Integral to the physiological and biochemical properties of the plant, the primary cell wall (PCW) is of great economical interest. Transglycanases are a class of cell-wall remodelling enzymes hypothesised to be involved – among other functions – in cellular elongation and strengthening of the PCW. At present only four transglycanases have been convincingly characterised but the potential existence of many more is likely. To detect potential novel transglycanase activity, broad spectrum fluorescent and radioactive assays were conducted using a variety of potential donor and acceptor substrates. Enzyme extracts were sourced from a range of plants that represented the majority of the plant kingdom. Beansprout extracts reproducibly displayed significant incorportation of radioactivity and fluorescence when incubated with an α-arabinan or β- galactan donor and labelled xyloglucan oligosaccharide (XGO) acceptor. However, further analysis indicated the presence of xyloglucan contamination in donor polysaccharides and thus the activity observed was xyloglucan endotransglucosylase (XET). It has been hypothesised in the literature that linkages formed between the hemicellulosic and pectic matrices may be due to the activity of a transglycanase. This study has found no evidence to support this. In addition, during identification of the gene responsible for mixed-linkage β- (1,3),(1,4)-glucan : xyloglucan endotransglycosylase (MXE) activity – observed in Equisetum – a heterologous Pichia pastoris expression system was developed allowing the synthesis of a novel recombinant hetero-transglycanase (HTG) conferring predominant MXE activity and of five previously unstudied recombinant XET-active xyloglucan endotransglycosylase/ hydrolases (XTHs).

By serial subcultivation on media containing egg-white lysozyme a highly resistant variant of M. lysodeikticus bacteriophage was selected. The parent strain was sensitive to 0.1 pg per ml. lysozyme while the variant was resistant to 4000 pg per ml. The two strains were examined in detail and it was found that the resistant strain differed genotypicelly from the sensitive strain in its ability to acetylate certain cell wall hydroxyl groups. This was the sole reason for lysozyme resistance. It was found that deacetylation of the resistant strain by ample chemical treatment restored its sensitivity to lysozyme while acetylation of the sensitive strain rendered it resistant.

The dematiaceous (melanised) fungus Lomentospora (Scedosporium) prolificans is a life-threatening opportunistic pathogen of immunocompromised humans, resistant to anti-fungal drugs. Melanin has been shown to protect human pathogenic fungi against antifungal drugs, oxidative killing and environmental stresses. To determine the protective role of melanin in L. prolificans to oxidative killing (H2O2), UV radiation and the polyene anti-fungal drug amphotericin B, targeted gene disruption was used to generate mutants of the pathogen lacking the dihydroxynaphthalene (DHN)-melanin biosynthetic enzymes polyketide synthase (PKS1), tetrahydroxynapthalene reductase (4HNR) and scytalone dehydratase (SCD1). Infectious propagules (spores) of the wild-type strain 3.1 were black/brown, whereas spores of the PKS-deficient mutant ΔLppks1::hph were white. Complementation of the albino mutant ΔLppks1::hph restored the black-brown spore pigmentation, while the 4HNR-deficient mutant ΔLp4hnr::hph and SCD-deficient mutant ΔLpscd1::hph both produced orange-yellow spores. The mutants ΔLppks1::hph and ΔLp4hnr::hph showed significant reductions in spore survival following H2O2 treatment, while spores of ΔLpscd1::hph and the ΔLppks1::hph complemented strain ΔLppks1::hph:PKS showed spore survivals similar to strain 3.1. Spores of the mutants ΔLp4hnr::hph and ΔLpscd1::hph and complemented strain ΔLppks1::hph:PKS showed spore survivals similar to 3.1 following exposure to UV radiation, but survival of ΔLppks1::hph spores was significantly reduced compared to the wild-type strain. Strain 3.1 and mutants ΔLp4hnr::hph and ΔLppks1::hph:PKS were resistant to amphotericin B while, paradoxically, the PKS1- and SCD1-deficient mutants showed significant increases in growth in the presence of the antifungal drug. Melanin was shown to play no role in the protection of the pathogen from immune cell recognition and killing by alveolar macrophages, with similar degrees of engulfment, and spore viabilities, of mutant and wild-type strains after phagocytosis. Contrary to expectations, the albino PKS-deficient mutant was significantly more virulent than the melanised wild-type strain during pathogenicity studies in the invertebrate infection model Galleria mellonella, with levels of virulence restored to near wild-type levels in the complemented strain ΔLppks1::hph:PKS. Taken together, the results presented in this thesis show that melanin protects L. prolificans from UV radiation and from oxidative killing by H2O2, consistent with its survival in extreme environmental habitats. However, melanin was 3 not found to play a role in the resistance of the pathogen to the antifungal drug amphotericin B, to protect the fungus from immune cell recognition or killing by alveolar macrophages, or to its pathogenicity.

No description available.

Lipoarabinomannan (LAM) and the mycolyl-arabinogalactan (mAG) complex are two major entities found in the cell wall of Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans. Given their important roles in the viability and virulence of the pathogen, enzymes involved in these pathways represent a rich source of potential therapeutic drug targets. As fundamental understanding of substrateenzyme interactions is often essential in the drug discovery process, the purpose of this study was to investigate the substrate specificities of an -(16)-mannosyltransferase (ManT) and a -(15,6)-galactofuranosyltransferase (GlfT2), two key enzymes involved in the biosynthesis of LAM and mAG, respectively. Although the ManT activity had been detected using an established radioactive assay, its substrate specificity remained poorly defined. The current study focused on the design, synthesis and evaluation of acceptor substrate analogs of ManT. Among those analogs prepared were those containing methoxy-, hydrogen-, and amino-substituted carbohydrate residues as well as epimeric derivatives. A homologous series of oxygen- and sulfur-linked mannosides were also prepared. Evaluation of these analogs revealed the steric requirements and hydrogen bonding interactions of the enzyme, and the effect of acceptor length on mannosyltransferase activity. Also, these results provided additional insight into the role of ManTs and allowed the current proposed pathway of LAM to be further revised. Another objective of the current study was to understand how GlfT2 catalyzes the alternating -(15) and -(16)-galactofuranosyl transfers in a single active site. A panel of mono- and dideoxy trisaccharide derivatives was synthesized, in which hydroxyl groups at either or both C-5 and C-6 positions on the sugar residues at the reducing ends were selectively removed. Biological evaluation of these analogs using a spectrophotometric assay, and structural analysis of some of the enzymatic products, showed that the removal of the hydroxyl group(s) in the acceptors appeared to have no dramatic effect on either GlfT2 activity or the regioselectivity of its galactosylation. These results suggest that groups other than the C-5 and C-6 hydroxyl groups of the acceptors are more critical for the enzyme catalysis. The identification of these key elements would be the further objective of this project. The results from these fundamental studies provide important information about how these enzymes interact with their substrates at the molecular level. More importantly, this work will serve as the basis for the further design of potential inhibitors, which are potential lead compounds for novel therapeutic agents that are active against tuberculosis.

Abstract In silico analysis showed that Arabidopsis thaliana gene AT5G09530 encodes a uniquely repetitive, proline-enriched protein that is conserved across species, and is likely secreted to the cell wall. Based on its most common amino acid repeat motif, I named the gene PELPK1 and its putative paralog PELPK2 (AT5G09520). Reporter (GUS) expression showed that the PELPK1 upstream genomic region is sufficient for expression in the aleurone layer during seed germination, and is induced throughout the plant by biotic factors (especially Pseudomonas syringae infection), defense chemicals (MeJa, salicylic acid), and mechanical wounding, consistent with the presence of conserved regulatory elements. Sub-cellular localization of a translational fusion of PELPK1 with GFP showed that the protein was secreted into seed-coat aleurone cells and to the cell walls of other tissues. Based on these results, it was concluded that the PELPK1 is a cell wall-associated protein and is most actively transcribed during radicle penetration of the seed coat and during pathogen and wounding responses. A proteomic survey of aleurone proteins failed to identify PELPK1, although several proteins not previously associated with this tissue were identified. Mutational analysis demonstrated that RNAi silencing significantly down-regulated the transcript abundance of PELPK1. Phenotypic analysis showed that RNAi plants exhibited significantly slower germination and root growth when the medium was supplemented with sucrose (100mM). Conversely, constitutive overexpression (OX) of PELPK1 enhanced seed germination and root elongation as compared to wild-type (WT). Analysis of soil-grown plants showed slower emergence and slower vegetative growth for RNAi lines, while OX plants exhibited faster emergence and enhanced vegetative growth and flowering as compared to WT. However, PELPK1 RNAi and OX lines did not differ from WT in response to treatment with pathogens. These results show that the abundance of PELPK1 is positively correlated with plant growth rate under some conditions. PELPK1 may influence growth through CW modification or other independent pathways. / Plant Biology

The cell wall is a multifunctional structure which is implicated in plant growth and development as well as responding to any environmental changes including biotic and abiotic stresses. One of the practical approaches in cell wall integrity studies is the modification of the quality and quantity of particular cell wall components or destroying the specific step in cell wall synthesis pathway using Cellulose Biosynthesis Inhibitors (CBIs). In this case, chemical screen for swollen organ phenotype has proved to be an important technique to identify the genes that are directly or indirectly involved in cellulose biosynthesis. In the present research, a number of synthetic CBIs were obtained through a chemical library screen from Chembridge Company for the root swollen phenotype which is believed to be the response to a defect in cellulose biosynthesis. Therefore, a genome-wide expression profiling based on Affymetrix ATH1 GeneChip arrays (contains 22810 probe sets) were applied to investigate the altered transcriptome of four different CBIs including CBI-15, 18, 22, and 27 and isoxaben in 5 day-old Arabidopsis thaliana seedlings. The results of this project revealed overlapped up and down-regulated genes as well as discriminate responses to each CBI. The most striking modification were found in genes involve in response to the stress as well as cell wall integrity and restructuring. Thus, the identification of regulated genes under CBIs treatment suggests a robust candidate group of genes that likely to be correlated to cell wall biosynthesis. / UOIT

Human systemic fungal infections are increasing, and causing high morbidity and mortality. Treatment is challenging because fungi share many metabolic pathways with mammals. Current antifungals are losing effectiveness due to drug resistance. In immunocompromised patients Aspergillus fumigatus causes systemic aspergillosis, the most important airborne fungal disease. Mortality from aspergillosis exceeds 50% even with aggressive treatment. We need novel antifungal drug targets. Fungal cell wall components are promising targets for antifungal therapy as they are essential for fungi and absent from humans. The sugar galactofuranose (Galf) is a 5-memberd ring form of galactose that is found in the cell walls of many fungi, but not in mammals. I used molecular biology and microscopy techniques to characterize Galf biosynthesis enzymes in the model species A. nidulans. I studied three enzymes that catalyze sequential steps in Galf biosynthesis: UgmA, UgtA and UgeA. UDP-galactopyranose mutase (UgmA) creates UDP-galactofuranose (UDP-Galf) from UDP galactopyranose (UDP-Galp) in the cytoplasm. The UDP-Galf transporter (UgtA) moves UDP Galf into membrane bound organelles for incorporation into cell wall compartments. Upstream of UgmA, UDP-glucose/galactose epimerase (UgeA) interconverts UDP-glucose into UDP-Galp, the UgmA substrate. Neither UgmA nor UgtA has a human counterpart; UgeA is in the Leloir galactose metabolism pathway that found in many organisms from bacteria to humans. None of UgeA, UgmA and UgtA is essential for viability of A. nidulans, but deleting any one of them substantially reduces colony growth and sporulation (Figure i). Wild type and Galf defective strains (ugeA∆, ugmA∆ and ugtA∆) were quantified for colony growth, cell morphometry, spore formation and germination, as well as wall architecture. The abundance of these proteins was regulated using the alcA promoter. Galf content was assessed by immunolocalization in the Galf defective strains, showing that those strains lacked immunodetectable Galf. Gene products were localized with fluorescent protein tags; both UgmA and UgeA were cytoplasmic, whereas UgtA was Golgi localized. Wall surfaces were imaged and force-probed using transmission electron microscopy and atomic force microscopy. Overall, Galf deletion strains had aberrant wall maturation, and poorly consolidated surfaces. Our results indicate that Galf is necessary for abundant sporulation, wild type growth and full maturation of Aspergillus cell wall. Galf deletion strains were assessed for sensitivity to antifungal agents in clinical use. They were significantly more sensitive to caspofungin and amphotericin B that target cell wall synthesis and cell membrane chemistry, respectively. Thus, anti-Galf drugs (once created) may be useful in combination with existing antifungal drugs. In summary, Galf biosynthesis pathway appears to be promising as an antifungal drug development target.

Growth anisotropy, in which cells grow predominantly in one direction, is common in plant cells, and an essential event for plant form and function. The direction and degree of growth anisotropy are governed by the mechanical properties of the primary cell wall. When aligned in a parallel manner, cellulose microfibrils accommodate great resistance in the direction of their alignment to expansion driven by isotropic turgor pressure. Using the Arabidopsis thaliana inflorescence stem as a model system, field emission scanning electron microscopy (FESEM) analysis demonstrated that the establishment of parallel arrangement of microfibrils is closely correlated with anisotropic cell expansion. In the novel anisotropy 1 (any1) mutant allele of the primary cellulose synthase CesA1, growth defects were correlated with random cellulose microfibril patterns in some inflorescence stem tissues. Microtubules have been considered to be the most likely candidates for controlling the orientation of cellulose microfibrils. Recent studies have indeed demonstrated a close association of the plasma membrane-localized cellulose-synthase-complexes (CSCs) that produce cellulose and cortical microtubules. Despite this close association, microtubule disruption did not cause cellulose microfibrils to lose parallel alignment in the radial and inner periclinal walls of cells in the inflorescence stem, suggesting that microtubules influence mechanical properties of cellulose microfibrils other than orientation. X-ray diffraction analysis demonstrated that cellulose crystallinity in wild-type plants declines at the growth-promoting temperature of 29°C, whereas crystallinity fails to adapt and remains high in mor1-1, the temperature-sensitive mutant whose microtubule arrays become disorganized at its restrictive temperature (29°C). This finding suggests that organized microtubules are involved in reducing cellulose crystallinity that normally accompanies increased cell expansion. Live-cell imaging of CSCs by tracking a yellow fluorescent protein (YFP)-tagged CesA6 subunit in hypocotyl cells demonstrated that dynamic and well-organized microtubules affect the velocity, the direction of movement, and the density of CSCs, suggesting that there is a close relationship between microtubules and CSCs. Together with the finding that microtubules also control the distribution of COBRA, a GPI-anchored wall protein that is essential for growth anisotropy, I discuss the variety of roles microtubules play in anisotropic growth.

Human systemic fungal infections are increasing, and causing high morbidity and mortality. Treatment is challenging because fungi share many metabolic pathways with mammals. Current antifungals are losing effectiveness due to drug resistance. In immunocompromised patients Aspergillus fumigatus causes systemic aspergillosis, the most important airborne fungal disease. Mortality from aspergillosis exceeds 50% even with aggressive treatment. We need novel antifungal drug targets. Fungal cell wall components are promising targets for antifungal therapy as they are essential for fungi and absent from humans. The sugar galactofuranose (Galf) is a 5-memberd ring form of galactose that is found in the cell walls of many fungi, but not in mammals. I used molecular biology and microscopy techniques to characterize Galf biosynthesis enzymes in the model species A. nidulans. I studied three enzymes that catalyze sequential steps in Galf biosynthesis: UgmA, UgtA and UgeA. UDP-galactopyranose mutase (UgmA) creates UDP-galactofuranose (UDP-Galf) from UDP galactopyranose (UDP-Galp) in the cytoplasm. The UDP-Galf transporter (UgtA) moves UDP Galf into membrane bound organelles for incorporation into cell wall compartments. Upstream of UgmA, UDP-glucose/galactose epimerase (UgeA) interconverts UDP-glucose into UDP-Galp, the UgmA substrate. Neither UgmA nor UgtA has a human counterpart; UgeA is in the Leloir galactose metabolism pathway that found in many organisms from bacteria to humans. None of UgeA, UgmA and UgtA is essential for viability of A. nidulans, but deleting any one of them substantially reduces colony growth and sporulation (Figure i). Wild type and Galf defective strains (ugeA∆, ugmA∆ and ugtA∆) were quantified for colony growth, cell morphometry, spore formation and germination, as well as wall architecture. The abundance of these proteins was regulated using the alcA promoter. Galf content was assessed by immunolocalization in the Galf defective strains, showing that those strains lacked immunodetectable Galf. Gene products were localized with fluorescent protein tags; both UgmA and UgeA were cytoplasmic, whereas UgtA was Golgi localized. Wall surfaces were imaged and force-probed using transmission electron microscopy and atomic force microscopy. Overall, Galf deletion strains had aberrant wall maturation, and poorly consolidated surfaces. Our results indicate that Galf is necessary for abundant sporulation, wild type growth and full maturation of Aspergillus cell wall. Galf deletion strains were assessed for sensitivity to antifungal agents in clinical use. They were significantly more sensitive to caspofungin and amphotericin B that target cell wall synthesis and cell membrane chemistry, respectively. Thus, anti-Galf drugs (once created) may be useful in combination with existing antifungal drugs. In summary, Galf biosynthesis pathway appears to be promising as an antifungal drug development target.