conclusions, studies using null mutants for lmcpa revealed that LmCPa was not at highest levels in the amastigote stage. Triton X-114 phase separation revealed the protein encoded by lmcpa to be hydrophobic, suggesting it could be membrane-associated. Stationary phase promastigotes of mutants null for the multicopy lmcpb genes showed a marked phenotype with respect to virulence, as their ability to infect macrophages in vitro was severely impaired. The infectivity was restored by re-expression of an enzymatically active internal copy of the gene array (g2.8), suggesting that the enzymes play a role in intracellular survival. Time course experiments demonstrated that the null mutants invaded macrophages in large numbers but were unable to survive in the majority of the cells. Despite their reduced virulence to PECs, the mutants were capable of infecting mice although the time taken for lesions to form was significantly longer than with wild type and the lesions were 100-fold smaller. Interestingly, lesion amastigotes of the mutants were able to infect macrophages in vitro as successfully as wild type, but took three months longer to produce lesions in mice. These data showed that the CPs encoded by lmcpb are important for parasite virulence, but are not essential for survival in the host. Double lmcpa/lmcpb null mutants were found to have a similar phenotype to the lmcpb null mutants in terms of their ability to infect macrophages in vitro, suggesting that lmcpa was not compensating for the loss of lmcpb in this phenotypic test, although it was found that they did not cause lesions in mice. Studies utilising null mutants re-expressing different copies of the lmcpb array revealed that individual isoenzymes differ in their substrate preferences and ability to complement the loss of virulence associated with the null mutant, suggesting that the individual isoenzymes have distinct roles in the parasite's interaction with the host. Immunogold labelling of the megasome in transfectants re-expressing an lmcpb CP gene with a truncated C-terminal extension (gl) demonstrated that the C-terminal extension is not necessary for intracellular targeting or activity. All life-cycle stages of the mutants null for the cathepsin B-like CP gene (lmcpc) could be cultured in vitro, demonstrating that this CP is not essential for the growth or differentiation of the parasite. However, they showed greatly reduced infectivity to macrophages in vitro, resulting in only a low percentage of the cells being infected. Re-expression of lmcpc in the null mutant increased the parasite's infectivity to macrophages in vitro. However, the null mutants for lmcpc formed lesions in mice at a rate comparable with wild type parasites, suggesting that although the CP encoded by lmcpc may play a role in the parasite-macrophage interaction it alone is not crucial for infectivity or virulence. Peptidyl-diazomethane CP inhibitors, which entered the parasites and inhibited LmCPb in situ, had little or no effect on parasite growth or differentiation axenically in vitro. However, at the same concentration, N-benzoyloxycarbonyl-phe-ala-diazomethane reduced the infectivity of wild type parasites to macrophages by 80%, a similar percentage to that seen with the lmcpb null mutant. However, further experiments suggested that LmCPb may not be the prime target of this inhibitor. With respect to drug target validation it is essential to determine which CPs are necessary for parasite survival. The overall results of this study suggest that while none summary of the CPs of L. mexicana are vital, some appear to have important and distinct roles in differentiation and intracellular survival.

During its transmission, Syphacia muris Yamaguti 1935, (Nermatoda: Oxyuroidea), undergoes 4 moults in the rat host to become an adult. Worms deposit up to 4000 eggs daily on the host's perianal region following a circadian rhythm. Most of the eggs are deposited in the day time, with a peak occurring around noon time. The rhythm is dependent upon the behaviour of the rat which is itself influenced by the lighting regime in the environment. Rats are nocturnal animals and normally feed and defaecate at night. Adult female worms of muris, therefore, release eggs during the day time when the rat is at rest so as to avoid the loss of eggs in the faeces at night. A technique is described to collect eggs from the perianal region of the rat for experimental infections. Eggs collected from around the peak of egg-laying activity produce the most worms in isolator-raised rats. In single infections, the worm burden increases with the dose size of eggs administered. But worm size is reduced in larger infections. Fecundity remains unaltered. In multiple infections a severe expulsion phase occurs in male rats. Females bear smaller worm burdens. An infection is transmitted to suckling rats about 12 days after birth so that litters already harbour worms on the day of weaning. The infection increases rapidly during the 32 days after weaning, thereafter it decreases and oscillates around a constant level. This oscillation may be attributed to periods of refraction and susceptibility to infection by the host. Infestation levels are remarkably similar in rats caged individually and in groups, indicating a degree of stability in the transmission strategy of Syphacia muris.

The present study has shown that age resistance to R. cesticillus, manifested by worm destrobilization, occurred in male chickens. In females, age resistance developed more rapidly than in males, until the birds were about 84 days old and from this age onwards the manifestations of resistance declined. The roles of immune responses, mucus secretion and host hormone balance in the development of age resistance to R. cesticillus were subsequently investigated. Host resistance to superinfection with R. cesticillus was demonstrated for the first time and this resistance was inhibited by the immunosuppressant dexamethasone. Further evidence for the immunogenicity of the cestode was provided by histological studies and by the demonstration of immunocytes and of ring precipitin and immunofluorescent antibodies. Experimental evidence suggested that these antibodies are not protective and it is probable the protective immune response is cellular. An in vitro culture system, described by Schiller (1970), kept young adult cestodes in an active condition for more than eight days, although growth was poor. Chicken intestinal mucus extracts were shown to be lethal to R.cesticillus in vitro and mucus from old birds appeared to have a greater effect than 'young' mucus. The effects of gonadal steroids on the course of infection of R. cesticillus were investigated by caponising male birds and implanting oestradiol or testosterone pellets. This procedure reproduced to some degree the patterns of infection of R. cesticillus observed in intact, untreated, mature birds. Scolex transplantation and the correlation of worm weights down the intestine with destrobilization strongly implicated intestinal environmental factors as being responsible for the phenomenon of worm destrobilization. The possible mechanisms whereby the immune response, mucus secretions and host hormone balance affect worm viability are discussed.

A study of the monthly incidence and intensity of infection of Apodemus sylvaticus and Clethrionomys glareolus with selected intestinal helminths has been made. The majority of the variation in these two factors was shown to he due to differences in the age and sex ratio of the host populations throughout the year. Multiple regression and correlation analyses were used to assess the effect of independent environmental variables on the incidence and intensity of infection. A competitive interaction was found to occur between the trichostrongylid nematode Nematospiroides dubius and the oxyurid Syphacia stroma in the small intestine of Apodemus sylvaticus, though such an interaction did not lead to the total exclusion of either species. The distribution of N. dubius in the small intestine of laboratory mice was found to Be highly aggregated with respect to Both the intestine and the worm population; this was thought to occur in response to a gradient in oxygen tension along the mouse gut. Growth curves of male and female N. dubius in male and female mice were typically sigmoid and the daily growth rates for each sex of worm were the same in Both sexes of mice. The growth of the free-living stages was negative after infectivity had been reached. The respiration rates of male and female N. dubius increased with age, whereas those of the free-living stages decreased after infectivity was reached. The size/metabolism relationship for free-living stages N. dubius indicated a slow-down in metabolism as soon as infectivity was reached; this is likely to be of adaptive significance to a non-feeding stage. The size/metabolism relationship of both male and female parasitic stages indicated that aerobic metabolism may be important in vivo.

This thesis investigates the epidemiology of intestinal parasites in wild populations of two social animals, olive baboons (Papio anubis) in Gombe Stream National Park, Tanzania, East-Africa, and lions (Panthera leo) in the Serengeti and Ngorongoro-Crater, Tanzania. These populations have been observed for over 20 years and detailed information on individual hosts was available for analysis with the parasitological data allowing to address questions about the relationship of host genetics and social behaviour to parasite infection. The baboons were infected with seven different helminths as well as two types of protozoans. Fifteen morphologically different parasites were found in the lions. All baboons and almost all lions were parasitized. Parasite distribution in both host species was overdispersed. Spatial differences in parasite infection in the baboons and lions emerged as the strongest effect on heterogeneity of infection. Parasites of both host species showed seasonal and temporal variation. Parasite-parasite associations did not appear to have a strong impact on overall patterns of infection in either baboons or lions. Across all parasite taxa, age (with one exception in baboons), sex, reproductive status and group size had little significant influence on parasite burden. For baboons age-prevalence and age-severity profiles resembled those for the same parasites in humans. Correlations between baboon social rank and parasite burden were equivocal. Parasite infection was not correlated with size of baboon female genital swelling. Two lion populations were compared, an inbred and an outbred. Only one parasite, Spirometra spp., had a significantly higher prevalence in the inbred population, contrary to expectations. Results of this study suggest that any effects on levels of infection in these wild populations due to social behaviour, genetics, sex and reproductive effort may be masked by the stronger influence of environmental and/or behavioural components of exposure, at least in the short term. This implies that the importance of factors such as genetics or social behaviour on infection may not always be apparent and may be dependent on the details of the local ecology of both host and parasite at the time the system is studied.

Malaria control programs in the past decade have been successful in decreasing the global burden of Plasmodium falciparum, the more virulent of the Plasmodium species causing malaria in humans. However, as the public health community gears towards elimination and eradication of malaria, there is need for an attention shift towards research of neglected Plasmodium species. A central event in malaria pathogenesis is the invasion of host red blood cells (RBCs) mediated by specific interactions between parasite ligands and RBC receptors. These interactions, also called invasion pathways, can be major determinants of host tropism. Restriction in invasion due to tropism is attributed to limiting disease severity, as it limits the proliferation of the parasite in vivo. In this dissertation, we determine the host cell tropism of Plasmodium knowlesi and Plasmodium vivax, two understudied Plasmodium species. The zoonotic P. knowlesi is now the major cause of malaria in parts of South East Asia, and P. vivax is the most widespread species worldwide causing substantial morbidity. After a review of the current literature on biological and clinical features of these species and their invasion pathways in Chapter 1, we determined the tropism of P. knowlesi in human RBCs in Chapter 2. We used density-based enrichment methods to test the invasion of P. knowlesi into human RBCs of varying age. Incorporating mathematical modeling and experimental adaptation, we demonstrated that an expansion of host cell niche is required for the parasite to reach densities observed clinically. We also obtained parasite lines adapted to efficient proliferation in human RBCs. In Chapter 3, we investigated the molecular basis of increased invasion of human RBCs through whole genome sequencing analysis of newly human-adapted lines, as well as historical strains we adapted to in vitro culture. We showed that different genetic changes in P. knowlesi can lead to the upregulation of PkDBPα-DARC pathway, and have provided evidence of major divergence in invasion ligands in recent field isolates. Finally, in Chapter 4, we studied the variation in human RBC preference of patient isolates for P. knowlesi and P. vivax. We confirmed that recent human P knowlesi isolates also vary in the niche of susceptible RBCs, with a subset exhibiting a lack of restriction in invading human RBCs. We also evaluated ex vivo P. vivax patient isolates for their host RBC preference. We determined that P. vivax field isolates differ in their level of reticulocyte preference. We further found an association between increased reticulocyte preference and schizont maturation. This body of work aims to contribute to our overall understanding of human RBC tropism of Plasmodium species of public health importance and the implication this has for parasite adaptation to humans. / Biological Sciences in Public Health

Approximately one third of the world’s population is at risk of contracting malaria. The World Health Organization estimates there were over 200 million news cases of malaria in 2015, resulting in nearly 500,000 deaths from this preventable disease. The majority of fatalities occur in Sub-Saharan Africa, where Plasmodium falciparum malaria causes severe disease in children under the age of five and pregnant women. In the last decade, increased anti-malaria interventions have resulted in substantial decreases in cases and fatalities. However, the recent emergence of artemisinin drug resistance in Southeast Asia threatens these gains, and the loss of another first-line antimalarial therapy would be a devastating setback. The first goal of this work was to identify genetic markers of artemisinin drug resistance. Identifying the genetic determinants and molecular mechanisms of artemisinin resistance is crucial for understanding the emergence of this phenomenon and tracking the spread of these drug resistant parasites. Over the course of four years, we used an in vitro drug resistance selection approach to generate three independent artemisinin-resistant lines. Here we characterize those lines, and present Pfcoronin, a kelch13-like protein, as a novel candidate marker for artemisinin resistance. This study identifies additional non-kelch13 molecular markers of artemisinin resistance, increases our understanding of how this resistance is acquired, and sheds light on the molecular mechanisms of artemisinin resistance in the parasite. In contrast to in vitro selection, natural selection of parasites occurs during natural infection. Investigation of specific genes under selection in the parasite will increase our understanding of biological processes that provide a fitness advantage, and potentially identify novel pathways for therapeutic development. Here, we focused on the acyl Co-A synthetase (ACS) gene family, previously shown to be under recent positive selection in P. falciparum. The signatures of recent positive selection identified in natural parasite populations suggest that particular ACS alleles may confer a selective advantage. Using molecular genetics approaches, we show distinct expression and localization patterns for individual ACS isoforms, and identify a growth defect in the ACS5 knockout line. Follow up studies characterize the fatty acid and metabolic profiles of individual ACS knockout lines, and point to a role for ACS5 in central carbon metabolism in P. falciparum. Our investigation of the ACS gene family and their role in P. falciparum growth and metabolism led us to hypothesize a link between ACS activity and central carbon metabolism. In the final chapter, we explore the basic fatty acid and glucose requirements for P. falciparum growth in vitro, and present a metabolic profile for these starved parasites. Under starvation conditions, we were able to demonstrate fatty acid oxidation activity in the parasite. This is an unexpected finding, as this pathway was not previously annotated in the genome. Taken together, these two projects tell a story of the selective pressures acting on P. falciparum parasites. Investigating in vitro selected artemisinin-resistant lines provides important insights into genetic markers and acquisition of resistance. Molecular and biochemical characterization of a gene family under natural selection in P. falciparum increases our understanding of important metabolic pathways that support parasite growth. / Biological Sciences in Public Health

Indiana University-Purdue University Indianapolis (IUPUI) / Toxoplasma gondii is an important human pathogen that infects millions of people worldwide and causing severe and potentially lethal disease in immunocompromised individuals. Recently, a homologue for the autophagy protein Atg8 (TgAtg8) was identified in Toxoplasma that is required for both canonical and noncanonical processes essential for parasite viability. Importantly, TgAtg8 functionality requires its conjugation to phosphatidylethanolamine through the activity of the Atg8 conjugation system. In this thesis, we characterized the proteins that interact with TgAtg8 and TgAtg3, a component of the Atg8 conjugation system, to further define their functions in Toxoplasma and identify opportunities for targeted inhibition of Atg8-related processes. We previously identified that TgAtg8 is acetylated at lysine 23 (K23) and assessed the role of this modification in this thesis. Using mutagenesis, we showed that K23 acetylation did not modulate the interaction with TgAtg3, but appeared to promote TgAtg8 protein stability. Additionally, endogenous mutation of K23 to the nonacetylatable amino acid arginine resulted in severe impairment of parasite replication and spontaneous differentiation into bradyzoites. To gain insight into the role of TgAtg8 in Toxoplasma biology, we next characterized TgAtg8 and TgAtg3 interacting proteins using affinity purification and mass spectrometry. We identified a novel group of interacting proteins that are unique to Toxoplasma, including the dynamin-related protein DrpC. Functional characterization of DrpC identified a potential role of TgAtg8 in trafficking of membrane from the Golgi to the nascent daughter parasites during replication. Lastly, we examined a group of small molecules recently identified as Atg3-Atg8 inhibitors in Plasmodium falciparum and assessed their activity against Toxoplasma. Although the compounds effectively inhibited Toxoplasma replication, they did so through novel mechanisms of action unrelated to the disruption of the TgAtg3-Atg8 interaction. Together, this work provides insight into the function of the Atg8 conjugation system in Toxoplasma that will help guide the future development of novel therapeutics targeting Atg8-related processes.

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