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Aspergillus fumigatus is an opportunistic fungus known to cause a group of life-threatening infections collectively known as aspergillosis. In this thesis, multilocus sequence and microsatellite markers, among others, were used to study global and local A. fumigatus population structures. We examined the roles of sexual and asexual reproduction in the initiation of azole resistance globally. Furthermore, we investigated the origin of multi-triazole resistance in India and whether the use of fungicides on farms propagates resistance in environmental strains of clinical importance in Hamilton, Ontario. We characterized for the first time the A. fumigatus population in Cameroon while concomitantly screening for environmental resistance. Our results showed that sexual reproduction plays a key role in the development of triazole resistance globally. We found that multi-triazole resistance in India has multiple origins, which include mutational, recombinational and exotic origins. Our results provided little to no evidence that azole fungicides are the origin, or increase the frequency of triazole resistance in clinical A. fumigatus in Hamilton. Additionally, we identified a significantly unique A. fumigatus population in Cameroon. Our findings will potentially contribute towards developing effective long-term management strategies against aspergillosis. / Thesis / Doctor of Philosophy (PhD) / Aspergillus fumigatus is a mold capable of causing severe infection in humans. Infections caused by A. fumigatus can often be treated using antifungals. However, there have been several reported cases of treatment failure around the world over the last two decades. Generally speaking, treatment failure in patients is often associated with antifungal resistance in A. fumigatus. My thesis aims at better understanding the distribution and investigating the origin of resistance in A. fumigatus at both global and local levels. Here, we analyze A. fumigatus strains from 15 countries, including strains from Hamilton, Ontario. Our findings will potentially contribute towards establishing effective long-term management strategies against A. fumigatus infections locally and globally.

Aspergillus fumigatus is the major etiology of invasive aspergillosis, a leading cause of death in immunocompromised patients. Septins are conserved GTPases that could be mediating host-spore interactions in A. fumigatus. To test this hypothesis, we are using a combination of microscopy techniques and cell culture-based methods. Using Atomic Force Microscopy, we found that spores from the ∆aspB strain have a disorganized rodlet layer compared to the parent strain. Disorganized rodlet layer can lead to an increase in immune recognition of common pathogen associated molecular patterns (PAMPs), chitin and β-glucan. It is unknown whether the increase in immune recognition of ∆aspB spores is due to more exposure to the host or if they total quantity of PAMPs has increased. To test this, we used several staining methods to compare total and exposed levels of PAMPs in the conidia. Our work shows that there is an increase in exposed chitin and total levels of chitin and β-glucan. To evaluate how this plays a role in vitro, we first exposed macrophage-like cells (J744.1) to septin deletion strain’s spores and measured the TNF-a to determine spore immunogenicity using an ELISA. As expected, only the deletions that abolish all septin complexes had a significant increase in TNF-a. Then, we determine how effective macrophages were at killing the spores. We found that the deletion strains were more susceptible to killing by the macrophages. A murine study revealed that ∆aspB infection results in more lung inflammation than the wildtype. Taken together, these results suggest the septin cytoskeleton is involved in A. fumigatus spore cell wall organization and immune evasion.

Depuis quelques années, un des défis de la Recherche est de valoriser les co-produits agro-industriels. Une des voies permettant la valorisation de ces « déchets » est la fermentation en milieu solide.Le but de ce travail est de produire de l'acide itaconique par des souches d'Aspergilli (Aspergillus itaconicus et Aspergillus terreus) à partir de ressources renouvelables. Le substrat choisi dans cette étude est le son de blé, coproduit largement disponible en Champagne-Ardenne.L'acide itaconique a été classé dans le TOP 12 des molécules plateformes par le Department Of Energy Américain. Ces molécules plateformes peuvent être produites à partir de biomasse ligno-cellulosique et peuvent être utilisées à la place de molécules d'origine pétrochimique.Dans notre étude, nous n'avons pas mis en évidence de production d'acide itaconique par la souche Aspergillus terreus NRRL 1960 mais nous avons observé, pour la première fois, la production d'acide fumarique par fermentation en milieu solide. L'acide fumarique est tout aussi intéressant que l'acide itaconique puisqu'il fait également partie du TOP 12 des molécules plateformes. La production maximale obtenue est de 0,44 mg/g de matière sèche par fermentation en milieu solide sur son de blé humidifié à 70% et à pH 3, après 5 jours d'incubation à 30°C.De plus, nous avons montré qu'Aspergillus itaconicus NRRL 161 est capable de produire 6,77 mg d'acide itaconique/g de matière sèche par fermentation en milieu solide sur son de blé humidifié à 60% par une solution de saccharose à 400 g/L et à pH 3, après 4 jours d'incubation à 30°C.Dans une dernière partie, nous avons mis en évidence, chez Aspergillus itaconicus NRRL 161, la présence potentielle du gène codant pour la Cis-Aconitic acid Decarboxylase, enzyme clé dans la production d'acide itaconique. / Since a few years, one of research's challenges is to valorise agro-industrial by-products. One of the ways permitting the valorisation of these “wastes” is solid-state fermentation.The aim of this work is to produce itaconic acid with Aspergilli (Aspergillus itaconicus and Aspergillus terreus) strains from renewable resources. The chosen substrate in this study is wheat bran, by-product widely available in Champagne-Ardenne.Itaconic acid is classified among the TOP 12 of building blocks by the American Department Of Energy. Building blocks can be produced from ligno-cellulosic biomass and can be used instead of petrochemical-based molecules.In our study, we have not highlighted itaconic acid production by Aspergillus terreus NRRL 1960, but we have observed fumaric acid production by solid state fermentation. Fumaric acid is as interesting as itaconic acid since it also belongs to the TOP 12 of building blocks. Maximal production of fumaric acid is 0.44 mg/g dry matter by solid-state fermentation on wheat bran moistened at 70% and at pH 3, after 5 days of incubation at 30°C.Furthermore, we have shown that Aspergillus itaconicus NRRL 161 is able to produce 6.77 mg of itaconic acid/g dry matter by solid state fermentation on wheat bran moistened at 60% with sucrose solution at 400 g/L and at pH 3, after 4 days of incubation at 30°C.In a last part, we have highlighted, in Aspergillus itaconicus NRRL 161, the potential presence of the Cis-Aconitic acid Decarboxylase encoding gene, key enzyme in itaconic acid production.

by Wong Yuk Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 110-114). / Abstract --- p.i / Acknowledgement --- p.iv / Dedication --- p.v / Table of Contents --- p.vi / List of Tables --- p.xi / List of Figures --- p.xii / Chapter Chapter 1 --- Introduction and literature review / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Literature review --- p.5 / Chapter 1.2.1 --- General considerations --- p.5 / Chapter 1.2.2 --- Nature of glucosinolate --- p.6 / Chapter 1.2.3 --- Degradation of glucosinolates by myrosinase --- p.7 / Chapter 1.2.4 --- Toxicology of glucosinolate and hydrolysis products --- p.8 / Chapter 1.2.5 --- Plant myrosinase --- p.9 / Chapter 1.2.6 --- Fungal myrosinase --- p.11 / Chapter 1.2.7 --- Purification and properties of fungal myrosinase --- p.11 / Chapter Chapter 2 --- Screening of fungi with myrosinase activity and physiological studies of myrosinase production in Aspergillus oryzae / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and methods --- p.16 / Chapter 2.2.1 --- Fungal strains --- p.16 / Chapter 2.2.2 --- Media --- p.16 / Chapter 2.2.3 --- Screening --- p.17 / Chapter 2.2.4 --- Enzyme assay and protein determination --- p.18 / Chapter 2.2.4.1 --- Myrosinase assay --- p.18 / Chapter 2.2.4.2 --- Definition of myrosinase unit and specific activity --- p.19 / Chapter 2.2.4.3 --- Protein determination --- p.19 / Chapter 2.2.5 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.19 / Chapter 2.2.5.1 --- Incubation time --- p.20 / Chapter 2.2.5.2 --- Inducer concentration --- p.20 / Chapter 2.3 --- Results --- p.21 / Chapter 2.3.1 --- Screening --- p.21 / Chapter 2.3.1.1 --- Degradation of sinigrin in culture medium --- p.21 / Chapter 2.3.1.2 --- Confirmation of myrosinase activity --- p.21 / Chapter 2.3.2 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.21 / Chapter 2.3.2.1 --- Incubation time --- p.21 / Chapter 2.3.2.2 --- Inducer concentration --- p.22 / Chapter 2.4 --- Discussion --- p.23 / Chapter 2.4.1 --- Fungi selection in screening programme --- p.23 / Chapter 2.4.2 --- Medium composition --- p.23 / Chapter 2.4.3 --- Screening --- p.24 / Chapter 2.4.4 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.25 / Chapter 2.4.4.1 --- Incubation time --- p.25 / Chapter 2.4.4.2 --- Inducer concentration --- p.25 / Chapter Chapter 3 --- Purification and characterization of myrosinase in Aspergillus oryzae / Chapter 3.1 --- Introduction --- p.33 / Chapter 3.2 --- Materials and methods --- p.35 / Chapter 3.2.1 --- Reagents --- p.35 / Chapter 3.2.2 --- Fungal propagation --- p.35 / Chapter 3.2.3 --- Purification of the fungal myrosinase --- p.36 / Chapter 3.2.3.1 --- Preparation of crude extract --- p.36 / Chapter 3.2.3.2 --- Dialysis --- p.37 / Chapter 3.2.3.3 --- DEAE-Sepharose CL-6B ion-exchange chromatography --- p.37 / Chapter 3.2.3.4 --- Sephacryl S-200 molecular sieving chromatography --- p.37 / Chapter 3.2.3.5 --- FPLC Phenyl Superose hydrophobic interaction chromatography --- p.38 / Chapter 3.2.3.6 --- FPLC Mono P chromatofocusing --- p.38 / Chapter 3.2.4 --- Myrosinase assay and protein concentration determination --- p.39 / Chapter 3.2.4.1 --- Spot test for myrosinase activity --- p.39 / Chapter 3.2.4.2 --- Standard end-point assay --- p.40 / Chapter 3.2.4.3 --- Determination of protein concentration --- p.42 / Chapter 3.2.5 --- Physicochemical characterization of the myrosinase isozymes --- p.42 / Chapter 3.2.5.1 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.42 / Chapter 3.2.5.2 --- Protein staining and glycoprotein detection --- p.43 / Chapter 3.2.5.3 --- Chromatofocusing --- p.43 / Chapter 3.2.5.4 --- Gel filtration with FPLC Superose 6 --- p.44 / Chapter 3.2.6 --- Enzymatic properties --- p.44 / Chapter 3.2.6.1 --- Effect of pH on crude enzyme stability --- p.44 / Chapter 3.2.6.2 --- Effect of substrate concentration on enzyme activity --- p.45 / Chapter 3.2.6.3 --- Effect of pH on enzyme activity --- p.45 / Chapter 3.2.6.4 --- Effect of temperature on enzyme activity --- p.46 / Chapter 3.2.6.5 --- Effects of metallic ions on enzyme activity --- p.46 / Chapter 3.2.6.6 --- Effects of various compounds on enzyme activity --- p.46 / Chapter 3.2.6.7 --- Effects of various buffers on enzyme activity --- p.47 / Chapter 3.3 --- Results --- p.48 / Chapter 3.3.1 --- Fungal propagation --- p.48 / Chapter 3.3.2 --- Purification of fungal myrosinase in Aspergillus oryzae --- p.48 / Chapter 3.3.2.1 --- Extraction of the enzyme --- p.48 / Chapter 3.3.2.2 --- Dialysis --- p.49 / Chapter 3.3.2.3 --- DEAE-Sepharose ion-exchange chromatography --- p.49 / Chapter 3.3.2.4 --- Sephacryl S-200 molecular sieving chromatography --- p.50 / Chapter 3.3.2.5 --- FPLC Phenyl Superose hydrophobic interaction chromatography --- p.50 / Chapter 3.3.2.6 --- FPLC Mono P chromatofocusing --- p.51 / Chapter 3.3.3 --- Physicochemical characterization --- p.52 / Chapter 3.3.3.1 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.52 / Chapter 3.3.3.2 --- Chromatofocusing --- p.53 / Chapter 3.3.3.3 --- Gel filtration --- p.53 / Chapter 3.3.4 --- Enzymatic properties --- p.53 / Chapter 3.3.4.1 --- Effect of pH on the crude enzyme stability --- p.53 / Chapter 3.3.4.2 --- Effect of substrate concentration on enzyme activity --- p.54 / Chapter 3.3.4.3 --- Effect of pH on enzyme activity --- p.54 / Chapter 3.3.4.4 --- Effect of temperature on enzyme activity --- p.55 / Chapter 3.3.4.5 --- Effects of metallic ions on enzyme activity --- p.55 / Chapter 3.3.4.6 --- Effects of various compounds on enzyme activity --- p.56 / Chapter 3.3.4.7 --- Effects of various buffers on enzyme activity --- p.57 / Chapter 3.4 --- Discussion --- p.58 / Chapter 3.4.1 --- Purification of Aspergillus oryzae myrosinase --- p.58 / Chapter 3.4.1.1 --- Dialysis --- p.58 / Chapter 3.4.1.2 --- Enzyme purification --- p.58 / Chapter 3.4.2 --- Physicochemical properties --- p.60 / Chapter 3.4.2.1 --- Glycoprotein --- p.60 / Chapter 3.4.2.2 --- Molecular weights --- p.60 / Chapter 3.4.2.3 --- Isoelectric points --- p.61 / Chapter 3.4.3 --- Enzymatic properties --- p.61 / Chapter 3.4.3.1 --- pH and temperature optima --- p.61 / Chapter 3.4.3.2 --- Substrate affinity --- p.62 / Chapter 3.4.3.3 --- Inhibitions by various compounds and metallic ions --- p.63 / Chapter 3.4.3.4 --- Inhibitions by various buffer systems --- p.64 / Chapter Chapter 4 --- Summary --- p.106 / References --- p.110

Asthma is a common chronic inflammatory condition which affects over 300 million people worldwide. Thickening of the subepithelial layer is a key feature of asthmatic airways and the extent of thickening has been correlated with severity of asthma and increased exacerbations. Recent epidemiological studies have shown a link between fungal sensitisation primarily with Aspergillus fumigatus (A. fumigatus) and exacerbations of asthma leading to increased morbidity and mortality. The airway epithelium acts as an initial defence barrier to inhaled allergens such as A. fumigatus and emerging evidence suggests that as well as orchestrating an allergic immune response, it initiates aspects of airway wall remodelling including subepithelial thickening. However, induction of a profibrogenic response by the airway epithelium following exposure to inhaled fungi associated with severe asthma has not been well documented. The epithelial expression and production of the profibrotic growth factors, TGF-β1, TGF-β2, IL-6, endothelin-1 and periostin, selected as implicated in the aetiology of asthma and their profibrotic activity, were investigated in response to both A. fumigatus spores and culture filtrate in vitro. Furthermore, in vivo chronic inhalation models using either live spores or culture filtrate from two different strains of A. fumigatus (AF293 and CEA10) were used to determine the ability of the fungi to induce murine airway wall remodelling. In vitro, spores from both strains were able to induce the expression and production of IL-6 and endothelin-1 from human bronchial epithelial cells but none of the other profibrotic growth factors. In vivo, despite spores from both strains inducing expression and production of IL-6 and endothelin-1, only CEA10 spores caused significant subepithelial collagen deposition however, both strains induced α-SMA, a myofibroblast and smooth muscle marker around the airways. As a secreted factor was suspected of driving airway wall remodelling, subsequent studies used culture filtrate produced by the two strains, AF293, a low and CEA10, a high protease producer in basal medium. Only AF293 culture filtrate induced IL-6 and endothelin-1 from human bronchial epithelial cells in vitro. However, in vivo, culture filtrate from both strains was able to induce IL-6 and endothelin-1 expression, with AF293 causing a more profound subepithelial collagen deposition and significantly increased α-SMA abundance. It was hypothesised that epithelial-derived endothelin-1 drives airway wall remodelling and hence Endothelin receptor A was inhibited in the in vivo culture filtrate inhalation model. A significant reduction in subepithelial collagen deposition and α-SMA localisation around the airways was demonstrated in mice receiving an Endothelin receptor A antagonist compared with culture filtrate alone. This thesis indicates that A. fumigatus exposure can drive features of airway wall remodelling such as subepithelial fibrosis possibly through the epithelial production of profibrotic growth factor, endothelin-1.

Invasive aspergillosis (IA) is associated with high mortality rates and can be difficult and expensive to treat with current drugs. The drugs used to treat IA are also associated with undesirable, and often severe, side-effects of the patient. The main causative agent of this disease is the opportunistic pathogen Aspergillus fumigatus. This study identifies genes which play a role in a fungal-specific type of programmed cell death (PCD) in A. fumigatus, known as heterokaryon incompatibility. The development of drugs specifically targeting the products of these genes could lead to fewer side-effects than those arising from currently available anti-fungal drugs. The drug amphotericin B is currently used to treat IA and has been shown to induce an apoptotic-like phenotype in A. fumigatus; however, the sterols targeted are present in both fungal and mammalian cell membranes. HI is a fungal-specific self/non-self recognition system that results in rapid compartmentalisation and cell death of hyphal fusion sites if the two fusing fungi are not genetically compatible. The HI system could be exploited as a novel drug target against invasive fungal pathogens through targeting a component of the molecular pathway to induce cell death. In contrast to current drugs, novel drugs could target HI components to induce PCD without affecting non-desirable targets that cause side-effects. The non-self recognition systems used by Neurospora crassa, Aspergillus Nidulans and Podospora anserina are the well characterised, and they each differ significantly in their modes of action. BLAST searches found 30 homologues of HI genes from other the systems of characterised species in A. fumigatus, with 8 containing the fungal-specific het domain. The first assay to determine whether disruption of het genes could affect HI was to observe the barrage phenotype between incompatible A. fumigatus individuals. However, there was no barrage visible as the leading edge of colonies stopped growing when in close proximity to another colony. Instead, nitrate non-utilising (Nit) A. fumigatus mutant strains were generated using chlorate and pair-wise crosses of 46 environmentally and clinically isolates on nitrate-containing media resulted in the formation of 16 viable heterokaryons. All of the heterokaryons fell into exclusive compatibility groups where no intergroup crossing was possible. Homologous recombination was used to disrupt five of the identified het domain genes with gene replacement cassettes, generated through fusion-PCR, in an akuB(KU80Delta) A. fumigatus strain. The mutant strains displayed both detrimental growth on standard agar growth media and reduced ability to recognise non-self strains. Full and partial heterokaryons were formed during intergroup pair-wise compatibility crosses using the mutants and strains that the akuB(KU80Delta) parent strain was previously incompatible with. This was followed with a non-bias approach of gene disruption using the Fusarium oxysporum impala160 transposable element in a Nit A. fumigatus mutant. Inducing transposon mutagenesis through exposure to low temperature generated a mutant library of spores in which the transposon had disrupted different open reading frames at different locations across the A. fumigatus genome. The mutant spore library was also screened for the ability to form viable intergroup heterokaryons with strains belonging to different compatibility groups. PCR recovery and DNA sequencing was able to identify the locus of impala160 in three isolates able to form viable heterokaryons. The sequences revealed the transposable element had disrupted the same gene, AFUA_2G05070, in each of the three isolates. This gene encodes an uncharacterised conserved hypothetical protein which may be a critical component for non-self recognition in A. fumigatus HI, and a potential target for novel anti-fungal drugs to induce PCD.