There has been a significant increase in serious fungal infections coincident with the increase in immunocompromised conditions such as AIDS. There has been a parallel development of new antifungal agents. In particular, azoles that inhibit lanosterol demethylase and hence ergosterol biosynthesis. Most yeast species including Candida albicans are azole-sensitive. However, other Candida species and many molds have intrinsically low azole sensitivity. Moreover, even in sensitive fungi azoles generally lack fungicidal activity, and consequently recurrences are common. These can be minimized by long-term use, but this has selected for azole resistance in normally sensitive fungi. These in vivo limitations associated with azole use have in vitro correlates: most Candida isolates exhibit azole tolerance (trailing growth at inhibitory azole concentrations) and azole tolerant mutants arise at high frequency in vitro. Molecular correlates have also been identified: expression of ERG11 encoding lanosterol demethylase and of multidrug resistance (MDR) genes responsible for azole efflux are upregulated following azole exposure. These data suggest that fungi respond in specific ways to azoles that ultimately reduce their efficacy. The hypothesis that signalling pathways mediate at least one component of this 'azole response' is supported by studies with specific signal transduction inhibitors (STIs), which in combination with azoles inhibit azole tolerance. STI-azole combinations have considerable therapeutic potential. However, the underlying hypothesis has not been rigorously tested, and the actual fungal STI targets and the putative signalling pathways they function in remain undefined. Thus, the Specific Aims of this proposal are to: (1) Identify azole tolerance (ATO) genes by RNA expression and genetic analysis: (a) Representative STIs that inhibit azole tolerance will be examined for their effects on azole-dependent upregulation of MDR and ERG genes in C. albicans and S cerevisiae. (b) Representative STIs will be examined for their effects on azole-dependent changes in global S. cerevisiae gene expression using whole genome microarrays; C. albicans homologs will be similarly examined. (c) Additional regulated genes (other than the MDR and ERG genes already screened) identified by array analysis will be examined for their role in S. cerevisiae azole sensitivity using disruption and multcopy overexpression; parallel disruption studies will be done with selected C. albicans homologs. (2) Identify signalling pathways involved in azole tolerance and ERG upregulation. The role of calcium signalling in azole tolerance will be rigorously examined and the ATO targets of this pathway identified. Similarly, the role of the PKC (cell integrity) pathway will be examined and its ATO targets identified. Finally, the signalling pathway responsible for azole-dependent upregulation of ERG expression will be elucidated with a focus on Rox1p and Haplp transcription factors.
