title: Highly Dynamic and Specific Phosphatidylinositol 4,5-Bisphosphate, Septin, and Cell Wall Integrity Pathway Responses Correlate with Caspofungin Activity against Candida albicans.
abstract: Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] activates the yeast cell wall integrity pathway. Candida albicans exposure to caspofungin results in the rapid redistribution of PI(4,5)P2 and septins to plasma membrane foci and subsequent fungicidal effects. We studied C. albicans PI(4,5)P2 and septin dynamics and protein kinase C (PKC)-Mkc1 cell wall integrity pathway activation following exposure to caspofungin and other drugs. PI(4,5)P2 and septins were visualized by live imaging of C. albicans cells coexpressing green fluorescent protein (GFP)-pleckstrin homology (PH) domain and red fluorescent protein-Cdc10p, respectively. PI(4,5)P2 was also visualized in GFP-PH domain-expressing C. albicans mkc1 mutants. Mkc1p phosphorylation was measured as a marker of PKC-Mkc1 pathway activation. Fungicidal activity was assessed using 20-h time-kill assays. Caspofungin immediately induced PI(4,5)P2 and Cdc10p colocalization to aberrant foci, a process that was highly dynamic over 3 h. PI(4,5)P2 levels increased in a dose-response manner at caspofungin concentrations of ≤4× MIC and progressively decreased at concentrations of ≥8× MIC. Caspofungin exposure resulted in broad-based mother-daughter bud necks and arrested septum-like structures, in which PI(4,5)P2 and Cdc10 colocalized. PKC-Mkc1 pathway activation was maximal within 10 min, peaked in response to caspofungin at 4× MIC, and declined at higher concentrations. The caspofungin-induced PI(4,5)P2 redistribution remained apparent in mkc1 mutants. Caspofungin exerted dose-dependent killing and paradoxical effects at ≤4× and ≥8× MIC, respectively. Fluconazole, amphotericin B, calcofluor white, and H2O2 did not impact the PI(4,5)P2 or Cdc10p distribution like caspofungin did. Caspofungin exerts rapid PI(4,5)P2-septin and PKC-Mkc1 responses that correlate with the extent of C. albicans killing, and the responses are not induced by other antifungal agents. PI(4,5)P2-septin regulation is crucial in early caspofungin responses and PKC-Mkc1 activation.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA hab42@pitt.edu.
authors.forename: Hassan
authors.initials: H
authors.lastname: Badrane
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: M Hong
authors.initials: MH
authors.lastname: Nguyen
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA VA Pittsburgh Healthcare System, Pittsburgh, Pennsylvania, USA.
authors.forename: Cornelius J
authors.initials: CJ
authors.lastname: Clancy
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology*; D001557:Benzenesulfonates / Q000494:pharmacology; D002176:Candida albicans / Q000187:drug effects*; D000077336:Caspofungin; D018797:Cell Cycle Proteins / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism*; D054714:Echinocandins / Q000494:pharmacology*; D015725:Fluconazole / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide / Q000378:metabolism; D055666:Lipopeptides / Q000494:pharmacology*; D008164:Luminescent Proteins; D008826:Microbial Sensitivity Tests; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010766:Phosphorylation; D000072417:Protein Domains / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D058112:Septins / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D000666:Amphotericin B; D000935:Antifungal Agents; D001557:Benzenesulfonates; D000077336:Caspofungin; D018797:Cell Cycle Proteins; D054714:Echinocandins; D015725:Fluconazole; D005656:Fungal Proteins; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D055666:Lipopeptides; D008164:Luminescent Proteins; D020928:Mitogen-Activated Protein Kinases; D019269:Phosphatidylinositol 4,5-Diphosphate; D011493:Protein Kinase C; D058112:Septins; D000097573:Red Fluorescent Protein; C007061:C.I. Fluorescent Brightening Agent 28; C092603:MKC1 protein, Candida albicans