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      Integration of the tricarboxylic acid (TCA) cycle with cAMP signaling and Sfl2 pathways in the regulation of CO 2 sensing and hyphal development in Candida albicans

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          Abstract

          Morphological transitions and metabolic regulation are critical for the human fungal pathogen Candida albicans to adapt to the changing host environment. In this study, we generated a library of central metabolic pathway mutants in the tricarboxylic acid (TCA) cycle, and investigated the functional consequences of these gene deletions on C. albicans biology. Inactivation of the TCA cycle impairs the ability of C. albicans to utilize non-fermentable carbon sources and dramatically attenuates cell growth rates under several culture conditions. By integrating the Ras1-cAMP signaling pathway and the heat shock factor-type transcription regulator Sfl2, we found that the TCA cycle plays fundamental roles in the regulation of CO 2 sensing and hyphal development. The TCA cycle and cAMP signaling pathways coordinately regulate hyphal growth through the molecular linkers ATP and CO 2. Inactivation of the TCA cycle leads to lowered intracellular ATP and cAMP levels and thus affects the activation of the Ras1-regulated cAMP signaling pathway. In turn, the Ras1-cAMP signaling pathway controls the TCA cycle through both Efg1- and Sfl2-mediated transcriptional regulation in response to elevated CO 2 levels. The protein kinase A (PKA) catalytic subunit Tpk1, but not Tpk2, may play a major role in this regulation. Sfl2 specifically binds to several TCA cycle and hypha-associated genes under high CO 2 conditions. Global transcriptional profiling experiments indicate that Sfl2 is indeed required for the gene expression changes occurring in response to these elevated CO 2 levels. Our study reveals the regulatory role of the TCA cycle in CO 2 sensing and hyphal development and establishes a novel link between the TCA cycle and Ras1-cAMP signaling pathways.

          Author summary

          Energy metabolism through the TCA cycle and mitochondrial electron transport are critical for the human fungal pathogen Candida albicans to survive and propagate in the host. This is, in part, due to the fact that C. albicans is a Crabtree-negative species, and thus exclusively uses respiration when oxygen is available. Here, we investigate the roles of the TCA cycle in hyphal development and CO 2 sensing in C. albicans. Through the use of ATP and the cellular signaling molecule CO 2, the TCA cycle integrates with the Ras1-cAMP signaling pathway, which is a central regulator of hyphal growth, to govern basic cellular biological processes. Together with Efg1, a downstream transcription factor of the cAMP signaling pathway, the heat shock factor-type transcription regulator Sfl2 controls CO 2-induced hyphal growth in C. albicans. Deletion of SFL2 results in the loss of global transcriptional responses under elevated CO 2 levels. Our study indicates that the TCA cycle not only occupies the central position of cellular metabolism but also regulates other biological processes such as CO 2 sensing and hyphal development through integration with the Ras1-cAMP signaling pathway in C. albicans.

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          Most cited references 54

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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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            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.

               Lara Kohler,  G Fink,  H. Liu (1994)
              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

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: ValidationRole: Writing – original draft
                Role: Data curationRole: Investigation
                Role: ConceptualizationRole: ValidationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: ValidationRole: Writing – original draft
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                7 August 2017
                August 2017
                : 13
                : 8
                Affiliations
                [1 ] State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
                [2 ] University of Chinese Academy of Sciences, Beijing, China
                [3 ] Department of Molecular and Cell Biology, University of California, Merced, California, United States of America
                University College Dublin, IRELAND
                Author notes

                The authors have declared that no competing interests exist.

                Article
                PGENETICS-D-17-01211
                10.1371/journal.pgen.1006949
                5567665
                28787458
                © 2017 Tao et al

                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.

                Page count
                Figures: 11, Tables: 1, Pages: 31
                Product
                Funding
                Funded by: Chinese national natural science foundation
                Award ID: 31625002 and 31370175
                Award Recipient :
                Funded by: Chinese national natural science foundation
                Award ID: 31570139
                Award Recipient :
                This work was supported by grants from the Chinese National Natural Science Foundation (31625002 and 31370175 to GH and 31570139 to LT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Metabolism
                Metabolic Processes
                Citric Acid Cycle
                Biology and Life Sciences
                Organisms
                Fungi
                Yeast
                Candida
                Candida Albicans
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
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                Medicine and Health Sciences
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                Biology and Life Sciences
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                Physical Sciences
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                Biology and Life Sciences
                Genetics
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                Biology and Life Sciences
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                Biochemistry
                Proteins
                DNA-binding proteins
                Transcription Factors
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Transcription Factors
                Biology and Life Sciences
                Biochemistry
                Proteins
                Regulatory Proteins
                Transcription Factors
                Physical Sciences
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                Custom metadata
                vor-update-to-uncorrected-proof
                2017-08-22
                The RNA-seq dataset has been deposited into the NCBI Gene Expression Omnibus (GEO) portal (accession# GSE102039).

                Genetics

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