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      Candida albicans White and Opaque Cells Undergo Distinct Programs of Filamentous Growth

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          Abstract

          The ability to switch between yeast and filamentous forms is central to Candida albicans biology. The yeast-hyphal transition is implicated in adherence, tissue invasion, biofilm formation, phagocyte escape, and pathogenesis. A second form of morphological plasticity in C. albicans involves epigenetic switching between white and opaque forms, and these two states exhibit marked differences in their ability to undergo filamentation. In particular, filamentous growth in white cells occurs in response to a number of environmental conditions, including serum, high temperature, neutral pH, and nutrient starvation, whereas none of these stimuli induce opaque filamentation. Significantly, however, we demonstrate that opaque cells can undergo efficient filamentation but do so in response to distinct environmental cues from those that elicit filamentous growth in white cells. Growth of opaque cells in several environments, including low phosphate medium and sorbitol medium, induced extensive filamentous growth, while white cells did not form filaments under these conditions. Furthermore, while white cell filamentation is often enhanced at elevated temperatures such as 37°C, opaque cell filamentation was optimal at 25°C and was inhibited by higher temperatures. Genetic dissection of the opaque filamentation pathway revealed overlapping regulation with the filamentous program in white cells, including key roles for the transcription factors EFG1, UME6, NRG1 and RFG1. Gene expression profiles of filamentous white and opaque cells were also compared and revealed only limited overlap between these programs, although UME6 was induced in both white and opaque cells consistent with its role as master regulator of filamentation. Taken together, these studies establish that a program of filamentation exists in opaque cells. Furthermore, this program regulates a distinct set of genes and is under different environmental controls from those operating in white cells.

          Author Summary

          Candida albicans is the most common human fungal pathogen, capable of growing as a commensal organism or as an opportunistic pathogen. Perhaps the best-studied aspect of C. albicans biology is the transition between the single-celled yeast form and the multicellular filamentous form. This transition is necessary for virulence, as cells locked in either state are avirulent. Here, we demonstrate that the yeast-filament transition is tightly regulated by another morphological switch, the white-opaque phenotypic switch. White cells undergo filamentation in response to a wide range of established physiological cues, while opaque cells do not. We further show that opaque cells can indeed undergo filamentation, but that they do so in response to different environmental cues than those of white cells. We define the genetic regulation of filamentous growth in opaque cells, as well as the transcriptional profile of these cell types, and contrast them with the established program of filamentation in white cells. Our results reveal a close relationship between the white-opaque switch and the yeast-hyphal transition, and provide further evidence of the morphological plasticity of this pathogen. They also establish that epigenetic switching allows two fungal cell types with identical genomes to respond differently to environmental cues.

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          Most cited references59

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          Cluster analysis and display of genome-wide expression patterns.

          A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.
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            Nonfilamentous C. albicans mutants are avirulent.

            Candida albicans and Saccharomyces cerevisiae switch from a yeast to a filamentous form. In Saccharomyces, this switch is controlled by two regulatory proteins, Ste12p and Phd1p. Single-mutant strains, ste12/ste12 or phd1/phd1, are partially defective, whereas the ste12/ste12 phd1/phd1 double mutant is completely defective in filamentous growth and is noninvasive. The equivalent cph1/cph1 efg1/efg1 double mutant in Candida (Cph1p is the Ste12p homolog and Efg1p is the Phd1p homolog) is also defective in filamentous growth, unable to form hyphae or pseudohyphae in response to many stimuli, including serum or macrophages. This Candida cph1/cph1 efg1/efg1 double mutant, locked in the yeast form, is avirulent in a mouse model.
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              The SAT1 flipper, an optimized tool for gene disruption in Candida albicans.

              The construction of Candida albicans mutants by targeted gene disruption usually depends on the use of nutritional markers for the selection of prototrophic transformants from auxotrophic host strains, but it is becoming increasingly evident that this strategy may cause difficulties in the interpretation of mutant phenotypes. Here, we describe a new method for inactivating both alleles of a target gene in C. albicans wild-type strains to obtain homozygous null mutants. The SAT1 flipping method relies on the use of a cassette that contains a dominant nourseothricin resistance marker (caSAT1) for the selection of integrative transformants and a C. albicans-adapted FLP gene that allows the subsequent excision of the cassette, which is flanked by FLP target sequences, from the genome. Two rounds of integration/excision generate homozygous mutants that differ from the wild-type parent strain only by the absence of the target gene, and reintegration of an intact gene copy for complementation of mutant phenotypes is performed in the same way. Transformants are obtained after only 1 day of growth on a selective medium, and integration into the target locus occurs with high specificity after adding homologous flanking sequences on both sides of the cassette. FLP-mediated excision of the SAT1 flipper cassette is achieved by simply growing the transformants for a few hours in medium without selective pressure, and nourseothricin-sensitive (NouS) derivatives can easily be identified by their slower growth on indicator plates containing a low concentration of nourseothricin. We demonstrate the use of the system by deleting the OPT1 gene, which encodes an oligopeptide transporter, in the C. albicans model strain SC5314. The null mutants became resistant to the toxic peptide KLLEth, and reintroduction of an intact OPT1 copy restored susceptibility. The SAT1 flipping method provides a highly efficient method for gene disruption in C. albicans wild-type strains, which eliminates currently encountered problems in the genetic analysis of this important human fungal pathogen.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                March 2013
                March 2013
                7 March 2013
                : 9
                : 3
                : e1003210
                Affiliations
                [1 ]Department of Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island, United States of America
                [2 ]Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America
                Carnegie Mellon University, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: HS ADH RJB. Performed the experiments: HS ADH MPH. Analyzed the data: HS ADH MPH RJB. Wrote the paper: HS ADJ RJB.

                Article
                PPATHOGENS-D-12-02196
                10.1371/journal.ppat.1003210
                3591317
                23505370
                97668cb1-31c0-4a0e-b33d-bbc4f4135944
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 4 September 2012
                : 14 January 2013
                Page count
                Pages: 19
                Funding
                ADH and ADJ were supported by an NIH grant to ADJ (AI49187). HS and RJB were supported by the NIH (AI081560 and AI081704), by NSF (MCB1021120) and RJB was also supported by a PATH Award from the Burroughs Wellcome Fund. MPH is supported by a training grant for Graduate Assistance in Areas of National Need (P200A100100). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Genetics
                Gene Expression
                Molecular Genetics
                Genomics
                Genome Expression Analysis
                Microbiology
                Host-Pathogen Interaction
                Microbial Pathogens
                Pathogenesis
                Medicine
                Infectious Diseases
                Fungal Diseases

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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