cAMP-independent signal pathways stimulate hyphal morphogenesis in Candida albicans

Abstract

The fungal pathogen Candida albicans can transition from budding to hyphal growth, which promotes biofilm formation and invasive growth into tissues. Stimulation of adenylyl cyclase to form cAMP induces hyphal morphogenesis. The failure of cells lacking adenylyl cyclase (cyr1Δ) to form hyphae has suggested that cAMP signaling is essential for hyphal growth. However, cyr1Δ mutants also grow slowly and have defects in morphogenesis, making it unclear whether hyphal inducers must stimulate cAMP, or if normal basal levels of cAMP are required to maintain cellular health needed for hyphal growth. Interestingly, supplementation of cyr1Δ cells with low levels of cAMP enabled them to form hyphae in response to the inducer N-acetylglucosamine (GlcNAc), suggesting that a basal level of cAMP is sufficient for stimulation. Furthermore, we isolated faster-growing cyr1Δ pseudorevertant strains that can be induced to form hyphae even though they lack cAMP. The pseudorevertant strains were not induced by CO₂ , consistent with reports that CO₂ directly stimulates adenylyl cyclase. Mutational analysis showed that induction of hyphae in a pseudorevertant strain was independent of RAS1, but was dependent on the EFG1 transcription factor that acts downstream of protein kinase A. Thus, cAMP-independent signals contribute to the induction of hyphal responses.

Figure 1. Deletion of cAMP pathway members results in hyphal defects

Hyphal formation was induced by growing cells in synthetic medium containing 100 mM GlcNAc at 37°C for the indicated time. Deletion of CSC25 (csc25Δ) caused a mild defect in hyphal growth, deletion of RAS1 (ras1Δ) caused a stronger defect, and deletion of CYR1 (cyr1Δ) completely abrogated it. The wild type (DIC185), csc25Δ (SP53-1), ras1Δ (SP54-1), cyr1Δ (SP60-66) strains were derived from BWP17 and the CR216 cyr1Δ strain was derived from the CAI4 strain of C. albicans as described previously (Rocha et al., 2001). Bars, 10 μm.

Figure 2. Addition of non-inducing levels of db-cAMP to cyr1Δ restores hyphal switching in response to GlcNAc

A. Cells of cyr1Δ strain CR216 were cultured in rich YP medium containing 100 mM galactose plus the indicated amount of dibutyryl cAMP (db-cAMP). The cells were incubated at 37°C for 2 h, GlcNAc was added to a final concentration of 10 mM, and then the cells were incubated at 37°C for 3 h before being photographed. Bars, 10 μm. B. Quantification of the average number of filamentous cells induced in the experiments described in panel A. Pre-incubation of cyr1Δ cells with non-inducing concentrations of db-cAMP restored the ability of GlcNAc to stimulate hyphal growth. The results represent the average of three different experiments, with at least 300 cells counted per experiment. Error bars indicate SD.

Figure 3. Faster growing pseudorevertants spontaneously arise from cyr1Δ cultures over time

A. Cells streaked onto YPD agar plate that were derived from a representative colony of cyr1Δ (CR216) cells that had been incubated at room temperature for 3 weeks. At least two populations of cells are evident: small colonies similar to the cyr1Δ parental cells and large colonies of pseudorevertants. B. Percent of cyr1Δ (CR216) colonies incubated for the indicated time that gave rise to mixed populations of large and small cells upon restreaking onto a fresh agar medium plate. Colonies that, when restreaked, gave at least 1 large colony were scored as positive. Results represent the average for 30 colonies assayed this way in each of 3 independent experiments. Error bars indicate SD. C. Growth rate comparison between the parental cyr1Δ cells and pseudorevertants derived from them. Indicated strains were streaked on YPD and grown for 3 days at 30°C. Both the previously reported published cyr1Δ strain CR216 and a newly made cyr1Δ strain we constructed (SP60-66) gave rise to pseudorevertants that grew at approximately similar rates. Pseudorevertant PR1 was derived from CR216 while PR2 was derived from SP60-66. D. GlcNAc induction of hyphal growth. Cells grown in SC-URA plus 100 mM dextrose medium, were washed and then resuspended in the same medium containing either 100 mM dextrose or 100 mM GlcNAc and cultured at 37°C for 90 min. As expected, wild-type cells formed hyphae but the cyr1Δ strains were completely defective. Interestingly, the faster growing pseudorevertants derived from both cyr1Δ strains were also stimulated to undergo filamentous growth in GlcNAc medium. Bars, 10 μm.

Figure 4. Pseudorevertants do not contain detectable cAMP

A. Wild type, cyr1Δ, and derived pseudorevertant cells were grown overnight in rich YPD medium, cAMP was extracted, and then cAMP levels were assayed using a GE Amersham cAMP EIA-Immunoassay kit with the non-acetylation protocol. The pseudorevertant strains PR1 and PR2 showed low signals that were at or below the limit of detection, indicating that no cAMP could be detected, similar to their parental strains cyr1Δ strain CR216 and cyr1Δ strain SP60-66. B. The strains described above were grown in synthetic SC-URA medium with 100 mM dextrose and the induced with 30% serum at 37° C for 30 min. cAMP was extracted as in panel A and assayed using the more sensitive acetylation protocol to detect cAMP. The cyr1Δ strains and their derived pseudorevertants contained no detectable cAMP under these conditions, similar to the results obtained with rich medium in panel A. The results for each strain represent the average cAMP value obtained from the analysis of at least four independent colonies. The dashed line indicates the limit of detection of the assay. Error bars indicate SD.

Figure 5. Pseudorevertants undergo true hyphal growth

A. Wild-type cells (DIC185), and pseudorevertant cells derived from both cyr1Δ strains (CR216 and SP60-66) were cultured overnight in 100 mM GlcNAc at 37°C and then photographed at low magnification. Bar, 30 μm. B. Hyphal cells from the cultures in panel A were stained with Hoechst 33342 to detect DNA (Blue) and Pontamine Fast Scarlet 34 (Red) to detect cell wall chitin. Hyphal cells from all three strains displayed characteristics of true hyphae including a first septum distal to the mother cell (arrow). Bar, 10 μm. C. Upper panels show a close-up view of a single septum. Lower panels show the septum image merged with Red staining from Pontamine Fast Scarlet 34 to verify the presence of the septum. Bar, 1 μm.

Figure 6. Pseudorevertants induce hyphal morphology and gene expression in response to many inducers, but not CO₂

Strains carrying the HWP1-GFP reporter gene were incubated under the indicated hyphal inducing conditions and then photographed using light microscopy to detect cell morphology and fluorescence microscopy to determine if GFP was produced. As expected, all of the inducers stimulated the wild type control strain to form hyphae and produce GFP, whereas the cyr1Δ strain showed no detectable hyphae or GFP. In contrast, the pseudorevertant strain PR3 showed induction of filamentous cells and GFP levels similar to the wild type under all conditions except for CO₂. All inductions were performed at 37°C. GlcNAc induction was performed using SC-URA medium with 100 mM GlcNAc and was visualized at 90 min. Serum induction was performed in SC-URA plus 100 mM dextrose and 30% bovine calf serum and was visualized after 90 min. Hyphal induction with alkaline pH was performed by culturing cells overnight in SC medium buffered to pH 8 with 150 mM HEPES. CO₂ induction was performed by culturing cells overnight in a chamber with 5% CO₂.

Figure 7. Pseudorevertants show increased Phloxine B staining, but not other characteristics of Opaque cells

A. The cyr1Δ strain CR216 was incubated at room temperature on YPD agar medium for the indicated time, restreaked onto fresh medium, and then the resulting colonies were assessed to determine the percent of faster-growing large colonies rather than the expected small colonies. The results represent the average for 6 colonies aged for two weeks, 59 colonies aged for three weeks, and 91 colonies aged for four weeks. B. The cyr1Δ cells (CR216) were incubated 3 weeks on YPD agar plates and then streaked onto a YPD plate containing 45 μg ml⁻¹ Phloxine B. Larger pseudorevertant colonies were observed to stain magenta (arrow), similar to that expected for cells in the Opaque phase. C. Wild type (DIC185), a cyr1Δ pseudorevertant (PR1) derived from strain CR216, and its wor1Δ derivative (PR1-wor1Δ) were streaked onto YPD agar plate containing 45 μg ml⁻¹ Phloxine B, showing that deletion of WOR1 did not change Phloxine B staining of the pseudorevertant. D. The strains described in panel C were streaked for single colonies on YPD and incubated at 30° C for 2 days. E. The strains described in panel C were grown in liquid YPD media at 30° C. Panels D and E show that deletion of WOR1 does not affect the growth rate of the pseudorevertant strain PR1.

Figure 8. Pseudorevertant strain PR2 induces hyphal growth independently of RAS1, but dependent on EFG1

A. Photographs of cells grown in dextrose medium or induced to undergo hyphal morphogenesis in medium containing 50 mM GlcNAc or 10% bovine serum. Cells were induced at 37°C with GlcNAc for 3h and with serum for 2 h. B. Graph indicating the percent of filamentous cells after growth in conditions described in panel A. Values represent the average of three independent assays with at least 200 cells counted for each condition. The strains used included a wild type control (DIC185), pseudorevertant (PR2), PR2 with EFG1 deleted (PR2 efg1Δ; strain HS628), the PR2 efg1Δ strain with EFG1 restored (PR2 efg1Δ + EFG1; strain HS6281), and PR2 with RAS1 deleted (PR2 ras1Δ; strain HS631).