<scp>ChIP</scp>‐seq reveals a role for <scp>CrzA</scp> in the <scp><i>A</i></scp><i>spergillus fumigatus</i> high‐osmolarity glycerol response (<scp>HOG</scp>) signalling pathway

Castro, Patrícia Alves de; Chen, Chenxi; Almeida, Ricardo Sérgio; Freitas, Fernanda Zanolli; Bertolini, Maria Célia; Morais, Enyara Rezende; Brown, Neil Andrew; Ramalho, Leandra Náira Zambelli; Hagiwara, Daisuke; Mitchell, Thomas K.; Goldman, Gustavo H.

doi:10.1111/mmi.12785

Cited by 45 publications

(68 citation statements)

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“…In C. albicans , chitin biosynthesis is regulated via protein kinase C (PKC) signaling, the high-osmolarity glycerol (HOG) pathway, and calcium/calcineurin signaling (19). A similar situation can be envisaged for A. fumigatus , where CrzA has also been shown to be involved in the regulation of HOG pathway intermediates (26), therefore providing a link between calcium signaling and the HOG pathways. Furthermore, in silico analysis of mycelia grown in subinhibitory caspofungin concentrations predicted a cross talk between the HOG MAPK SakA and the CWI MAPK MpkA (30).…”

Section: Discussionmentioning

confidence: 64%

“…For the EMSAs, DNA fragments of approximately 200 bp, encompassing the CrzA calcineurin-dependent reporter element (CDRE) consensus binding sequence of the chsA , chsC , chsG , and csmB genes (Fig. 3A) were used as probes, and the recombinant glutathione S -transferase (GST)::CrzA (25, 26) was produced in Escherichia coli and purified. DNA-protein complexes with reduced mobility were observed for all four DNA fragments, indicating in vitro binding of CrzA to these DNA sequences (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These results are in agreement with reference 19, which predicted regulation of two chitin synthase-encoding genes by CrzA, although direct evidence for gene promoter binding was not provided. In a previous study, ChIP-seq (chromatin immunoprecipitation coupled to DNA sequencing) of CrzA did not identify any cell wall biosynthesis gene targets, which is probably due to the different growth conditions (calcium- versus caspofungin-treated mycelia) used (26). In agreement with the promoter binding studies, CrzA translocation to the nucleus is calcineurin dependent in the presence of different concentrations of caspofungin.…”

Section: Discussionmentioning

confidence: 96%

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The Aspergillus fumigatus CrzA Transcription Factor Activates Chitin Synthase Gene Expression during the Caspofungin Paradoxical Effect

Ries

Rocha

Castro

et al. 2017

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Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis (IA), a life-threatening disease in immunocompromised humans. The echinocandin caspofungin, adopted as a second-line therapy in combating IA, is a β-1,3-glucan synthase inhibitor, which, when used in high concentrations, reverts the anticipated A. fumigatus growth inhibition, a phenomenon called the “caspofungin paradoxical effect” (CPE). The CPE has been widely associated with increased chitin content in the cell wall due to a compensatory upregulation of chitin synthase-encoding genes. Here, we demonstrate that the CPE is dependent on the cell wall integrity (CWI) mitogen-activated protein kinase MpkAMPK1 and its associated transcription factor (TF) RlmARLM1, which regulate chitin synthase gene expression in response to different concentrations of caspofungin. Furthermore, the calcium- and calcineurin-dependent TF CrzA binds to and regulates the expression of specific chitin synthase genes during the CPE. These results suggest that the regulation of cell wall biosynthetic genes occurs by several cellular signaling pathways. In addition, CrzA is also involved in cell wall organization in the absence of caspofungin. Differences in the CPE were also observed between two A. fumigatus clinical isolates, which led to the identification of a novel basic leucine zipper TF, termed ZipD. This TF functions in the calcium-calcineurin pathway and is involved in the regulation of cell wall biosynthesis genes. This study therefore unraveled additional mechanisms and novel factors governing the CPE response, which ultimately could aid in developing more effective antifungal therapies.

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Section: Discussionmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 96%

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The Aspergillus fumigatus CrzA Transcription Factor Activates Chitin Synthase Gene Expression during the Caspofungin Paradoxical Effect

Ries

Rocha

Castro

et al. 2017

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“…Preliminary quality analysis of the raw FASTQ files was conducted using FastQC [46], quality filtering was then performed using fastq-mcf [47] to remove sequencing adapters (adapter sequences supplied by Illumina, Inc) and trim low quality bases. Tophat2 [48] was used to map the processed reads to the H99 genome and transcriptome as determined from the genome annotation (version 2, downloaded from the Broad Institute) [49], using the following parameters: "—transcriptome-max-hits 1—max-multihits 1—max-intron-length 4000—library-type fr-unstranded—no-coverage-search".…”

Section: Methodsmentioning

confidence: 99%

Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans

et al. 2017

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Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in C. neoformans appears to be adapted to promote fungal virulence.

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“…We have identified among several genes regulated by calcium stress, flcA, encoding the putative flavin adenine dinucleotide (FAD) transmembrane transporter (the predicted homolog of the S. cerevisiae flavin carrier FLC2). 39,40 The Candida albicans homolog of this gene was identified when expressed in S. cerevisiae because it increased the heme transport. Since S. cerevisiae cannot grow very well on media with heme as a single iron source, the overexpression of the C. albicans homolog, CaFLC1, improved considerably its growth on heme iron as single iron source.…”

Section: Introductionmentioning

confidence: 99%

The putative flavin carrier family FlcA-C is important forAspergillus fumigatusvirulence

et al. 2016

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Aspergillus fumigatus is an opportunistic fungal pathogen and the most important species causing pulmonary fungal infections. The signaling by calcium is very important for A. fumigatus pathogenicity and it is regulated by the transcription factor CrzA. We have previously used used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) aiming to identify gene targets regulated by CrzA. We have identified among several genes regulated by calcium stress, the putative flavin transporter, flcA. This transporter belongs to a small protein family composed of FlcA, B, and C. The DflcA null mutant showed several phenotypes, such as morphological defects, increased sensitivity to calcium chelating-agent ethylene glycol tetraacetic acid (EGTA), cell wall or oxidative damaging agents and metals, representative of deficiencies in calcium signaling and iron homeostasis. Increasing calcium concentrations improved significantly the DflcA growth and conidiation, indicating that DflcA mutant has calcium insufficiency. Finally, DflcA-C mutants showed reduced flavin adenine dinucleotide (FAD) and were avirulent in a low dose murine infection model.

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ChIP‐seq reveals a role for CrzA in the Aspergillus fumigatus high‐osmolarity glycerol response (HOG) signalling pathway

Cited by 45 publications

References 83 publications

The Aspergillus fumigatus CrzA Transcription Factor Activates Chitin Synthase Gene Expression during the Caspofungin Paradoxical Effect

The Aspergillus fumigatus CrzA Transcription Factor Activates Chitin Synthase Gene Expression during the Caspofungin Paradoxical Effect

Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans

The putative flavin carrier family FlcA-C is important forAspergillus fumigatusvirulence

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