Signaling through Lrg1, Rho1 and Pkc1 Governs Candida albicans Morphogenesis in Response to Diverse Cues

Abstract

The capacity to transition between distinct morphological forms is a key virulence trait for diverse fungal pathogens. A poignant example of a leading opportunistic fungal pathogen of humans for which an environmentally responsive developmental program underpins virulence is Candida albicans. C. albicans mutants that are defective in the transition between yeast and filamentous forms typically have reduced virulence. Although many positive regulators of C. albicans filamentation have been defined, there are fewer negative regulators that have been implicated in repression of filamentation in the absence of inducing cues. To discover novel negative regulators of filamentation, we screened a collection of 1,248 C. albicans homozygous transposon insertion mutants to identify those that were filamentous in the absence of inducing cues. We identified the Rho1 GAP Lrg1, which represses filamentous growth by stimulating Rho1 GTPase activity and converting Rho1 to its inactive, GDP-bound form. Deletion of LRG1 or introduction of a RHO1 mutation that locks Rho1 in constitutively active, GTP-bound state, leads to filamentation in the absence of inducing cues. Deletion of the Rho1 downstream effector PKC1 results in defective filamentation in response to diverse host-relevant inducing cues, including serum. We further established that Pkc1 is not required to sense filament-inducing cues, but its kinase activity is critical for the initiation of filamentous growth. Our genetic analyses revealed that Pkc1 regulates filamentation independent of the canonical MAP kinase cascade. Further, although Ras1 activation is not impaired in a pkc1Δ/pkc1Δ mutant, adenylyl cyclase activity is reduced, consistent with a model in which Pkc1 functions in parallel with Ras1 in regulating Cyr1 activation. Thus, our findings delineate a signaling pathway comprised of Lrg1, Rho1 and Pkc1 with a core role in C. albicans morphogenesis, and illuminate functional relationships that govern activation of a central transducer of signals that control environmental response and virulence programs.

Fig 1. Lrg1 negatively regulates filamentation via Rho1.

(A) Lrg1 is a repressor of filamentous growth. Strains were subcultured to log phase in YPD at 30°C for 4 hrs, and cells were imaged by DIC microscopy. The scale bar indicates 10 μm. (B) Top panel: Strains were subcultured to log phase in YPD at 30°C for 4 hrs, and cells were imaged by DIC microscopy. The scale bar indicates 10 μm. Bottom panel: 1 μL of a saturated overnight culture was spotted on YPD plates. Plates were incubated at 30°C for 48 hrs and images were taken using a Zeiss stereoscope. Scale bar indicates 1 mm. (C) Activated Rho1 promotes filamentation. Overnight cultures were subcultured for 24 hrs in the presence or absence of 0.05 μg/ml of doxycycline to achieve transcriptional repression of the wild-type allele of RHO1 that is under the control of the tetO promoter. Strains were subcultured in YPD at 30°C, with or without 0.05 μg/ml of doxycycline, for 4 hrs. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm.

Fig 2. Pkc1 is a master regulator of filamentous growth.

(A) Pkc1 acts downstream of Lrg1. Strains were subcultured to log phase in YPD at 30°C for 4 hrs. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm. (B) Homozygous deletion of PKC1 blocks filamentation in response to diverse cues. Strains were subcultured to log phase in the specified conditions for 4 hrs. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm. (C) Deletion of PKC1 blocks the upregulation of filament-specific transcripts HWP1 and IHD1. Strains were subcultured to log phase in YPD at 30°C or YPD + 10% serum at 37°C, and the transcript levels of HWP1 and IHD1 was monitored by qRT-PCR and normalized to GPD1. Data are plotted as means ± SD for triplicate samples and are representative of two independent experiments.

Fig 3. Pkc1 kinase activity is critical for filamentation.

Strains were subcultured for 4 hrs with either 10 μM of the Hsp90 inhibitor geldanamycin, 5 μM of the ATP analog 1-NA-PP1 that inhibits the gatekeeper allele, or both. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm.

Fig 4. The MAP kinase cascade downstream of Pkc1 is not required for filamentous growth.

Strains were subcultured for 4 hrs in YPD at 30°C, YPD + 10% serum at 37°C, or YPD + 10μM geldanamycin (GdA: Hsp90 inhibitor) at 30°C. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm.

Fig 5. Pkc1 acts downstream of Ras1 to regulate filamentation.

(A) Hyperactivation of Ras1 does not rescue filamentation in a mutant lacking Pkc1. Strains were grown in YPD at 30°C or YPD + 10% serum at 37°C for 3.5 hrs. Cells were imaged by DIC microscopy. The scale bar indicates 15 μm. (B) Deletion of PKC1 does not affect Ras1 activation. Strains were grown in YPD at 30°C or YPD + 10% serum at 37°C for 3.5 hrs. The total Ras1 protein and GTP-Ras1 fraction were resolved by SDS-PAGE gel. The GTP-Ras1:total Ras1 ratio is shown as a percentage.

Fig 6. Pkc1 acts upstream of cAMP signaling.

(A) Farnesol inhibits filamentation induced by LRG1 deletion. Strains were grown in YPD at 30°C or YPD at 37°C, in the absence or presence of 200 μM farnesol for 3.5 hrs. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm. (B) Cyr1-dependent changes in gene expression require functional Pkc1. Strains were grown in YPD at 30°C or YPD + 10% serum at 37°C for 3.5 hrs at 200 rpm. Total RNA was analyzed on the nanostring ncounter system. Shown is the fold change of expression at 30°C vs. 37°C. Data are plotted as means ± SD for two independent experiments. (C) Cyr1-dependent Nrg1 degradation requires functional Pkc1. Strains were grown in YPD at 30°C or YPD + 10% serum at 37°C for 5 min, 35 min, or 65 min. Total proteins were resolved by SDS-PAGE gel and the blot was hybridized with α-HA to detect Nrg1 and α-tubulin to monitor tubulin as loading control.

Fig 7. Pkc1 and Ras1 converge on regulating cAMP signaling, and Pkc1 governs morphogenesis through additional effectors.

(A) Hyperactivation of Ras1 partially restores HWP1 transcript levels in a mutant lacking Pkc1. Strains were subcultured to log phase in YPD at 30°C or YPD + 10% serum at 37°C. The transcript levels of HWP1 and IHD1 in YPD + 10% serum at 37°C were first normalized to GPD1 and then normalized to the level in YPD at 30°C. Data are plotted as means ± SD for triplicate samples and are representative of two independent experiments. ***, p < 0.005 (Student t test). (B) Activation of cAMP signaling does not rescue filamentation in a mutant lacking Pkc1. Strains were grown in YPD at 30°C, YPD + 10% serum with or without 10 mg/mL of dibutyryl cAMP at 30°C for 3.5 hrs. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm.

Fig 8. A schematic diagram depicting the regulation of filamentation by Rho1-Pkc1 signaling and cAMP-PKA signaling pathways.

The Ras1-cAMP-PKA signaling cascade is a known master regulator of C. albicans filamentous growth. The Rho1-Pkc1 cell wall integrity pathway is previously reported as a key modulator of cell wall integrity through activation of a MAPK cascade that terminates with Mkc1. Key proteins involved in both signaling cascades are depicted along with black arrows showing connections between these regulators. Our work identifies a novel role for Pkc1 in governing C. albicans morphogenesis by directly or indirectly regulating Cyr1 function (red dashed arrow) and through distinct effector(s) remain to be identified (red dashed arrow).