Downloaded from orbit.dtu.dk on: Oct 09, 2021 Aspergillus hydrophobins - Identification, classification and characterization Jensen, Britt Guillaume Publication date: 2012 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Jensen, B. G. (2012). Aspergillus hydrophobins - Identification, classification and characterization. Department of Systems Biology, Technical University of Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Preface PhD Thesis Britt Guillaume Jensen I Aspergillus hydrophobins – Identification, classification and characterization Preface This thesis is submitted to the Technical University of Denmark and describes the results obtained during my PhD study. The work was carried out at the Center for Microbial Biotechnology (CMB) at the Department of Systems Biology from January 1st 2009 to March 1st 2012. The project was financed by the Technical University of Denmark. First and foremost, I would like to thank my main supervisor Kristian Fog Nielsen for excellent guidance, constructive criticism and interesting discussions. My co-supervisor (previous main supervisor), Ib Søndergaard, is thanked for the initial opportunity to engage in the PhD study and very useful help and guidance. Both are highly appreciated for their endless support both in scientific and private matters. Special thanks go to my co-supervisor and former office mate, Mona Højgaard Pedersen, for endless discussions, guidance and memorable journeys to Paris and California. Thank you for having been my “hydrophobin partner in crime”. I would also like to thank my co-supervisors Jens Christian Frisvad, Lars Jelsbak and Susanne Jacobsen for invaluable help, guidance and exiting discussions throughout the years. As part of my PhD project several molecular biology techniques have been used. A big “merci beaucoup” goes to Jakob Blæsbjerg Nielsen for teaching me the ways and for your everlasting patience in the lab. Also big thanks to Anita and Olivera for being such great office mates. Thanks for the laughs and continuous supply of cookies and liquorice. Furthermore, a big thanks to the rest of CMB for providing such an inspiring environment. My collaboration with Søren Dohn, Anja Boisen and Thomas Pedersen at Department of Micro- and Nanotechnology is also highly appreciated; Søren for unravelling the world of cantilevers and Thomas for hours in front of the SEM. Finally, I would like to thank my family and friends for their endless support and encouragement. To my husband Peter, a big thanks for your everlasting patience and interest in my project. Thank you for always being there. Britt Guillaume Jensen Kgs. Lyngby, September 2012 Preface PhD Thesis Britt Guillaume Jensen II Aspergillus hydrophobins – Identification, classification and characterization Summary PhD Thesis Britt Guillaume Jensen III Aspergillus hydrophobins – Identification, classification and characterization Summary Aspergillus species are a diverse group of fungi found ubiquitously in nature, and include species used in industry as well as human pathogens causing pulmonary infections. For immunocompromised individuals these infections are often fatal, due to lack of adequate treatment. Hydrophobins, a class of small hydrophobic proteins, are uniquely found in filamentous fungi including Aspergillus species. Little is known about their roles in Aspergillus or their possible involvement in disease. Aspergillus species and the bacterium Pseudomonas aeruginosa both commonly colonize the lungs of cystic fibrosis (CF) patients, but limited research has dealt with Aspergillus-P. aeruginosa interactions. Whether, hydrophobins are involved in the Aspergillus-P. aeruginosa interaction is not known. The overall aim of this PhD study was to provide an overview of Aspergillus hydrophobins and to achieve a deeper understanding of the roles of hydrophobins in Aspergillus species. Furthermore, Aspergillus-P. aeruginosa interactions were examined. In recent years the genomes of the main Aspergillus species have become available providing a unique amount of data for research. Using bioinformatics, Aspergillus species were found to display a varying number of putative hydrophobins ranging from two in A. oryzae to eight in A. niger. Over 50 % of the Aspergillus hydrophobins could not be classified into the two original classes (I and II) defined by the physical properties and hydropathy patterns of hydrophobins, but displayed intermediate forms. The majority of the identified Aspergillus hydrophobins were either class I hydrophobins or intermediate forms. Only a single class II hydrophobin was found in A. terreus. To characterize hydrophobins in Aspergillus species, a number of different A. nidulans hydrophobin deletion strains (rodA∆, dewA∆, AN0940∆, AN1837∆, AN6401∆) were constructed. Deletion of the hydrophobins rodA or dewA resulted in reduced expression of other hydrophobins. Interestingly, deletion of both hydrophobins rodA and dewA resulted in a synthetic genetic interaction and an increased expression of the hydrophobin AN7539. The deletion strains were additionally used to test a number of previously proposed biological functions of hydrophobins. Lack of single hydrophobins had no effect on colony surface hydrophobicity or the ability of the strains to breach from an aqueous environment. Phenotypes were only apparent in rodA∆ strains, known to be involved in rodlet formation, and the dewA∆ strain, thus the role of the other hydrophobins in A. nidulans proved hard to Summary PhD Thesis Britt Guillaume Jensen IV Aspergillus hydrophobins – Identification, classification and characterization determine. If the hydrophobins AN0940, AN1837 and AN6401 are located on the conidia surface, they do not play dominant roles. These hydrophobins may only be found on the mycelium surface or secreted to the surrounding environment An examination of the interactions between different Aspergilli and P. aeruginosa revealed that all tested Aspergilli were suppressed by P. aeruginosa. An increase in production of phenazines, a class of anti-fungal compounds produced by P. aeruginosa, was observed in the contact zone of the two organisms. However, A. fumigatus differed from the other Aspergilli by not stimulating production of phenazines. Using P. aeruginosa mutants, factors involved in the rpoN pathway were found to be involved in the interaction. Furthermore, common late stage CF mutations in P. aeruginosa seemed to alter the interaction pattern rendering the bacterium more susceptible to A. fumigatus. Hydrophobins did not seem to play a role in the interaction as no differences could be observed between a control strain and hydrophobin deletion strains. Dansk resumé PhD Thesis Britt Guillaume Jensen V Aspergillus hydrophobins – Identification, classification and characterization Dansk resumé Slægten Aspergillus er en broget gruppe af svampe, som findes overalt i naturen, og som inkluderer både arter benyttet i industrien, og sygdomsfremkaldende arter impliceret i specielt humane lungeinfektioner. Disse svampeinfektioner kan, på grund af manglende behandlingsmuligheder, være fatale for immunkompromitterede individer. Hydrofobiner er en gruppe af små hydrofobe proteiner, som kun eksisterer i filamentøse svampe som Aspergillus. Der eksisterer kun begrænset viden omkring hydrofobinernes rolle i Aspergillus arter og deres potentielle involvering i sygdomsmekanismer. Både Aspergillus arter og bakterien Pseudomonas aeruginosa koloniserer ofte lungerne af cystisk fibrose (CF) patienter, men der er på nuværende tidspunkt begrænset viden omkring interaktionen mellem Aspergillus og P. aeruginosa. Hvorvidt, hydrofobiner spiller en rolle i Aspergillus-P. aeruginosa interaktioner er endnu ukendt. Formålet med dette PhD projekt var at identificere Aspergillus hydrophobiner og få en dybere forståelse for hydrofobiners rolle i Aspergillus arter. Desuden blev Aspergillus-P. aeruginosa interaktioner undersøgt. I de seneste år er de vigtigste Aspergillus genomer blevet tilgængelige, hvilket har resulteret i en guldmine af data. Ved brug af bioinformatik, blev et vekslende antal af hydrofobiner i Aspergillus arterne fundet, varierende fra to i A. oryzae til otte i A. niger. Hydrofobiner er oprindeligt blevet opdelt i to klasser (I og II) på baggrund af deres fysiske egenskaber og hydropati-plot, men over 50 % af de fundne Aspergillus hydrofobiner kunne ikke inddeles i de to klasser. Disse hydrofobiner synes at være intermediære former. Af de fundne Aspergillus hydrofobiner, var størstedelen enten klasse I eller intermediære hydrofobiner. Kun et enkelt hydrofobin i A. terreus blev fundet til at være et klasse II hydrofobin. Forskellige A. nidulans hydrofobin deletions stammer (rodA∆, dewA∆, AN0940∆, AN1837∆, AN6401∆) blev konstrueret for at karakterisere