Two-component histidine phosphotransfer protein Ypd1 is not essential for viability in Candida albicans

Abstract

Prokaryotes and lower eukaryotes, such as yeasts, utilize two-component signal transduction pathways to adapt cells to environmental stress and to regulate the expression of genes associated with virulence. One of the central proteins in this type of signaling mechanism is the phosphohistidine intermediate protein Ypd1. Ypd1 is reported to be essential for viability in the model yeast Saccharomyces cerevisiae. We present data here showing that this is not the case for Candida albicans. Disruption of YPD1 causes cells to flocculate and filament constitutively under conditions that favor growth in yeast form. To determine the function of Ypd1 in the Hog1 mitogen-activated protein kinase (MAPK) pathway, we measured phosphorylation of Hog1 MAPK in ypd1Δ/Δ and wild-type strains of C. albicans. Constitutive phosphorylation of Hog1 was observed in the ypd1Δ/Δ strain compared to the wild-type strain. Furthermore, fluorescence microscopy revealed that green fluorescent protein (GFP)-tagged Ypd1 is localized to both the nucleus and the cytoplasm. The subcellular segregation of GFP-tagged Ypd1 hints at an important role(s) of Ypd1 in regulation of Ssk1 (cytosolic) and Skn7 (nuclear) response regulator proteins via phosphorylation in C. albicans. Overall, our findings have profound implications for a mechanistic understanding of two-component signaling pathways in C. albicans, and perhaps in other pathogenic fungi.

FIG 1

(a) Schematic representation (size not to scale) of Ypd1 (282 amino acids) depicting the conserved histidine phosphotransfer (HPt) domain. The sequence prediction was done by using SMART (http://smart.embl-heidelberg.de/). (b) Sequence alignment of the HPt domains of C. albicans Ypd1 and S. cerevisiae, S. pombe, C. neoformans, A. nidulans, A. fumigatus, N. crassa, and D. discoideum histidine phosphotransfer proteins. The conserved histidine residue within the HPt domain is boxed. The boxed histidine amino acid is the putative site of phosphorylation.

FIG 2

Growth curve of C. albicans wild-type (WT), ypd1Δ/Δ, and ypd1Δ/Δ::YPD1 strains. Overnight cultures were transferred to fresh YPD medium to a starting OD of 0.1, and growth was measured every hour until stationary growth phase was reached.

FIG 3

Observation of flocculation of ypd1Δ/Δ mutant cells. (a) Log-phase cultures of WT (YPD1/YPD1), ypd1Δ/Δ, and ypd1Δ/Δ::YPD1 strains of C. albicans at 30°C. Flocculation of ypd1Δ/Δ cells is indicated by the formation of clumps of cells settled at the bottom of the tube (arrows). (b) Microscopic observation indicates a mix of hyphae and yeast cells clumped together in the ypd1Δ/Δ mutant compared to the WT and gene-reconstituted strains.

FIG 4

Growth of C. albicans WT (YPD1/YPD1), ypd1Δ/Δ, and ypd1Δ/Δ::YPD1 strains in RPMI growth medium at 37°C for 3 h. (a) Extensive flocculation is observed in ypd1Δ/Δ mutant cells. (b) Microscopic observation of ypd1Δ/Δ cells indicates a thick mesh of hyphae clumped together, while WT and gene-reconstituted cells form small germ tubes.

FIG 5

Growth of WT (YPD1/YPD1), ypd1Δ/Δ, and ypd1Δ/Δ::YPD1 strains of C. albicans at 30°C for 48 h on YPD agar (control plate) (a) and YPD agar containing 0.025% SDS (b). Five-microliter cell dilutions (5 × 10⁵ to 5 × 10¹ cells) were spotted on each plate.

FIG 6

Subcellular localization of Ypd1-GFP. C. albicans strains expressing GFP-tagged Ypd1 were grown to log phase and prepared for microscopy as described in Materials and Methods. The Ypd1-GFP cells were stained with DAPI and MitoTracker Red. (a) Ypd1-GFP with DAPI. Merged DAPI and GFP fluorescence images indicate nuclear and cytosolic localization of Ypd1. (b) Ypd1-GFP and MitoTracker Red. BF, bright field. All the images have similar levels of contrast.

FIG 7

Detection of phosphorylation of Hog1 MAPK by Western blot analysis. C. albicans strain SN425 (YPD1/YPD1) and the ypd1Δ/Δ mutant were grown to log phase in YPD broth and treated with H₂O₂. Samples were taken at the indicated times (minutes; given above the lanes). Western blots were performed using either a phospho-p38 MAPK monoclonal antibody or an α-tubulin antibody. The phospho-p38 MAPK antibody detects endogenous levels of Hog1 MAP kinase only when the kinase is phosphorylated. The α-tubulin antibody was used to detect equal loading of protein samples.