CANTRAIN · Host-pathogen interaction systems as tools to identify antifungal targets in C. Albicans and C. Dubliniensis

Final Activity Report Summary - CANTRAIN (Host-pathogen interaction systems as tools to identify antifungal targets in C. Albicans and C. Dubliniensis)

1. We have bridged the gap between environmental stimuli inducing infection mechanism and the signal transduction pathways triggered by these stimuli. We have characterised a G protein-coupled receptor that is important for the yeast-to-hyphae transition on solid medium. Currently, we are characterising the pathway by which this receptor triggers this morphogenesis. In this aspect, we have recently identified a genetic interaction between the phosphodiesterases and this receptor, a phenotype that does not occur in Saccharomyces cerevisiae. We have also characterised the CaSPS-sensing pathway, a pathway by which amino acids induce their own uptake or induce the expression of a number of proteases. 2. Several potential new targets for antifungal drug discovery in both C. albicans and C. dubliniensis have been identified. These include the fungal specific phosphodiesterase Pde2, the trehalose-6-phosphate phosphatase Tps2, various components of the SPS sensing system, the AUF genes, which are genes belonging to one family that are upregulated during adhesion on human tissue. 3. In order to find these novel targets and also in order to characterise the pathways involved, we have developed proteomics and transcriptome analysis under various conditions. These include in vitro, ex vivo and in vivo host-pathogen interaction systems. One example is a newly developed in vivo biofilm system using a subcutaneous rat model system. This analysis also includes a comparative genomics approach comparing C. albicans and C. dublinienis. Two important differences that we have observed are the role of methionine for morphogenesis in these two species and the different substrate specificities of the potassium transporters. Finally, novel reporter constructs and a C. albicans specific two-hybrid system have been developed. 4. Apart from the pathogen, we have also looked at the host site in order to understand the reaction of the host upon an infection. Both macrophage and dendritic cell systems have been investigated and the results have been submitted for publication. 5. We have also developed different screening assays in order to screen for novel antifungal drugs. An example of a screening system is based on the G protein-coupled receptor Gpr1 that we mentioned above. A deletion of this receptor results in sensitivity to a low concentration of histatin 5, a naturally occurring antifungal. We are using a wild type strain in the presence of histatin 5 to find compounds that inhibit the growth, which are possible antagonists of this Gpr1 protein. We have also developed novel types of screening for antifungal compounds where in one screen, the antifungal efficacy as well as toxicology for human cells can be determined. Training part Apart from the biannual workshops were all the ESR and ER students presented their results, we have also organised several training courses in which most of them participated. These include: - real time PCR course; - in vitro biofilm course; - host-pathogen interaction of C. albicans using human epithelial models; - bioinformatics and microarray analysis course o C. albicans - macrophage interaction course; - microarray analysis / bioinformatics; - antifungal drug screening using robotics; - FEBS advanced practical and lecture course. Potential applications CANTRAIN will provide the pharmaceutical industry with new, partially validated antufungal targets and for some targets already lead compounds. In addition, CANTRAIN will provide trained personnel to carry out these tasks in the industry or in academia. Novel technologies, such as proteome and microarray analyses during host-pathogen interactions will also be developed.

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