Tooth decay is a serious health risk and a significant contributor to health care costs in both industrialized and developing nations. Tooth decay is the end result of a change in the balance of plaque ecology towards more acidogenic and aciduric bacterial species. The primary force facilitating this change is an increase in the amount and frequency of simple carbohydrate, in particular, sucrose ingestion by the host. The acidity of plaque increases after host ingestion of fermentable carbohydrates and this promotes demineralization of the tooth enamel which is restored by salivary buffering and mineral deposition. Frequent and prolonged periods of low plaque pH drive cycles of enamel homeostasis towards demineralization, which ultimately leads to the formation of dental caries. Streptococcus mutans is the main etiologic agent in the development of dental caries. The cariogenic potential of S. mutans is based on their ability to produce and tolerate large amounts of acid and to adhere to and accumulate large numbers on the surface of a tooth. They are capable of efficiently fermenting a variety of simple carbohydrates and can produce high concentrations of acid, even in a low pH environment. However, it is the ability of S. mutans to rapidly synthesize copious amounts of water-insoluble and water-soluble glucan from dietary sucrose, which allow the bacteria to accumulate large enough numbers to dominate the dental plaque and significantly lower the plaque pH. Synthesis of glucan is mediated by glucosyltransferase enzymes and is crucial to sucrose-dependent adherence and to the cariogenicity of S. mutans. S. mutans also makes four non-GTF glucan-binding proteins: GbpA and GbpD are secreted and released proteins that contain a region that is homologous to the glucan-binding domains of the Gtf enzymes, and GbpC is a cell wall bound protein that confers the property of dextran-dependent aggregation during stressful conditions, and GbpB whose glucan-binding properties appear secondary to its role in cell-wall metabolism. It was hypothesized that Gbps A, C, and D primarily function to shape the architecture of S. mutans biofilms which in turn affects the cariogenicity of S. mutans. To test this hypothesis, a panel of Gbp mutants was constructed from S. mutans strain UA130 that encompasses all deletions of Gbps (GbpA, GbpC and GbpD) individually and in combination. Specific pathogen-free rats were infected with the WT S. mutans UA130 strain along with each of the Gbp mutants, were fed a high sucrose diet for seventy days, and were then scored for caries. Significant attenuation of caries was observed in some but not all gbp mutants. Biofilms were also grown and analyzed via confocal microscopy and COMSTAT image analysis software. Architectural differences were found with all of the gbp mutants when compared to the wild-type, most notably the mutant strains lost significant biofilm depth. Several of the architectural parameters correlated with caries attenuation. It was concluded that deletion of one or more Gbps resulted in a partial loss of the cohesive properties of S. mutans biofilms and changes in biofilm architecture. In several cases this resulted in significant attenuation of cariogenicity but not a complete loss. The architectural changes that resulted from this loss of biofilm cohesiveness and the specific combinations of Gbp deletions that lead to significant attenuation suggested specific roles for each Gbp in biofilm formation. Furthermore, the attenuation of Gbp mutant strains could not be explained by differences in acidogenicity or aciduricity among the mutants. Therefore it was concluded that Gbps A, C and D make profound contributions to biofilm architecture and changes in biofilm architecture, as a result of loss of Gbp-mediated cohesion, affects S. mutans cariogenicity.

Starch is a water-insoluble glucose biopolymer used as an energy cache in plants and is synthesized and degraded in a diurnal cycle. Reversible phosphorylation of starch granules regulates the solubility and, consequentially, the bioavailability of starch glucans to degradative enzymes. Glucan phosphatases release phosphate from starch glucans and their activity is essential to the proper diurnal metabolism of starch. Previously, the structural basis of glucan phosphatase activity was entirely unknown. The work in this dissertation outlines the structural mechanism of activity of two plant glucan phosphatases called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). The crystal structures of SEX4 and LSF2 were determined with and without phosphoglucan ligands bound, revealing the basis of their interaction with an endogenous substrate. The data show that SEX4 and LSF2 interact with starch glucans via distinctive mechanisms. SEX4 binds glucan chains via an aromatic-rich pocket spanning its Carbohydrate Binding Module (CBM) and catalytic Dual Specificity Phosphatase (DSP) domains. Conversely, LSF2 lacks a CBM and, instead, binds glucans at two non-catalytic surface-binding sites that are located distally from its active site. In addition, it was previously reported that SEX4 and LSF2 act upon distinct phospho-glucan substrates: SEX4 preferentially dephosphorylates the C6-position of starch glucans and LSF2 exclusively dephosphorylates the C3- position. The data herein reveal that SEX4 and LSF2 contain differences in their active site topology that serve to position the glucan chain in opposite orientations, therefore accounting for the differences in substrate specificity. Using these insights, SEX4 was engineered with reversed substrate specificity, i.e. preferential C3-specific activity. Previous work has established the interaction between phosphatases and protein, lipid, and nucleic acids; however, the current study represents the first insights into phosphatase interaction with carbohydrate substrates. In addition, the insights gained provide a model that will be used in future studies with the mammalian glucan phosphatase laforin, which is linked to neurodegeneration and the fatal epileptic disorder Laforaʼs Disease.

Systemic fungal infection is a life-threatening problem. Anti-fungal drugs are the most effective clinical strategy to cure such infections. However, most current anti-fungal drugs either have high toxicity or have a narrow spectrum of effect. Meanwhile, anti-fungal drugs are losing their clinical efficacy due to emerging drug resistance. To protect us from these deadly pathogenic fungi, scientists need to study new drug targets and to solve problems related to drug resistance. The cell wall is essential for fungal cell survival and is absent from animal cells, so it is a promising reservoir for screening safe and effective drug targets. Alpha-1,3-glucan is one of the major cell wall carbohydrates and is important for the virulence of several pathogenic fungi. In this thesis, molecular biology and microscopy techniques were used to investigate the function and the synthesis process of α-1,3-glucan in the model fungus A. nidulans. My results showed that α-1,3-glucan comprises about 15% of A. nidulans cell wall dry weight, but also that α-1,3-glucan does not have an important role in cell wall formation and cell morphology. Deletion of α-1,3-glucan only affects conidial adhesion and cell sensitivity to calcofluor white. In contast, elevated α-1,3-glucan content can cause severe phenotypic defects. To study the α-1,3-glucan synthesis process, I systematically characterized four proteins, including two α-1,3-glucan synthases (AgsA and AgsB) and two amylase-like proteins (AmyD and AmyG). Results showed AgsA and AgsB are both functional synthases. AgsB is the major synthase due to its constant expression. AgsA mainly functions in conidiation stages. AmyG is a cytoplasmic protein that is critical for α-1,3-glucan synthesis, likely being required for an earlier step in the synthesis process. In contrast to the other three proteins, AmyD has a repressive effect on α-1,3-glucan accumulation. These results shed light on therapeutic strategies that might be developed against α-1,3-glucan. I also developed a strategy to investigate drug resistance mutations. The tractability of A. nidulans and the power of next generation sequencing enabled an easy approach to isolate single mutation strains and to identify the causal mutations from a genome scale efficiently. I suggest this strategy has applications to study the drug resistance mechanisms of current anti-fungal drugs and even possibly future ones.

The degradation of carbohydrates is essential to many biological processes such as cell wall remodelling, host-pathogen defense, and energy synthesis in the form of ATP. Several of these processes utilize carbohydrate-active enzymes to accomplish these goals. Studying the degradation of polysaccharides by carbohydrate-active enzymes synthesized by microbes has allowed us to further understand biomass conversion. A portion of these polysaccharides consists of β-1,3-linked glucose (i.e. β-1,3-glucan), which is found in plants, fungi, and brown macroalgae. The hydrolysis of β-1,3-glycosidic linkages is catalyzed by β-1,3-glucanases, which are present in six different glycoside hydrolase (GH) families: 16, 17, 55, 64, 81, and 128. These enzymes play important biological roles including carbon utilization, cell wall modeling, and pathogen defense. This study focuses on a gene from Bacillus halodurans encoding for a multi-modular protein (BhLam81) consisting of a glycoside hydrolase from family 81 (BhGH81), a carbohydrate-binding module (CBM) from family 6 (BhCBM6), and a CBM from family 56 (BhCBM56). Previously, thorough structural and substrate-specific characterization has been carried out on BhCBM6. This CBM binds the non-reducing end of β-1,3-glucan. A member of CBM family 56 has been shown to recognize and bind the insoluble β-1,3-glucan, pachyman, however it is structurally uncharacterized. A glycoside hydrolase belonging to family 81 from Saccharomyces cerevisiae has been previously shown to degrade the β-1,3-glucans, laminarin and pachyman, however the structure of this enzyme was not determined. Recently, a member of GH family 81 has been structurally characterized; however, substrate-specificity was not determined in that study. Therefore, this study concentrated on two goals: Determining the substrate-specificity of BhGH81 and BhCBM56, and solving the structure of BhGH81 and BhCBM56 in order to gain insight into the molecular details of how they recognize and act on their substrate(s). The deoxyribonucleic acid (DNA) encoding for these modules were dissected by restriction digest from B. halodurans genomic DNA and recombinantly expressed in Escherichia coli (E. coli) as separate constructs. Both BhGH81 and BhCBM56 were purified and their crystal structures obtained. BhGH81 and BhCBM56 were solved to 2.5 Å resolution by single-wavelength anomalous dispersion (SAD) and to 1.7 Å resolution by multi-wavelength anomalous dispersion (MAD), respectively. In order to determine the substrate-specificity of BhGH81 and BhCBM56 and speculate on the molecular details of how they recognize and act on their substrate(s), substrate-specificity tests were combined with structural analysis for both of these modules. By using qualitative depletion assays, quantitative depletion assays, and affinity electrophoresis, it was revealed that BhCBM56 binds both insoluble and soluble β-1,3-glucan. The crystal structure of BhCBM56 revealed that it is a β-sandwich composed of two antiparallel β-sheets consisting of five β-strands each. By comparing BhCBM56 to a β-1,3-glucan binding protein from Plodia interpunctella (βGRP) a putative substrate-binding cleft on the concave side of the β-sandwich created by a platform of hydrophobic residues surrounded by several polar and charged residues was revealed. This comparison also allowed for speculation of the amino acids (W1015, H965, and D963) that are potentially essential for recognition of β-1,3-glucan substrates by BhCBM56. Activity of BhGH81 on β-1,3-glucans was confirmed by both thin-layer chromatography and product analysis using high performance anion exchange chromatography. The high performance anion exchange chromatography of BhGH81 hydrolysis suggested it has both exo and endo modes of action. The crystal structure of BhGH81 revealed that it consists of domains A, B, and C: A β-sandwich domain (A), a linker domain (B), and an (α/α)6-barrel domain (C). This structure revealed a putative substrate-binding cleft on one side of the (α/α)6-barrel with a blind canyon active site topology. It also revealed two putative catalytic residues, E542 and E546. All GHs from family 81 characterized so far, hydrolyze β-1,3-glucan in an endo acting manner. By comparing the structure of BhGH81 acquired in this study to a cellulase from Thermobifida fusca, which has an endo-processive mode of action, we can speculate that BhGH81 also has an endo-processive mode of action. The structural and biochemical analysis of BhGH81 and BhCBM56 in this study has aided in further understanding the molecular details both GH family 81 and CBM family 56 proteins, as well as the degradation of β-1,3-glucan by multimodular enzymes. Understanding these molecular details could be important for industrial applications such as, engineering a microbial platform for more efficient biofuel production. / Graduate

Plant primary cell walls are hydrated extracellular complexes composed largely of polysaccharides: cellulose, hemicellulose and pectin. Cell wall constituents and composition vary in cell-, environment-, and species-dependent manners. For example, within land plant hemicelluloses xyloglucan is ubiquitous while mixedlinkage (1→3),(1→4)-β-D-glucan (MLG) is found only in the Poales and Equisetum. Glycosyl hydrolase 16 (GH16) enzyme family members include numerous enzymes with pertinence to the understanding of the ‘lives’ of cell wall hemicelluloses. However, despite this, the details of the interactions between GH16 enzymes and their substrates have often not been elucidated. Likewise, the true preferences of many of these enzymes and the range of substrates which they can utilise remain to be fully explored. By providing a greater wealth of information for the correlation of enzyme structure with reaction catalysed, such an understanding would enable better predictions of the activities of novel enzymes. Crucially, this would also allow better identification of roles performed by these enzymes in planta as well as of the potential applications of these enzymes. This work sought to further our understanding of the interactions between GH16 enzymes and their substrates by the study of five activities exhibited by GH16 enzymes – xyloglucan endotransglucosylase (XET), xyloglucan endoglucanase/hydrolase (XEG/XEH), mixed-linkage glucan : xyloglucan endotransglucosylase (MXE), lichenase and cellulose : xyloglucan endotransglucosylase (CXE). All of the analysed activities act on xyloglucan and/or MLG. Of particular focus is the novel enzyme MXE from the evolutionarily isolated genus Equisetum (horsetail), which acts on both. Notable findings include: identification of MXE/CXE gene; determination of the substrate specificity of MXE; defining of the sites of attack of lichenase, XEG, XET and MXE; discovery of novel xyloglucan structures and discrepancies between the xyloglucan present in different barley organs.

Photobacterium profundum SS9 is a γ-proteobacterium which grows optimally at 15°C and 28 MPa (a psychrophilic piezophile) and can grow over a range of temperatures (2-20oC) and pressures (0.1-90 MPa). Previous research had demonstrated that P. profundum SS9 adapts its membrane proteins and phospholipids in response to growth conditions. In this study, methodology was developed for growing P. profundum SS9 under cold temperatures and high pressures in both liquid and solid cultures. The effect of changing growth conditions on cell envelope polysaccharides was then investigated. The lipopolysaccharide (LPS) profile of a rifampicin resistant P. profundum SS9 derivative, SS9R, was shown to change at 0.1 MPa with respect to temperature and at 15°C with respect to pressure. Compositional analysis showed that the LPS was almost entirely composed of glucose. This provides evidence that, under these conditions, the major polysaccharide produced by P. profundum SS9 is a glucan. Two putative polysaccharide mutants, FL26 & FL9, were previously isolated from a screen for cold-sensitive mutants of P. profundum SS9R. Both mutants displayed an increased sensitivity to cold temperatures on solid medium and were unaffected in their growth at high pressure. FL26 was found to exhibit an LPS alteration similar to previously published O-antigen ligase mutants, providing evidence that this mutant is likely to lack O-antigen ligase. Interestingly, FL26 was also shown to have a reduced ability to form biofilms and had increased swimming motility. This suggests that there are a number of changes which occur in FL26 in the absence of O-antigen. FL9 was found to have an altered LPS and capsular polysaccharide (CPS), similar to an E. coli wzc mutant. In E. coli, Wzc is involved in the polymerisation and transport of CPS, disruption of which can also lead to LPS alterations. The LPS and CPS alterations may lead to the cold-sensitivity phenotype, either individually or in combination. In conclusion, alterations in the cell envelope polysaccharides were shown to affect cold temperature sensitivity on solid agar. Cold-sensitivity is most likely directly related to the LPS alterations and stability of the membrane under cold temperatures. Exopolysaccharides (EPS) have previously been shown to affect desiccation and freezethaw resistance, making it is possible that the CPS plays a similar role in this case.

The opportunistic fungus Aspergillus fumigatus has emerged as one of the most common fungal human pathogens, causing severe and usually fatal systemic infections that account for more than 200,000 cases annually with mortality rates usually exceeding 50%. During infection, the virulence of A. fumigatus highly depends on its capacity to rapidly respond to external stress encounters in the human niche, such as the host immunological response and the activity of antifungal drugs. The echinocandin, caspofungin, is one of most commonly used antifungal drugs to treat intolerant or refractive patients suffering from invasive aspergillosis. Caspofungin disrupts the catalytic subunit of the β-1,3-glucan synthase complex, Fks1, resulting in the reduced production of the main cell wall component of A. fumigatus, the polysaccharide β-1,3-glucan. Despite its clinical relevance in patients with aspergillosis, caspofungin displays attenuated activity at high concentrations, a phenomenon known as ‘the paradoxical effect’. Little is known about the paradoxical growth of A. fumigatus during caspofungin treatment. Therefore, in this thesis, I investigated the key cellular and molecular responses of A. fumigatus upon caspofungin treatment, particularly during paradoxical growth by live-cell imaging. High-resolution confocal live-cell microscopy revealed that treatment with either low (0.5 µg/ml) or high (4 µg/ml) concentrations of caspofungin for 36 h caused similar abnormalities in A. fumigatus, including wider, hyperbranched hyphae, increased septation and repeated hyphal tip lysis. Regenerative intrahyphal growth occurred as a rapid adaptation to the lytic effects of caspofungin on hyphal tips and the dynamic relocation of Fks1 to vacuoles was a key feature observed in response to caspofungin treatment. The reduced amount of β-1,3-glucan resulting from caspofungin treatment was compensated by increased α-1,3-glucan and chitin content in mature hyphal tips. Interestingly, all lysed cells recovered by regenerative intrahyphal growth. However, after 48 h treatment, only cells exposed to high caspofungin concentrations developed paradoxical growth in leading hyphae. This response was associated with a relocalization of Fks1 at hyphal tips. Consistently, cells undergoing paradoxical growth showed normal morphology and ceased to undergo cell lysis, as well as having a normal content of β-1,3-glucan and α-1,3-glucan but not chitin, which remained high. Notably, the localization of the regulatory subunit of the β-1,3-glucan synthase complex, Rho1, was unaffected by caspofungin, but it was required for the development of paradoxical growth. Interestingly, the gene expression of the β-1,3-glucan synthase complex was downregulated by caspofungin treatment. In addition, caspofungin activity induced the nuclear translocation of the Ca+2 regulated transcription factor CrzA to nuclei and only hyphal tip cells in which this translocation occurred underwent cell lysis. Finally, similarly high concentrations of caspofungin also induced paradoxical growth of Aspergillus fumigatus during human A549 alveolar cell invasion. This thesis outlines several critical adaptations that occur at the cellular, subcellular and molecular levels at different times during exposure to high and low concentration of caspofungin.

Plant cell walls determine cellular shape and provide structural support for the entire plant. Polysaccharides, comprising the major components of the wall, are actively remodelled throughout development. Xyloglucan endotransglucosylase (XET)/hydrolase (XTH, EC 2.4.1.207) cleaves xyloglucan (XyG), the donor substrate, and attaches a portion to another XyG chain, the acceptor substrate. Recently, a novel transglucosylase called mixed-linkage β-glucan (MLG) : XyG endotransglucosylase (MXE) was discovered in horsetails (Equisetum spp.) that could attach a portion of MLG to XyG, resulting in a hetero-polymer product. My aims were to further investigate the nature of this activity, biochemically characterize the enzyme, and explore its physiological role. MXE activity was attributable to an enzyme unlike Equisetum XTHs. MXE had a p1 of 4.1 (XTHs were 6.6-9), a pH optimum of 6.3 (XTHs preferred 5.5), and had higher activity using smaller oligosaccharide acceptor substrates like XXXGol (XTHs were more active using XLLGol). Importantly, the MXE protein was shown to utilize both MLG and XyG as donor substrates, and therefore have both MXE and XET activity. Also, the enzyme was capable of using various glucan oligosaccharides (O) as substrates, including MLGO, XyGO, and cello-O, but not laminari-O. By using a novel ex vivo approach, the proportion of extractable MXE product to XET product was found to increase in older tissues. Transglucosylase products were localized in sclerenchyma and structural parenchyma by in situ assays, implying a strenghening function for MXE. Surprisingly, another novel activity was discovered that could covalently attach cellulose to XyG, and termed cellulose : xyloglucan endotransglucosylase (CXE). This activity was attributed to the MXE enzyme, implying that the protein is a promiscuous endotransglucosylase. The presence of CXE in other plants has not yet been tested. Besides being a novel discovery in plant cell biology, the modification of cellulose has applications in a number of industries.

No description available.

C. albicans is a polymorphic fungal pathogen which has the ability to shift from yeast to hyphae. C. albicans cell wall is composed of glucan, chitin, mannoprotein and mannan. It is not possible, using standard extraction methods, to isolate mannan from C. albicans hyphae. To isolate hyphal mannan, we developed a simplified alkali extraction method. Using this method it was determined that hyphal mannan has a much lower molecular weight, a smaller polymer distribution and altered conformation structure when compared to yeast mannan. The hyphal mannan was found to contain little to no acid-labile portion with only α-Man-PO4 groups and no long chains of β-1, 2-linked mannosyl repeat units, when compared to the yeast mannan. It was concluded that the C. albicans hyphal mannan is substantially different from the mannan found in the yeast form. This is an entirely new observation that extends the existing knowledge about the structural biology of C. albicans hyphae and may provide insights into the role of hyphae in pathogenesis.