Candida albicans Csy1p is a nutrient sensor important for activation of amino acid uptake and hyphal morphogenesis

Abstract

Candida albicans is an important human pathogen that displays a remarkable ability to detect changes in its environment and to respond appropriately by changing its cell morphology and physiology. Serum- and amino acid-based media are known to induce filamentous growth in this organism. However, the mechanism by which amino acids induce filamentation is not yet known. Here, we describe the identification and characterization of the primary amino acid sensor of C. albicans, Csy1. We show that Csy1p plays an important role in amino acid sensing and filamentation. Loss of Csy1p results in a lack of amino acid-mediated activation of amino acid transport and a lack of induction of transcription of specific amino acid permease genes. Furthermore, a csy1Delta/csy1Delta strain, lacking Csy1p, is defective in filamentation and displays altered colony morphology in serum- and amino acid-based media. These data provide the first evidence that C. albicans utilizes the amino acid sensor Csy1p to probe its environment, coordinate its nutritional requirements, and determine its morphological state.

FIG. 1.

Induction of amino acid transport in wild-type C. albicans. (A) Time-dependent [³H]valine transport in the wild-type strain (CAEB-1) in ammonium-based medium lacking (SD) or containing (SDH) 10 mM histidine. (B) [³H]valine transport of wild-type cells in minimal medium without (shaded bar) or with (solid bars) 10 mM l-α-amino acids. (C) Uptake of [³H]-labeled valine, phenylalanine, lysine, glutamine, and isoleucine in minimal medium lacking (shaded bars) or containing (solid bars) histidine.

FIG. 2.

Transcription levels of AAP genes in wild-type and csy1Δ/csy1Δ strains in the presence of histidine. Northern blot analysis was performed on RNA isolated from wild-type (WT) (CAEB-1) and csy1Δ/csy1Δ (CAEB-5) strains grown to mid-log phase in minimal medium in the absence (−) or presence (+) of 10 mM histidine. The probes used are derived from the CAN1, orf 6.7739, orf 6.4609, and ACT1 genes as described in Materials and Methods.

FIG. 3.

Csy1p sequence alignment. (A) Alignment of C. albicans Csy1p with the S. cerevisiae amino acid sensor Ssy1p, the branched-chain AAP Bap2p, and the tyrosine transporter Tat1p. (B) Sequence alignment of the N-terminal extensions of Ssy1p and Csy1p. Dark shading, identical residues; light shading, similar residues. The predicted membrane-spanning domains (TM1 to TM12) for Csy1p are indicated by solid lines above the aligned proteins and are numbered sequentially.

FIG. 4.

Generation and molecular characterization of the csy1Δ/csy1Δ knockout. A CSY1 gene knockout was generated using the strategy described in Materials and Methods. (A) Schematic representation of the CSY1 genomic locus before and after replacement with ARG4 and URA3 cassettes. O1 and O2 are the two oligonucleotides used to confirm CSY1 gene disruption by PCR analysis. (B) Southern blot analysis of wild-type (WT), heterozygote (CSY1/csy1Δ), and homozygote (csy1Δ/csy1Δ) strains. Genomic DNAs were digested with SpeI. The probe used is a 0.7-kb fragment of CSY1. (C) Agarose gel electrophoresis showing the PCR products obtained with oligonucleotides O1 and O2 (see panel A) and genomic DNAs isolated from the wild-type, csy1Δ/csy1Δ, and csy1Δ/csy1Δ+CSY1 strains as templates.

FIG. 5.

Comparison of amino acid transport and sensitivity to the amino acid analog l-glutamic acid γ-hydrazide in the wild-type and csy1Δ/csy1Δ strains. (A) Uptake of amino acids in the wild-type (WT) (CAEB-1) and csy1Δ/csy1Δ (CAEB-5) strains. Cells were grown in SD medium lacking or supplemented with 10 mM histidine and were assayed as described in Materials and Methods. Standard deviations are shown. Uptake values were normalized to those of the wild-type strain in SD medium. (B) Growth of wild-type (CAEB-1), CSY1/csy1Δ (CAEB-3), csy1Δ/csy1Δ (CAEB-5), and csy1Δ/csy1Δ+CSY1 (CAEB-6) strains on SD plates containing 25 mM l-glutamic acid γ-hydrazide and incubated at 37°C for 1 week.

FIG. 6.

csy1Δ/csy1Δ mutants are defective in filamentation and colony morphology. Wild-type (WT) (CAEB-1), csy1Δ/csy1Δ (CAEB-5), and csy1Δ/csy1Δ+CSY1 (CAEB-6) strains were grown at 37°C on solid plates containing either 10% serum (A) or Lee's medium (B). Colony morphologies (A1 to 3; B1 to 6) and cell morphologies (A4 to 6; B7 to 9) were assessed by light microscopy.