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      The Zn 2Cys 6-type transcription factor LeuB cross-links regulation of leucine biosynthesis and iron acquisition in Aspergillus fumigatus

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          Abstract

          Both branched-chain amino acids (BCAA) and iron are essential nutrients for eukaryotic cells. Previously, the Zn 2Cys 6-type transcription factor Leu3/LeuB was shown to play a crucial role in regulation of BCAA biosynthesis and nitrogen metabolism in Saccharomyces cerevisiae and Aspergillus nidulans. In this study, we found that the A. fumigatus homolog LeuB is involved in regulation of not only BCAA biosynthesis and nitrogen metabolism but also iron acquisition including siderophore metabolism. Lack of LeuB caused a growth defect, which was cured by supplementation with leucine or iron. Moreover, simultaneous inactivation of LeuB and HapX, a bZIP transcription factor required for adaptation to iron starvation, significantly aggravated the growth defect caused by inactivation of one of these regulators during iron starvation. In agreement with a direct role in regulation of both BCAA and iron metabolism, LeuB was found to bind to phylogenetically conserved motifs in promoters of genes involved in BCAA biosynthesis, nitrogen metabolism, and iron acquisition in vitro and in vivo, and was required for full activation of their expression. Lack of LeuB also caused activation of protease activity and autophagy via leucine depletion. Moreover, LeuB inactivation resulted in virulence attenuation of A. fumigatus in Galleria mellonella. Taken together, this study identified a previously uncharacterized direct cross-regulation of BCCA biosynthesis, nitrogen metabolism and iron homeostasis as well as proteolysis.

          Author summary

          Adaptation to the host niche is an essential attribute of pathogens. Here we found that the Zn 2Cys 6-type transcription factor LeuB cross-regulates branched-chain amino acid biosynthesis, nitrogen metabolism, iron acquisition via siderophores, and proteasome activity in the mold Aspergillus fumigatus. Lack of this regulatory circuit impaired virulence in an insect infection model. Mammals do neither express Zn 2Cys 6-type transcription factors nor have the capacity to produce branched-chain amino acids or siderophores. Consequently, this regulatory circuit is a paradigm for fungal pathogen-specific adaptation to the host niche.

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          Most cited references41

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          Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis.

          Aspergillus species are globally ubiquitous saprophytes found in a variety of ecological niches. Almost 200 species of aspergilli have been identified, less than 20 of which are known to cause human disease. Among them, Aspergillus fumigatus is the most prevalent and is largely responsible for the increased incidence of invasive aspergillosis (IA) in the immunocompromised patient population. IA is a devastating illness, with mortality rates in some patient groups reaching as high as 90%. Studies identifying and assessing the roles of specific factors of A. fumigatus that contribute to the pathogenesis of IA have traditionally focused on single-gene deletion and mutant characterization. In combination with recent large-scale approaches analyzing global fungal responses to distinct environmental or host conditions, these studies have identified many factors that contribute to the overall pathogenic potential of A. fumigatus. Here, we provide an overview of the significant findings regarding A. fumigatus pathogenesis as it pertains to invasive disease.
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            Fusion PCR and gene targeting in Aspergillus nidulans.

            We describe a rapid method for the production of fusion PCR products that can be used, generally without band purification, to transform Aspergillus nidulans. This technique can be used to replace genes; tag genes with fluorescent moeties or epitope tags; or replace endogenous promoters with regulatable promoters, by introducing an appropriate selective cassette (e.g., fluorescent protein + selectable marker). The relevant genomic fragments and cassette are first amplified separately by PCR using primers that produce overlapping ends. A second PCR using 'nested' primers fuses the fragments into a single molecule with all sequences in the desired order. This procedure allows a cassette to be amplified once, frozen and used subsequently in many fusion PCRs. Transformation of nonhomologous recombination deficient (nkuADelta) strains of A. nidulans with fusion PCR products results in high frequencies of accurate gene targeting. Fusion PCR takes less than 2 d. Protoplast formation and transformation takes less than 1 d.
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              Siderophore Biosynthesis But Not Reductive Iron Assimilation Is Essential for Aspergillus fumigatus Virulence

              The ability to acquire iron in vivo is essential for most microbial pathogens. Here we show that Aspergillus fumigatus does not have specific mechanisms for the utilization of host iron sources. However, it does have functional siderophore-assisted iron mobilization and reductive iron assimilation systems, both of which are induced upon iron deprivation. Abrogation of reductive iron assimilation, by inactivation of the high affinity iron permease (FtrA), has no effect on virulence in a murine model of invasive aspergillosis. In striking contrast, A. fumigatus l-ornithine-N 5-monooxygenase (SidA), which catalyses the first committed step of hydroxamate-type siderophore biosynthesis, is absolutely essential for virulence. Thus, A. fumigatus SidA is an essential virulence attribute. Combined with the absence of a sidA ortholog—and the fungal siderophore system in general—in mammals, these data demonstrate that the siderophore biosynthetic pathway represents a promising new target for the development of antifungal therapies.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draft
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – review & editing
                Role: Formal analysisRole: Funding acquisitionRole: Investigation
                Role: Formal analysisRole: Investigation
                Role: Formal analysisRole: Investigation
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Validation
                Role: Formal analysisRole: InvestigationRole: Methodology
                Role: Investigation
                Role: Conceptualization
                Role: Formal analysisRole: InvestigationRole: Methodology
                Role: Formal analysisRole: InvestigationRole: Methodology
                Role: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: ResourcesRole: Validation
                Role: Formal analysisRole: InvestigationRole: Methodology
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                26 October 2018
                October 2018
                : 14
                : 10
                : e1007762
                Affiliations
                [1 ] Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, China
                [2 ] Department of Laboratory Medicine, Shaoyang University, Shaoyang, China
                [3 ] Division of Molecular Biology/Biocenter, Medical University of Innsbruck, Innrain, Innsbruck, Austria
                [4 ] Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich Schiller University Jena, Jena, Germany
                [5 ] Transfer Group Anti-infectives, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany
                [6 ] Division of Hygiene & Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria
                Oregon State University, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-4653-4932
                http://orcid.org/0000-0002-9257-8648
                http://orcid.org/0000-0001-9503-9634
                http://orcid.org/0000-0002-8814-4193
                http://orcid.org/0000-0003-2472-9878
                http://orcid.org/0000-0002-2891-7326
                Article
                PGENETICS-D-18-01527
                10.1371/journal.pgen.1007762
                6221358
                30365497
                b609cbbf-85a7-477d-8ff7-f6a65eefd249
                © 2018 Long et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 31 July 2018
                : 12 October 2018
                Page count
                Figures: 7, Tables: 0, Pages: 23
                Funding
                Funded by: Deutsche Forschungsgemeinschaft
                Award ID: DFG BR 1130/14-1
                Award Recipient :
                Funded by: Deutsche Forschungsgemeinschaft
                Award ID: DFG HO 2596/1-1
                Award Recipient :
                Funded by: Austrian Research funds
                Award ID: FWF I1346-B22
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 81330035
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31770086
                Award Recipient :
                Funded by: Program for Jiangsu excellent scientific and technological innovation team
                Award ID: 17CXTD00014
                Award Recipient :
                Funded by: the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions
                Award ID: PAPD
                Award Recipient :
                Funded by: FWF-Doctoral Program HOROS
                Award ID: W1253
                Award Recipient :
                Funded by: Medical University of Innsbruck
                Award ID: MUI start 19970
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31470193
                Award Recipient :
                Funded by: Shaoyang University Science Fund
                Award ID: 17ZX24
                Award Recipient :
                This work was financially supported by the National Natural Science Foundation of China (NSFC) (grant 81330035 and grant 31770086 to LL and grant 31470193 to SZ), Program for Jiangsu excellent scientific and technological innovation team (17CXTD00014), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and Shaoyang University Science Fund (17ZX24 to NL), and this work was also supported in part by the joint D-A-CH program “Novel molecular mechanisms of iron sensing and homeostasis in filamentous fungi” (Austrian Research funds FWF I1346-B22 to HH, Deutsche Forschungsgemeinschaft DFG BR 1130/14-1 to AAB, and DFG HO 2596/1-1 to PH), the FWF-Doctoral Program HOROS (W1253 to HH) and the Medical University of Innsbruck Fund (MUI start 19970 to UB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Amino Acids
                Aliphatic Amino Acids
                Leucine
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Amino Acids
                Aliphatic Amino Acids
                Leucine
                Biology and Life Sciences
                Biochemistry
                Proteins
                Amino Acids
                Aliphatic Amino Acids
                Leucine
                Biology and Life Sciences
                Biochemistry
                Biosynthesis
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Fungal Molds
                Aspergillus
                Aspergillus Fumigatus
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Fungal Pathogens
                Aspergillus Fumigatus
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Fungal Pathogens
                Aspergillus Fumigatus
                Biology and Life Sciences
                Mycology
                Fungal Pathogens
                Aspergillus Fumigatus
                Biology and Life Sciences
                Biochemistry
                Metabolism
                Nitrogen Metabolism
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzymes
                Proteases
                Biology and Life Sciences
                Biochemistry
                Proteins
                Enzymes
                Proteases
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Model Organisms
                Saccharomyces Cerevisiae
                Research and Analysis Methods
                Model Organisms
                Saccharomyces Cerevisiae
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Yeast
                Saccharomyces
                Saccharomyces Cerevisiae
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Yeast and Fungal Models
                Saccharomyces Cerevisiae
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Transcription Factors
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Transcription Factors
                Biology and Life Sciences
                Biochemistry
                Proteins
                Regulatory Proteins
                Transcription Factors
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Fungal Molds
                Aspergillus
                Aspergillus Nidulans
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Yeast and Fungal Models
                Aspergillus Nidulans
                Custom metadata
                vor-update-to-uncorrected-proof
                2018-11-07
                All relevant data are within the manuscript and its Supporting Information files.

                Genetics
                Genetics

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