Transcriptional regulation by protein kinase A in Cryptococcus neoformans

Abstract

A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for anti-cryptococcal therapy.

Figure 1. Comparison of Heat Shock Sensitivity for the WT Strain, and the pka1 and pkr1 Mutants

(A) Cells (10⁵) were subjected to heat shock treatment at 50 °C for the indicated time and spotted on YPD plates. The plates were incubated for 3 d at 30 °C. (B) Enumeration of colony forming units (CFUs) for the strains after heat shock. The data are expressed as the percent of the starting number of cells recovered after heat shock and represent the mean values ± standard deviation (SD) from three independent experiments.

Figure 2. Suppression of Capsule Formation by Secretion Inhibitors

(A) Sensitivity of the WT strain H99 and the pka1 and pkr1 mutants to trafficking inhibitors. Photographs of cells on medium containing the ferrous iron chelator BPS, and the chelator plus monensin, are included to demonstrate growth of the strains under low iron conditions similar to those used in the broth cultures (B) to measure capsule size. (B) Suppression of capsule formation in both the WT strain and the pkr1 mutant after treatment with trafficking inhibitors at the indicated concentrations. Cells were cultured in LIM at 30 °C and examined at 72 h after addition of the inhibitors indicated. Chemicals and concentrations are indicated at the top and the strains are noted on the left of the photographs. Bar = 10 μM. (C and D) Sixty cells from the WT (H99) strain (C) and the pkr1 mutant (D) were measured to determine cell diameter and capsule radius with and without treatment with the trafficking inhibitors (as shown in [B]). The capsule sizes of the treated cells were statistically different (p <0.001) from the sizes for the untreated cells in all cases. Each bar represents the average of 60 measurements.

Figure 3. Lithium Chloride and Glycerol Treatments Influence Capsule Size

(A) Sensitivity of the WT strain H99 and the pka1 and pkr1 mutants to growth on YPD medium containing LiCl. The strains are listed on the left side of the panel. The plates were incubated at 30 °C or 37 °C for 5 d before being photographed. (B) Sixty cells from the WT (H99) strain treated with LiCl at the indicated concentrations were measured for cell diameter and capsule radius. Each bar represents the average of measurements for 60 cells. The capsule sizes of the cells treated with 10, 30, and 75 mM LiCl were statistically different (p <0.001) from the sizes for the untreated cells. (C) Examination of capsule formation in the WT strain, and the pkr1 and ova1 mutants, in a range of LiCl concentrations. Bar = 10 μM. (D) Capsule formation in the WT and the pkr1 and ova1 mutants in the presence of glycerol at two concentrations (5% and 10%). Bar = 10 μM. In (B–D), cells were cultured in LIM at 30 °C for 16 to 72 h with addition of glycerol or LiCl as indicated. (E) Sixty cells from the WT (H99) strain grown with the indicated concentrations glycerol were measured for cell diameter and capsule radius. Each bar represents the average of 60 measurements. The capsule sizes of the cells with either concentration of glycerol were statistically different (p <0.001) from the sizes for the untreated cells.

Figure 4. Sensitivity of PKA Mutants to Caffeine

Ten-fold serial dilutions of the WT and the pka1 and pkr1 mutants were grown on YPD medium containing caffeine at 30 °C and 37 °C. The strains used for all treatments are listed on the left side of the panel. The plates were incubated for 5 d before being photographed.

Figure 5. Multiple Alignment of Ova1 with TFS1 and Other PEBP Family Members

The amino acid sequence alignment was performed with Clustal W (http://www.ch.embnet.org); identical residues are boxed in black and similar residues are shown in gray. The phosphatidylethanolamine-binding domain is indicated by a bold arrow spanning residues 91–204 of Ova1. Cn, C. neoformans var. grubii; Ov, Onchocerca volvulus; Sc, Saccharomyces cerevisiae; Um, Ustilago maydis.

Figure 6. Northern Analysis of OVA1 Expression in WT and Mutant Strains in Media with Two Different Levels of Iron

Cells were grown in LIM (designated −) and LIM + Fe (designated +). The Northern blot was prepared from total RNA and hybridized with a DNA fragment from the OVA1 gene. The same filter was hybridized with a DNA fragment from the actin gene (ACT1) as a loading control. Note that the hybridization pattern is consistent with the SAGE data, and also indicates that iron does not significantly influence OVA1 transcript levels in any of the strains. The numbers at the bottom indicate the ratio of the hybridization signals for the OVA1 and ACT1 genes, as determined from the scanned images. The expression of OVA1 was also confirmed by a quantitative real-time PCR analysis (Figure S2).

Figure 7. Influence of the ova1 Mutation on Capsule and Melanin Formation, and Epistasis with pka1

(A) Capsule size is shown by staining with India ink for the WT and mutant strains. Cells were cultured either in liquid LIM or on DMEM plates (for capsule induction) at 30 °C. The photographs are of cells cultured for 48 h in LIM, and the same results were obtained in several independent trials. Bar = 10 μM. (B) Sixty cells from the cultures used for (A) were measured for cell diameter and capsule radius. Each bar represents the average of 60 measurements. The capsule sizes were statistically different (p <0.001) for all pair-wise comparisons except WT versus pka1ova1, and pka1 versus pka1ova1+OVA1. (C) Melanin formation was tested in the strains indicated by inoculation of cultures on L-DOPA or YPD plates (as a control to demonstrate equal growth) and incubation for 2 d at 30 °C. (D) Sensitivity of the pka1 mutant and the pka1 ova1 double mutant to LiCl. Cells were washed and 10-fold serial dilutions of a 10⁶ cells/ml suspension were plated on either YPD or YPD + 75 mM LiCl.