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No description available.

Candida albicans is a commensal fungal organism that forms part of the normal human oral microbial flora. Although very few individuals will ever develop infection from C. albicans, it has the potential to cause a range of serious mucocutaneous infections. In healthy individuals, C. albicans is able to colonise mucosal surfaces as a commensal organism. However, in certain susceptible individuals changes occur within the host to enable C. albicans to go from being a commensal to a pathogen. Little is known about the local host innate immune defence mechanisms of the oral mucosa that are important in providing protection and maintenance of a commensal relationship. The majority of in vitro studies to investigate the host - C. albicans interaction have used an oral mucosal model based on the TR146 buccal cancer cell line, which lacks the presence of a fibroblast containing connective tissue. This mucosa model was compared for suitability against two full thickness oral mucosa models based upon primary cells seeded onto a fibroblast containing connective tissue; one generated 'in house' and the other a commercially available model (MatTek). Normal oral mucosa was used as a comparison. Immunohistochemical staining and infection studies showed that the mucosal model based upon the TR146 buccal cancer cell line resembled least the oral mucosa in vivo, whilst both full thickness oral mucosal models were more advanced and more closely resembled the oral mucosa in vivo. Infection of the full thickness oral mucosa models with a mutant strain of C. albicans, in which morphogenic properties could be controlled, was performed. Over 48 hours, hyphal-infected models displayed high levels of C. albicans invasion and showed a time-dependent increase in cell death compared to yeast-infected models. In addition, hyphal-infected models demonstrated a rapid host response by inducing cytokine release and affecting the expression of a large number of genes, as shown by microarray analysis, in particular those involved with a pro-inflammatory response. In comparison yeast-infected models caused a slower cytokine release response and affected the expression of fewer genes. The hyphal-infected tissue also demonstrated a greater host antimicrobial peptide (human l3-defensin) response when compared to yeast-infected tissue. Overall this study showed that the oral mucosa responds differently to yeast and hyphal forms of C. albicans with respect to gene, cytokine and antimicrobial peptide expression. These data suggested that the oral mucosa may have detected yeast and hyphae using different mechanisms that may initiate different intracellular signalling XI

This thesis describes the design and construction of a one-hybrid system for the analysis of transcriptional regulators in <i>C. albicans.</i> In <i>Saccharomyces cerevisiae, Escherichia coli </i>LexA fusions have been used to examine the mechanisms of action of transcription factors by targeting them artificially to reporter genes containing the EcLexA operator.  However, the EcLexA ORF is unlikely to be functional in <i>C. albicans</i> because it contains twelve CUG codons, which are decoded as serine instead of leucine in this fungus.  Therefore we used the <i>Staphylococcus aureus lexA </i>ORF, which lacks CUG codons, to develop an equivalent tool for <i>C. albicans.  </i>SaLexA is used in combination with a <i>StlacZ </i>reporter carrying the SalexA operator sequence.  The SaLexA system was validated using two transcription factors: CaNrg1 and CaGcn4.  The SaLexA-CaNrg1 fusion repressed the reporter in <i>C. albicans </i>confirming that CaNrg1 is a repressor.  Also, the SaLexA-CaGcn4 fusion activated the reporter confirming that CaGcn4 is an activator. Following these successful validation experiments, the one-hybrid system was used to show that CaNrg1-mediated repression is dependent upon the Sssn6-Tup1 co-repressor complex.  The system was also used to examine the transcriptional activities of the <i>C. albicans </i>APSES proteins Efg1 and Efh1, which contribute to the regulation of morphogenesis in <i>C. albicans.  </i>Efg1 was shown to act as a transcriptional repressor, whereas Efh1 acts as a transcriptional activator.

Candida albicans, a successful human pathogen, displays the phenomenon of glucoseenhanced oxidative stress resistance (Rodaki et al., 2009), which is not observed in other yeast species tested. The molecular bases of the phenomenon are not clear. It was suggested that glucose signalling might play a major role. Therefore, the impact of specific C. albicans mutations was tested to determine which of three known major glucose signalling pathways are required for glucose-enhanced oxidative stress resistance. Two major glucose signalling pathways were found to contribute to the phenomenon (the glucose repression pathway and the cAMP pathway), and a third pathway (the SRR pathway) is not essential for this response. The next step was to identify targets of these pathways that might contribute to the phenotype. First, it was tested whether known oxidative stress systems contribute to the GEXSR. The selected targets represented almost all main systems involved in redox control and ROS detoxification (catalase, superoxide dismutases, thiredoxins, and glutathione peroxidases) which seemed to contribute not significantly to the GEXSR. The exceptions to this were glutathione reductase (Glr1) and glutaredoxin (Grx2/Ttr1), inactivation of which affected manifestation of the phenomenon. This reinforced the view that the GSH/GSSG balance plays a key role in the GEXSR. Second, comparative analyses of transcriptomic profiles of C. albicans glucose- and lactategrown cells in response to oxidative stress and glucose treatment correspondingly revealed a small set of commonly up-regulated genes: UCF1, RNR22, MOH1, orf19.3302, and HSP21/orf19.822 (Enjalbert et al., 2006; Rodaki et al., 2009). Each potential GEXSR-specific gene appeared to be regulated in a distinct manner by the major glucose signalling pathways. The ectopic expression of potential GEXSR targets did not provide any experimental evidence to support their roles in this response. That might be related to inefficient expression from ACT1 promoter under the experimental conditions tested, and also caused by other effects.