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Adhesion of the opportunistic fungal pathogen, Candida albicans, to host surfaces is mediated through the mannoprotein-rich surface. This thesis examines the role of cell wall mannoproteins in the host-fungus interactions and the mechanism by which these proteins are tethered to the major structural polysaccharides of the wall. The mannosyl modifications of cell surface proteins are involved in host interaction. Periodate oxidation of fungal surface carbohydrates reduced adhesion to epithelial cells confirming that mannan is a component adhesion of C. albicans. Strains of C. albicans disrupted in MNT1, 2 and 3 genes encoding mannosyl transferases, were reduced in adhesion to both epithelial cells and Matrigel. Adhesion was also influenced by the Ura-status of C. albicans strains, which differs as a result of targeted gene disruption by the URA-blaster strategy. Ura-strains were less adhesive but this was not due to altered growth rate and could not be alleviated by adding excess uridine. Therefore current methods for gene deletion have to be questioned when considering adhesion as a virulence factor. Strains defective in glycosylation were altered in covalently associated cell wall proteins (CWPs) in terms of mobility during electrophoresis. The greatest alterations observed were in the CWPs of C. albicans strains with severe glycosylation defects, such as in Dochl/Doch1 and Dpmt1/Dpmt1 mutants, defective in N- and O-linked glycosylation, respectively. These strains also secreted more mannoprotein were altered in morphology or gross cell wall structure, and had elevated cell wall chitin. Therefore glycosylation is required for normal CWP incorporation and cell wall construction. For the first time C. albicans was shown to link a CWP, other than Pir-CWP, to the cell wall network via an alkali-sensitive linkage. The nature of this type of linkage remains unknown, however an O-mannan chain, whose synthesis is not initiated by Pmt1p, may be involved. Pir-CWP incorporation was increased in a Dpmt1/Dpmt1 null mutant and could be partially attributed to increased expression. Pir-CWP expression and incorporation was pH-dependent and may be regulated in response to the different pH at different host niches.

The aim of this project as to construct a chsI null mutant strain of Candida albicans and analyse the mutant phenotype in order to elucidate the function of CaChs1p. The inability to generate such a strain by conventional gene disruption suggested CaCHSI was important or essential for growth. This was verified by creating a conditional chsI mutant strain (KWC340) by placing the only wild type copy of CaCHSI under the control of a regulatable maltase (MRPI) promoter. When strain KWC340 was grown in conditions that were repressing for the MRPI promoter CaCHSI mRNA was not detected by Northern analysis and cells grew as multinucleate chains. Calcoflour staining indicated these cells lacked a primary septum and this was confirmed by examining thin sections by electron microscopy. WGA-gold labelling of thin sections of three chs mutants of C. albicans confirmed that CaChs1p synthesises chitin of the primary septum, CaChs3p synthesises lateral cell wall chitin and the chitin ring at the site of bud emergence whereas chitin localisation of the chs2 mutant was indistinguishable from wild type cells. All three chs mutants formed chlamydospores. Measurement of chitin synthase activity in vitro indicated that CaChs2p encodes the major enzyme activity. This activity was similar to ScChs1p in respect to nikkomycin sensitivity, zymogenicity and preference for divalent cations suggesting Cachs2p is the functional homologue of ScChs1p. This project addressed the conflicting reports on the chitin content of chs2 mutant hyphal cells. Chitin contents were measured in three ways. The deacetylation/deamination protocol of Ride & Drysdale used by Gow et al. (1994) showed a 44% reduction in hyphal chitin in the chs2 mutant. Measurement of total glucosamine by acid hydrolysis as employed by Mio et al. (1996) determined wild type chitin levels in chs2 hyphae as did the third method which measures GlcNAC released from cells by the action of two enzymes, a chitinase and N-acetylglucosaminidase.

The antibiotic-resistant bacteria seriously threaten public heath. Scientists of both public and private sectors are devoted to develop new drugs to overcome this problem. Marine species are highly diversity and have been shown to be a potential sources to find efficient compounds. The medicine application of the natural products from ocean increases in last decade. The purpose of the project was to isolate antibiotic-producers from sea-bottom mud. Three filament microorganisms from marine environment, Acremonium altematum T1-1, Aspergillus ochraceus N7 and Nocardiopsis dassonvillei F5, can produce bioactivity compounds. All compounds purified using silica chromatographic separation from culture extraction and determination of their structure by NMR spectrum and X-ray diffraction pattern for crystals. A. altematum T1-1 produced 5,6-dihydropenicillic acid (DHPA), A. ochraceus N7 produced penicillic acid (PA), and N. dassonvillei F5 produced iodinin. The structures of DHPA and PA were identified as derivatives of five-member lactone, and iodinin is a derivative compound of phenazine. The optimal growth of DHPA -producer A. altematum T1-1 was in 6% salinity at 30¢J and best DHPA production was in 0% salinity at 25¢J. The optimal growth of PA-producer A. ochraceus N7 was in 6% salinity at 25¢J, and best PA production conditions was below 3% salinity at 25 ¢J. The optimal growth of iodinin-producer N. dassonvillei F5 was in 6% salinity at 30¢J and best iodinin production condition was 1-3% salinity at 25¢J. Only PA has highly inhibited of B. cereus at the MIC of 75 mg/ml. The PA had cytotoxicity against cancer cell lines of P-388, HT-29 and A549 while iodinin inhibited P-388 and A549. The ED50 on the swimming ability of rotifer was 1.03 and 2.5 mg/ml for PA and DHPA, respectively. It revealed that the growth of these two mold can occur in regular marine environment and produce toxin to kill rotifer. The growth of N. dassonvilleiand F5 and iodinin caused the death and virus-invasion of shrimp.

The understanding of the phenomenon of thermophily requires investigations of both thermophilic prokaryotes and eukaryotes. In the eukaryotes, thermophily is exhibited only by a few species of fungi which can grow up to 60°C. A comparative study of homologous enzymes from thermophilic and mesophilic fungi and the analysis of the observed differences is a useful approach not only in discerning the mechanisms in thermophily but also in understanding the features of fungal growth and metabolism. Because of the availability of background information of invertase from some mesophilic sources and the convenience of assaying the enzyme, it was chosen for the projected study in a thermophilic fungus, Themomyces lanuginosus. The behaviour of invertase in the thermophilic fungus differed from invertases of mesophilic organisms in several respects, e.g., in the thermophilic fungus the enzyme was induced only in the presence of its substrate; it was intracellular and it was unstable both in mycelia and in cell-free extracts. The enzyme specific activity was maximum in 6 h-sucrose-grown mycelia following, which it progressively declined before maximal increase in biomass occurred and much of the inducer (sucrose) was still present in the growth medium. Further, invertase activity in cell-free extracts was unstable; it was completely inactivated during storage for 3 days at O°C. The enzyme activity was stabilised by the addition of thiol compounds, dithiothreitol (DTT) and glutathione (GSH) to cell-& extracts. In contrast, the addition of disulphides and thiol-modifying compounds rapidly inactivated the enzyme indicating the involvement of free sulphydryl group(s) in enzyme activity. The enzyme activity was reciprocally modulated by reduced (GSH) and oxidized (GSSG) glutathione, suggesting that invertase may be regulated by thiol/disulphide exchange reaction. Such a modulation of invert- activity has not been reported hr any other invertase. This observation suggested that the enzyme in the thermopbilic fungus is different from invert- that have been studied from mesophilic sources, notably from yeast and Neurospora. To obtain more information on this unusual behaviour of invertase of T. lanuginosus, an attempt was made to purify the enzyme and study its physico-chemical properties. Invertase was purified by ammonium sulphate fractionation of cellular proteins, ion-exchange and thiol-affinity chromatography followed by preparative electrophoresis. The final preparation of invertase after the electroelution step gave a single band on a native PAGE. However, the same preparation of invertase resolved into five bands of different molecular mass. The heterogeneity of the enzyme preparation on SDS-PAGE raised two possibilities with respect to the purity of the enzyme: (1) the final preparation contained multiple invertases of different molecular mass, or (2) the invertase preparation was associated with contaminating proteins. To distinguish between these two possibilities, proteins from induced (sucrose-grown) and non-induced (glucose-grown) mycelia were compared after identical steps of purification. The rationale of this experiment was that if heterogeneity of invertase is due to multiple forms of the same enzyme, they would most likely be absent in the non-induced mycelia. When the final preparations of proteins from both the mycelia were analysed on SDS-PAGE, it was observed that certain proteins were present in both the induced and the non-induced mycelia, suggesting that they might be the contaminating proteins present in the invertase purified by the above procedures. Some physico-chemical properties of invertase were studied. The purified enzyme was unstable during storage, losing activity completely in five days at O°C. Addition of DTT or glutathione did not prevent this loss of enzyme activity. This response of purified invertase preparations to DTT was quite opposite to that in cell-free extracts where invertase activity was stabilised by thiol compounds. To elucidate the reason for this difference in the behaviour of invertase in cell-free extracts and in pursed preparations, the approach taken was to first inactivate the enzyme in both1 type of preparations and then attempt to reactivate it. Dialysis of cell-free extracts had been found to cause an accelerated and complete inadivation oft he enzyme. The same treatment also inactivated freshly purified invertase, but to a lesser extent (60%). Whereas addition of DTT completely reatored the enzyme activity in the dialysed cell-bee extracts, it caused only a marginal revival of activity in dialysed invertase. This change in the response of purified invertase to DTT suggested that some cellular proteins were required br the reactivation of the enzyme by DTT that had been removed during the purification of invertase. A cellular protein was identified that reactivated inactive invertase in the presence of DTT. This protein was given the acronym "PRIA" for 'protein which restores i nvertase activity'. The mechanism of reactivation involved the conversion of the inactive invertase molecules into an active form. A model has been proposed to explain the requirement of UPRIA" for the reactivation of invertase. The salient features of this model are : (i) invert= requires free sulphydryl group(s) for activity, (ii) inactivation of invertase involves the formation of intramolecular disulphide bond(s) in the enzyme, (iii) the disulphide bond(s) is inaccessible to reduction by DTT, (iv) interaction of invertase and "PRIA” results in a conformational change in the enzyme that exposes the disulphide bond(s), rendering it susceptible to reduction by DTT and converting inactive invertase into active enzyme molecules. A surprising observation was the resistance of purified invertase to inactivation by the disulphides, GSSG, CoASSCoA and cystine. This was in marked contrast to their effective inhibition of invertase in the cell-& extracts. The experimental analysis of this unexpected resistance of purified invertase to disulphides revealed that following thioldnity chromatography on a Afegeldol column, invertase became resistant to disulphide inactivation. Moreover, the purified invertase was more stable during storage and to dialysis treatments in contrast to invertase activity in the cell-free extracts. These obsemtions suggested that invertase was altered- presumably it underwent a conformational change during the -el-501 chromatography step; possibly, the interaction of invertase with the gel matrix resulted in some cysteine residues in the enzyme becoming inaccessible to oxidation, thereby conferring resistance to inactivation by disulphides. The in vitro modulation of invertase activity by GSH and GSSG suggested the possibility that the enzyme may be regulated by a similar mechanism in the fungal mycelium. To substantiate this, GSH and GSSG levels in the mycelia were estimated. The GSH/GSSG ratio dememed in the mycelia between 6 and 18 h of growth and this was correlated with the decline in invertase activity. The fall in the GSHIGSSG ratio suggested that the intracellular environment waa becoming progressively oxidised during growth. Because NADPH participates in maintaining the cellular glutathione in a reduced state by the glutathione reduct- reaction, NADPH and NADPt levels were estimated. The NADPH/NADPt ratio declined by a factor of four between 6 and 36 h of growth and this decrease was positively correlated with the decrease in the flux of glucose through the pentose phosphate pathway. Incorporation of 'H-thymidine in mycelia indicated that with age of the culture, the number of growing hyphal tipslunit weight of mycelia declined. An attempt was made to integrate the changes in various biochemical parameters with the pattern of invertase development in T. lanuginosus when grown in a medium containing sucrose, i.e. invertase activity appeared rapidly as soon as perceptible growth occurred but it did not increase in parallel with the increase in biomass. Rather, the activity started to decline at approximately 6 h at which time growth was quantitatively mall. Since invertase activity in T. lanuginosus was induced by sucrose which is transported inside by a specific transporter, the development of invertase activity was linked to the uptake of sucrose by the fungal mycelia. It was considered likely that the sucrose transporter in T. lanuginosus, is localised at the hyphal tip where the entry of sucrose induces invertase. He enzyme is kept active in the hyphal tip because of a reductive environment due to a high GSHIGSSG ratio as a result of high NADPH levels. The latter serves to maintain GSH in a reduced state by the glutathione reductase reaction. In mature hyphae, lower generation of NADPH will result in lower GSHIGSSG ratio that will inactivate invertase by thiol oxidation. According to this model, the early burst of invertase activity in sucrose grown T. lanuginosus mycelia is due to the initiation of branch initials whereas the fall in enzyme activity is because of the decline in the proportion of hyphal tips per unit mass of mycelium as elongation growth and wall thickening occurs.

Thesis (M.S.)--Oregon State University, 2002. / Typescript (photocopy). Includes bibliographical references. Also available on the World Wide Web.

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