Candida albicans VPS11 is required for vacuole biogenesis and germ tube formation

Abstract

The Candida albicans vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C vps (vacuolar protein sorting) mutants of Saccharomyces cerevisiae are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study C. albicans homologues of the S. cerevisiae class C VPS genes have been identified. Deletion of a C. albicans VPS11 homologue resulted in a number of phenotypes that closely resemble those of the class C vps mutants of S. cerevisiae, including the absence of a vacuolar compartment. The C. albicans vps11Delta mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore, vps11Delta strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.

FIG. 1.

C. albicans Vps11p and Vps18p have conserved C-terminal RING domains. C-terminal sequences from S. cerevisiae and C. albicans Vps11p proteins (A) and from S. cerevisiae and C. albicans Vps18p proteins (B) were aligned using MultAlin (11). A series of cysteine and histidine residues (highlighted on a solid background) which conform to the H2 type RING consensus sequence are conserved in both sets of homologues. Other conserved residues are highlighted on a shaded background. Amino acid residue numbers are given at each end of the sequences.

FIG. 2.

Deletion of C. albicans VPS11. (A) Line drawing of C. albicans VPS11 locus and location of region deleted in vps11Δ::ARG4 and vps11Δ::URA3. (B) Southern blot analysis of constructed heterozygotes VPS11/vps11Δ (GPS1 and -2) and vps11Δ/vps11Δ strains (GPD1 to -4). The parental strain, BWP17 (VPS11/VPS11), is shown in the leftmost lane. (C) RNA isolated from cells grown to log phase (1 × 10⁷ to 2 × 10⁷ cells/ml) in YPD medium was subjected to Northern blot analysis using a VPS11-specific probe. Two low-abundance RNA species of approximately 3.0 and 3.6 kb were detected in each of the parent strain (BWP17), the prototrophic control strain (YJB6284), and three revertant strains (GPR1 to -3). No VPS11 transcript was detected in the double-mutant strains GPH1 to -3. The blot was stripped and reprobed for the actin transcript as a loading and RNA integrity control.

FIG. 3.

The C. albicans vps11Δ mutant is sensitive to osmotic and temperature stress. Serial dilutions of strains BWP17 (VPS11/VPS11), GPS1 (VPS11/vps11Δ), GPD1 (vps11Δ/vps11Δ), GPR1 (vps11Δ/vps11Δ/VPS11), GPR2 (vps11Δ/vps11Δ/VPS11), GPH1 (vps11Δ/vps11Δ), GPH2 (vps11Δ/vps11Δ), and YJB6284 (VPS11/VPS11) were spotted onto YPD + uri agar and incubated as described in Materials and Methods. Cell suspensions were also spotted onto YPD + uri agar supplemented with either 2.5 M glycerol or 1 M NaCl₂ and incubated at 30°C.

FIG. 4.

C. albicans VPS11 is involved in vacuole biogenesis. Strains YJB6284 (VPS11/VPS11), GPH1 (vps11Δ/vps11Δ), and GPR1 (vps11Δ/vps11Δ/VPS11) were stained with the vacuole-specific dye carboxy-DCFDA to analyze vacuole morphology. (A) YJB6284 cells have one to three distinctly stained subcellular vacuole compartments. (B) The double mutant GPH1 exhibits diffuse staining throughout the cytoplasm, indicating the absence of a vacuole compartment. (C) The revertant strain GPR1 has a staining pattern similar to that of YJB6284, indicating that vacuole biogenesis is restored.

FIG. 5.

The C. albicans vps11Δ mutant has reduced secreted lipase and protease activities. Shown are results for strains YJB6284 (VPS11/VPS11), GPH1 (vps11Δ/vps11Δ), and GPR1 (vps11Δ/vps11Δ/VPS11). (A) SAP expression was induced in liquid culture using BSA as a nitrogen source before SAP activity was assessed on BSA-plus-yeast extract agar. The white precipitation zone indicates SAP-mediated BSA hydrolysis. (B) Cell suspensions were spotted onto egg yolk medium in order to detect lipase activity, indicated by a white precipitation zone at the colony periphery.

FIG. 6.

C. albicans VPS11 is required for normal filamentation. (A) Cell suspensions of BWP17 (VPS11/VPS11), GPH1 (vps11Δ/vps11Δ), GPR1 (vps11Δ/vps11Δ/VPS11), and YJB6284 (prototrophic control strain) were spotted onto M199 agar medium and incubated at 37°C to induce filamentation. After 4 days, BWP17, YJB6284, and GPR1 all had well defined filamentous borders. No filamentous border was observed for the double mutant GPH1. (B) Prototrophic strains YJB6284, GPH1, and GPR1 grown in YPD medium were subcultured to YPD medium with 10% (vol/vol) fetal bovine serum and incubated at 37°C to induce filamentation.