Three prevacuolar compartment Rab GTPases impact Candida albicans hyphal growth

Abstract

Disruption of vacuolar biogenesis in the pathogenic yeast Candida albicans causes profound defects in polarized hyphal growth. However, the precise vacuolar pathways involved in yeast-hypha differentiation have not been determined. Previously we focused on Vps21p, a Rab GTPase involved in directing vacuolar trafficking through the late endosomal prevacuolar compartment (PVC). Herein, we identify two additional Vps21p-related GTPases, Ypt52p and Ypt53p, that colocalize with Vps21p and can suppress the hyphal defects of the vps21Δ/Δ mutant. Phenotypic analysis of gene deletion strains revealed that loss of both VPS21 and YPT52 causes synthetic defects in endocytic trafficking to the vacuole, as well as delivery of the virulence-associated vacuolar membrane protein Mlt1p from the Golgi compartment. Transcription of all three GTPase-encoding genes is increased under hyphal growth conditions, and overexpression of the transcription factor Ume6p is sufficient to increase the transcription of these genes. While only the vps21Δ/Δ single mutant has hyphal growth defects, these were greatly exacerbated in a vps21Δ/Δ ypt52Δ/Δ double mutant. On the basis of relative expression levels and phenotypic analysis of gene deletion strains, Vps21p is the most important of the three GTPases, followed by Ypt52p, while Ypt53p has an only marginal impact on C. albicans physiology. Finally, disruption of a nonendosomal AP-3-dependent vacuolar trafficking pathway in the vps21Δ/Δ ypt52Δ/Δ mutant, further exacerbated the stress and hyphal growth defects. These findings underscore the importance of membrane trafficking through the PVC in sustaining the invasive hyphal growth form of C. albicans.

Fig 1

The active conformation of the Vps21p GTPase supports C. albicans hyphal growth. Inactive (VPS21^S24N) and active (VPS21^Q69L) conformational mutant alleles of VPS21 were introduced into the vps21Δ/Δ mutant strain. Cell suspensions of each strain were applied as spots to M199 agar and incubated at 37°C for 4 days. Similar results were found after incubation on 10% FBS agar at 37°C (data not shown). WT, wild type.

Fig 2

Ypt52p and Ypt53p can suppress vps21Δ/Δ mutant hyphal growth and stress defects. A wild-type allele of either VPS21 or YPT52 or an ACT1p-YPT53 construct was introduced into the vps21Δ/Δ mutant. Similar constructs encoding putatively inactive (YPT52^S26N and YPT53^T27N) and active (YPT52^Q73L and YPT53^Q75L) conformational mutant alleles of either gene, were also introduced into the vps21Δ/Δ mutant strain (A and B only). (A and B) Cell suspensions of each strain were applied as spots to 10% FBS agar and incubated at 37°C for 4 days. Similar results were found after incubation on M199 agar at 37°C (see Fig. S3A in the supplemental material). (C and D) Cell suspensions of each strain were prepared by serial dilution, applied to YPD agar or to YPD agar supplemented with 25 μg/ml Congo red or 0.05% SDS, and incubated at 30°C for 2 days.

Fig 3

Ypt52p and Ypt53p colocalize with the late endosomal Rab GTPase Vps21p. GFP-VPS21, mCherry (mCh)-YPT52, and mCh-YPT53 gene fusions were each expressed from C. albicans ACT1p. Strains harboring ACT1p-GFP-VPS21 and either ACT1p-mCh-YPT52 or ACT1p-mCh-YPT53 were grown as either yeast cells in YPD medium at 30°C (A) or as hyphae in M199 medium at 37°C (B) and observed by phase-contrast (PC) and fluorescence microscopy with a 100× objective (A) or a 40× objective (B). Representative images are shown with Pearson's correlation coefficient (r) values expressed as means ± standard deviations (n = 30 to 50 cells). DIC, differential interference contrast.

Fig 4

Ypt52p affects vacuole morphology in C. albicans yeast. Each strain was grown as yeast in YPD broth at 30°C, and vacuoles were stained with CMAC. Live cells were then observed by phase-contrast and fluorescence microscopy. Merged images are shown.

Fig 5

Vps21p and Ypt52p facilitate endocytic and biosynthetic trafficking to the C. albicans vacuole. The vacuolar membrane protein Mlt1p was tagged with GFP in each mutant and a wild-type control strain to evaluate biosynthetic trafficking from the Golgi compartment to the vacuole. Each strain was then pulse-chase labeled with FM4-64 to simultaneously assess trafficking from the plasma membrane via endocytosis. Cells were observed by fluorescence microscopy to determine FM4-64 (center left panels) and Mlt1-GFP (center right panels) distribution and by Nomarski optics (far left panels). Merged FM4-64-GFP images are also shown (far right panels).

Fig 6

Loss of both Vps21p and Ypt52p causes synthetic defects in C. albicans hyphal growth. (A) Cell suspensions of each strain were applied as spots to 10% FBS agar and incubated at 37°C for 4 days. Similar results were found after incubation on M199 agar at 37°C (data not shown). (B) Selected strains are shown at higher magnification after 7 days of incubation on FBS agar at 37°C. (C) Cell suspensions of each strain were applied as spots to M199 agar and incubated at 37°C for 5 days. Similar results were found after incubation on FBS agar at 37°C (data not shown). Please see the text for an explanation of * and **.

Fig 7

Loss of both Vps21p and Ypt52p causes profound defects in vacuole formation during C. albicans hyphal growth. Each strain was induced to form hyphae for 3.5 h in M199 medium, and vacuoles were stained with CMAC. Live cells were then observed by phase-contrast and fluorescence microscopy. Merged images are shown.

Fig 8

Loss of AP-3 trafficking dramatically increases vacuolar size in the endosome-deficient vps21Δ/Δ ypt52Δ/Δ mutant yeast but exacerbates the hyphal growth defects. Vacuole morphology was determined by CMAC staining of yeast cells grown in YPD at 30°C (top panels) or hyphal cells grown in M199 medium at 37°C (bottom panels). Hyphal growth on 10% FBS agar was also compared (center panels).