Abstract
The morphogenetic transition between yeast and filamentous forms of the human fungal pathogen Candida albicans is regulated by a variety of signaling pathways. How these pathways interact to orchestrate morphogenesis, however, has not been as well characterized. To address this question and to identify genes that interact with the Regulation of Ace2 and Morphogenesis (RAM) pathway during filamentation, we report the first large-scale genetic interaction screen in C. albicans. Our strategy for this screen was based on the concept of complex haploinsufficiency (CHI). A heterozygous mutant of CBK1 (cbk1Δ/CBK1), a key RAM pathway protein kinase, was subjected to transposon-mediated, insertional mutagenesis. The resulting double heterozygous mutants (6,528 independent strains) were screened for decreased filamentation on Spider Medium (SM). From the 441 mutants showing altered filamentation, 139 transposon insertion sites were sequenced, yielding 41 unique CBK1-interacting genes. This gene set was enriched in transcriptional targets of Ace2 and, strikingly, the cAMP-dependent protein kinase A (PKA) pathway, suggesting an interaction between these two pathways. Further analysis indicates that the RAM and PKA pathways co-regulate a common set of genes during morphogenesis and that hyper-activation of the PKA pathway may compensate for loss of RAM pathway function. Our data also indicate that the PKA–regulated transcription factor Efg1 primarily localizes to yeast phase cells while the RAM–pathway regulated transcription factor Ace2 localizes to daughter nuclei of filamentous cells, suggesting that Efg1 and Ace2 regulate a common set of genes at separate stages of morphogenesis. Taken together, our observations indicate that CHI–based screening is a useful approach to genetic interaction analysis in C. albicans and support a model in which these two pathways regulate a common set of genes at different stages of filamentation.
Highlights
Candida albicans is a member of the resident flora of the gastrointestinal tract and is the most common fungal pathogen in humans
We demonstrate that libraries of C. albicans strains containing heterozygous mutations in two different genes can be generated and used to study genetic interactions in C. albicans on a large scale
We applied this approach to the study of the RAM (Regulation of Ace2 and Morphogenesis) signaling network during the morphogenetic transition of C. albicans from yeast to filamentous growth
Summary
Candida albicans is a member of the resident flora of the gastrointestinal tract and is the most common fungal pathogen in humans. C. albicans genetically restricted to the yeast form by constitutive expression of NRG1 are able to establish infection in mice but no disease results until the expression of NRG1 is repressed and the organism is able to form filaments. Many mutants that are unable to undergo morphogenesis display other phenotypes. Many transcription factors that are required for morphogenesis regulate a host of other genes and display pleiomorphic phenotypes. The complicated nature of the relationship between morphogenesis has been further highlighted by the elegant study recently reported by Noble et al [5]. Noble et al generated a bar-coded collection of homozygous deletion mutants and used it in a signature-tagged mutagenesis study of infectivity in a mouse model [5].
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