Systematic analysis of pH adaptation in fungi

Abstract

A critical trait for fungal pathogens is ability to adapt to the host environment. In Candida albicans and Aspergillus nidulans it has been demonstrated that alkaline adaptation, mediated through the pH-responsive transcription factor Rim101/PacC, is critical for fungal virulence. Despite this, as Rim101/PacC is a transcription factor, it is likely that components of the downstream transcriptional response to pH, rather than Rim101/PacC itself, mediate this virulence phenotype. In this thesis a systematic approach is adopted to investigate the mechanisms of pH adaptation in two fungal pathogens, Candida glabrata and Aspergillus fumigatus, as well as the model organism Saccharomyces cerevisiae. Specifically comparative microarray analysis of the pH response at pH4 and pH8 was performed in all three fungi, in order to identify the conserved components of the translational response to pH. Furthermore, in order to better understand pH adaptation in S. cerevisiae, phenotypic analysis of genome-wide knockout libraries at pH4 and pH8 was also undertaken. As S. cerevisiae rim101 strains also demonstrate salt sensitivity parallel phenotypic and microarray analysis of yeast adaptation to lithium chloride was also performed. Finally, in order to identify genes under the control of the pH-responsive transcription factor Rim101, comparative microarray analysis of RIM101 and rim101 strains was also carried out at pH4 and pH8. These studies demonstrated that phenotypic and transcriptional components of pH adaptation appear to be distinct from eachother. In order to further investigate this molecular network modelling methods were employed using molecular interaction data available In S. cerevisiae. These approaches demonstrated a close network relationship between phenotypic and transcriptional components of alkaline adaptation. Comparative analysis of the yeast response to lithium chloride and alkaline pH further suggest that Rim101 and components of the Rim signalling pathway are critical to salt, but not alkaline, adaptation. Furthermore, Rlm101 appears to act predominantly as a transcriptional repressor at alkaline pH. The transcriptional response to pH was compared in S. cerevisiae, C. glabrata and A. fumigatus by microarray analysis at pH4 and pH8 in each organism, followed by identification of crossspecies pH functional orthologues by reciprocal blast analysis. This approach enabled the identification of species conserved components of the pH transcriptional response. Iron and copper homeostatic genes were consistently upregulated at alkaline pH in all three organisms, and relatively conserved in S. cerevisiae and C. glabrata. Fudhermore, at alkaline pH there is strong repression of many ribosomal genes consistent with the observation that yeast growth is limited at alkaline pH. This raises the possibility that pH-dependent iron and copper acquisition are major determinants of the ability to grow at alkaline pH, and therefore within the host.