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Characterization and expression of fungal defensin in Escherichia coli and its antifungal mechanism by RNA-seq analysis

INTRODUCTION: Invasive fungal infections (IFIs) are fatally threatening to critical patients. The fungal defensin as an antifungal protein can widely inhibit fungi. METHODS: In this study, eight antifungal genes from different filamentous fungi were optimized by synonymous codon bias and heterologou...

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Autores principales: Chen, Yu-Pei, Li, Yingying, Chen, Fangfang, Wu, Hongtan, Zhang, Shudi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10306309/
https://www.ncbi.nlm.nih.gov/pubmed/37389349
http://dx.doi.org/10.3389/fmicb.2023.1172257
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author Chen, Yu-Pei
Li, Yingying
Chen, Fangfang
Wu, Hongtan
Zhang, Shudi
author_facet Chen, Yu-Pei
Li, Yingying
Chen, Fangfang
Wu, Hongtan
Zhang, Shudi
author_sort Chen, Yu-Pei
collection PubMed
description INTRODUCTION: Invasive fungal infections (IFIs) are fatally threatening to critical patients. The fungal defensin as an antifungal protein can widely inhibit fungi. METHODS: In this study, eight antifungal genes from different filamentous fungi were optimized by synonymous codon bias and heterologously expressed in Escherichia coli. RESULTS AND DISCUSSION: Only the antifungal protein (AFP) from Aspergillus giganteus was produced, whereas the AFP from its mutation of the chitin-binding domain could not be expressed, thereby suggesting the importance of the motif for protein folding. In addition, the recombinant AFP (rAFP, 100 μg/mL) pre-heated at 50°C for 1 h effectively inhibited Paecilomyces variotii CICC40716 of IFIs by 55%, and no cell cytotoxicity was observed in RAW264.7 cells. After being pre-heated at 50°C for 8 h, the fluorescence emission intensity of the rAFP decreased and shifted from 343 nm to 335 nm. Moreover, the helix and β-turn of the rAFP gradually decreased with the pre-heated treatment temperature of 50°C via circular dichroism spectroscopy. Propidium iodide staining revealed that the rAFP could cause damage to the cell membrane. Moreover, the corresponding differentially expressed genes (DEGs) for downregulation such as amino sugar and nucleotide sugar metabolism, as well as mitogen-activated protein kinase (MAPK) signaling pathway involved in the cell wall integrity were found via the RNA-seq of rAFP treatment. By contrast, the upregulated DEGs were enriched in response to the oxidative stress of Biological Process by the Gene Ontology (GO) database. The encoding proteins of laccase, multicopper oxidase, and nitroreductase that contributed to reactive oxygen species (ROS) scavenging could be recognized. These results suggested that the rAFP may affect the integrity of the cell wall and cell membrane, and promote the increase in ROS, thereby resulting in fungal death. Consequently, drug development could be based on the inhibitory effect of the rAFP on IFIs.
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spelling pubmed-103063092023-06-29 Characterization and expression of fungal defensin in Escherichia coli and its antifungal mechanism by RNA-seq analysis Chen, Yu-Pei Li, Yingying Chen, Fangfang Wu, Hongtan Zhang, Shudi Front Microbiol Microbiology INTRODUCTION: Invasive fungal infections (IFIs) are fatally threatening to critical patients. The fungal defensin as an antifungal protein can widely inhibit fungi. METHODS: In this study, eight antifungal genes from different filamentous fungi were optimized by synonymous codon bias and heterologously expressed in Escherichia coli. RESULTS AND DISCUSSION: Only the antifungal protein (AFP) from Aspergillus giganteus was produced, whereas the AFP from its mutation of the chitin-binding domain could not be expressed, thereby suggesting the importance of the motif for protein folding. In addition, the recombinant AFP (rAFP, 100 μg/mL) pre-heated at 50°C for 1 h effectively inhibited Paecilomyces variotii CICC40716 of IFIs by 55%, and no cell cytotoxicity was observed in RAW264.7 cells. After being pre-heated at 50°C for 8 h, the fluorescence emission intensity of the rAFP decreased and shifted from 343 nm to 335 nm. Moreover, the helix and β-turn of the rAFP gradually decreased with the pre-heated treatment temperature of 50°C via circular dichroism spectroscopy. Propidium iodide staining revealed that the rAFP could cause damage to the cell membrane. Moreover, the corresponding differentially expressed genes (DEGs) for downregulation such as amino sugar and nucleotide sugar metabolism, as well as mitogen-activated protein kinase (MAPK) signaling pathway involved in the cell wall integrity were found via the RNA-seq of rAFP treatment. By contrast, the upregulated DEGs were enriched in response to the oxidative stress of Biological Process by the Gene Ontology (GO) database. The encoding proteins of laccase, multicopper oxidase, and nitroreductase that contributed to reactive oxygen species (ROS) scavenging could be recognized. These results suggested that the rAFP may affect the integrity of the cell wall and cell membrane, and promote the increase in ROS, thereby resulting in fungal death. Consequently, drug development could be based on the inhibitory effect of the rAFP on IFIs. Frontiers Media S.A. 2023-06-14 /pmc/articles/PMC10306309/ /pubmed/37389349 http://dx.doi.org/10.3389/fmicb.2023.1172257 Text en Copyright © 2023 Chen, Li, Chen, Wu and Zhang. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Chen, Yu-Pei
Li, Yingying
Chen, Fangfang
Wu, Hongtan
Zhang, Shudi
Characterization and expression of fungal defensin in Escherichia coli and its antifungal mechanism by RNA-seq analysis
title Characterization and expression of fungal defensin in Escherichia coli and its antifungal mechanism by RNA-seq analysis
title_full Characterization and expression of fungal defensin in Escherichia coli and its antifungal mechanism by RNA-seq analysis
title_fullStr Characterization and expression of fungal defensin in Escherichia coli and its antifungal mechanism by RNA-seq analysis
title_full_unstemmed Characterization and expression of fungal defensin in Escherichia coli and its antifungal mechanism by RNA-seq analysis
title_short Characterization and expression of fungal defensin in Escherichia coli and its antifungal mechanism by RNA-seq analysis
title_sort characterization and expression of fungal defensin in escherichia coli and its antifungal mechanism by rna-seq analysis
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10306309/
https://www.ncbi.nlm.nih.gov/pubmed/37389349
http://dx.doi.org/10.3389/fmicb.2023.1172257
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