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.

FIG 1

Live cell imaging of PI(4,5)P₂ accumulations. (A) Confocal image of live C. albicans SC5314 cells expressing CaPH×2-GFP exposed to caspofungin (1× MIC), highlighting the early accumulation of PI(4,5)P₂ foci (arrows) in the vicinity of the plasma membrane. As indicated, the image was acquired 3 min after exposure to caspofungin. Acq. Time, acquisition time. (B) Time-lapse images of SC5314 cells expressing CaPH×2-GFP during a 3-h exposure to caspofungin (4× MIC), demonstrating the chaotic and highly dynamic nature of aberrant PI(4,5)P₂ localization (arrows). Images were acquired at 41, 53, 73, and 89 min after exposure to caspofungin (from left to right and top to bottom, respectively, beginning at the top left).

FIG 2

The caspofungin-induced redistribution of PI(4,5)P₂ is independent of Mkc1 protein kinase. Wild-type DAY286 and mkc1-null mutant VIC1177 strains expressing CaPH×2-GFP were imaged by confocal microscopy for 3 h following exposure to caspofungin (90 ng/ml and 15 ng/ml, respectively). The PI(4,5)P₂ redistribution in both strains was similar to that observed in strain SC5314 in response to caspofungin. Arrows, PI(4,5)P₂ foci. The acquisition times after caspofungin exposure are indicated. VIC1177 was more sensitive to caspofungin than DAY286; both strains were more sensitive than SC5314. Experiments involving live cell imaging of the response to caspofungin were performed at concentrations ranging from 15 to 600 ng/ml (see also the time lapse videos in the supplemental material).

FIG 3

Live imaging of PI(4,5)P₂ within C. albicans cells exposed to caspofungin. Wild-type SC5314 cells expressing CaPH×2-GFP were imaged by confocal microscopy for 3 h following exposure to a range of caspofungin concentrations (0.25× to 64× MIC). Images of representative fields taken from immediately prior to drug exposure (time zero [T₀]) and 1, 2, and 3 h after exposure are shown. Images of control cells grown in the absence of caspofungin and cells exposed to 1×, 4×, and 32× MIC are also presented. In control cells, PI(4,5)P₂ was distributed regularly throughout the plasma membrane. The PI(4,5)P₂ intensity in control cells diminished over the 3-h time frame, due to a dampening of the signal that was consistently observed during growth in the experimental system. In caspofungin-exposed cells, the PI(4,5)P₂ intensity increased over time (the results are quantified in Fig. 4), and wide mother-daughter cell necks, thick septa, and aberrant septum-like structures were apparent (detailed in Fig. 5). Abnormal PI(4,5)P₂ accumulations were present in the vicinity of the plasma membrane following caspofungin exposure, as shown in greater detail in Fig. 1. Time-lapse videos are provided in the supplemental material.

FIG 4

Quantification of PI(4,5)P₂ levels. GFP intensities over 3 h were measured with Imaris software. The graph presents normalized intensities captured every 3 min in response to the range of caspofungin exposures, with the results being presented as a percentage of the control (no treatment) intensity at time zero. As shown, PI(4,5)P₂ levels increased steadily until about 2.5 h at each concentration. The levels increased in a dose-response manner up to a caspofungin concentration of 4× MIC. PI(4,5)P₂ levels then decreased in a dose-response manner at concentrations ranging from 8× to 32× MIC. This pattern resembled the paradoxical fungicidal effects of caspofungin at similar concentrations against C. albicans SC5314 and other strains that are well described.

FIG 5

Cytokinesis and septation during caspofungin exposure. (A) Time-lapse images obtained every 6 min after exposure to caspofungin (4× MIC) reveal broad mother-daughter necks and thick, bowed septa during cytokinesis. (B) Time-lapse images obtained every 3 min after exposure to caspofungin (4× MIC) demonstrate aberrant septation in certain cells. Unidirectional septum-like structures which become arrested in the cytoplasm are visualized.

FIG 6

Colocalization of PI(4,5)P₂ and Cdc10p during caspofungin exposure. C. albicans SC5314 cells coexpressing CaPH×2-GFP and RFP-Cdc10 were imaged by confocal microscopy for 3 h following exposure to 4× MIC caspofungin. PI(4,5)P₂ and Cdc10p colocalize (yellow signals) at numerous foci within abnormally wide bud necks and at aberrant plasma membrane foci. Arrows, several examples of colocalization. The acquisition times after caspofungin exposure are indicated. DIC, differential interference contrast; CaRFP, C. albicans-optimized RFP.

FIG 7

Cell wall integrity pathway activation in response to caspofungin. (A) C. albicans cells were exposed to caspofungin (CSF; 1× MIC) for 3 h, and Western blotting was performed at the indicated time points using anti-phospho-p44/42 MAP kinase to detect the phosphorylated form of Mkc1p. After caspofungin exposure, Mkc1p was phosphorylated within minutes. Phosphorylation reached a peak at 10 min, after which it was diminished over 3 h. Data were confirmed in two independent experiments. (B) In a dose-ranging experiment, C. albicans cells were exposed to caspofungin (0.5× to 64 × MIC) for 10 min. The intensities of the Western blot bands (bottom) were measured with Adobe Photoshop software, and a graph of the mean values from two independent experiments was plotted (top). There was a dose-response of pathway activation that reached a maximum at 4× MIC. At higher concentrations, pathway activation was attenuated, consistent with paradoxical effects. The bar graph shows mean values and error bars from replicate experiments. In both panels, Mkc1p was detected as a loading control.

FIG 8

Caspofungin time-kill experiments. Time-kill experiments were conducted under in vitro conditions similar to those used for live cell imaging. Killing of C. albicans over 20 h was similar at caspofungin concentrations ranging from 0.5× to 4× MIC. Killing was attenuated at concentrations ranging from 8× to 64× MIC, consistent with the paradoxical fungicidal effects that were demonstrated previously under slightly different growth conditions (5, 8).

FIG 9

Live imaging of PI(4,5)P₂ within C. albicans cells exposed to various agents. Experiments were conducted as described in the legend to Fig. 3, and images of cells exposed to caspofungin (4× MIC) and the indicated other drugs for 3 h are shown. The abnormalities in PI(4,5)P₂ levels, distribution, cell morphology, and cytokinesis and septation that were observed in response to caspofungin were not observed in the presence of the other agents.

FIG 10

Cell wall integrity pathway activation in response to various agents. Western blot experiments were performed as described in the legend to Fig. 5, using cells exposed to caspofungin and other agents. Caspofungin caused greater activation of the cell wall integrity pathway than the other agents, as measured by the intensity of Mkc1p phosphorylation. Mkc1p was detected as a loading control. CASPO, caspofungin; FLC, fluconazole; AMB, amphotericin B; CW, calcofluor white.