Interrogation of the cell wall integrity pathway in Aspergillus niger identifies a putative negative regulator of transcription involved in chitin deposition

ABSTRACT Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity and protein tyrosine phosphatases (PTPs) in yeasts. InSaccharomyces cerevisiae, two PTPs, Ptp2 and Ptp3, inactivate the MAPKs, Hog1 and Fus3, with different specificities. To further examine the functions and substrate specificities of Ptp2 and Ptp3, we tested whether they could inactivate a third MAPK, Mpk1, in the cell wall integrity pathway. In vivo and in vitro evidence indicates that both PTPs inactivate Mpk1, but Ptp2 is the more effective negative regulator. Multicopy expression of PTP2, but not PTP3, suppressed growth defects due to the MEK kinase mutation, BCK1-20, and the MEK mutation,MKK1-386, that hyperactivate this pathway. In addition, deletion of PTP2, but not PTP3, exacerbated growth defects due to MKK1-386. Other evidence supported a role for Ptp3 in this pathway. Expression of MKK1-386 was lethal in the ptp2Δ ptp3Δ strain but not in either single PTP deletion strain. In addition, the ptp2Δ ptp3Δ strain showed higher levels of heat stress-induced Mpk1-phosphotyrosine than the wild-type strain or strains lacking either PTP. The PTPs also showed differences in vitro. Ptp2 was more efficient than Ptp3 at binding and dephosphorylating Mpk1. Another factor that may contribute to the greater effectiveness of Ptp2 is its subcellular localization. Ptp2 is predominantly nuclear whereas Ptp3 is cytoplasmic, suggesting that active Mpk1 is present in the nucleus. Last, PTP2 but not PTP3 transcript increased in response to heat shock in a Mpk1-dependent manner, suggesting that Ptp2 acts in a negative feedback loop to inactivate Mpk1.

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The fission yeast cell wall stress sensor-like proteins Mtl2 and Wsc1 act by turning on the GTPase Rho1p but act independently of the cell wall integrity pathway

MicrobiologyOpen ◽

10.1002/mbo3.113 ◽

2013 ◽

Vol 2 (5) ◽

pp. 778-794 ◽

Cited By ~ 10

Author(s):

Sandra Cruz ◽

Sofía Muñoz ◽

Elvira Manjón ◽

Patricia García ◽

Yolanda Sanchez

Keyword(s):

Cell Wall ◽

Yeast Cell ◽

Fission Yeast ◽

Wall Stress ◽

Cell Wall Integrity ◽

Yeast Cell Wall ◽

Stress Sensor ◽

Fission Yeast Cell ◽

Cell Wall Stress ◽

Cell Wall Integrity Pathway

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Zinc oxide nanoparticles induce toxicity by affecting cell wall integrity pathway, mitochondrial function and lipid homeostasis in Saccharomyces cerevisiae

Chemosphere ◽

10.1016/j.chemosphere.2018.09.028 ◽

2018 ◽

Vol 213 ◽

pp. 65-75 ◽

Cited By ~ 16

Author(s):

Piyoosh Kumar Babele ◽

Pilendra Kumar Thakre ◽

Ramesh Kumawat ◽

Raghuvir Singh Tomar

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Zinc Oxide ◽

Mitochondrial Function ◽

Zinc Oxide Nanoparticles ◽

Cell Wall Integrity ◽

Lipid Homeostasis ◽

Oxide Nanoparticles ◽

Cell Wall Integrity Pathway

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Aspergillus fumigatus G-Protein Coupled Receptors GprM and GprJ Are Important for the Regulation of the Cell Wall Integrity Pathway, Secondary Metabolite Production, and Virulence

mBio ◽

10.1128/mbio.02458-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 1

Author(s):

Aílton Pereira da Costa Filho ◽

Guilherme Thomaz Pereira Brancini ◽

Patrícia Alves de Castro ◽

Clara Valero ◽

Jaire Alves Ferreira Filho ◽

...

Keyword(s):

Cell Wall ◽

G Protein ◽

Galleria Mellonella ◽

G Protein Coupled Receptors ◽

Cell Wall Integrity ◽

Fungal Development ◽

Content Type ◽

Cell Wall Integrity Pathway ◽

Mycotoxin Biosynthesis ◽

G Protein Coupled

ABSTRACT G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. IMPORTANCE A. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

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Mpt5p, a Stress Tolerance- and Lifespan-Promoting PUF Protein in Saccharomyces cerevisiae, Acts Upstream of the Cell Wall Integrity Pathway

Eukaryotic Cell ◽

10.1128/ec.00188-06 ◽

2006 ◽

Vol 6 (2) ◽

pp. 262-270 ◽

Cited By ~ 30

Author(s):

Mark S. Stewart ◽

Sue Ann Krause ◽

Josephine McGhie ◽

Joseph V. Gray

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Life Span ◽

Stress Tolerance ◽

Synthetic Medium ◽

Cell Wall Integrity ◽

Puf Proteins ◽

Downstream Target ◽

Cell Wall Integrity Pathway ◽

Puf Protein

ABSTRACT Pumilio family (PUF) proteins affect specific genes by binding to, and inhibiting the translation or stability of, their transcripts. The PUF domain is required and sufficient for this function. One Saccharomyces cerevisiae PUF protein, Mpt5p (also called Puf5p or Uth4p), promotes stress tolerance and replicative life span (the maximum number of doublings a mother cell can undergo before entering into senescence) by an unknown mechanism thought to partly overlap with, but to be independent of, the cell wall integrity (CWI) pathway. Here, we found that mpt5Δ mutants also display a short chronological life span (the time cells stay alive in saturated cultures in synthetic medium), a defect that is suppressed by activation of CWI signaling. We found that Mpt5p is an upstream activator of the CWI pathway: mpt5Δ mutants display the appropriate phenotypes and genetic interactions, display low basal activity of the pathway, and are defective in activation of the pathway upon thermal stress. A set of mRNAs that specifically bind to Mpt5p was recently reported. One such putative target, LRG1, encodes a GTPase-activating protein for Rho1p that directly links Mpt5p to CWI signaling: Lrg1p inhibits CWI signaling, LRG1 mRNA contains a consensus Mpt5p-binding site in its putative 3′ untranslated region, loss of Lrg1p suppresses the temperature sensitivity and CWI signaling defects of mpt5Δ mutants, and LRG1 mRNA abundance is inhibited by Mpt5p. We conclude that Mpt5p is required for normal replicative and chronological life spans and that the CWI pathway is a key and direct downstream target of this PUF protein.

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Cell fusion in yeast is negatively regulated by components of the cell wall integrity pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e18-04-0236 ◽

2019 ◽

Vol 30 (4) ◽

pp. 441-452 ◽

Cited By ~ 2

Author(s):

Allison E. Hall ◽

Mark D. Rose

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Cell Wall ◽

Yeast Cell ◽

Cell Fusion ◽

Cell Walls ◽

Cell Wall Integrity ◽

Fusion Genes ◽

Cell Wall Degradation ◽

Cell Wall Integrity Pathway

During mating, Saccharomyces cerevisiae cells must degrade the intervening cell wall to allow fusion of the partners. Because improper timing or location of cell wall degradation would cause lysis, the initiation of cell fusion must be highly regulated. Here, we find that yeast cell fusion is negatively regulated by components of the cell wall integrity (CWI) pathway. Loss of the cell wall sensor, MID2, specifically causes “mating-induced death” after pheromone exposure. Mating-induced death is suppressed by mutations in cell fusion genes ( FUS1, FUS2, RVS161, CDC42), implying that mid2Δ cells die from premature fusion without a partner. Consistent with premature fusion, mid2Δ shmoos had thinner cell walls and lysed at the shmoo tip. Normally, Cdc42p colocalizes with Fus2p to form a focus only when mating cells are in contact (prezygotes) and colocalization is required for cell fusion. However, Cdc42p was aberrantly colocalized with Fus2p to form a focus in mid2Δ shmoos. A hyperactive allele of the CWI kinase Pkc1p ( PKC1*) caused decreased cell fusion and Cdc42p localization in prezygotes. In shmoos, PKC1* increased Cdc42p localization; however, it was not colocalized with Fus2p or associated with cell death. We conclude that Mid2p and Pkc1p negatively regulate cell fusion via Cdc42p and Fus2p.

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