Pkc1 acts downstream of Ras1 to regulate filamentation.

<p><b>(A)</b> 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>(B)</b> Deletion of <i>PKC1</i> 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.</p

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

<p>The Ras1-cAMP-PKA signaling cascade is a known master regulator of <i>C</i>. <i>albicans</i> 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 <i>C</i>. <i>albicans</i> morphogenesis by directly or indirectly regulating Cyr1 function (red dashed arrow) and through distinct effector(s) remain to be identified (red dashed arrow).</p

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

<p>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.</p

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

<p><b>(A)</b> Hyperactivation of Ras1 partially restores <i>HWP1</i> 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 <i>HWP1</i> and <i>IHD1</i> in YPD + 10% serum at 37°C were first normalized to <i>GPD1</i> 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. ***, <i>p</i> < 0.005 (Student <i>t</i> test). <b>(B)</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.</p

Lrg1 negatively regulates filamentation via Rho1.

<p><b>(A)</b> 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>(B) Top panel:</b> 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>Bottom panel:</b> 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. <b>(C)</b> 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 <i>RHO1</i> that is under the control of the <i>tetO</i> 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.</p

Pkc1 acts upstream of cAMP signaling.

<p><b>(A)</b> Farnesol inhibits filamentation induced by <i>LRG1</i> 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>(B)</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.</p

Pkc1 kinase activity is critical for filamentation.

<p>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.</p

Pkc1 is a master regulator of filamentous growth.

<p><b>(A)</b> 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>(B)</b> Homozygous deletion of <i>PKC1</i> 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. <b>(C)</b> Deletion of <i>PKC1</i> blocks the upregulation of filament-specific transcripts <i>HWP1</i> and <i>IHD1</i>. Strains were subcultured to log phase in YPD at 30°C or YPD + 10% serum at 37°C, and the transcript levels of <i>HWP1</i> and <i>IHD1</i> was monitored by qRT-PCR and normalized to <i>GPD1</i>. Data are plotted as means ± SD for triplicate samples and are representative of two independent experiments.</p

Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space

<div><p>The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen <i>Candida albicans</i> is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of <i>C</i>. <i>albicans HSF1</i> induces morphogenesis in the absence of external cues. <i>HSF1</i> depletion compromises Hsp90 function, thereby driving filamentation. <i>HSF1</i> overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.</p></div

Global Analysis of the Fungal Microbiome in Cystic Fibrosis Patients Reveals Loss of Function of the Transcriptional Repressor Nrg1 as a Mechanism of Pathogen Adaptation

<div><p>The microbiome shapes diverse facets of human biology and disease, with the importance of fungi only beginning to be appreciated. Microbial communities infiltrate diverse anatomical sites as with the respiratory tract of healthy humans and those with diseases such as cystic fibrosis, where chronic colonization and infection lead to clinical decline. Although fungi are frequently recovered from cystic fibrosis patient sputum samples and have been associated with deterioration of lung function, understanding of species and population dynamics remains in its infancy. Here, we coupled high-throughput sequencing of the ribosomal RNA internal transcribed spacer 1 (ITS1) with phenotypic and genotypic analyses of fungi from 89 sputum samples from 28 cystic fibrosis patients. Fungal communities defined by sequencing were concordant with those defined by culture-based analyses of 1,603 isolates from the same samples. Different patients harbored distinct fungal communities. There were detectable trends, however, including colonization with <i>Candida</i> and <i>Aspergillus</i> species, which was not perturbed by clinical exacerbation or treatment. We identified considerable inter- and intra-species phenotypic variation in traits important for host adaptation, including antifungal drug resistance and morphogenesis. While variation in drug resistance was largely between species, striking variation in morphogenesis emerged within <i>Candida</i> species. Filamentation was uncoupled from inducing cues in 28 <i>Candida</i> isolates recovered from six patients. The filamentous isolates were resistant to the filamentation-repressive effects of <i>Pseudomonas aeruginosa</i>, implicating inter-kingdom interactions as the selective force. Genome sequencing revealed that all but one of the filamentous isolates harbored mutations in the transcriptional repressor <i>NRG1</i>; such mutations were necessary and sufficient for the filamentous phenotype. Six independent <i>nrg1</i> mutations arose in <i>Candida</i> isolates from different patients, providing a poignant example of parallel evolution. Together, this combined clinical-genomic approach provides a high-resolution portrait of the fungal microbiome of cystic fibrosis patient lungs and identifies a genetic basis of pathogen adaptation.</p></div