Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1

Abstract

Candida albicans is an opportunistic human fungal pathogen and a member of the mucosal microbiota. To survive in the host and cause disease, C. albicans utilizes several virulence traits, including the ability to respond and adapt to diverse stressors, as well as the morphogenetic switch between yeast and filamentous morphologies. While complex cellular circuitry governs these virulence attributes, the following two kinase-mediated signaling pathways play particularly critical roles in controlling these processes: the Hog1 mitogen-activated protein kinase (MAPK) cascade and the protein kinase A (PKA) pathway. Here, we describe the construction of C. albicans strains harboring substitutions in the ATP-binding pockets of Hog1 and the catalytic subunits of PKA, Tpk1, and Tpk2 to render their activities sensitive to the addition of bulky ATP analogs. Specifically, inhibition by the ATP analog 1NM-PP1 resulted in phenotypes characteristic of the corresponding homozygous deletion mutants for each kinase gene. These strains represent a toolset for the rapid and specific inhibition of PKA and Hog1 kinase activity to further understand their roles in regulating C. albicans morphogenesis and stress responses. IMPORTANCE As an opportunistic pathogen in humans, the fungus Candida albicans relies on virulence traits to cause disease. They include the ability to transition from yeast to filamentous morphologies and the ability to grow in diverse environmental stress conditions, including nutrient limitation, as well as osmotic and heat shock. Previous work identified the following two kinases that play a critical role in regulating these responses: Hog1 and PKA. Here, we generated versions of each kinase that are sensitive to inhibition by a bulky ATP analog, 1NM-PP1. In the presence of the analog, kinase activity is inhibited rapidly and specifically, facilitating the analysis of both kinases in regulating C. albicans morphogenesis and stress responses. Together, these strains represent an important toolset to further our understanding of C. albicans biology and virulence.

Keywords: 1NM-PP1; ATP; Candida albicans; Hog1; genetic resource; protein kinase A.

FIG 1

Inhibition of AS Tpk1 and Tpk2 by 1NM-PP1 blocks C. albicans growth and hyphal morphogenesis. (A) A flowchart outlining the strain construction strategy used to make the TPK1 and TPK2 AS mutants. A black arrow indicates deletion or complementation at both alleles using a transient CRISPR strategy (23). Two orange arrows indicate the sequential complementation of both alleles using homologous recombination. The marker code utilizes gray to indicate auxotrophy for the indicated amino acid or sensitivity to the indicated drug. Red indicates prototrophy for the amino acid or resistance to the drug indicated. (B) Treatment with 1NM-PP1 inhibits the growth of AS TPK1 or TPK2 strains. C. albicans strains were grown overnight to saturation in YPD and then diluted to an optical density at 600 nm (OD₆₀₀) of 0.002 into fresh YPD containing either 2.5 μM 1NM-PP1 or dimethyl sulfoxide (DMSO) vehicle control. Strains were grown at 30°C for 24 h and growth was assessed by OD₆₀₀ (see color bar). (C) Treatment with 2.5 μM 1NM-PP1 inhibits filamentation of the AS TPK2 strain. Strains were grown at 37°C in either YPD with 10% serum; Spider medium; or yeast nitrogen base supplemented with 11 mM glucose, 2% casamino acid, and 5 mM GlcNAc (YNBNAG) for 4 h. (D) Treatment with 5 μM 1NM-PP1 inhibits filamentation of the AS TPK1 and TPK2 strains in RPMI with 10% serum and 5% CO₂ at 37°C. Strains were grown for 4 h. (E) TPK1 expression and TPK2 expression are not significantly different in wild-type or AS C. albicans strains. Transcript levels were normalized to ACT1. Data are presented as mean ± SD of technical triplicates. (F) A strain of C. albicans with both AS TPK1 and TPK2 alleles exhibited analog-specific inhibition of growth. C. albicans strains were grown as described in B. (G) Treatment with 1NM-PP1 inhibits filamentation in a strain of C. albicans with both AS TPK1 and TPK2 alleles. Strains were grown in YNBNAG at 37°C for 4 h. (H) Silver staining of the purified Tpk2/Bcy1 holoenzyme. Strains were grown to log phase in YPD, and then the PKA holoenzyme was purified using a flag-affinity purification as described previously (24). HF represents His6-Flag3. The purified product was run on a 10% SDS-PAGE gel and silver stained. *, impurities. (I) PKA kinase assays were performed using the Peptag kit (Promega). The nonphosphorylated peptide contains one positive charge (+1) and migrates toward the cathode. The phosphorylated peptide contains one negative charge (−1) and migrates toward the anode. Products were resolved on a 0.8% agarose gel in 50 mM Tris-HCl (pH 8.0). The gel was imaged with an Alexa 568 channel. In all panels, the acronym AS stands for analog sensitive. All experiments were performed in biological duplicate.

FIG 2

Inhibition of AS Hog1 by 1NM-PP1 results in hypersensitivity to osmotic stress and hyperfilamentation. (A) A flowchart outlining the strain construction strategy used to make the HOG1 mutants. The orange arrow indicates the sequential complementation of both alleles using conventional homologous recombination with a recyclable cloNAT-resistant cassette. The marker availability is coded as described in Fig. 1A. (B) A growth defect was observed in the AS HOG1 strain treated with 1 M NaCl and 1NM-PP1. C. albicans strains were grown overnight to saturation in YPD and then diluted to an OD₆₀₀ of 0.002 into fresh YPD in the absence or presence of 2.5 μM 1NM-PP1 or 1 M NaCl, as indicated. Strains were grown at 30°C for 24 h, and growth was assessed by OD₆₀₀ (see color bar). (C) RHR2 transcript levels are elevated in response to osmotic stress. The AS allele of HOG1 rescued the increased expression in RHR2 transcript levels only in the absence of 1NP-PP1. The indicated strains grown in YPD were treated with NaCl (0.8 M for 15 min) after 15-min exposure to DMSO or 1 μM 1NM-PP1. Transcript levels were normalized to ACT1 and are reported as fold change relative to the absence of NaCl. Data are presented as mean ± SD of technical triplicates. (D) 1NM-PP1 treatment induces filamentation of the AS HOG1 strain. Strains were grown at 34°C for 8 h. The inset depicts the morphology of cultures in the absence of 1NM-PP1. In all panels, the acronym AS stands for analog sensitive. All experiments were performed in biological duplicate.