Rapid redistribution of phosphatidylinositol-(4,5)-bisphosphate and septins during the Candida albicans response to caspofungin

Abstract

We previously showed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and septin regulation play major roles in maintaining Candida albicans cell wall integrity in response to caspofungin and other stressors. Here, we establish a link between PI(4,5)P2 signaling and septin localization and demonstrate that rapid redistribution of PI(4,5)P2 and septins is part of the natural response of C. albicans to caspofungin. First, we studied caspofungin-hypersusceptible C. albicans irs4 and inp51 mutants, which have elevated PI(4,5)P2 levels due to loss of PI(4,5)P2-specific 5'-phosphatase activity. PI(4,5)P2 accumulated in discrete patches, rather than uniformly, along surfaces of mutants in yeast and filamentous morphologies, as visualized with a green fluorescent protein (GFP)-pleckstrin homology domain. The patches also contained chitin (calcofluor white staining) and cell wall protein Rbt5 (Rbt5-GFP). By transmission electron microscopy, patches corresponded to plasma membrane invaginations that incorporated cell wall material. Fluorescently tagged septins Cdc10 and Sep7 colocalized to these sites, consistent with well-described PI(4,5)P2-septin physical interactions. Based on expression patterns of cell wall damage response genes, irs4 and inp51 mutants were firmly positioned within a group of caspofungin-hypersusceptible, septin-regulatory protein kinase mutants. irs4 and inp51 were linked most closely to the gin4 mutant by expression profiling, PI(4,5)P2-septin-chitin redistribution and other phenotypes. Finally, sublethal 5-min exposure of wild-type C. albicans to caspofungin resulted in redistribution of PI(4,5)P2 and septins in a manner similar to those of irs4, inp51, and gin4 mutants. Taken together, our data suggest that the C. albicans Irs4-Inp51 5'-phosphatase complex and Gin4 function upstream of PI(4,5)P2 and septins in a pathway that helps govern responses to caspofungin.

Fig 1

PI(4,5)P2 accumulates in discrete patches in irs4 and inp51 mutants. C. albicans SC5314 (wild-type) and irs4 and inp51 mutants expressing CaPH-GFP were grown in yeast (A to C, respectively; YPD medium at 30°C) or hyphal (D to F, respectively; YPD medium + 5% fetal bovine serum [FBS] at 37°C) morphologies in 35-mm-diameter glass-bottom dishes and imaged using confocal microscopy. As shown, CaPH-GFP was distributed uniformly in SC5314 (A and D) but concentrated in discrete patches in irs4 (B and E) and inp51 (C and F) mutants.

Fig 2

PI(4,5)P2 and chitin colocalize at aberrant patches in irs4 and inp51 mutants. PI(4,5)P2 was localized in C. albicans SC5314 and irs4 and inp51 mutants using a CaPH-GFP reporter (A, D, and G, respectively), as in the experiments shown in Fig. 1. Chitin was localized in the same cells by calcofluor white staining (B, E, and H, respectively). Overlay images revealed that CaPH-GFP and calcofluor white colocalized to aberrant sites in irs4 (F) and inp51 (I) mutants (cyan color).

Fig 3

The GPI-anchored cell wall protein Rbt5 colocalizes with chitin at aberrant patches in mutant cells. C. albicans CAI4 and irs4 and inp51 mutants expressing an Rbt5-GFP fusion were visualized by confocal microscopy as in earlier experiments. inp51 mutant cells are shown as a representative. Rbt5-GFP was distributed normally in CAI4 cells (A) but was concentrated within aberrant patches (white arrows) in the inp51 mutant (B and C [higher-resolution images]). Rbt5-GFP colocalized with calcofluor white in the inp51 mutant (D, cyan color, arrowhead). The irs4 mutant resembled the inp51 mutant in all experiments.

Fig 4

Mutant cells exhibit plasma membrane and cell wall abnormalities that correspond to sites of chitin accumulation. C. albicans SC5314 exhibited normal plasma membrane and cell wall (A) and septa (B) by transmission electron microscopy. The inp51 mutant, on the other hand, had striking invaginations of the plasma membrane and cell wall (invaginations in panels C and E are shown at higher power [arrowheads] in panels D and F, respectively). Immunogold-labeled wheat germ agglutinin, which binds chitin, was distributed normally in SC5314 cells (G, black dots) but accumulated within the plasma membrane and cell wall invaginations of the mutant (H). The inp51 mutant is shown as a representative; similar results were obtained for the irs4 mutant.

Fig 5

Septins and chitin colocalize in mutant cells. C. albicans SC5314 and irs4 and inp51 mutants expressing Cdc10-GFP were visualized by confocal microscopy, and chitin was localized by calcofluor white staining. Cdc10-GFP and calcofluor white were distributed normally in SC5314 cells (A and B, respectively), but were concentrated within aberrant patches in the inp51 mutant (E and F, respectively). As evident in the overlay images, Cdc10-GFP colocalized with calcofluor white in the inp51 mutant (G and H [higher resolution], cyan color and arrowheads).

Fig 6

Septins and PI(4,5)P2 colocalize in mutant cells. C. albicans inp51 colocalized Cdc10-RFP and CaPH-GFP (yellow color and arrowheads in overlay images in B; higher-resolution images in C and D). Images of irs4 mutants expressing Cdc10-RFP and CaPH-GFP, and both mutants expressing Sep7-RFP and CaPH-GFP, were similar (not shown).

Fig 7

C. albicans irs4 and inp51 mutants display cell wall damage response gene expression profiles that are similar to those of septin regulatory protein kinase mutants. The expression of 14 damage response genes was analyzed in mutant strains in the absence of cell wall stress. The expression of TDH3, a housekeeping gene involved in glycolysis, was used to normalize expression between strains. Strains were further normalized to their complemented strain to reduce strain background errors. Resultant values were log base 2 transformed. As shown, irs4 and inp51 mutants were firmly positioned within the septin regulatory protein kinase functional group by hierarchical analysis and clustered most tightly with gin4. The cell wall-related protein kinase mutant ire1 was an outlier from the other mutants. Blue and yellow on heat map refer to log base 2 transformed values of −2 and 2, respectively.

Fig 8

The gin4 mutant (A) demonstrates aberrant chitin and PI(4,5)P2-containing patches. CaPH-GFP (B) and calcofluor white (C) colocalized in aberrant patches in C. albicans gin4 mutant cells (D, cyan color and arrowheads in overlay image), similarly to irs4 and inp51 mutants. gin4 mutants were created using the SAT-flipper method and C. albicans SC5314.

Fig 9

Brief exposure to caspofungin results in redistribution of PI(4,5)P2 and septins to aberrant patches in wild-type C. albicans cells. A 5-min exposure to caspofungin at the MIC (0.125 μg/ml) did not kill a wild-type C. albicans strain (A) but resulted in redistribution of CaPH-GFP (B) and Cdc10-RFP (C) to aberrant patches (D, overlay image). PI(4,5)P2-septin colocalization was similar to that seen in irs4, inp51, and gin4 mutant cells in the absence of cell wall stress.

Fig 10

Model for the PI(4,5)P2-septin cell wall regulatory network. Exposure of C. albicans to caspofungin inhibits β-1,3-d-glucan synthesis and creates cell wall stress (box 1). Upon sensing this stress, cells rapidly redistribute PI(4,5)P2 and septins (box 3). PI(4,5)P2 accumulation activates the PKC-MAPK cell wall integrity pathway, and PI(4,5)P2 and septins direct the deposit of chitin and other cell wall components at sites of colocalization (box 4). The redistribution of PI(4,5)P2 and septins in response to caspofungin is similar to that observed in gin4, irs4, and inp51 mutants, which are hypersusceptible to caspofungin and exhibit highly similar cell wall damage response gene expression profiles. Therefore, the data suggest that (i) Gin4 and the Irs4/Inp51 5′-phosphatase complex function upstream of PI(4,5)P2 and septins in a pathway that relays the cell wall stress signal (box 2), and (ii) an intact Gin4-Irs4/Inp51-PI(4,5)P2-septin pathway is required for the maintenance of cell wall integrity in the face of ongoing caspofungin exposure (box 5). Cell wall regulation (CWR) through the pathway is part of the natural response to caspofungin and, as our earlier mouse data indicated (2), necessary for the progression of invasive candidiasis after the initial stages of tissue colonization (box 6). White and blue boxes represent components of this model that have been established or suggested by our data, respectively.