The Candida albicans pescadillo homolog is required for normal hypha-to-yeast morphogenesis and yeast proliferation

Abstract

A single species, Candida albicans, causes half of all invasive fungal infections in humans. Unlike other fungal pathogens, this organism switches between growth as budding yeast and as pseudohyphal and hyphal filaments in host organs and in vitro. Both cell types play a role in invasive disease: while hyphal and pseudohyphal filaments penetrate host cells and tissues, yeast cells are likely to facilitate dissemination through the bloodstream and establishment of distant foci of infection. Many regulators of the yeast-to-hypha switch have emerged from intensive investigations of this morphogenetic step, but the hypha-to-yeast switch remains poorly understood. Using a forward genetic approach, a novel putative regulator involved in the hypha-to-yeast switch was identified, the C. albicans pescadillo homolog, PES1. In eukaryotes from yeast to human, pescadillo homologs are involved in cell cycle control and ribosome biogenesis, and are essential. We find a pescadillo homolog to act in fungal morphogenesis, specifically in lateral yeast growth on filamentous cells. We also find essentiality of PES1 in C. albicans to be dependent on cell type, because hyphal cells, but not yeast cells, tolerate its loss. PES1 is therefore critical for completion of the C. albicans life cycle, in which the fungus switches between filamentous and yeast growth. Consistent with these in vitro findings, PES1 is required for C. albicans virulence in an in vivo insect model of infection.

Fig. 1.

Lateral yeast are produced under conditions that induce hyphal tip growth and hyphal branching. Yeast cells of SC5314 (PES1/PES1) were inoculated into Spider medium, permitted to settle on a fibronectin-coated coverslip, and incubated at 37 °C for 6.5 h. Cells of the same culture on the same coverslip were photographed. White arrow, original yeast from which hyphal outgrowth occurred. Black arrow, hyphal branch or tip. Black star, lateral yeast. (Scale bar, 10 μm.)

Fig. 2.

Hyphae-inducing conditions permit growth when expression of PES1 is repressed from tetO. Strains were grown overnight in YPD without doxycycline at 30 °C and diluted to an initial density of OD₆₀₀ 0.2. Fivefold dilutions were spotted onto YPD (A), YPD with 20 μg/ml doxycycline (B), Spider (C), Spider with 20 μg/ml doxycycline (D), 20% calf serum 2% glucose (E), and 20% calf serum 2% glucose with 20 μg/ml doxycycline (F). Plates were incubated at 37 °C for 4 days. (1) JKC915 (HIS1/his1::FRT-tetR PES1/PES1). (2) JKC956 (HIS1/his1::FRT-tetR PES1/pes1::FRT). (3) JKC962 (HIS1/his1::FRT-tetR PES1/pes1::FRT). (4) JKC1137 (HIS1/his1::FRT-tetR pes1::FRT/FRT-tetO-PES1). (5) JKC1143 (HIS1/his1::FRT-tetR pes1::FRT/FRT-tetO-PES1).

Fig. 3.

PES1 is expressed in both yeast and hyphal cells, and control of PES1 expression from the tetO promoter is independent of yeast- or hyphae-inducing conditions. Strains were grown overnight in YPD at 30 °C, diluted to an initial density of OD₆₀₀ 0.2, and incubated in four conditions: YPD at 30 °C (samples 1-6 and 13-18), YPD with 10% calf serum at 37 °C (samples 7-12 and 19-24), YPD with 100 μg/ml doxycycline at 30 °C (samples 25-30), and YPD with 10% calf serum and 100 μg/ml doxycycline at 37 °C (samples 31-36). Cells were harvested at the indicated time points, and RNA preparations were blotted and probed with PES1. GSP1 was used as a loading control probe. (1-12) SC5314 (PES1/PES1). (13-36) JKC1137 (HIS1/his1::FRT-tetR pes1::FRT/FRT-tetO-PES1).

Fig. 4.

Lateral yeast growth is defective during Pes1 inactivation. Wild-type and pes1Δ/PES1^W416R strains were grown for 9 days at 24 °C on YP 2% galactose (A), and on RPMI1640, buffered to pH 7.5 with 165 mM Mops (B). (1, 3, and 5) SC5314 (PES1/PES1). (2 and 4) JKC1155 (pes1::FRT/FRT-PES1^W416R). (6) JKC1160 (pes1::FRT/FRT-PES1^W416R). (1 and 2) Tips of young filaments. (3–6) Filaments emanating from colony rim. Arrows, clusters of lateral yeast. (Scale bar, 100 μm.)

Fig. 5.

PES1 overexpression leads to decreased hyphal growth and increased lateral yeast growth. Strains were streaked on Spider medium without doxycycline and incubated at 37 °C for 4 days. (1) JKC915 (HIS1/his1::FRT-tetR PES1/PES1). (2) JKC962 (HIS1/his1::FRT-tetR PES1/pes1::FRT). (3) JKC1143 (HIS1/his1::FRT-tetR pes1::FRT/FRT-tetO-PES1). (Scale bar, 100 μm.)

Fig. 6.

PES1 depletion leads to loss of virulence in a Galleria mellonella model. C. albicans pes1Δ/pMAL2-PES1 mutant and wild-type strains were grown overnight at 30 °C in YPD to repress PES1 expression in the mutant, or in YP 2% maltose (YPM) to induce PES1 expression in the mutant, and resuspended in PBS to an inoculum of 10⁶ cells. Sixteen larvae per condition were injected and kept at 37 °C. (1) Larvae injected with PBS only. (2) Larvae injected with JKC681 (pes1::FRT/FRT-pMAL2-PES1) pregrown in YPM. (3) Larvae injected with JKC681 (pes1::FRT/FRT-pMAL2-PES1) pregrown in YPD. (4) Larvae injected with SC5314 (PES1/PES1) pregrown in YPM. (5) Larvae injected with SC5314 (PES1/PES1) pregrown in YPD. The P value for difference in survival curves between larvae injected with JKC681 pregrown in YPD and with JKC681 pregrown in YPM is 0.0001 (Mantel-Cox test). The P value for difference in survival curves between larvae injected with SC5314 pregrown in YPD and with SC5314 pregrown in YPM is 0.547.