Staurosporine Induces Filamentation in the Human Fungal Pathogen Candida albicans via Signaling through Cyr1 and Protein Kinase A

Abstract

Protein kinases are key regulators of signal transduction pathways that participate in diverse cellular processes. In fungal pathogens, kinases regulate signaling pathways that govern drug resistance, stress adaptation, and pathogenesis. The impact of kinases on the fungal regulatory circuitry has recently garnered considerable attention in the opportunistic fungal pathogen Candida albicans, which is a leading cause of human morbidity and mortality. Complex regulatory circuitry governs the C. albicans morphogenetic transition between yeast and filamentous growth, which is a key virulence trait. Here, we report that staurosporine, a promiscuous kinase inhibitor that abrogates fungal drug resistance, also influences C. albicans morphogenesis by inducing filamentation in the absence of any other inducing cue. We further establish that staurosporine exerts its effect via the adenylyl cyclase Cyr1 and the cyclic AMP (cAMP)-dependent protein kinase A (PKA). Strikingly, filamentation induced by staurosporine does not require the known upstream regulators of Cyr1, Ras1 or Pkc1, or effectors downstream of PKA, including Efg1. We further demonstrate that Cyr1 is capable of activating PKA to enable filamentation in response to staurosporine through a mechanism that does not require degradation of the transcriptional repressor Nrg1. We establish that staurosporine-induced filamentation is accompanied by a defect in septin ring formation, implicating cell cycle kinases as potential staurosporine targets underpinning this cellular response. Thus, we establish staurosporine as a chemical probe to elucidate the architecture of cellular signaling governing fungal morphogenesis and highlight the existence of novel circuitry through which the Cyr1 and PKA govern a key virulence trait. IMPORTANCE The impact of fungal pathogens on human health is devastating. One of the most pervasive fungal pathogens is Candida albicans, which kills ~40% of people suffering from bloodstream infections. Treatment of these infections is extremely difficult, as fungi are closely related to humans, and there are limited drugs that kill the fungus without host toxicity. The capacity of C. albicans to transition between yeast and filamentous forms is a key virulence trait. Thus, understanding the genetic pathways that regulate morphogenesis could provide novel therapeutic targets to treat C. albicans infections. Here, we establish the small molecule staurosporine as an inducer of filamentous growth. We unveil distinct regulatory circuitry required for staurosporine-induced filamentation that appears to be unique to this filament-inducing cue. Thus, this work highlights the fact that small molecules, such as staurosporine, can improve our understanding of the pathways required for key virulence programs, which may lead to the development of novel therapeutics.

Keywords: Candida albicans; cyclic AMP; kinase inhibitor; morphogenesis; staurosporine; virulence.

FIG 1

Staurosporine induces filamentation independent of Pkc1. SN95 wild-type (WT) cells were subcultured to log phase in YPD at 30°C, YPD plus 0.5 μg/ml staurosporine at 30°C, YPD plus 10 μM geldanamycin at 30°C, Spider medium at 37°C, or 10% serum at 37°C. Cells were imaged by DIC microscopy. The scale bar indicates 20 μm. White arrows highlight representative filaments with obvious constrictions along the filament. Red arrows highlight representative filaments with a widening at the bud neck that narrows toward the tip.

FIG 2

Staurosporine-induced filamentation requires Cyr1 and PKA. (A) Farnesol inhibits filamentation induced by staurosporine. SN95 wild-type cells were grown to log phase at 30°C in YPD or YPD plus 0.5 μg/ml staurosporine in the absence or presence of 200 μM farnesol. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm. (B) Cyr1 and PKA are the only components of the cAMP signaling pathway tested that are required for filamentation induced by staurosporine. A CAI4 wild-type strain or mutants lacking components of the cAMP signaling pathway were subcultured to stationary phase in YPD at 30°C in the presence or absence of 0.5 μg/ml staurosporine (STS). Cells were imaged by DIC microscopy. The scale bar indicates 20 μm.

FIG 3

Staurosporine induces a distinct gene expression program from other filament-inducing cues and promotes filamentation independent of Nrg1 degradation. (A) SN95 wild-type cells were subcultured to log phase in YPD at 30°C, YPD plus 10% serum at 37°C, YPD plus 200 mM hydroxyurea at 30°C, or YPD plus 0.5 μg/ml staurosporine at 30°C. cDNA was prepared from total RNA for qRT-PCR. The transcript levels of filament-specific transcripts (ECE1, HGC1, HWP1, and RBT1) and yeast-specific transcripts (YWP1 and NRG1) were monitored by qRT-PCR and normalized to GPD1. The fold change in gene expression under each condition relative to YPD at 30°C is plotted as the mean ± standard deviation from triplicate samples and is representative of two independent experiments. (B) Nrg1 protein persists in filaments induced by staurosporine. SN95 wild-type cells expressing native levels of HA-tagged Nrg1 were subcultured to log phase in YPD plus 10% serum at 37°C, YPD plus 200 mM hydroxyurea at 30°C, or YPD plus 0.5 μg/ml staurosporine at 30°C. Total protein was resolved by SDS-PAGE, and the blot was hybridized with anti-hemagglutinin to detect Nrg1 and anti-PSTAIRE to monitor Cdc28 as a loading control. (C) SN95 wild-type cells were grown to log phase under identical conditions as described for panel B. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm.

FIG 4

Dibutyryl cAMP does not rescue staurosporine-induced filamentation of a cyr1Δ/cyr1Δ mutant. A CAI4 wild-type strain and a cyr1Δ/cyr1Δ mutant were grown to log phase at 37°C in YPD plus 10% serum or at 30°C in YPD plus 0.5 μg/ml staurosporine in the absence or presence of 100 mM dibutyryl cAMP, as indicated. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm.

FIG 5

Mfg1 and Flo8 are not required for polarized growth in response to staurosporine. Strains were grown to log phase at 30°C in YPD, 30°C in YPD plus 0.5 μg/ml staurosporine, or 37°C in YPD plus 10% serum. Cells were imaged by DIC microscopy. The scale bar indicates 20 μm.

FIG 6

Septin ring formation and chitin-containing septum formation are aberrant in filaments formed in response to staurosporine. (A) Strains were subcultured to log phase in YPD at 30°C, YPD plus 0.5 μg/ml staurosporine at 30°C, or YPD plus 10% serum at 37°C. Shown are representative fluorescence microscopy images of SN95 wild-type cells expressing native levels of GFP-tagged Cdc10 to visualize septin (left panels) and RFP-tagged Hhf1 to visualize DNA (middle panels). The scale bar represents 10 μm. The fluorescence microscopy images were merged with DIC images (right panels). (B) Strains were subcultured to log phase in YPD at 30°C in the presence or absence of 0.5 μg/ml staurosporine. Shown are representative fluorescence microscopy images of SN95 wild-type cells expressing native levels of GFP-tagged Nop1 to visualize nuclei (middle panels) and stained with calcofluor white to visualize chitin (left panels). The scale bar represents 10 μm. The fluorescence microscopy images were merged with DIC images (right panels).