Abstract
Fungal cells trigger adaptive mechanisms to survive in situations that compromise cell wall integrity. We show here that the global transcriptional response elicited by inhibition of the synthesis of β-1,3-glucan by caspofungin, encompasses a set of genes that are dependent on Slt2, the MAPK of the Cell Wall Integrity (CWI) pathway, and a broad group of genes regulated independently of Slt2. Genes negatively regulated by the cyclic AMP/Protein Kinase A (PKA) signaling pathway were overrepresented in the latter group. Moreover, cell wall stress mediated by inhibition of β-1,3-glucan synthesis, but not by other cell wall interfering compounds, negatively regulated PKA signaling as indicated by the nuclear localisation of Msn2, cellular glycogen accumulation, a decrease of intracellular cAMP levels and a severe decrease in both the activation of the small GTPase Ras2 and the phosphorylation of known substrates of PKA. All these effects relied on the plasma membrane-spanning sensor of the CWI pathway Wsc1. In addition, caspofungin induced a reduction in the cytosolic pH, which was dependent on the extracellular region of Wsc1. Therefore, alterations of the β-1,3-glucan network in the fungal cell wall, induce, through Wsc1, the activation of the CWI pathway and parallel inhibition of PKA signaling.
Highlights
The Cell Wall Integrity (CWI) pathway, which is the main route responsible for maintaining cell wall homeostasis, has been extensively studied in the model yeast Saccharomyces cerevisiae[9]
Since we have previously described how all the transcriptional induction was dependent on the CWI-pathway sensor Wsc[1], we employed real-time PCR assays. Quantitative RT-PCR (RT-qPCR) to study the effect the deletion of ROM2, the principal guanine nucleotide exchange factor (GEF) of the small G-protein Rho[1] associated with Wsc[1] transduction, had on the induction of representative Slt2-dependent (MLP1, PRM5, and AFR1) and independent (ALD3, CTT1, and HXT5) genes in response to CAS
This response is mainly mediated by the cell wall integrity MAPK signalling cascade governed by the MAPK Slt[2] and the transcription factor Rlm[1]
Summary
The Cell Wall Integrity (CWI) pathway, which is the main route responsible for maintaining cell wall homeostasis, has been extensively studied in the model yeast Saccharomyces cerevisiae[9]. An intermediate component in the PKA-mediated regulation of gene expression is the Rim[15] protein kinase, which is a regulator of four major transcription factors: Gis[1], Rph[1], and the partially redundant Msn2/Msn[4]. These activate gene expression associated to diauxic shift and stress response[28, 29]. Information about the global transcriptional response to the cellular damage caused by CAS in wild-type yeast cells is available, little is known about the regulatory mechanisms involved, apart from the activation of the CWI pathway[11,12,13]. This work improves upon current knowledge on the molecular mechanisms involved in the cellular adaptation response to the cell wall damage caused by inhibition of the beta-1,3-glucan synthase
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