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A Reliable Assay to Evaluate the Virulence of Aspergillus nidulans Using the Alternative Animal Model Galleria mellonella (Lepidoptera)

Authors: Caroline Mota Fernandes
Caroline Mota FernandesAffiliation: Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
For correspondence: cmota@biof.ufrj.br
Bio-protocol author page: a4626
, Fernanda Lopes Fonseca
Fernanda Lopes FonsecaAffiliation: Centro de Desenvolvimento Tecnológico em Saúde, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
Bio-protocol author page: a4627
, Gustavo Henrique Goldman
Gustavo Henrique GoldmanAffiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil
Bio-protocol author page: a4628
, Marcos Dias Pereira*
Marcos Dias PereiraAffiliation: Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
For correspondence: marcosdp@iq.ufrj.br
Bio-protocol author page: a4629
 and Eleonora Kurtenbach*
Eleonora KurtenbachAffiliation: Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Bio-protocol author page: a4630
 (*contributed equally to this work),  date: 6/5/2017, 284 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2329.

[Abstract] The greater wax moth Galleria mellonella has emerged as an effective heterologous host to study fungal pathogenesis and the efficacy of promising antifungal drugs (Mylonakis et al., 2005; Li et al., 2013). Here, a methodology describing the Aspergillus nidulans infection in G. mellonella larvae, along ...

Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia

Authors: Anupam Jhingran
Anupam JhingranAffiliation: Infectious Disease Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA
Bio-protocol author page: a3498
, Shinji Kasahara
Shinji KasaharaAffiliation: Infectious Disease Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA
Bio-protocol author page: a3499
 and Tobias M Hohl
Tobias M HohlAffiliation 1: Infectious Disease Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA
Affiliation 2: Immunology Program, Sloan Kettering Institute; Memorial Sloan Kettering Cancer Center, New York, USA
For correspondence: hohlt@mskcc.org
Bio-protocol author page: a3500
,  date: 9/20/2016, 1779 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1927.

[Abstract] Aspergillus fumigatus is a ubiquitous fungal pathogen that forms airborne conidia. The process of restricting conidial germination into hyphae by lung leukocytes is critical in determining infectious outcomes. Tracking the outcome of conidia-host cell encounters in vivo is technically challenging and ...

An Improved and Simplified Radial Gel Diffusion Assay for the Detection of Xylanase Activity

Authors: Raviraj M. Kalunke
Raviraj M. KalunkeAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
Bio-protocol author page: a3295
, Ilaria Moscetti
Ilaria MoscettiAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
Bio-protocol author page: a3296
, Silvio Tundo
Silvio TundoAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
Bio-protocol author page: a3297
 and Renato D’Ovidio
Renato D’OvidioAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
For correspondence: dovidio@unitus.it
Bio-protocol author page: a3298
,  date: 7/5/2016, 1630 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1863.

[Abstract] Xylanase (E.C. 3.2.1.8) degrades β-1, 4 xylan by cleaving β-1, 4 glycosidic linkages randomly, resulting in the generation of xylose and xylo-oligosaccharides. Xylanases are produced by organisms including fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans and insects. Xylanases present ...

Aspergillus terreus Infection of Fruits and Terrein Quantification by HPLC Analysis

Authors: Markus Gressler
Markus GresslerAffiliation 1: Department of Mycology, Pasteur Institute, Paris, France
Affiliation 2: Microbial Biochemistry and Physiology, Leibniz Institute for Natural Product Research and Infection Biology (Hans Knöll Institute), Jena, Germany
Bio-protocol author page: a3241
 and Matthias Brock
Matthias BrockAffiliation 1: School of Life Sciences, University of Nottingham, Nottingham, UK
Affiliation 2: Microbial Biochemistry and Physiology, Leibniz Institute for Natural Product Research and Infection Biology (Hans Knöll Institute), Jena, Germany
For correspondence: Matthias.brock@nottingham.ac.uk
Bio-protocol author page: a3242
,  date: 6/20/2016, 1244 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1845.

[Abstract] The opportunistic fungal human and plant pathogen Aspergillus terreus (A. terreus) can be isolated from sea water, soil or decaying organic matter such as rotting leaves and fruits. While growing on fruits A. terreus produces secondary metabolites such as terrein, which may ease its penetration into ...

In vitro Deneddylation Assay

Authors: Anna M. Köhler
Anna M. KöhlerAffiliation: Abteilung für Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik und Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Göttingen, Germany
Bio-protocol author page: a2960
, Cindy Meister
Cindy MeisterAffiliation: Abteilung für Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik und Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Göttingen, Germany
Bio-protocol author page: a2961
 and Gerhard H. Braus
Gerhard H. BrausAffiliation: Abteilung für Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik und Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Göttingen, Germany
For correspondence: gbraus@gwdg.de
Bio-protocol author page: a2962
,  date: 3/20/2016, 1634 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1756.

[Abstract] Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated ...

Establishing a Biofilm Co-culture of Pseudomonas and Aspergillus for Metabolite Extraction

Authors: He Zheng
He ZhengAffiliation: Departments of Civil and Environmental Engineering, Northwestern University, Evanston, USA
Bio-protocol author page: a2747
, Nancy P. Keller
Nancy P. KellerAffiliation: Departments of Bacteriology, and Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, USA
For correspondence: npkeller@wisc.edu
Bio-protocol author page: a319
 and Yun Wang
Yun WangAffiliation: Departments of Civil and Environmental Engineering, Northwestern University, Evanston, USA
For correspondence: yun-wang@northwestern.edu
Bio-protocol author page: a2748
,  date: 12/5/2015, 2375 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1667.

[Abstract] Filamentous fungi and bacteria form mixed-species biofilms in nature and diverse clinical contexts (Frey-Klett et al., 2011; Peleg et al., 2010). The interactions between fungi and bacteria, often mediated by secreted metabolites, have important ramifications for the biology of the interacting partners ...

XTT Assay of Antifungal Activity

Authors: Flávio V. Loures
Flávio V. LouresAffiliation 1: Department of Medicine, University of Massachusetts Medical School, Massachusetts, United States of America
Affiliation 2: Department of Immunology, Institute of Biomedical Sciences, São Paulo University, São Paulo, Brazil
For correspondence: loures@icb.usp.br
Bio-protocol author page: a2409
 and Stuart M. Levitz
Stuart M. LevitzAffiliation: Department of Medicine, University of Massachusetts Medical School, Massachusetts, USA
Bio-protocol author page: a2410
,  date: 8/5/2015, 2428 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1543.

[Abstract] XTT assay is a colorimetric method that uses the tetrazolium dye, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) to quantify cell-mediated damage to fungi. Actively respiring fungal cells convert the water-soluble XTT to a water-soluble, orange colored formazan product ...

Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans

Authors: Takuji Oka
Takuji OkaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
For correspondence: oka@bio.sojo-u.ac.jp
Bio-protocol author page: a1613
, Yukako Katafuchi
Yukako KatafuchiAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1614
, Kohsai Fukuda
Kohsai FukudaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1615
, Keisuke Ekino
Keisuke EkinoAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1616
, Masatoshi Goto
Masatoshi GotoAffiliation: Department of Bioscience and Biotechnology, Kyushu University, Fukuoka City, Japan
Bio-protocol author page: a1617
 and Yoshiyuki Nomura
Yoshiyuki NomuraAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1618
,  date: 9/5/2014, 2647 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1222.

[Abstract] GfsA is a fungal β-galactofuranosyltransferase involved in the biosynthesis of O-glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant protein of this enzyme from two heterologous host organisms. However, GfsA could not be expressed as a recombinant protein in ...

Determination of D-galactofuranose Content of Galactomannoproteins in Aspergillus nidulans

Authors: Takuji Oka
Takuji OkaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
For correspondence: oka@bio.sojo-u.ac.jp
Bio-protocol author page: a1613
, Yukako Katafuchi
Yukako KatafuchiAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1614
, Kohsai Fukuda
Kohsai FukudaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1615
, Keisuke Ekino
Keisuke EkinoAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1616
, Masatoshi Goto
Masatoshi GotoAffiliation: Department of Bioscience and Biotechnology, Kyushu University, Fukuoka City, Japan
Bio-protocol author page: a1617
 and Yoshiyuki Nomura
Yoshiyuki NomuraAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1618
,  date: 9/5/2014, 2137 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1223.

[Abstract] Galactofuranose (Galf) is a component of several polysaccharides and glycoconjugates in certain species of filamentous fungi. Galf residues are frequently found in Aspergillus glycoproteins, including N-glycans and O-mannose glycans that modify many cell wall proteins and extracellular enzymes. It is ...

Transport Assays in Aspergillus nidulans

Authors: Emilia Krypotou
Emilia Krypotou Affiliation: Faculty of Biology, University of Athens, Athens, Greece
Bio-protocol author page: a1008
 and George Diallinas
George DiallinasAffiliation: Faculty of Biology, University of Athens, Athens, Greece
For correspondence: diallina@biol.uoa.gr
Bio-protocol author page: a1009
,  date: 11/20/2013, 2705 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.971.

[Abstract] Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent Km and Vmax values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol, we describe a rapid and easy method for performing uptake assays in the ...
1 2 

Western Analysis of Histone Modifications (Aspergillus nidulans)

Authors: Alexandra Soukup
Alexandra SoukupAffiliation: Department of Genetics, University of Wisconsin, Madison, USA
Bio-protocol author page: a532
 and Nancy P. Keller
Nancy P. KellerAffiliation: Department of Bacteriology and Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, USA
For correspondence: npkeller@wisc.edu
Bio-protocol author page: a319
,  date: 4/5/2013, 4937 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.424.

[Abstract] Western blotting allows for the specific detection of proteins and/or modifications of proteins by an antibody of interest. This protocol utilizes a crude nuclei extraction protocol for Aspergillus nidulans to enrich for histones and other nuclear proteins prior to gel electrophoresis. Post translational ...

Transport Assays in Aspergillus nidulans

Authors: Emilia Krypotou
Emilia Krypotou Affiliation: Faculty of Biology, University of Athens, Athens, Greece
Bio-protocol author page: a1008
 and George Diallinas
George DiallinasAffiliation: Faculty of Biology, University of Athens, Athens, Greece
For correspondence: diallina@biol.uoa.gr
Bio-protocol author page: a1009
,  date: 11/20/2013, 2705 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.971.

[Abstract] Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent Km and Vmax values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol, we describe a rapid and easy method for performing uptake assays in the ...

Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans

Authors: Takuji Oka
Takuji OkaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
For correspondence: oka@bio.sojo-u.ac.jp
Bio-protocol author page: a1613
, Yukako Katafuchi
Yukako KatafuchiAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1614
, Kohsai Fukuda
Kohsai FukudaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1615
, Keisuke Ekino
Keisuke EkinoAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1616
, Masatoshi Goto
Masatoshi GotoAffiliation: Department of Bioscience and Biotechnology, Kyushu University, Fukuoka City, Japan
Bio-protocol author page: a1617
 and Yoshiyuki Nomura
Yoshiyuki NomuraAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1618
,  date: 9/5/2014, 2647 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1222.

[Abstract] GfsA is a fungal β-galactofuranosyltransferase involved in the biosynthesis of O-glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant protein of this enzyme from two heterologous host organisms. However, GfsA could not be expressed as a recombinant protein in ...

XTT Assay of Antifungal Activity

Authors: Flávio V. Loures
Flávio V. LouresAffiliation 1: Department of Medicine, University of Massachusetts Medical School, Massachusetts, United States of America
Affiliation 2: Department of Immunology, Institute of Biomedical Sciences, São Paulo University, São Paulo, Brazil
For correspondence: loures@icb.usp.br
Bio-protocol author page: a2409
 and Stuart M. Levitz
Stuart M. LevitzAffiliation: Department of Medicine, University of Massachusetts Medical School, Massachusetts, USA
Bio-protocol author page: a2410
,  date: 8/5/2015, 2428 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1543.

[Abstract] XTT assay is a colorimetric method that uses the tetrazolium dye, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) to quantify cell-mediated damage to fungi. Actively respiring fungal cells convert the water-soluble XTT to a water-soluble, orange colored formazan product ...

Determination of D-galactofuranose Content of Galactomannoproteins in Aspergillus nidulans

Authors: Takuji Oka
Takuji OkaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
For correspondence: oka@bio.sojo-u.ac.jp
Bio-protocol author page: a1613
, Yukako Katafuchi
Yukako KatafuchiAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1614
, Kohsai Fukuda
Kohsai FukudaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1615
, Keisuke Ekino
Keisuke EkinoAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1616
, Masatoshi Goto
Masatoshi GotoAffiliation: Department of Bioscience and Biotechnology, Kyushu University, Fukuoka City, Japan
Bio-protocol author page: a1617
 and Yoshiyuki Nomura
Yoshiyuki NomuraAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1618
,  date: 9/5/2014, 2137 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1223.

[Abstract] Galactofuranose (Galf) is a component of several polysaccharides and glycoconjugates in certain species of filamentous fungi. Galf residues are frequently found in Aspergillus glycoproteins, including N-glycans and O-mannose glycans that modify many cell wall proteins and extracellular enzymes. It is ...

In vitro Deneddylation Assay

Authors: Anna M. Köhler
Anna M. KöhlerAffiliation: Abteilung für Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik und Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Göttingen, Germany
Bio-protocol author page: a2960
, Cindy Meister
Cindy MeisterAffiliation: Abteilung für Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik und Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Göttingen, Germany
Bio-protocol author page: a2961
 and Gerhard H. Braus
Gerhard H. BrausAffiliation: Abteilung für Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik und Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Göttingen, Germany
For correspondence: gbraus@gwdg.de
Bio-protocol author page: a2962
,  date: 3/20/2016, 1634 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1756.

[Abstract] Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated ...

An Improved and Simplified Radial Gel Diffusion Assay for the Detection of Xylanase Activity

Authors: Raviraj M. Kalunke
Raviraj M. KalunkeAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
Bio-protocol author page: a3295
, Ilaria Moscetti
Ilaria MoscettiAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
Bio-protocol author page: a3296
, Silvio Tundo
Silvio TundoAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
Bio-protocol author page: a3297
 and Renato D’Ovidio
Renato D’OvidioAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
For correspondence: dovidio@unitus.it
Bio-protocol author page: a3298
,  date: 7/5/2016, 1630 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1863.

[Abstract] Xylanase (E.C. 3.2.1.8) degrades β-1, 4 xylan by cleaving β-1, 4 glycosidic linkages randomly, resulting in the generation of xylose and xylo-oligosaccharides. Xylanases are produced by organisms including fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans and insects. Xylanases present ...

Aspergillus terreus Infection of Fruits and Terrein Quantification by HPLC Analysis

Authors: Markus Gressler
Markus GresslerAffiliation 1: Department of Mycology, Pasteur Institute, Paris, France
Affiliation 2: Microbial Biochemistry and Physiology, Leibniz Institute for Natural Product Research and Infection Biology (Hans Knöll Institute), Jena, Germany
Bio-protocol author page: a3241
 and Matthias Brock
Matthias BrockAffiliation 1: School of Life Sciences, University of Nottingham, Nottingham, UK
Affiliation 2: Microbial Biochemistry and Physiology, Leibniz Institute for Natural Product Research and Infection Biology (Hans Knöll Institute), Jena, Germany
For correspondence: Matthias.brock@nottingham.ac.uk
Bio-protocol author page: a3242
,  date: 6/20/2016, 1244 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1845.

[Abstract] The opportunistic fungal human and plant pathogen Aspergillus terreus (A. terreus) can be isolated from sea water, soil or decaying organic matter such as rotting leaves and fruits. While growing on fruits A. terreus produces secondary metabolites such as terrein, which may ease its penetration into ...
1 
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