Highly Dynamic and Specific Phosphatidylinositol 4,5-Bisphosphate, Septin, and Cell Wall Integrity Pathway Responses Correlate with Caspofungin Activity against Candida albicans

Abstract

ABSTRACT Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] activates the yeast cell wall integrity pathway. Candida albicans exposure to caspofungin results in the rapid redistribution of PI(4,5)P 2 and septins to plasma membrane foci and subsequent fungicidal effects. We studied C. albicans PI(4,5)P 2 and septin dynamics and protein kinase C (PKC)-Mkc1 cell wall integrity pathway activation following exposure to caspofungin and other drugs. PI(4,5)P 2 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)P 2 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)P 2 and Cdc10p colocalization to aberrant foci, a process that was highly dynamic over 3 h. PI(4,5)P 2 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)P 2 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)P 2 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 H 2 O 2 did not impact the PI(4,5)P 2 or Cdc10p distribution like caspofungin did. Caspofungin exerts rapid PI(4,5)P 2 -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)P 2 -septin regulation is crucial in early caspofungin responses and PKC-Mkc1 activation.