Arv1 lipid transporter function is conserved between pathogenic and nonpathogenic fungi

Abstract

The lipid transporter Arv1 regulates sterol trafficking, and glycosylphosphatidylinositol and sphingolipid biosyntheses in Saccharomyces cerevisiae. ScArv1 contains an Arv1 homology domain (AHD) that is conserved at the amino acid level in the pathogenic fungal species, Candida albicans and Candida glabrata. Here we show S. cerevisiae cells lacking Arv1 are highly susceptible to antifungal drugs. In the presence of drug, Scarv1 cells are unable to induce ERG gene expression, have an altered pleiotrophic drug response, and are defective in multi-drug resistance efflux pump expression. All phenotypes are remediated by ectopic expression of CaARV1 or CgARV1. The AHDs of these pathogenic fungi are required for specific drug tolerance, demonstrating conservation of function. In order to understand how Arv1 regulates antifungal susceptibility, we examined sterol trafficking. CaARV1/CgARV1 expression suppressed the sterol trafficking defect of Scarv1 cells. Finally, we show that C. albicansarv1/arv1 cells are avirulent using a BALB/c disseminated mouse model. We suggest that overall cell survival in response to antifungal treatment requires the lipid transporter function of Arv1.

Figure 1

A sequence alignment of various eukaryotic AHDs. The zinc-binding motif is underlined. Identical and conserved amino acids are indicated.

Figure 2

The effects of expressing CaARV1 or CgARV1 on growth in the presence of nystatin. Cells were dropped onto the appropriate drop out minimal medium plates at 10-fold serial dilutions (1 X 10⁶ cells in the first well). Cells were grown in the absence or presence of 0.5 µg/ml nystatin at 30°C for 48 hr. (A) The expression of various forms of ScArv1 and their effect on growth. (B) The expression of various forms of CaArv1 and their effect on growth. (C) The expression of various forms of CgArv1 and their effect on growth. (D) The growth of various CaARV1 strains at 35°C for 48 hr.

Figure 3

The effects on mRNA expression levels due to loss of ScArv1. Cells were grown to exponential phase (1 X 10⁷ cells) at 30°C in YEPD liquid medium. Wild type and Scarv1 cells were then grown in the absence and presence of 50 µg/ml lovastatin for 16 hr, and mRNA levels of various genes were determined using qRT-PCR. The relative expression values are the average of three independent experiments.

Figure 4

The effect of loss of ScArv1 on mRNA expression levels in fluconazole-treated cells. Cells were grown to exponential phase at 30°C in liquid YEPD medium. Wild type and Scarv1 cells were grown at 30°C in the absence or presence of 50 µg/ml fluconazole for 6 hr, and mRNA levels of various genes were determined using qRT-PCR. The relative expression values are the average of three independent experiments.

Figure 5

The effects on protein levels due loss of ScArv1. Cells were grown to exponential phase at 30°C in liquid YEPD medium. Wild type and Scarv1 cells were then grown at 30°C in the absence or presence of 50 µg/ml of lovastatin and 50 µg/ml fluconazole for 16 hr and 6 hr, respectively. Protein expression levels were determined by western analysis. Epitope tagged Erg1 and epitope-tagged Erg11 were detected using monoclonal anti-Myc antibodies; Erg25 was detected using anti-Erg25 polyclonal antibodies (A) WT and Scarv1 cells harboring an empty vector control, (B) Scarv1 cells harboring a full-length CaARV1, (C) Scarv1 cells harboring a full-length CgARV1. The western blots are typical representations of three independent experiments. Left panels represent protein levels in the absence and presence of lovastatin; right panels represent protein levels in the absence and presence of fluconazole. Asterisk, possible Erg1 degradative product.

Figure 6

The effect of loss of ScArv1 on ScPdr5 and ScSnq2 protein levels. Cells were grown to exponential phase at 35°C in YEPD medium. Wild type and Scarv1 cells were then grown at 30°C in the absence or presence of 50 µg/ml of lovastatin or 50 µg/ml fluconazole for 16 hr and 6 hr, respectively. Protein expression levels were determined by western analysis. Epitope-tagged Pdr5 and Snq2 were detected using monoclonal anti-Myc antibodies. (A) Pdr5; (B) Snq2. The western blots are typical representations of three independent experiments. Left panels represent protein levels in the absence and presence of lovastatin; right panels represent protein levels in the absence and presence of fluconazole.

Figure 7

mRNA levels in antifungal-treated Scarv1 cells expressing CaARV1 or CgARV1. Cells were grown to exponential phase at 30°C in the appropriate drop out minimal medium. Wild type or Scarv1 cells expressing CaARV1 or CgARV1 were then grown at 30°C in the presence of 50 µg/ml lovastatin (A–D) and fluconazole (E–H) for 16 hr and 6 hr, respectively, and the mRNA levels of various genes were subsequently determined using qRT-PCR. The relative values are the average of three independent experiments.

Figure 8

Percentage of various lipid microdomains in various ScARV1 cells. Cells were grown to exponential phase at 30°C in the appropriate drop out minimal medium. Cells were fixed with EM-grade formaldehyde for 10 min at 23°C. Fixed cells were incubated with 10 µg/ml filipin complex in the dark for 15 min at 23°C. Unesterified microdomains were visualized using fluorescence microscopy. Values are expressed as percentages of total cells.

Figure 9

The effects on mRNA expression levels due to loss of CaArv1. Cells were grown to exponential phase at 30°C in YEPD liquid medium. Wild type and arv1 cells were then grown in the absence and presence of fluconazole for 6 hr, and mRNA levels of various genes were determined using qRT-PCR. The relative expression values are the average of three independent experiments.

Figure 10

Filipin staining of various C. albicans strains. All strains were grown to exponential phase in YEPD at 30°C. Cells were then transferred to serum containing medium and grown at 37°C. After shift to serum, cells were fixed at the indicated times with 3.7% EM grade formaldehyde, and subsequently filipin-stained in order to visualize unesterified sterol localization. The arrows point out striated sterol containing structures. Insets are enlargements of the indicated picture; arrows with asterisks indicate where the picture has been enlarged. Pictures were taken using a 2 second exposure time.

Figure 11

Disseminated candidiasis mouse model studies. For disseminated candidiasis studies, BALB/c mice were tail vein injected with 1 X 10⁵ cells of CaARV1/ARV1, CaARV1/arv1, and CaARV1/arv1 strains that were grown to exponential phase at 30°C. Mice were checked every day for signs of disseminated candidiasis, such as hypothermia and coat ruffling. The mice were sacrificed at this point and counted as deceased on that day. The average days survived; solid line, ARV1/ARV1; segmented line, ARV1/arv1; dotted line, arv1/arv1.