85
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Calcineurin Is Required for Pseudohyphal Growth, Virulence, and Drug Resistance in Candida lusitaniae

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Candida lusitaniae is an emerging fungal pathogen that infects immunocompromised patients including HIV/AIDS, cancer, and neonatal pediatric patients. Though less prevalent than other Candida species, C. lusitaniae is unique in its ability to develop resistance to amphotericin B. We investigated the role of the calcium-activated protein phosphatase calcineurin in several virulence attributes of C. lusitaniae including pseudohyphal growth, serum survival, and growth at 37°C. We found that calcineurin and Crz1, a C. albicans Crz1 homolog acting as a downstream target of calcineurin, are required for C. lusitaniae pseudohyphal growth, a process for which the underlying mechanism remains largely unknown in C. lusitaniae but hyphal growth is fundamental to C. albicans virulence. We demonstrate that calcineurin is required for cell wall integrity, ER stress response, optimal growth in serum, virulence in a murine systemic infection model, and antifungal drug tolerance in C. lusitaniae. To further examine the potential of targeting the calcineurin signaling cascade for antifungal drug development, we examined the activity of a calcineurin inhibitor FK506 in combination with caspofungin against echinocandin resistant C. lusitaniae clinical isolates. Broth microdilution and drug disk diffusion assays demonstrate that FK506 has synergistic fungicidal activity with caspofungin against echinocandin resistant isolates. Our findings reveal that pseudohyphal growth is controlled by the calcineurin signaling cascade, and highlight the potential use of calcineurin inhibitors and caspofungin for emerging drug-resistant C. lusitaniae infections.

          Related collections

          Most cited references42

          • Record: found
          • Abstract: found
          • Article: not found

          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.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Calcineurin is required for virulence of Cryptococcus neoformans.

            Cyclosporin A (CsA) and FK506 are antimicrobial, immunosuppressive natural products that inhibit signal transduction. In T cells and Saccharomyces cerevisiae, CsA and FK506 bind to the immunophilins cyclophilin A and FKBP12 and the resulting complexes inhibit the Ca2+-regulated protein phosphatase calcineurin. We find that growth of the opportunistic fungal pathogen Cryptococcus neoformans is sensitive to CsA and FK506 at 37 degrees C but not at 24 degrees C, suggesting that CsA and FK506 inhibit a protein required for C. neoformans growth at elevated temperature. Genetic evidence supports a model in which immunophilin-drug complexes inhibit calcineurin to prevent growth at 37 degrees C. The gene encoding the C. neoformans calcineurin A catalytic subunit was cloned and disrupted by homologous recombination. Calcineurin mutant strains are viable but do not survive in vitro conditions that mimic the host environment (elevated temperature, 5% CO2 or alkaline pH) and are no longer pathogenic in an animal model of cryptococcal meningitis. Introduction of the wild-type calcineurin A gene complemented these growth defects and restored virulence. Our findings demonstrate that calcineurin is required for C. neoformans virulence and may define signal transduction elements required for fungal pathogenesis that could be targets for therapeutic intervention.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections.

              The number of immunocompromised patients with invasive fungal infections continues to increase and new antifungal therapies are not keeping pace with the growing incidence of these infections and their associated mortality. Calcineurin inhibition is currently used to exert effective immunosuppression following organ transplantation and in treating various other conditions. However, the calcineurin pathway is also intricately involved in the growth and pathogenesis of the three major fungal pathogens of humans, Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, and the exploitation of fungal calcineurin pathways holds great promise for the future development of novel antifungal agents. This Review summarizes our current understanding of calcineurin biology in these fungal species, and its exciting potential role in treating invasive fungal infections.
                Bookmark

                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                31 August 2012
                : 7
                : 8
                : e44192
                Affiliations
                [1 ]Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, United States of America
                [2 ]Department of Chemistry, Duke University, Durham, North Carolina, United States of America
                [3 ]Department of Microbiology and Parasitology, University of Perpetual Help – Dr. Jose G. Tamayo Medical University, Biñan, Laguna, Philippines
                [4 ]Environment and Biotechnology Division, Department of Science and Technology, Bicutan, Philippines
                [5 ]National Institutes of Health-Philippines, University of the Philippines, Manila, Philippines
                Yonsei University, Republic of Korea
                Author notes

                Competing Interests: This work was supported by pilot funds from Astellas Pharma Inc. and Merck & Co. Inc. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

                Conceived and designed the experiments: AB MN JH YC. Performed the experiments: JZ FS UB YC. Analyzed the data: JZ FS UB AB MN JH YC. Wrote the paper: JZ JH YC.

                Article
                PONE-D-12-18208
                10.1371/journal.pone.0044192
                3432075
                22952924
                d0e9227b-27c5-4f0e-a9b5-57f298d3f55b
                Copyright @ 2012

                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
                : 20 June 2012
                : 30 July 2012
                Page count
                Pages: 14
                Funding
                This work was supported in part by the Duke University Center for AIDS Research (CFAR), a National Institutes of Health (NIH)–funded program (2P30 AI064518-06 to Y.-L.C.) and NIH/National Institute of Allergy and Infectious Diseases R01 grant AI50438 (J.H.). This work was also supported by Pilot funds from Astellas Pharma Inc. and Merck & Co. Inc. (J.H. and Y.-L.C.). The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Genetics
                Gene Function
                Molecular Genetics
                Microbiology
                Mycology
                Fungal Physiology
                Fungal Structure
                Fungi
                Yeast
                Medical Microbiology
                Microbial Growth and Development
                Microbial Mutation
                Microbial Pathogens
                Microbial Physiology
                Pathogenesis
                Model Organisms
                Animal Models
                Mouse
                Medicine
                Infectious Diseases
                Fungal Diseases

                Uncategorized
                Uncategorized

                Comments

                Comment on this article