The Candida albicans ESCRT pathway makes Rim101-dependent and -independent contributions to pathogenesis

Abstract

Candida albicans is an opportunistic pathogen that colonizes diverse mucosal niches with distinct environmental characteristics. To adapt to these different sites, C. albicans must activate and attenuate a variety of signal transduction pathways. A mechanism of signal attenuation is through receptor endocytosis and subsequent vacuolar degradation, which requires the endosomal sorting complex required for transport (ESCRT) pathway. This pathway comprises several polyprotein complexes (ESCRT-0, -I, -II, -III, and -DS) that are sequentially recruited to the endosomal membrane. The ESCRT pathway also activates the Rim101 transcription factor, which governs expression of genes required for virulence. Here, we tested the hypothesis that the ESCRT pathway plays a Rim101-independent role(s) in pathogenesis. We generated deletion mutants in each ESCRT complex and determined that ESCRT-I, -II, and -III are required for Rim101 activation but that ESCRT-0 and ESCRT-DS are not. We found that the ESCRT-0 member Vps27 and ESCRT-DS components are required to promote epithelial cell damage and, using a murine model of oral candidiasis, found that the vps27Delta/Delta mutant had a decreased fungal burden compared to that of the wild type. We found that a high-dose inoculum can compensate for fungal burden defects but that mice colonized with the vps27Delta/Delta strain exhibit less morbidity than do mice infected with the wild-type strain. These results demonstrate that the ESCRT pathway has Rim101-independent functions for C. albicans virulence.

Fig. 1.

Model of ESCRT pathway and Rim101 pathway intersection. The Rim101 pathway intersects with the ESCRT pathway. ESCRT-0, -I, -II, and -III complexes are recruited to the endosomal membrane. ESCRT-III member Snf7 can interact either with Rim101 pathway members, resulting in proteolytic activation of Rim101, or with downstream ESCRT members, resulting in MVB formation. Yeast ESCRT complex components are listed below, and designations in bold are of those investigated in this study.

Fig. 2.

PCR genotyping of vps27Δ/Δ strains. VPS27 was amplified in a PCR using Vps27 5det and Vps27 3det primers (Table 2). Size markers in kilobases are noted on the left, and genotypes of each band are labeled on the right. Strains shown are VPS27/VPS27 (DAY185), VPS27/Δ (DAY1176), vps27Δ/Δ (DAY1160), and vps27Δ/Δ + VPS27 (DAY1264) strains. WT, wild type.

Fig. 3.

FM 4-64 trafficking defects of C. albicans ESCRT mutants. (A) Exponentially growing cultures were exposed to 16 mM FM 4-64 for 15 min on ice. Cells were washed with M199 pH 8, resuspended in fresh medium, and grown for 60 min at 30°C. Eighty microliters of culture was added to 10 μl each of 100 mM NaF and 100 mM NaN₃ before microscopic examination. Strains analyzed include wild-type (WT) (DAY185), vps27Δ/Δ (DAY1160), hse1Δ/Δ (DAY1218), mvb12Δ/Δ (DAY1162), vps28Δ/Δ (DAY1161), vps36Δ/Δ (DAY1163), vps22Δ/Δ (DAY1217), vps20Δ/Δ (DAY1157), snf7Δ/Δ (DAY763), vps4Δ/Δ (DAY1155), bro1Δ/Δ (DAY1156), and doa4Δ/Δ (DAY1158) strains. Arrows denote class E-like exclusion bodies within the cell. (B) Complementation of the vps27Δ/Δ mutant. FM 4-64 staining was performed as described above using WT (DAY185), vps27Δ/Δ (DAY1160), and vps27Δ/Δ + VPS27 (DAY1264) strains.

Fig. 4.

Effect of ESCRT mutants in Rim101-dependent growth assays. Fivefold dilutions of overnight YPD cultures were spotted onto YPD, YPD pH 9, or YPD plus LiCl agar medium. Plates were incubated at 37°C for 2 days before being photographed. Strains analyzed are the same as those in Fig. 3A.

Fig. 5.

ESCRT mutant filamentation phenotypes. (Top) Three microliters of overnight YPD cultures was spotted on M199 pH 8 agar plates, and plates were incubated for 6 days at 37°C before being photographed. (Bottom) Three microliters of overnight YPD cultures was spotted onto M199 pH 4 agar plates, and plates were incubated for 6 days at 37°C before being photographed. Strains analyzed are the same as those in Fig. 3A.

Fig. 6.

Rim101 processing at alkaline and acidic pH. (A) Overnight YPD cultures were inoculated into 40 ml M199 pH 4 and grown for 5 h at 30°C. Cell pellets were collected and resuspended in 50 ml M199 pH 8 and grown for 30 min at 30°C, protein purified, and analyzed by Western blotting. Rim101-V5 was detected after 1 h of incubation with anti-V5-HRP antibody (Invitrogen) by chemiluminescence. Strains analyzed included wild-type (WT) (DAY1212), vps27Δ/Δ (DAY1178), hse1Δ/Δ (DAY1221), mvb12Δ/Δ (DAY1193), vps28Δ/Δ (DAY1183), vps36Δ/Δ (DAY1188), vps22Δ/Δ (DAY1222), vps20Δ/Δ (DAY1257), snf7Δ/Δ (DAY568), vps4Δ/Δ (DAY576), bro1Δ/Δ (DAY1219), and doa4Δ/Δ (DAY1260) strains. Molecular mass markers (in kilodaltons) are indicated to the right. (B) Overnight YPD culture was inoculated into 40 ml M199 pH 4 and grown for 5 h at 30°C. Protein preparations and Western blot analysis were done as described above.

Fig. 7.

ESCRT mutants have growth defects on iron-depleted medium. YPD plus BPS cultures were diluted 1:50 in PBS, and 5-fold serial dilutions were spotted onto YPD and YPD plus BPS agar plates. Plates were incubated at 37°C for 2 days before being photographed. Strains investigated included wild-type (WT) (DAY185), ftr1Δ/Δ (DAY750), rim101Δ/Δ (DAY25), vps27Δ/Δ (DAY1160), hse1Δ/Δ (DAY1218), mvb12Δ/Δ (DAY1162), vps28Δ/Δ (DAY1161), vps36Δ/Δ (DAY1163), vps22Δ/Δ (DAY1217), vps20Δ/Δ (DAY1157), snf7Δ/Δ (DAY763), vps4Δ/Δ (DAY1155), bro1Δ/Δ (DAY1156), and doa4Δ/Δ (DAY1158) strains.

Fig. 8.

(A) ESCRT mutants show defects in epithelial cell damage. C. albicans cells (10⁵) were incubated for 10 h with ⁵¹Cr-labeled FaDu cells. Supernatant was collected and compared to that of uninfected FaDu cells. Samples were run in triplicate for each assay. Graph shows damage conferred by each mutant relative to a simultaneously tested wild-type (WT) strain. Mutants were tested in at least three separate assays. *, P < 0.05. Strains analyzed included wild-type (DAY185), ftr1Δ/Δ (DAY750), rim101Δ/Δ (DAY25), vps27Δ/Δ (DAY1160), hse1Δ/Δ (DAY1218), mvb12Δ/Δ (DAY1162), vps28Δ/Δ (DAY1161), vps36Δ/Δ (DAY1163), vps22Δ/Δ (DAY1217), vps20Δ/Δ (DAY1157), snf7Δ/Δ (DAY763), vps4Δ/Δ (DAY1155), bro1Δ/Δ (DAY1156), and doa4Δ/Δ (DAY1158) strains. (B) Complementation of the vps27Δ/Δ mutant using wild-type (DAY185), vps27Δ/Δ (DAY1160), and vps27Δ/Δ + VPS27 (DAY1264) strains.

Fig. 9.

A vps27Δ/Δ mutant is attenuated in a mouse model of oral candidiasis. (A) Tongue fungal burden and animal weight loss after low-dose inoculation. Cortisone-immunosuppressed animals were infected with 50 μl of 10⁵ CFU/ml of wild-type (WT) (DAY185), rim101Δ/Δ (DAY25), vps27Δ/Δ (DAY1160), and snf7Δ/Δ (DAY763) C. albicans. At 3 DPI half the animals were harvested and their tongues were collected for determination of fungal burden. The remaining animals were reimmunosuppressed with cortisone and harvested at 6 DPI for determination of fungal burden. *, P < 0.05. (B) Tongue fungal burden and animal weight loss after high-dose inoculation. The experiment was run identically but with an infectious dose of 50 μl of 10⁶ CFU/ml C. albicans.