Malaria is still a significant public health problem in the Tropics, with an estimated 200 million cases a year and more than 1 million deaths, mostly in young children in sub-Saharan Africa. Plasmodium falciparum is the parasite responsible for the majority of the morbidity and mortality due to malaria. We know from the historical use of malaria to treat neurosyphilis that there were several different strains of P. falciparum, some of which were more pathogenic and had higher multiplication rates than others. High multiplication rates of P. falciparum isolates have been associated with severe disease in Thailand, but not in Kenya or Mali. In determining what differences exist between fast- and slow-growing malaria parasites, and understanding their relationship with clinical outcome, we may discover a way of targeting those parasites that cause most disease. This thesis describes a genetic analysis of the determinants of growth and pathogenicity in the rodent malaria parasite, Plasmodium chabaudi. The use of rodent malaria parasite strains for genetic analysis has several experimental, ethical and financial advantages over the use of human malaria parasites. In addition, rodent malaria parasite strains also vary significantly in their growth and pathogenicity, making them excellent candidates for a genetic analysis of these characteristics. The first section of this thesis is concerned with the characterisation of the growth, pathogenicity and transmissibility of two strains, DS and DK, of the rodent malaria parasite P. c. adami. The DS strain is fast-growing, pathogenic, non-selective in its invasion of red blood cells and a poor transmitter to mosquitoes. The DK strain is slow-growing, non-pathogenic, selective in its invasion of red blood cells and a good transmitter to mosquitoes. In the second section of this thesis is a detailed study of the growth characteristics of DS and DK in mixed infections, relative to their growth in single infections. Both sections provide information relevant for the main objective of this thesis, but also contribute to the body of work on pathogenicity and transmissibility, and pathogenicity and strain behaviour in mixed infections, which has been carried out in rodent malaria parasites to-date. The third section of the thesis contains the results of a genetic analysis of the difference in growth between P. c. adami strains DS and DK, using the Linkage Group Selection (LGS) technique. On several occasions, DS and DK were crossed in the mosquito vector and, following selection for fast growth in mice, the cross progeny were initially screened with genome-wide, quantitative AFLP markers. Markers specific to the slow-growing parent DK which were greatly reduced in intensity after selection were found on P. chabaudi chromosomes 6, 7 and 9. This result suggests that the difference in growth between the two strains is determined by multiple genetic loci. The selection on chromosomes 7 and 9 was then looked at in greater detail, using SNP-based markers quantified by Pyrosequencing™. It was found, consistently, that a region at one end of DS chromosome 9 was inherited as a single, non-recombining unit in cross progeny selected for fast growth. As this was the region most strongly selected against, it suggests that a gene (or genes) in this region has a major role in the determination of growth characteristics, and therefore pathogenicity, in P. c. adami. Narrowing down this region further, in order to identify the candidate gene(s), remains a key future objective.

Doctor of Philosophy / Department of Plant Pathology / Christopher R. Little / Understanding the genetic structure of fungal pathogens enables the prediction of evolutionary forces that drive pathogen evolution, which assists informed decision-making regarding disease management. The genetic structure of Fusarium thapsinum and F. andiyazi, two important pathogens that cause grain mold and stalk rot of sorghum (Sorghum bicolor), are little understood. The genetic structure and pathogenicity of a F. thapsinum population from sorghum in Kansas were evaluated with amplified fragment length polymorphisms (AFLPs), vegetative compatibility groups (VCGs), sexual cross-fertility, and seedling pathogenicity. Two sympatric populations and a genetically intermediate "hybrid" group were identified in Kansas. Seedling pathogenicity of strains ranged from non-pathogenic to pathogenic, which may be partially attributable to genetic variability in the F. thapsinum populations. Genetic relatedness between populations of F. thapsinum from sorghum in Kansas, Australia, Thailand, and three African countries (Cameroon, Mali, and Uganda) were evaluated with AFLP markers and sexual crosses. Genetic diversity was high in all locations, but female fertility is very low. These results are consistent with the hypothesis that both sexual and asexual modes of reproduction are important components of the life cycle of F. thapsinum in these populations. More strains from Kansas and Africa were available for analysis than from Australia and Thailand, so the Kansas and Africa populations dominated the genetic structure observed. The two smaller populations from Australia and Thailand were more closely related to the Kansas population than they were to the African population. The three non-African populations contained information from the African population and from other, as yet unidentified, source population(s). Identifying the population(s) from which this genetic diversity originated is an important unanswered question. Stalk rot of sorghum was evaluated by inoculating stalk rot sensitive and stalk rot resistant sorghum lines with six genetically diverse F. thapsinum strains from Kansas under field and greenhouse conditions. One susceptible line (Tx7000) and two resistant lines (SC599 and BTx399) were evaluated in the field but only Tx7000 and SC599 were evaluated in the greenhouse. Disease severity was measured by major lesion length and the number of nodes crossed by the lesion. There were differences in aggressiveness amongst the F. thapsinum strains in both the greenhouse and field evaluations. This study provides the first evidence for differences in stalk rot aggressiveness amongst F. thapsinum strains and highlights the importance of challenging germplasm with well-characterized strains that represent the genetic spectrum of the entire population. The genetic diversity within F. andiyazi populations and some closely related strains was evaluated with AFLP markers. Phylogenetic and STRUCTURE analyses of the AFLP markers grouped the 81 F. andiyazi strains into three distinct clusters. The clusters were not based on the geographic origin of the strains. These results indicate the presence of at least one and possibly two undescribed sister taxa of F. andiyazi. More work is needed to further characterize these sister species of F. andiyazi and to understand their role in sorghum pathogenicity. There is genetic variation in global populations of F. thapsinum and the observed variation could be associated with variation in both seedling and adult plant pathogenicity. The study of F. andiyazi populations validated the need to properly identify and characterize Fusarium spp. associated with sorghum from different regions of the world.

It was of interest to clone key genes involved in O-glycosylation with a view to using reverse genetics to establish their function. The Candida homolog of the S. cerevisiae MNT1 gene (Hausler and Robbins, 1992) was cloned by heterologous probing of a genomic DNA library. The CaMNT1 gene was found to be regulated differentially in response to the environment and exhibited a transitory increase in the level of transcription during early germ tube formation. Low stringency Southern analysis of C. albicans genomic DNA identified several CaMNT1 homologs suggesting CaMNT1 is part of a multigene family whose members are presumed to be yeast Golgi mannosyltransferases. In order to demonstrate that specific glycosyl residues were actively involved in the host-fungus interaction, the CaMNT1 gene was disrupted in two strains using the ura-blaster technique. Disruption at the CaMNT1 locus led to a 90% reduction in -1,2-mannosyltransferase activity when -methyl mannoside was used as an acceptor, but had no obvious influence on viability, growth rate, germ tube formation or proteinase production. CaMnt1 appears to be involved in O-glycosylation since the Camnt1 null mutant strain accumulated intracellularly the O-glycosylated enzyme chitinase. Mannosyltransferase-deficient Camnt1 mutants were significantly reduced in their ability to adhere to human buccal epithelial cells in vitro and were attenuated in virulence in systemic models of candidosis. O-linked mannan may therefore be important for direct interactions with epithelial surfaces or for the stabilization and function of cell surface adhesins. The low virulence potential displayed by Camnt1 mutants clearly demonstrates the important role glycosylation plays in the virulence of C. albicans. Given that O-glycosylation differs significantly between yeast and man, this protein modification may constitute a novel target for antifungal agents.

PhD, Faculty of Health Sciences, University of the Witwatersrand / The South African gold-mining workforce has an unusually high incidence of Mycobacterium kansasii disease, yet little is known about the possible sources of M. kansasii infection, genetic diversity and the basis for this organism’s pathogenicity. The purpose of this study was to investigate these issues in a gold-mining environment. Five M. kansasii isolates and 10 other potentially pathogenic mycobacteria were cultured mainly from showerhead biofilms. PCR-restriction analysis (PRA) of the hsp65 gene on 191 clinical and on the 5 environmental M. kansasii isolates revealed 160 subtype I (157 clinical and 3 environmental), 8 subtype II (clinical) and 6 subtype IV (5 clinical and 1 environmental) strains. Twenty-two isolates (21 clinical and 1 environmental) did not show the typical M. kansasii PRA patterns. After confirmation by DNA sequencing as belonging to the M. kansasii species, the results suggested that these isolates were probably new subtypes of M. kansasii. In contrast to the clonal population structure found amongst the subtype I isolates from studies in other countries, DNA fingerprinting of 114 subtype I clinical and environmental isolates showed genetic diversity amongst the isolates. One of the 2 environmental isolates showed 100% identity with a clinical isolate, suggesting that water distribution systems are the possible sources of M. kansasii infection for the miners. An investigation into the genetic differences between clinical (subtype I) and environmental (III, IV and V) isolates, using Hybridisation Monitored Differential Analysis (HMDA), identified 45 open reading frames (ORFs) encoding predominantly membrane-associated proteins that include six potential virulence factors, two family members of transcription regulators for drug and xenobiotic metabolism, three family members of multidrug efflux systems, a number of proteins associated with lipid and carbohydrate metabolism and transport, and a number of hypothetical proteins with unknown function. Immunological analysis of M. kansasii isolates, using the Lymphocyte Transformation and Cytometric Bead Array assays, showed that M. kansasii modulates immune responses through suppression of lymphocyte blastogenesis and by altering the expression of Th1/Th2/Th17 cytokines by human lymphocytes in vivo for its own survival. This study demonstrated for the first time that water distribution systems in South Africa are possible sources of M. kansasii infection, and showed that subtype I strains of M. kansasii from the study region display genetic diversity and have unique or divergent genes not found in other subtypes. It also demonstrated that immunosuppression is one of the pathogenic mechanisms employed by M. kansasii.

by Chan Chi Wai, Michael. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 238-272). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

A triglyceride lipase gene FgLip1 was identified in the genome of <i>Fusarium graminearum</i> strain PH-1. Yeast cells overexpressing FgLip1 showed lipolytic activity against a broad range of triglyceride substrates. Northern blot analyses revealed that expression of FgLip1 was activated in planta during the fungal infection process and under starvation conditions <i>in vitro</i>. FgLip1 expression was strongly induced in minimal medium supplemented with wheat germ oil, but only weakly induced by olive oil and triolein. Saturated fatty acids were the strongest inducers for FgLip1 expression and this induction was proportionally suppressed by the presence of unsaturated fatty acids. To determine the potential function of FgLip1, gene replacement was conducted on strain PH-1. When compared to wild-type PH-1, ∆FgLip1 mutants showed greatly reduced lipolytic activities at the early stage of incubation on minimal medium supplemented with either saturated or unsaturated lipid as the substrate, indicating that FgLip1 encodes a secreted lipase for exogenous lipid hydrolysis. The ∆FgLip1 mutants exhibited growth deficiency on both liquid and solid minimal media supplemented with the saturated triglyceride tristearin as the sole carbon source, suggesting that FgLip1 is required for utilization of this substance. No variation in disease symptoms between the ∆FgLip1 mutants and the wild-type strain was observed on susceptible cereal hosts including wheat, barley and corn. To delineate the promoter region responsible for the specific regulation of FgLip1 expression, a series of deletions of FgLip1 5 upstream region were fused with the open reading frame of a green florescent protein (GFP) gene and the constructs were introduced into <i>F. graminearum</i>. GFP expression in the resulting transformants indicated that a 563-bp FgLip1 promoter sequence was sufficient to regulate expression of the FgLip1 gene and regulatory elements responsible for gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements within the 563-372 bp region were mutated using a linker-scanning mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be responsible for FgLip1 basal expression, glucose suppression and fatty acid induction, respectively.

Iron is an essential component of various proteins and pigments for both plants and pathogenic fungi. However, redox cycling between the ferric and ferrous forms of iron can also catalyse the production of dangerous free radicals and iron homeostasis is therefore tightly regulated. During pathogen attack, plants quickly produce large amounts of reactive oxygen species at the site of attempted pathogen ingress. This so-called oxidative burst has received considerable attention, but no single enzyme has been shown to account for the phenomenon. Using inductively coupled plasma mass spectrometry and histochemistry, I show that iron is secreted to the apoplast of the diploid wheat <i>Triticum monococcum</i> during attack by the powdery mildew fungus <i>Blumeria graminis</i> f.sp. <i>tritici</i>. This iron accumulates at cell wall appositions synthesised de novo beneath sites of pathogen attack. I further show, using histochemistry and pharmaceutical inhibitors, that this apoplastic iron accumulation is required for production of H2O2 in the oxidative burst. To understand the impact of this massive change in iron homeostasis on gene transcription, I employ a 187 gene targeted macroarray platform and establish that iron overload induces the expression of iron homeostasis-related genes and defence-related genes through iron itself and iron-mediated H2O2 production, respectively. To illustrate how the plant is able to withstand the negative effects of its own oxidative defences, I characterise a novel quinone redox cycle, and show that simultaneous induction of a protective quinone reductase isoform and downregulation of reactive oxygen-producing quinone reductase isoform prevents the spread of reactive oxygen during pathogen attack. Finally, in an effort to understand the impact of iron on fungal pathogenicity, I investigate iron uptake in the head blight pathogen, <i>Fusarium graminearum</i>. Fungi use at least two separate systems to take up iron, one based on enzymatic iron reduction and the other based on the synthesis and secretion of small iron chelators termed siderophores. Using mutants disrupted in either of two modes of iron uptake, I establish that siderophore production is essential for full F. graminearum virulence on wheat. This thesis exposes iron as an important component of both plant defence and fungal virulence.

A triglyceride lipase gene FgLip1 was identified in the genome of <i>Fusarium graminearum</i> strain PH-1. Yeast cells overexpressing FgLip1 showed lipolytic activity against a broad range of triglyceride substrates. Northern blot analyses revealed that expression of FgLip1 was activated in planta during the fungal infection process and under starvation conditions <i>in vitro</i>. FgLip1 expression was strongly induced in minimal medium supplemented with wheat germ oil, but only weakly induced by olive oil and triolein. Saturated fatty acids were the strongest inducers for FgLip1 expression and this induction was proportionally suppressed by the presence of unsaturated fatty acids. To determine the potential function of FgLip1, gene replacement was conducted on strain PH-1. When compared to wild-type PH-1, ∆FgLip1 mutants showed greatly reduced lipolytic activities at the early stage of incubation on minimal medium supplemented with either saturated or unsaturated lipid as the substrate, indicating that FgLip1 encodes a secreted lipase for exogenous lipid hydrolysis. The ∆FgLip1 mutants exhibited growth deficiency on both liquid and solid minimal media supplemented with the saturated triglyceride tristearin as the sole carbon source, suggesting that FgLip1 is required for utilization of this substance. No variation in disease symptoms between the ∆FgLip1 mutants and the wild-type strain was observed on susceptible cereal hosts including wheat, barley and corn. To delineate the promoter region responsible for the specific regulation of FgLip1 expression, a series of deletions of FgLip1 5 upstream region were fused with the open reading frame of a green florescent protein (GFP) gene and the constructs were introduced into <i>F. graminearum</i>. GFP expression in the resulting transformants indicated that a 563-bp FgLip1 promoter sequence was sufficient to regulate expression of the FgLip1 gene and regulatory elements responsible for gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements within the 563-372 bp region were mutated using a linker-scanning mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be responsible for FgLip1 basal expression, glucose suppression and fatty acid induction, respectively.

Iron is an essential component of various proteins and pigments for both plants and pathogenic fungi. However, redox cycling between the ferric and ferrous forms of iron can also catalyse the production of dangerous free radicals and iron homeostasis is therefore tightly regulated. During pathogen attack, plants quickly produce large amounts of reactive oxygen species at the site of attempted pathogen ingress. This so-called oxidative burst has received considerable attention, but no single enzyme has been shown to account for the phenomenon. Using inductively coupled plasma mass spectrometry and histochemistry, I show that iron is secreted to the apoplast of the diploid wheat <i>Triticum monococcum</i> during attack by the powdery mildew fungus <i>Blumeria graminis</i> f.sp. <i>tritici</i>. This iron accumulates at cell wall appositions synthesised de novo beneath sites of pathogen attack. I further show, using histochemistry and pharmaceutical inhibitors, that this apoplastic iron accumulation is required for production of H2O2 in the oxidative burst. To understand the impact of this massive change in iron homeostasis on gene transcription, I employ a 187 gene targeted macroarray platform and establish that iron overload induces the expression of iron homeostasis-related genes and defence-related genes through iron itself and iron-mediated H2O2 production, respectively. To illustrate how the plant is able to withstand the negative effects of its own oxidative defences, I characterise a novel quinone redox cycle, and show that simultaneous induction of a protective quinone reductase isoform and downregulation of reactive oxygen-producing quinone reductase isoform prevents the spread of reactive oxygen during pathogen attack. Finally, in an effort to understand the impact of iron on fungal pathogenicity, I investigate iron uptake in the head blight pathogen, <i>Fusarium graminearum</i>. Fungi use at least two separate systems to take up iron, one based on enzymatic iron reduction and the other based on the synthesis and secretion of small iron chelators termed siderophores. Using mutants disrupted in either of two modes of iron uptake, I establish that siderophore production is essential for full F. graminearum virulence on wheat. This thesis exposes iron as an important component of both plant defence and fungal virulence.

The family Adenoviridae consists of five genera, including the genus Aviadenovirus, which infects avian species. The genus Aviadenovirus currently comprises five fowl (Fowl adenovirus A-E), one falcon (Falcon adenovirus A), and one goose (Goose adenovirus) adenovirus species. Fowl adenoviruses (FAdVs) have a worldwide distribution. Some are associated with diseases such as inclusion body hepatitis (IBH), while FAdV species C serotype 4 (FAdV-4) has been associated with hydropericardium-hepatitis syndrome (HHS). In this study, the complete nucleotide sequence of fowl adenovirus serotype 8 (FAdV-8) was determined. The full genome was 44,055 nucleotides (nt) in length, with an organization similar to that of the FAdV-1 and FAdV-9 genomes. No regions homologous to early regions E1, E3, and E4 of mastadenoviruses were recognized Pathogenicity of FAdV-8 and FAdV-4 were studied in specific-pathogen-free chickens following oral and intramuscular inoculations. Pathogenicity was determined on the basis of clinical signs and gross and histological lesions. Additionally, virus shedding and viral genome copy numbers in liver, cecal tonsil, and bursa of Fabricius were determined. The role of interleukins (IL) in the pathogenicity of and immune response to FAdVs is unknown. Therefore, in a chicken experiment, interferon-γ, IL-10, IL-18, and IL-8 gene expression was evaluated following FAdV-8 and FAdV-4 infection. Cytokine gene expression was examined in the liver, spleen, and cecal tonsils. This study explored the ability of fowl adenoviruses to subvert the host cell’s secretion of cytokines in response to infection as an important viral mechanism for immune evasion during infection. Variations in virulence of FAdVs are likely to be determined by the fiber alone as shown by Pallister et al. (1996). Therefore, we compared and analyzed the nt and amino acid (aa) sequences of the fiber gene of pathogenic and non-pathogenic FAdVs representing species groups D (FAdV-11) and E (FAdV-8). According to our data, virulence might not be associated only with sequence of the fiber gene. This work is a continuation of our efforts towards better understanding of the molecular biology of FAdVs and the pathogenesis of the disease, with an emphasis on the role of interleukins, an unknown area.