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Submitted in complete fulfillment for the Degree of Master of Science (Applied Science), Durban University of Technology, Durban, South Africa, 2016. / β-xylosidases catalyse the hydrolyses of xylooligosaccharides into the monosaccharide sugar, xylose. In this study we report the production of xylose under different conditions in Pichia pastoris and Saccharomyces. cerevisiae, and its conversion to bioethanol using Pichia stipitis. The aim of this study was to change the characteristics of the A. niger 90196 β-xylosidase through random mutagenesis and increase expression under the control of different promoter systems in yeasts P. pastoris and S. cerevisiae. The recombinant library created through random mutagenesis was screened for changes in activity and subsequently pH and temperature stability. One variant showed an increase in enzyme expression, thermostability, and a change in amino acid sequence at residue 226. The enzyme was then cloned, expressed and characterized in P. pastoris GS115 and S. cerevisiae. β-xylosidase was constitutively expressed in P. pastoris using the GAP promoter and the inducible AOX promoter. In S. cerevisiae the enzyme was expressed using the constitutive PGK promoter and inducible ADH2 promoter systems. Enzyme functionality with the different expression systems was compared in both hosts. The GAP system was identified as the highest-producing system in P. pastoris, yielding 70 U/ml after 72 hours, followed by the PGK system in S. cerevisiae, with 8 U/ml. A 12% SDS-PAGE gel revealed a major protein band with an estimated molecular mass of 120 kDA, and the zymogram analysis revealed that this band is a fluorescent band under UV illumination, indicating enzyme activity. Stability characteristics was determined by expressing the enzyme at different pH and temperatures. Under the control of the GAP promoter in P. pastoris, enzyme activity peaked at pH4 while retaining 80% activity between pH 3 – 5. Highest activity of 70 U/ml xylosidase was recorded at 60ºC. Due to the high enzyme production in P. pastoris, the co-expression of this enzyme with a fungal xylanase was evaluated. The xylanase gene from Thermomyces lanuginosus was cloned with the GAP promoter system and expressed together with the β-xylosidase recombinant in P. pastoris. Enzyme activities of the co-expressed recombinant revealed a decrease in enzyme activity levels. The co-expressed xylanase production decreased by 26% from 136 U/ml to 100 U/ml while the xylosidase expression decreased 86% from 70 U/ml to 10 U/ml. The xylose produced from the hydrolysis of birchwood xylan was quantified by HPLC. The monosaccharide sugar was used in a separate saccharification and fermentation strategy by P. stipitis to produce bioethanol, quantified by gas chromatography. Bioethanol production peaked at 72 h producing 0.7% bioethanol from 10 g/l xylose. In conclusion a β-xylosidase from Aspergillus niger was successfully expressed in P. pastoris and was found to express large quantities of xylosidase, that has not been achieved in any prior research to date. The enzyme was also successfully co-expressed with a Thermomyces xylanase and is now capable of bioethanol production through xylan hydrolysis. This highlights potential use in industrial applications in an effort to reduce the world dependence on petroleum and fossil fuels. However the technical challenges associated with commercialization of bioethanol production are still significant. / M

Neste trabalho foi estudada a influência da forma de preparo do inóculo na morfologia do microrganismo e na produção de glicoamilase por Aspergillus awamori, em cultivo submerso. Foram realizados ensaios variando-se a concentração de esporos utilizada no preparo do inóculo para o fermentador (pré-cultivo de células em incubador rotativo a 200 rpm e 35 oC, por 24 horas) no intervalo entre 9,5 exp 03 e 1,8 exp 07 esporos/mL. Os inóculos preparados com concentração de esporos entre 9,5 exp 03 e 9,5 exp 05 esporos/mL apresentaram-se na forma de uma suspensão de “pellets" e conduziram a um crescimento na forma filamentosa, em fermentador. A produção de glicoamilase, nos ensaios com concentração inicial de substrato de 40 g/L, realizados em fermentador, não foi influenciada pela concentração de esporos nessa faixa, mantendo-se entre 2100 e 2200 U/L. O aumento da concentração de esporos utilizada no preparo do inóculo para 1,8 exp 07 esporos/mL conduziu à formação de um inóculo na forma filamentosa contendo muitos aglomerados de esporos não germinados, que levaram a um crescimento, em fermentador, na forma de “pellets", reduzindo a produção de glicoamilase para cerca de 1600 U/L e mostrando que o crescimento na forma de “pellets" não é indicado para a produção de glicoamilase. Foram estudadas, também, outras formas de preparo do inóculo, variando-se as condições de germinação dos esporos (pH e tempo de pré-cultivo do inóculo). A forma de preparo do inóculo que conduziu a uma maior produção de glicoamilase, em fermentador, foi o cultivo de esporos em incubador rotativo, a pH 2,5 durante 7 horas, o que evitou a aglomeração de esporos durante a etapa de germinação e a formação de “pellets" em fermentador, conduzindo a um crescimento na forma filamentosa e a uma alta produção de glicoamilase. / The influence of the inoculum preparation on Aspergillus awamori morphology and glucoamylase synthesis in submerged cultures has been investigated. A series of runs were performed, varying the spore concentration of the inoculum (inoculum size) in the range from 9.5 exp 03 to 1.8 exp 07 spores/mL. The inoculum was cultivated in shaker, at 35 oC and 200 rpm, for 24 hours. The inoculum prepared with a spore concentration in the range from 9.5 exp 03 to 9.5 exp 05 spores/mL was composed by a pellet suspension. This pellet suspension led to a filamentous growth in fermenter, but did not influence the glucoamylase production, which reached values from 2,100 to 2,200 U/L. The use of a higher spore concentration (1.8 exp 07 spores/mL) produced an inoculum composed by dispersed hyphae and many spores agglomerates, which led to pellet formation in the fermenter and reduced glucoamylase production to 1,600 U/L. Thus, pellet formation is not recommended in the process of glucoamylase synthesis. Some forms of inoculum preparation have been studied, varying spore germination conditions (pH and time of inoculum culture). The form of inoculum preparation that led to the highest glucoamylase activity in fermenter was the spore germination in shaker at pH 2.5 for 7 hours, which avoided pellet formation in the reactor and conducted to a high glucoamylase production.

The Crystal Structure of Sl Nuclease from Aspergillus oryzae S 1 nuclease from Aspergillus oryzae is a glycoprotein of 32 kDa molecular weight. The protein has two enzymatic activities: it is an endo-exonuclease with high specificity for single stranded nucleic acids, and it has an additional 3' -nucleotidase activity. S 1 nuclease is widely used in molecular biology as a single-strand specific nuclease due to its high stability and efficiency. It cleaves single-stranded regions of nucleic acids producing 5' -nucleotides without significant side-reactions. The crystal structure of S 1 nuclease has been determined to 1.7 A resolution by molecular replacement based on the known structure of PI nuclease from Penicillinum citrinum, which has 49 % sequence identity compared to S 1. The overall fold and the active site of S 1 nuclease is basically identical to that of PI nuclease, and also very similar to Phospholipase C from Bacillus cereus and alpha-toxin from Clostridium perfringens. The characteristic feature of this family of enzymes is a trinuclear zinc cluster in their active sites. A BLAST search in the sequence databases revealed several other protein sequences from bacteria, protozoa and plants possessing an approximately 30 % sequence identity compared to S 1 nuclease, but showing an almost complete conservation of structurally and functionally important residues. Soaking and co-crystallisation experiments with substrate analogues have been carried out in order to obtain an enzyme-substrate complex. These efforts have not resulted in the structure determination of any complexes under crystallisation conditions: no binding of substrate has been observed. Nevertheless, an enzyme mechanism has been proposed based on structural data of S 1 nuclease and nucleases with similar active sites. The Crystal Structure of an Sm-Related Protein from Archaeoglobus fulgidus In eukaryotes Sm and Sm-like proteins are the core components of the small nuclear ribonucleoprotein particles (snRNPs), which are involved in a variety of functions including rRNA processing, tRNA maturation and pre-mRNA processing. The Sm proteins are 70 to 120 amino acids long and share a common bi-partite signature sequence. The spliceosome, where the transesterification reaction of splicing occurs, is assembled by several snRNPs named after their constituting snRNA: U1, U2, U4, U5 and U6. An snRNA contains a short single stranded, uridine rich sequence motif, where the Sm proteins bind, but the three-dimensional arrangement of the Sm proteins and the mode of binding is unknown. In humans there are seven different canonical Sm proteins, which according to biochemical and electron microscopic studies seem to form a seven membered ring in vitro. Recently two crystal structures of human Sm protein dimers have been published. Interestingly Sm-related protein sequences have been found in the available genomic database of various Archaebacteria based on sequence homology. In contrast with eukaryotes only one or two Sm-related protein sequences have been identified in one organism. Their function is currently unknown, since analogous pre-mRNA splicing does not occur in Archaebacteria. Two Sm-related proteins of Archaeoglobus fulgidus have been cloned and expressed as fusion proteins. One of them called AF-Sm2 has been o crystallised utilising ammonium sulphate as precipitant and solved to 1.95 A resolution by SIRAS using a single mercury derivative. AF-Sm2 crystallises in a hexagonal space group (P6) and contains one molecule per asymmetric unit. The 77 residue long protein has a very similar fold compared to the solved human Sm protein structures: a short N-terminal a-helix followed by a five stranded, strongly bent, U-shaped ~-sheet resulting in a barrel-like overall fold. Six AF-Sm2 molecules form a ring in the crystal structure mediated by extensive hydrophobic and hydrogen-bonding interactions. Gel filtration experiments have indicated a pH dependence of oligomerisation in accordance with the crystallisation experiences. Currently the target of the Sm-related proteins of Archaeoglobus fulgidus and the stochiometry of oligomerisation in vivo is completely unknown.

Aspergillus fumigatus is increasingly recognised as a cystic fibrosis (CF) pathogen with reported infection rates up to 50% and associated with increased hospitalisations and lung deterioration. Research investigating immune responses of CF phagocytes against A. fumigatus has been scarce and the role of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in anti-Aspergillus immune responses is not known. The studies in this thesis aimed to explore innate immune responses by CF phagocytes against A. fumigatus. Peripheral blood mononuclear cells (PBMC), monocytes (MNC) and polymorphonuclear cells (PMN) were isolated from blood samples of CF patients with at least one copy of the F508del mutation. CF phagocytes efficiently cleared A. fumigatus similar to healthy controls. Microtubuleassociated proteins 1A/1B light chain 3B (LC3) expression was found to be attenuated in CF PMN and MNC, indicating a disbalance in the autophagy or LC3-associated phagocytosis pathway. Regarding inflammatory responses, it was found that upon phagocytosis a resting A. fumigatus conidia, CF phagocytes produce up to 4-fold more reactive oxygen species (ROS) compared to controls. The excessive ROS was then shown not to be necessary for adequate killing, suggesting the increased ROS to be redundant in the antifungal response. Patient metrics obtained from the clinic showed that the excessive ROS production correlated to exacerbations and lung function. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that CF PMN only express 20-25% of the 4 haemoglobin subunits compared to healthy controls. Combining the LC-MS data suggests that CF PMN are under hypoxic stress. In conclusion, the effective clearance of A. fumigatus by CF phagocytes comes at the cost of an excessive respiratory burst which correlates to disease severity. Our data indicate that CF PMN are under hypoxic stress while circulating in the blood stream, which is likely to contribute to the hyperinflammatory phenotype observed upon interaction with A. fumigatus.

Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. In healthy individuals, local pulmonary host defence mechanisms can efficiently eliminate the fungus without any overt symptoms. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. However, local host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control the invasive fungal disease. In different immunocompromised murine models, myeloid but not lymphoid cells were strongly recruited upon infection. Notably, neutrophils and macrophages were recruited to infected lungs in different immunosuppressed regimens. Other myeloid cells, particularly dendritic cells and monocytes were only recruited in the corticosteroid model after infection. Lymphoid cells, particularly CD4+ or CD8+ T-cells and NK cells were highly reduced upon immunosuppression and were not recruited after A. fumigatus infection. Importantly, adoptive CD11b+ myeloid cell transfer rescued immunosuppressed mice from lethal A. fumigatus infection. These findings illustrate that CD11b+ myeloid cells are critical for anti-A. fumigatus defence under immunocompromised conditions. Despite improved antifungal agents, invasive A. fumigatus lung infections cause a high rate morbidity and mortality in neutropenic patients. Granulocyte transfusions have been tested as an alternative therapy for the management of high-risk neutropenic patients with invasive A. fumigatus infections. To increase the granulocyte yield for transfusion, donors are treated with corticosteroids. Yet, the efficacy of granulocyte transfusion and the functional defence mechanisms of granulocytes collected from corticosteroid treated donors remain largely elusive. We aimed to assess the efficacy of granulocyte transfusion and functional defence mechanisms of corticosteroid treated granulocytes using mouse models. In this thesis, we show that transfusion of granulocytes from corticosteroid treated mice did not protect cyclophosphamide immunosuppressed mice against lethal A. fumigatus infection in contrast to granulocytes from untreated mice. Upon infection, increased levels of inflammatory cytokines helped to recruit granulocytes to the lungs without any recruitment defects in corticosteroid treated and infected mice or in cyclophosphamide immunosuppressed and infected mice that have received the granulocytes from corticosteroid treated mice. However, corticosteroid treated human or mouse neutrophils failed to form neutrophil extracellular traps (NETs) in in vitro and in vivo conditions. Further, corticosteroid treated granulocytes exhibited impaired ROS production against A. fumigatus. Notably, corticosteroids impaired the β-glucan receptor Dectin-1 (CLEC7A) on mouse and human granulocytes to efficiently recognize and phagocytize A. fumigatus, which markedly impaired fungal killing. We conclude that corticosteroid treatment of granulocyte donors for increasing neutrophil yields or patients with ongoing corticosteroid treatment could result in deleterious effects on granulocyte antifungal functions, thereby limiting the benefit of granulocyte transfusion therapies against invasive fungal infections. / Der Mensch kommt über die Atemluft in regelmäßigem Kontakt mit Sporen des saprophyitschen Pilzes Aspergillus fumigatus. Glücklicherweise eliminieren die lokalen Abwehrmechanismen der Lunge den Pilz in gesunden Individuen sehr effektiv und ohne offenkundige Symptome. In immunkomprimierten Patienten hingegen verursacht A. fumigatus verheerende Infektionen. Allerdings ist die lokale Immunreaktion gegen A.fumigatus-vermittelte Infektionen der Lunge unter immunsuppressiven Bedingungen immer noch nicht ausreichend definiert. In Anbetracht der dynamischen Veränderungen an Immunzellunterpopulationen im Gewebe nach immunsuppressiver Therapie haben wir die zeitliche und örtliche pulmonale Immunreaktion nach A. fumigatus infektion untersucht, um die grundlegenden immunologischen Geschehnisse aufzudecken, die in dieser Situation zur unzureichenden Kontrolle des Pilzes führen. In anderen immunsupprimierten Mausmodellen fand eine starke Rekrutierung myeloider Zellen nach Infektion statt. In besonderem Maße wurden nach der Infektion Neutrophile und Makrophagen in die Lunge immunsupprimierter Mäuse rekrutiert. Andere myeloide Zellen, insbesondere dendritische Zellen und Monozyten, wurden nur im Corticosteroid-Modell nach Infektion rekrutiert. Lymphoide Zellen, insbesondere CD4+ oder CD8+ Zellen und NK Zellen, waren nach Immunsuppression stark reduziert und wurden nach Infektion mit A. fumigatus nicht rekrutiert. Adoptiver Zelltransfer von CD11b+ myeloiden Zellen stellte die Abwehr immunsupprimierter Mäuse gegen A. fumigatus wieder her, was die wesentliche Bedeutung dieser Zellen in der Immunabwehr unterstreicht. Diese Erkenntnisse verdeutlichen, dass CD11b+ myeloide Zellen unter immunkomprimierten Bedingungen entscheidend für die Abwehr gegen A-fumigatus sind. ...

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published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

MP1, a gene previously identified in P. marneffei by cDNA library screening, encodes a secreted cell wall mannoprotein Mp1p. Thirteen MP1 homologues named MPLP1 to 13 were previously identified in P. marneffei by BLAST analysis. Two MP1 homologues namely AFMP1 and AFMP2 which encodes Afmp1p and Afmp2p were previously identified by expressed sequence tag library screening in Aspergillus fumigatus – an important fungal pathogen closely related to P. marneffei. Mp1p, Afmp1p and Afmp2p have previously been reported to be immunogenic. Mp1p was also reported to bind fatty acid and was suggested to contribute to virulence in a MP1 knockout P. marneffei strain in a mouse model and a cell line model although the Koch’s postulates has yet been met to establish MP1 as a novel virulence factor. With reference to sequence identity of Afmp proteins to Mp1p, Afmp proteins were speculated to have functions similar to Mp1p. BLAST searches against the A. fumigatus genome identified two novel AFMPs namely AFMP3 and AFMP4. Sequence analysis of Afmp3p and Afmp4p revealed the presence of putative N-terminal signal peptide and substantial sequence identity to Mp1p, Afmp1p and Afmp2p. Two MP1 knockdown P. marneffei mutants were constructed to demonstrate suppression of MP1 expression alone can result in loss of virulence and also the dosage effect of MP1 expression on P. marneffei virulence towards mice. Subsequent mice challenge experiments using MP1 like protein (MPLP) knockdown strains suggested MP1 to be the most important virulence factor among all its homologues in P. marneffei. Histopathology examinations of organs from challenged mice suggested survival disadvantages in mice for P. marneffei mutants with knockdown of MP1 and effect of MP1 on granuloma formation in infected mice. Mice challenge experiments using AFMP1 to 4 knockdown A. fumgiatus mutants suggested significant decrease in virulence of A. fumigatus upon AFMP4 knockdown and complete protection of challenged mice upon knockdown of AFMP1 to 4. Histopathology examinations of organs from challenged mice suggested survival disadvantages in mice for A. fumigatus mutants with knockdown of AFMPs and effect of AFMPs on granuloma formation in infected mice. Mice experiments using Pichia pastoris expressing MP1 or AFMP4 suggested the effects of MP1 and AFMP4 on virulence are not caused by factors specific to P. marneffei or A. fumigatus. It was shown using a human peripheral blood mononuclear cells model that Mp1p and Afmp4p confer intracellular survival advantage to P. marneffei and A. fumigatus upon infection. Expression of Mp1p or Afmp4p in P. pastoris also confers survival advantage to this nonpathogenic yeast in human peripheral blood mononuclear cells. Reduction in proinflammatory prostaglandin E2 production were noticed in human peripheral blood mononuclear cells infected by P. marneffei, A. fumigatus or P. pastoris strains that expressed Mp1p or Afmp4p. Such reduction in eicosanoids production also coincides with the inhibition of apoptosis as shown by enzyme activity of caspase-8, caspase-9 and caspase-3 in human peripheral blood mononuclear cells. These findings suggest two novel virulence factors – Mp1p and Afmp4p, which confer survival advantages to P. marneffei and A. fumigates respectively. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy