The Aspergillus fumigatus Phosphoproteome Reveals Roles of High-Osmolarity Glycerol Mitogen-Activated Protein Kinases in Promoting Cell Wall Damage and Caspofungin Tolerance

Mattos, Eliciane Cevolani; Silva, Lilian Pereira; Valero, Clara; Castro, Patrícia Alves de; Reis, Thaila Fernanda dos; Ribeiro, Laryssa Freitas; Marten, Mark R.; Silva‐Rocha, Rafael; Westmann, Cauã Antunes; Silva, Carlos Henrique Tomich de Paula da; Taft, Carlton A.; Al-Furaiji, Narjes; Bromley, Michael; Mortensen, Uffe Hasbro; Benz, J. Philipp; Brown, Neil Andrew; Goldman, Gustavo H.

doi:10.1128/mbio.02962-19

Cited by 32 publications

(26 citation statements)

References 82 publications

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“…Among these TFs, nine are important not only for caspofungin tolerance but also for resistance. Six of them (ΔcbfA, ΔzipD, ΔnctC, ΔznfA, ΔatfA, and ΔrlmA) have been previously identified as being associated with calcium sensitivity and caspofungin sensitivity and displaying reduced CPE (22,23,28,42). ZipD and RlmA are important for osmotic and cell wall stresses, collaborating for cell wall construction and the activation of several pathways important for osmotic stability (22,23).…”

Section: Discussionmentioning

confidence: 99%

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

Valero

Colabardini

Chiaratto

et al. 2020

mBio

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Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus. Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug β-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance. IMPORTANCE Aspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive β-1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance.

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Section: Discussionmentioning

confidence: 99%

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

Valero

Colabardini

Chiaratto

et al. 2020

mBio

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“…The Δ asgA was significantly more sensitive to cell wall perturbing agents. In agreement with its role in cell wall maintentance, AgsA was shown to have increased phosphorylation upon CR exposure [35]. We are currently performing additional work to decipher the relationship between AgsA and MpkA in the presence of cell wall stress.…”

Section: Discussionmentioning

confidence: 71%

Aspergillus fumigatusG-protein coupled receptors GprM and GprJ are important for the regulation of the cell wall integrity pathway, secondary metabolite production, and virulence

Filho

Brancini

Castro

et al. 2020

Preprint

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G-protein coupled receptors (GPCRs) are extracellular signalling receptors that sense environmental cues to coordinate a biological response. Fungi sense their environment primarily through GPCR-mediated signalling pathways, which in turn regulate fungal development, metabolism, virulence and mycotoxin biosynthesis. A. fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that presents 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 when 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 when compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. RNA-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, and that this regulation occurs, 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 signalling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. This work further contributes to unravelling functions of A. fumigatus GPCRs and shows that GprM and GprJ are essential for CWI, secondary metabolite production and virulence.

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“…The Δ asgA was significantly more sensitive to cell wall-perturbing agents but did not produce a dark pigment in the supernatants when grown in liquid medium, suggesting it is not involved in the repression of the melanin production. In agreement with its role in cell wall maintenance, AgsA was shown to have increased phosphorylation upon CR exposure ( 35 ). We are currently performing additional work to decipher the relationship between AgsA and MpkA in the presence of cell wall stress.…”

Section: Discussionmentioning

confidence: 76%

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

Filho

Brancini

Castro

et al. 2020

mBio

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View full text Add to dashboard Cite

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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The Aspergillus fumigatus Phosphoproteome Reveals Roles of High-Osmolarity Glycerol Mitogen-Activated Protein Kinases in Promoting Cell Wall Damage and Caspofungin Tolerance

Cited by 32 publications

References 82 publications

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

Aspergillus fumigatusG-protein coupled receptors GprM and GprJ are important for the regulation of the cell wall integrity pathway, secondary metabolite production, and virulence

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

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