Phosphoproteomic and transcriptomic analyses reveal multiple functions for Aspergillus nidulans MpkA independent of cell wall stress

Chelius, Cynthia; Ribeiro, Laryssa Freitas; Huso, Walker; Kumar, Jyothi; Lincoln, Stephen R.; Tran, Bao; Goo, Young Ah; Srivastava, Ranjan; Harris, Steven D.; Marten, Mark R.

doi:10.1016/j.fgb.2019.01.003

Cited by 8 publications

(17 citation statements)

References 88 publications

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“…Strains and Media-Aspergillus nidulans A1405 (Fungal Genetics Stock Center; FGSC) was used as the control strain. Frozen stocks were spread on MAGV plates (2% malt extract, 1.5% agar, 2% glucose, 2% peptone, and 1 ml/L Hutner's trace elements and vitamin solution) and incubated for 2 days at 28°C (29). 1E7 spores were harvested and inoculated into 50 ml of YGV (pH 3.3) (0.5% yeast extract, 1% glucose, and 1 ml/L Hutner's trace elements and vitamin solution).…”

Section: Methodsmentioning

confidence: 99%

“…One hundred micrograms of protein was exposed to SDS-page electrophoresis and transferred to a nitrocellulose membrane (Life technologies, Carlsbad, CA). Western blotting preparation steps were performed as described in Chelius et al, 2019. Two biological replicates were modeled using Michaelis-Menten kinetics.…”

Section: Methodsmentioning

confidence: 99%

“…Samples harvested from shake flask culture were prepared for phosphoproteomic analysis following methods of Chelius et al, 2019. Briefly, frozen biomass was crushed by mortar and pestle with liquid nitrogen, mixed with 1:1 v/v TNE buffer, and the BCA assay (Pierce, Rockford, IL) was used to determine protein concentration.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, frozen biomass was crushed by mortar and pestle with liquid nitrogen, mixed with 1:1 v/v TNE buffer, and the BCA assay (Pierce, Rockford, IL) was used to determine protein concentration. The Filter aided sample preparation (FASP) was used to digest protein (29) and then 500 g protein was incubated with Trypsin Gold (Promega, Madison, WI) (50 g protein: 1 g trypsin) overnight in a 37°C water bath. The samples were lyophilized to dryness and phosphopeptides were collected using the Pierce High-Select TiO 2 Phosphopeptide Enrichment Kit (Pierce, Rockford, IL) protocol.…”

Section: Methodsmentioning

confidence: 99%

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Dynamic Transcriptomic and Phosphoproteomic Analysis During Cell Wall Stress in Aspergillus nidulans

Chelius

Huso

Reese

et al. 2020

Molecular & Cellular Proteomics

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The fungal cell-wall integrity signaling (CWIS) pathway regulates cellular response to environmental stress to enable wall repair and resumption of normal growth. This complex, interconnected, pathway has been only partially characterized in filamentous fungi. To better understand the dynamic cellular response to wall perturbation, a β-glucan synthase inhibitor (micafungin) was added to a growing A. nidulans shake-flask culture. From this flask, transcriptomic and phosphoproteomic data were acquired over 10 and 120 minutes, respectively. To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets. Over 1800 genes were dynamically expressed and 430 phosphorylation sites had changing phosphorylation levels upon micafungin exposure. Twelve kinases had altered phosphorylation and phenotypic profiling of all non-essential kinase deletion mutants revealed putative connections between PrkA, Hk-8-4, and Stk19 and the CWIS pathway. Our collective data implicate actin regulation, endocytosis, and septum formation as critical cellular processes responding to activation of the CWIS pathway, and connections between CWIS and calcium, HOG, and SIN signaling pathways.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Dynamic Transcriptomic and Phosphoproteomic Analysis During Cell Wall Stress in Aspergillus nidulans

Chelius

Huso

Reese

et al. 2020

Molecular & Cellular Proteomics

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“…Four MAPKs have been identified in A. fumigatus : (i) MpkA, which is important for the CWI pathway and oxidative stress ( 22 , 24 , 25 ); (ii) MpkB, which regulates the pheromone response/filamentous growth pathway, conidiation, and dihydroxynaphthalene (DHN) melanin production ( 26 , 27 ); and (iii) SakA and (iv) MpkC, which are paralogues that regulate the HOG pathway ( 28 ). MpkC and SakA interact physically and play roles in caspofungin tolerance and carbon source utilization, respectively ( 24 , 29 – 33 ).…”

Section: Introductionmentioning

confidence: 99%

Putative Membrane Receptors Contribute to Activation and Efficient Signaling of Mitogen-Activated Protein Kinase Cascades during Adaptation of Aspergillus fumigatus to Different Stressors and Carbon Sources

Silva

Frawley

Assis

et al. 2020

mSphere

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The high-osmolarity glycerol (HOG) response pathway is a multifunctional signal transduction pathway that specifically transmits ambient osmotic signals. Saccharomyces cerevisiae Hog1p has two upstream signaling branches, the sensor histidine kinase Sln1p and the receptor Sho1p. The Sho1p branch includes two other proteins, the Msb2p mucin and Opy2p. Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Here, we investigated the roles played by A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p putative homologues during the activation of the mitogen-activated protein kinase (MAPK) HOG pathway. The shoA, msbA, and opyA singly and doubly null mutants are important for the cell wall integrity (CWI) pathway, oxidative stress, and virulence as assessed by a Galleria mellonella model. Genetic interactions of ShoA, MsbA, and OpyA are also important for proper activation of the SakAHog1p and MpkASlt2 cascade and the response to osmotic and cell wall stresses. Comparative label-free quantitative proteomics analysis of the singly null mutants with the wild-type strain upon caspofungin exposure indicates that the absence of ShoA, MsbA, and OpyA affects the osmotic stress response, carbohydrate metabolism, and protein degradation. The putative receptor mutants showed altered trehalose and glycogen accumulation, suggesting a role for ShoA, MsbA, and OpyA in sugar storage. Protein kinase A activity was also decreased in these mutants. We also observed genetic interactions between SlnA, ShoA, MsbA, and OpyA, suggesting that both branches are important for activation of the HOG/CWI pathways. Our results help in the understanding of the activation and modulation of the HOG and CWI pathways in this important fungal pathogen. IMPORTANCE Aspergillus fumigatus is an important human-pathogenic fungal species that is responsible for a high incidence of infections in immunocompromised individuals. A. fumigatus high-osmolarity glycerol (HOG) and cell wall integrity pathways are important for the adaptation to different forms of environmental adversity such as osmotic and oxidative stresses, nutrient limitations, high temperatures, and other chemical and mechanical stresses that may be produced by the host immune system and antifungal drugs. Little is known about how these pathways are activated in this fungal pathogen. Here, we characterize four A. fumigatus putative homologues that are important for the activation of the yeast HOG pathway. A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p are genetically interacting and are essential for the activation of the HOG and cell wall integrity pathways. Our results contribute to the understanding of A. fumigatus adaptation to the host environment.

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Micafungin-induced Cell Wall Damage Stimulates Microcycle Conidiation in Aspergillus nidulans

Reese

Chelius

Riekhof

et al. 2021

Preprint

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Fungal cell wall receptors relay messages about the state of the cell wall to the nucleus through the Cell Wall Integrity Signaling (CWIS) pathway. The ultimate role of the CWIS pathway is to coordinate repair of cell wall damage and to restore normal hyphal growth. Echinocandins such as micafungin represent a class of antifungals that trigger cell wall damage by affecting synthesis of beta-glucans, filamentous fungal response to these antifungals are fundamentally unknown. To obtain a better understanding of the dynamics of the CWIS response and its multiple effects, we have coupled dynamic transcriptome analysis with morphological studies of Aspergillus nidulans hyphae responding to micafungin. Our results reveal that expression of the master regulator of asexual development, BrlA, is induced by micafungin exposure. Further study showed that micafungin elicits microcycle conidiation in a BrlA-dependent manner, and that this response is abolished in the absence of MpkA. Our results suggest that microcycle conidiation may be a general response to cell wall perturbation which in some cases would enable fungi to tolerate or survive otherwise lethal damage.

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Phosphoproteomic and transcriptomic analyses reveal multiple functions for Aspergillus nidulans MpkA independent of cell wall stress

Cited by 8 publications

References 88 publications

Dynamic Transcriptomic and Phosphoproteomic Analysis During Cell Wall Stress in Aspergillus nidulans

Dynamic Transcriptomic and Phosphoproteomic Analysis During Cell Wall Stress in Aspergillus nidulans

Putative Membrane Receptors Contribute to Activation and Efficient Signaling of Mitogen-Activated Protein Kinase Cascades during Adaptation of Aspergillus fumigatus to Different Stressors and Carbon Sources

Micafungin-induced Cell Wall Damage Stimulates Microcycle Conidiation in Aspergillus nidulans

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