Abstract
Grf10, a homeodomain-containing transcription factor, regulates adenylate and one-carbon metabolism and morphogenesis in the human fungal pathogen Candida albicans Here, we identified functional domains and key residues involved in transcription factor activity using one-hybrid and mutational analyses. We localized activation domains to the C-terminal half of the Grf10 protein by one-hybrid analysis and identified motifs using bioinformatic analyses; one of the characterized activation domains (AD1) responded to temperature. The LexA-Grf10 fusion protein activated the lexAop-HIS1 reporter in an adenine-dependent fashion, and this activation was independent of Bas1, showing that the adenine limitation signal is transmitted directly to Grf10. Overexpression of LexA-Grf10 led to filamentation, and this required a functioning homeodomain, consistent with Grf10 controlling the expression of key filamentation genes; filamentation induced by LexA-Grf10 overexpression was independent of adenine levels and Bas1. Alanine substitutions were made within the conserved interaction regions (IR) of LexA-Grf10 and Grf10 to investigate roles in transcription. In LexA-Grf10, the D302A mutation activated transcription constitutively, and the E305A mutation was regulated by adenine. When these mutations were introduced into the native gene locus, the D302A mutation was unable to complement the ADE phenotype and did not promote filamentation under hypha-inducing conditions; the E305A mutant behaved as the native gene with respect to the ADE phenotype and was partially defective in inducing hyphae. These results demonstrate allele-specific responses with respect to the different phenotypes, consistent with perturbations in the ability of Grf10 to interact with multiple partner proteins.IMPORTANCE Metabolic adaptation and morphogenesis are essential for Candida albicans, a major human fungal pathogen, to survive and infect diverse body sites in the mammalian host. C. albicans utilizes transcription factors to tightly control the transcription of metabolic genes and morphogenesis genes. Grf10, a critical homeodomain transcription factor, controls purine and one-carbon metabolism in response to adenine limitation, and Grf10 is necessary for the yeast-to-hypha morphological switching, a known virulence factor. Here, we carried out one-hybrid and mutational analyses to identify functional domains of Grf10. Our results show that Grf10 separately regulates metabolic and morphogenesis genes, and it contains a conserved protein domain for protein partner interaction, allowing Grf10 to control the transcription of multiple distinct pathways. Our findings contribute significantly to understanding the role and mechanism of transcription factors that control multiple pathogenic traits in C. albicans.
Highlights
Grf10, a homeodomain-containing transcription factor, regulates adenylate and one-carbon metabolism and morphogenesis in the human fungal pathogen Candida albicans
Functional Mapping of Activator Grf10 of C. albicans first region, the central region (CR), is located between amino acids 154 and 248 and is conserved among hemiascomycetes, species that are closely related to Candida spp. [19, 20]; the second region is the interaction region (IR), is located between amino acids 272 and 351, and contains residues that are conserved across a wider range of fungal species, including both ascomycetes and basidiomycetes (Fig. 1B; see Fig. S1 in the supplemental material)
Because Grf10 promotes gene expression [12], we hypothesized that it contains activation domain(s), as opposed to transcriptional repression domains; unlike other functional domains, the sequences of activation domains are poorly conserved and are defined experimentally for individual transcription factors [23]
Summary
A homeodomain-containing transcription factor, regulates adenylate and one-carbon metabolism and morphogenesis in the human fungal pathogen Candida albicans. Fitness attributes are required to support cellular growth and survival, while virulence attributes are required to increase the likelihood of C. albicans to cause and establish infections Transcription regulators, such as Efg, Tup, Gcn, and Ace, have been showed by transcriptomic analyses to regulate the expression of metabolic and virulence genes in C. albicans [7,8,9,10]. These studies underscore the critical role of transcription factors in coordinating the expression of fitness- and virulence-related genes for C. albicans to respond and survive in changing environments
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