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      Stimulation of Chitin Synthesis Rescues Candida albicans from Echinocandins

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          Abstract

          Echinocandins are a new generation of novel antifungal agent that inhibit cell wall β(1,3)-glucan synthesis and are normally cidal for the human pathogen Candida albicans. Treatment of C. albicans with low levels of echinocandins stimulated chitin synthase ( CHS) gene expression, increased Chs activity, elevated chitin content and reduced efficacy of these drugs. Elevation of chitin synthesis was mediated via the PKC, HOG, and Ca 2+-calcineurin signalling pathways. Stimulation of Chs2p and Chs8p by activators of these pathways enabled cells to survive otherwise lethal concentrations of echinocandins, even in the absence of Chs3p and the normally essential Chs1p, which synthesize the chitinous septal ring and primary septum of the fungus. Under such conditions, a novel proximally offset septum was synthesized that restored the capacity for cell division, sustained the viability of the cell, and abrogated morphological and growth defects associated with echinocandin treatment and the chs mutations. These findings anticipate potential resistance mechanisms to echinocandins. However, echinocandins and chitin synthase inhibitors synergized strongly, highlighting the potential for combination therapies with greatly enhanced cidal activity.

          Author Summary

          Fungal pathogens are increasingly important agents of human disease and are also difficult to treat since few antifungal agents kill the invading organism. The cell wall of a fungus is essential for its viability and this can be attacked by a new generation of antifungal antibiotics called echinocandins. Echinocandins such as caspofungin are normally cidal for the human pathogen Candida albicans. These inhibit the synthesis of β(1,3)-glucan, a major strength-imparting polysaccharide in the cell wall. Treatment of C. albicans with echinocandins in vitro stimulated the formation of a second cell wall polysaccharide—chitin, which rescued the cells. Treatments that increased the chitin content of the C. albicans cell wall reduced the efficacy of echinocandins and could even induce the formation of novel structures such as a salvage septum that enabled the cells to continue to undergo cell division under otherwise lethal conditions. Combined treatments with echinocandins and chitin synthase inhibitors synergized strongly, highlighting the potential for potent combination therapies with enhanced fungicidal activity.

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          Most cited references57

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          Cell wall integrity signaling in Saccharomyces cerevisiae.

          The yeast cell wall is a highly dynamic structure that is responsible for protecting the cell from rapid changes in external osmotic potential. The wall is also critical for cell expansion during growth and morphogenesis. This review discusses recent advances in understanding the various signal transduction pathways that allow cells to monitor the state of the cell wall and respond to environmental challenges to this structure. The cell wall integrity signaling pathway controlled by the small G-protein Rho1 is principally responsible for orchestrating changes to the cell wall periodically through the cell cycle and in response to various forms of cell wall stress. This signaling pathway acts through direct control of wall biosynthetic enzymes, transcriptional regulation of cell wall-related genes, and polarization of the actin cytoskeleton. However, additional signaling pathways interface both with the cell wall integrity signaling pathway and with the actin cytoskeleton to coordinate polarized secretion with cell wall expansion. These include Ca(2+) signaling, phosphatidylinositide signaling at the plasma membrane, sphingoid base signaling through the Pkh1 and -2 protein kinases, Tor kinase signaling, and pathways controlled by the Rho3, Rho4, and Cdc42 G-proteins.
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            Isogenic strain construction and gene mapping in Candida albicans.

            Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle. Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing. To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus. The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations. The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura- strain otherwise isogenic with the parental clinical isolate. Subsequent mutation of the Ura- strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker. URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene. Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura- phenotype. These Ura- segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis. To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats. Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.
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              Echinocandin antifungal drugs.

              The echinocandins are large lipopeptide molecules that are inhibitors of beta-(1,3)-glucan synthesis, an action that damages fungal cell walls. In vitro and in vivo, the echinocandins are rapidly fungicidal against most Candida spp and fungistatic against Aspergillus spp. They are not active at clinically relevant concentrations against Zygomycetes, Cryptococcus neoformans, or Fusarium spp. No drug target is present in mammalian cells. The first of the class to be licensed was caspofungin, for refractory invasive aspergillosis (about 40% response rate) and the second was micafungin. Adverse events are generally mild, including (for caspofungin) local phlebitis, fever, abnormal liver function tests, and mild haemolysis. Poor absorption after oral administration limits use to the intravenous route. Dosing is once daily and drug interactions are few. The echinocandins are widely distributed in the body, and are metabolised by the liver. Results of studies of caspofungin in candidaemia and invasive candidiasis suggest equivalent efficacy to amphotericin B, with substantially fewer toxic effects. Absence of antagonism in combination with other antifungal drugs suggests that combination antifungal therapy could become a general feature of the echinocandins, particularly for invasive aspergillosis.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plpa
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                April 2008
                April 2008
                4 April 2008
                : 4
                : 4
                : e1000040
                Affiliations
                [1]School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, United Kingdom
                Johns Hopkins University School of Medicine, United States of America
                Author notes

                Conceived and designed the experiments: LW CM NG. Performed the experiments: LW CM ID ML AM. Analyzed the data: LW CM ID ML AM NG. Wrote the paper: LW CM NG.

                Article
                07-PLPA-RA-0660R3
                10.1371/journal.ppat.1000040
                2271054
                18389063
                0a0863e3-5beb-49d5-84bc-3656205dfbc6
                Walker 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.
                History
                : 19 September 2007
                : 7 March 2008
                Page count
                Pages: 12
                Categories
                Research Article
                Cell Biology/Cell Growth and Division
                Cell Biology/Microbial Growth and Development
                Cell Biology/Microbial Physiology and Metabolism
                Infectious Diseases/Antimicrobials and Drug Resistance
                Infectious Diseases/Fungal Infections
                Microbiology/Medical Microbiology
                Microbiology/Microbial Growth and Development
                Molecular Biology/Post-Translational Regulation of Gene Expression

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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