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      Multiple roles and diverse regulation of the Ras/cAMP/protein kinase A pathway in Candida albicans

      1 , 2 , 3 , 4 , 4 , 5 , 6 , 1 , 7
      Molecular Microbiology
      Wiley

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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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            Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans.

            Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels-which are susceptible to chromosome position effects-can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2Delta/leu2Delta, his1Delta/his1Delta; arg4Delta/arg4Delta, his1Delta/his1Delta; and arg4Delta/arg4Delta, leu2Delta/leu2Delta, his1Delta/his1Delta) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.
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              Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog.

              A Candida albicans gene (CPH1) was cloned that encodes a protein homologous to Saccharomyces cerevisiae Ste12p, a transcription factor that is the target of the pheromone response mitogen-activated protein kinase cascade. CPH1 complements both the mating defect of ste12 haploids and the filamentous growth defect of ste12/ste12 diploids. Candida albicans strains without a functional CPH1 gene (cph1/cph1) show suppressed hyphal formation on solid medium. However, cph1/cph1 strains can still form hyphae in liquid culture and in response to serum. Thus, filamentous growth may be activated in C. albicans by the same signaling kinase cascade that activates Ste12p in S. cerevisiae; however, alternative pathways may exist in C. albicans.
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                Author and article information

                Journal
                Molecular Microbiology
                Mol Microbiol
                Wiley
                0950382X
                January 2019
                January 2019
                November 04 2018
                : 111
                : 1
                : 6-16
                Affiliations
                [1 ]State Key Laboratory of Genetic Engineering, School of Life Sciences; Fudan University; Shanghai 200438 China
                [2 ]State Key Laboratory of Mycology, Institute of Microbiology; Chinese Academy of Sciences; Beijing 100101 China
                [3 ]University of Chinese Academy of Sciences; Beijing 100049 China
                [4 ]Department of Dermatology; Affiliated Hospital of Guizhou Medical University; Guiyang 550025 China
                [5 ]Institute of Molecular and Cell Biology; 61 Biopolis Drive, Proteos Singapore 138673 Singapore
                [6 ]Department of Biochemistry, Yong Loo Lin School of Medicine; National University of Singapore; Singapore 119228 Singapore
                [7 ]Institutes of Biomedical Sciences; Fudan University; Shanghai 200032 China
                Article
                10.1111/mmi.14148
                30299574
                9e05d9e5-e9c3-4fb1-ae64-e7ac137bee7f
                © 2018

                http://doi.wiley.com/10.1002/tdm_license_1.1

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