Candida glabrata is a significant and increasingly common pathogen of humans yet its mechanism of virulence remains unclear. Comparative genomic studies revealed that C. glabrata is more closely related to the non-pathogenic yeast Saccharomyces cerevisiae and that both these genomes are distinct from C. albicans. In order to explore C. glabrata virulence attributes, C. glabrata ORFs with no orthologue in S. cerevisiae were studied since these ORFs may have accompanied the adaptation of C. glabrata to the human host. Reciprocal best hit searches identified C. glabrata ORFs with no S. cerevisiae orthologue. A barcoded deletion library targeting 65 C. glabrata-specific ORFs was constructed. To functionally characterise the deletion library, mutants were tested for fitness and phenotypically screened to identify gene products required for growth in response to biologically relevant stresses. As such, novel phenotypes associated with the deletion of previously uncharacterised ORFs were uncovered. Mutants were also tested for infection-related properties including biofilm formation, antifungal agent susceptibility and for virulence in a Drosophila melanogaster infection model, resulting in the identification of two C. glabrata-specific ORFs, CAGL0K05687g and CAGL0H01749g, which were required for virulence. Three ORFs with notable phenotypes were taken forward for further characterisation. An adapted genome-wide synthetic genetic interaction approach was used to create genetic interaction networks for C. glabrata ORFs over-expressed in S. cerevisiae. Genetic interaction analysis of a C. glabrata chromatin remodeler CAGL0D05434g revealed a role for this ORF in metal ion homeostasis and DNA damage repair. Genetic interaction profiling for an oxidoreductase encoded by CAGL0K05687g was used to reveal mechanisms related to transport by which this ORF may be required for virulence.

No description available.

No description available.

This thesis had the purpose to understand pathogenicity in Candida albicans. The study focused on the previously known virulence factors. In addition, new genes related to virulence were investigated. To achieve this objective, two strains were initially compared; SC5314 (virulent strain) and RV4688 (attenuated strain) by transcript profiling and phenotypic analysis. Transcript profiling revealed that genes related to oxidative stress were down-regulated in the attenuated strain as compared to the virulent strain. RV4688 also formed hypha less readily and adhered less to buccal epithelia than SC5314. It was also determined that the virulence status of these two strains could not be changed. As suggested by microarrays, genes related to oxidative stress might be related to virulence in C. albicans. Therefore, the role of three different genes involved in stress response was analysed: SOD1, SOD2 and TTR1. The D sod1/sodl and D sod2/sod2 mutants were kindly provided by Dr. Sa-Ouk Kang. TTR1 was disrupted and subsequently reintegrated. To investigate the role of two genes in oxidative stress and virulence, a double mutant for SOD1 and TTRI was also generated and each gene was individually reintegrated. The oxidative stress mutants had delayed evagination to form germ tubes. The SOD1 and TTR1 genes seem to have specific roles to respond to the oxidative stress inducers menadione and diamide. However, both of them seem important for virulence in C. albicans, as the D sod1/sod1 and D ttr1/ttr1 mutants were attenuated for virulence in mice and quantitatively more killed by human neutrophils than the wild-type control. When different clinical isolates were characterized, it was demonstrated that SC5314 does not reflect what might happen with all the wild-type virulent strains because other clinical isolates showed clearly different phenotypes. Therefore, C. albicans virulence properties expression might be different in each strain.

Candida albicans is an opportunistic human pathogen. Several factors contribute to the virulence of this fungus. These include phenotypic switching, biofilm formation, the secretion of hydrolytic enzymes, the production of adhesion molecules, and the ability to undergo reversible morphological changes from yeast to (pseudo)hyphae in response to the host physiological conditions. Multiple signalling pathways link the relevant environmental signals to hyphal growth, and several downstream transcription factors promote or repress genes involved in the yeast-to-hypha transition. A number of genes are induced specifically during hyphal development. ALS3 encodes a hypha-specific cell wall protein of the Agglutinin-Like Sequence family of adhesins. The first aim of this project was to study the transcriptional regulation of ALS3. Previous work in our laboratory identified two sequences that bind the transcriptional repressor Nrgl (NREs), and that are required for repression under yeast inducing conditions. Moreover, a region of 150 bp between -471 and -321 was defined as being important for ALS3 activation (A1 region). However, no short sequence or known response element within this activation region, in isolation, was sufficient for hypha- specific activation of ALS3. In this study we identified an additional activation region (A2) located between -1438 and -1050. Furthermore, we found that the transcription factors Efgl, Bcrl and Teel are required for the activation of ALS3 under serum- inducing conditions. Also, Cphl contributes to the hypha-specific activation of ALS3, but it is not essential for expression. Teel does not act directly through the five putative Teel binding sites present in the ALS3 promoter, which are not functional, but mediates activation of ALS3 indirectly via Bcrl. Under conditions that promote yeast growth, ALS3 expression is repressed by Nrgl, Tupl and Rfgl, but not by Ssn6 or the mating factor al. The morphogenetic repressor Nrgl targets the global repressor Tupl to the promoters of hypha-specific genes and other genes to inhibit the yeast-to-hypha transition. The second aim of our project was to study the regulation of Nrgl. Previous work in our laboratory showed that, as expected, NRG1 mRNA levels decrease during hyphal development. However, the down-regulation of NRG1 was too slow to account for the rapid induction of hyphal growth. In this study we found that Nrgl is a phosphoprotein that is found phosphorylated in yeast cells and becomes rapidly and transiently dephosphorylated in serum-induced cells. In addition, Efgl is required for Nrgl- mediated repression via the NRE under non-inducing conditions, whereas the release of Nrgl-mediated repression under inducing conditions is dependent on components of the cAMP-PKA pathway. Furthermore, we found that the Nrgl-mediated repression of an NRE-containing reporter construct in yeast cells requires both Tupl and Ssn6, although this might not always be the case for native promoters. Altogether, the results of this project show that several signalling pathways converge at the promoter of the hypha-specific gene ALS3 to regulate its expression. The activity of one of the negative regulators of ALS3, the morphogenetic repressor Nrgl, appears to be modulated both at the transcriptional and post-translational level. Furthermore, the cAMP-PKA pathway seems to link positive and negative regulation of morphogenesis in C. albicans.

The genus Candida contains about 200 species, most of which cannot be differentiated on morphology alone, but they can be identified using physiological characteristics such as assimilation of different nitrogen sources, fermentation of sugars and inhibition of growth. The genus Candida is composed of an extremely heterogeneous group of organisms that grow as yeast forms (blastoconidia), and most members of the genus also produce filamentous growth forms (pseudohyphae, pseudomycelium or/and true hyphae). In addition, C. albicans and C. dubliniensis also have the ability to form true hyphae and chlamydospores. Thus, C. albicans is pleiomorphic and undergoes reversible morphogenetic transitions between budding, pseudohyphal and hyphal forms (Odds, 1988; Kerridge, 1993). Pseudohyphae range from relatively short to extended cells and unlike hyphae they have constrictions at their septa (Merson-Davies and Odds, 1989; Sudbery, 2001). C. albicans can also form chlamydospores, asexual spores that are formed by rounding off of a cell or cells from a suspensor cell. These generally appear under unfavourable conditions and in media of high carbon/nitrogen ratio (Kurtz et al., 1990). Candidiasis is an endogenous disease, but it can be transmitted laterally for example from health care workers to patients or between sexual partners, and vertically, as from mother to the neonate (Calderone, 2002b). In its commensal phase, C. albicans usually grows in the form of yeast cells (Poulain et al., 1985), whereas yeast cells, hyphae and pseudohyphae are all observed in infected tissue. C. albicans is found in the normal gastrointestinal flora of most healthy humans. Its normal habitat is the digestive tract of warm-blooded animals from which the fungal species is most frequently isolated. It has been found that 2 to 70% of individuals carry C. albicans in the oral cavity (Odds, 1988).

No description available.

To examine the functional significance of these STRE-, GCRE- and YRE-like elements in <i>C. albicans</i>, a basal promoter construct, based on the <i>C. albicans</i> <i>ADH1 </i>promoter, was constructed.  The <i>Renilla reniformis </i>luciferase gene (<i>RLUC</i>) was utilised as the reporter of choice in <i>C albicans</i>.  Copies of each type of enhancer element were then introduced upstream of the basal <i>ADH1b-RLUC</i> fusion.  Transcriptional activation by GCRE, STRE and two YRE was then studied under morphogenetic inducing conditions (serum and pH-inducing), and a range of stress conditions including temperature, amino acid starvation, glucose limitation, and exposure of H₂O₂, ethanol and heavy metal stresses.  The functionality of each element was further tested in <i>C. albans</i> mutants lacking the corresponding transcription factor. The GCRE, STRE, YRE1 and YRE2 elements did not activate transcription in response to morphogenesis induced by serum or Lee’s media.  The GCRE was shown to activate transcription in response to amino acid starvation in a Gcn4 dependent and Efg1 independent manner.  Surprisingly the well-characterised STRE mediated general stress response of <i>S. cerevisiae </i>did not appear to be conserved in <i>C. albicans</i>.  The STRE element did not appear to be functional as a stress responsive element in <i>C. albicans</i> under the range of stress conditions tested.  However, Nrg1, a represser of morphogenesis in <i>C. albicans</i> was shown to act through a STRE-related element, the NRE element (MVCCCT).  The hypha-specific gene <i>ALS8 </i>contains two Nrg1-responsive NREs (Murad <i>et al. </i>2001).  The fact that the core sequence of the NRE element is closely related to the STRE element raises the possibility of an evolutionary change of function for the STRE sequence in <i>C. albicans.  </i>The functionality of the YRE1 element appears to have been conserved in <i>C. albicans.  </i>As in <i>S. cerevisiae, </i>YRE1 mediates transcriptional responses to oxidative and heavy metal stresses in a Cap1-dependent manner.

Histological appearances of many moulds overlap and there is a need for a method to allow identification in tissue specimens.  Two methods for extracting fungal DNA from wax tissue sections, based on the TaKaRa DEXPAT ^TM kit and QIAamp^{<span style='font-family:Symbol'>Ò}DNA Mini Kit, were optimised and compared.  DNA was amplified by PCR using pan-fungal primers, and detected by Southern blot hybridisation with a probe specific for <i>Aspergillus fumigatus </i>and <i>A. flavus.  </i>The TaKaRa DEXPAT ^TM kit based method, with additional steps using lyticase and ethanol precipitation, was superior and was used to test sequential wax tissue specimens from 56 patients with IFI.  PCR products not hybridising with the probe were identified by sequencing.  The species was confirmed in all tissue culture positive cases (23 <i>A. fumigatus </i>or <i>A. flavus, </i>one <i>Chaetomium globosum</i> and one <i>Scedosporium apiospermum).  </i>Of culture negative cases, a diagnosis of <i>A. fumigatus</i> or <i>A. flavus</i> was established in 25 and emerging moulds in two (one probable <i>Alternaria </i>species and one unidentified).  Overall, emerging moulds were identified in 4 cases (7%) with a trend toward a temporal increase in these infections.  This method provides a valuable diagnostic tool for both patient management and future antifungal and epidemiological trials. Reasons for therapeutic failure in IFI are unclear.  Amphoterican B susceptibility of isolates cultured from tissue biopsies from patients who had received a median of 12 days therapy, were tested using a method  based on the NCCLS M38-A broth microdilution method.  The difficulty in treating IFI does not appear to be due to susceptibility of the isolates, but may be due to poor penetration of antifungal agents into infected tissue.

This thesis examines the role of surface mannosylation in adhesion to host surfaces and the manner in which the fungus compensates for loss of such glycosylation. This project firstly analysed the importance of <i>C. albicans</i> of surface carbohydrate in adhesion to human epithelia by disrupting the carbohydrate ring structure with sodium periodate.  This disruption led to reduced yeast adherence to the epithelial cells, implicating a role for surface mannosylation in this process.  An attempt to elucidate the relative role of <i>O</i>-linked glycans in this process proved that they do not play a major part in adhesion to buccal epithelia. Global transcript profiling was employed to study how gene expression is affected in mutants defective in glycosylation in order to identify genes which may be compensating for loss of mannan.  Of the genes implicated in this analysis, three were chosen for further study –<i>IFP7109, ECM22 </i>and <i>ECM331.  </i>A null mutant of <i>ECM22</i> mutant was constructed and subjected to phenotypic analysis but no cell wall or growth defective phenotype was found.  Reporter gene analysis was used to examine the transcriptional response of the three genes to various environmental conditions and found that the genes responded to many agents that trigger the cell wall salvage pathway further suggesting a role in compensation to changes in cell wall composition.