A MAP kinase pathway is implicated in the pseudohyphal induction by hydrogen peroxide in Candica albicans

Abstract

Hydrogen peroxide (H(2)O(2)) functions as a ubiquitous intracellular messenger besides as an oxidative stress molecule. This dual role is based on the distinct cellular responses against different concentrations of H(2)O(2). Previously, we demonstrated that both low (> 1 mM) and high (4-10 mM) doses of exogenous H(2)O(2) induce filamentous growth with distinct cell morphology and growth rate in Candida albicans, suggesting the different transcription response. In this study, we revealed that the sub-toxic and toxic levels of H(2)O(2) indeed induced pseudohyphae, but not true hyphae. Supporting this, several hyphae-specific genes that are expressed in true hyphae induced by serum were not detected in either sub-toxic or toxic H(2)O(2) condition. A DNA microarray analysis was conducted to reveal the transcription profiles in cells treated with sub-toxic and toxic conditions of H(2)O(2). Under the sub-toxic condition, a small number of genes involved in cell proliferation and metabolism were up-regulated, whereas a large number of genes were up-regulated in the toxic condition where the genes required for growth and proliferation were selectively restricted. For pseudohyphal induction by sub-toxic H(2)O(2), Cek1 MAPK activating the transcription factor Cph1 was shown to be important. The absence of expression of several hyphae-specific genes known to be downstream targets of Cph1-signaling pathway for true hyphae formation suggests that the Cek1-mediated signaling pathway is not solely responsible for pseudohyphal formation by subtoxic H(2)O(2) and, but instead, complex networking pathway may exists by the activation of different regulators.

Fig. 1.

H₂O₂ toxicity. Cells were treated with various concentrations of H₂O₂ for the indicated periods of time. Fluorescence microscopy examination revealed healthy cells with annexin V (−) PI (−), apoptotic cells with annexin V (+) PI (−), and necrotic cells with annexin V (+) PI (+). Annexin is indicated in green and PI in red.

Fig. 2.

Characterization of pseudohyphae induced by H₂O_2. (A) Expression of hyphae-specific genes. Total RNAs were prepared from cells cultured in YPD containing 1 mM H₂O₂ for 180 min, 10 mM H₂O₂ for 30 min, and 10% serum for 180 min at 37°C. Fifteen micrograms of RNA were loaded per lane for Northern analysis. Transcripts for hyphae-specific genes were detected using probes amplified with gene-specific primers. Lanes: 1, 0 min; 2, 30 min; 3, 60 min; 4, 90 min; 5, 120 min; 6, 180 min. (B) Results of calcofluor staining. Above cells cultured for 30, 60, 90, 120, and 180 min were washed twice and resuspended in water, and incubated for 30 min with 20 μl of calcofluor (1 mg ml⁻¹ in water). The fluorescence was observed at 365 nm with a confocal laser scanning microscope.

Fig. 3.

Effect of CPH1 and EFG1 on pseudohyphal induction by H₂O₂. (A) Expression of CPH1 and EFG1. Total RNAs were prepared from cells cultured in YPD for yeast cells or YPD containing 1 mM H₂O₂ for 180 min, 10 mM H₂O₂ for 30 min, and 10% serum for 180 min at 37°C. Fifteen micrograms of RNA were loaded per lane for Northern analysis. Transcripts for CPH1 and EFG1 were detected using probes amplified with gene-specific primers. (B) Effect of CPH1 and EFG1 on hyphal induction by H₂O₂. Wild type and cph1-null (cph1Δ/cph1Δ), efg1-null (efg1Δ/efg1Δ), and double null (cph1Δ/cph1Δ efg1Δ/efg1Δ) were cultured in YPD containing 0, 1, 4, and 10 mM H₂O₂. Culture time was 180 min except for 10 mM (30 min).

Fig. 4.

CEK1 is responsible for the pseudohyphal induction by H₂O₂. The experimental conditions were same as in Fig. 4. (A) Expression of CST20, HST17, and CEK1. CST20 and HST17 are two genes immediately upstream of CEK1 in the MAPK pathway. (B) Phosphorylation of Cek1p. Western analysis was performed with total cell extracts. An anti-phospho-p44/p42 MAPK (Erk1/2) (Thr202/ Tyr204) antibody was used as a primary antibody that recognizes the phosphorylated form of Mkc1 and Cek1 kinases. Bands were visualized with an enhanced chemiluminescence system. (C) Effect of CEK1 on hyphal induction by H₂O₂. Wild type and cek1-null (cek1Δ/cek1Δ), and CEK1-revertant (CEK1-R) were used.