Rapid characterization of aquatic hyphomycetes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Julien Cornut, Sophie de Respinis, Mauro Tonolla, Orlando Petrini, Felix Bärlocher, Eric Chauvet, Andreas Bruder To cite this version: Julien Cornut, Sophie de Respinis, Mauro Tonolla, Orlando Petrini, Felix Bärlocher, et al.. Rapid characterization of aquatic hyphomycetes by matrix-assisted laser desorption/ionization time-of- flight mass spectrometry. Mycologia, Mycological Society of America, 2019, 111 (1), pp.177-189. 10.1080/00275514.2018.1528129. hal-02359264 HAL Id: hal-02359264 https://hal.archives-ouvertes.fr/hal-02359264 Submitted on 16 Jan 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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ISSN 0027-5514 Any correspondence concerning this service should be sent to the repository administrator: [email protected] Rapid characterization of aquatic hyphomycetes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Julien Cornuta,b*, Sophie De Respinis a*, Mauro Tonollaa,c, Orlando Petrini d, Felix Bärlocher e, Eric Chauvet f, and Andreas Bruder a,b aLaboratory of Applied Microbiology, University of Applied Sciences and Arts of Southern Switzerland, Via Mirasole 22A, 6501 Bellinzona, Switzerland; bInstitute of Earth Sciences, University of Applied Sciences and Arts of Southern Switzerland, Trevano Campus, 6952 Canobbio, Switzerland; cMicrobial Ecology Laboratory, Microbiology Unit, Department of Botany and Plant Biology, University of Geneva, Switzerland; dPOLE Pharma Consulting, Breganzona, Switzerland; eDepartment of Biology, Mount Allison University, Sackville, News Brunswick E4L1G7, Canada; fEcoLab, Université de Toulouse, CNRS, UPS, INPT, 31062 Toulouse, France ABSTRACT KEYWORDS Protein fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spec- Aquatic fungi; ITS trometry (MALDI-TOF MS) is a rapid, reliable, and economical method to characterize isolates of sequencing; mass terrestrial fungi and other microorganisms. The objective of our study was to evaluate the spectrometry; morphology; suitability of MALDI-TOF MS for the identification of aquatic hyphomycetes, a polyphyletic phylogeny; proteomics; taxonomy group of fungi that play crucial roles in stream ecosystems. To this end, we used 34 isolates of 21 aquatic hyphomycete species whose identity was confirmed by spore morphology and internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) nuc rDNA sequencing. We tested the efficiency of three protein extraction methods, including chemical and mechanical treatments using 13 different protocols, with the objective of producing high-quality MALDI-TOF mass spectra. In addition to extraction protocols, mycelium age was identified as a key parameter affecting protein extraction efficiency. The dendrogram based on mass-spectrum similarity indicated good and relevant taxonomic discrimination; the tree structure was comparable to that of the phylogram based on ITS sequences. Consequently, MALDI-TOF MS could reliably identify the isolates studied and provided greater taxonomic accuracy than classical morphological methods. MALDI-TOF MS seems suited for rapid characterization and identification of aquatic hyphomycete species. INTRODUCTION Descals 1981). With more than 300 species of aquatic Aquatic hyphomycetes (also known as freshwater hyphomycetes described to date (Nikolcheva and hyphomycetes, amphibious fungi, or Ingoldian fungi) Bärlocher 2004; Shearer et al. 2007), spore morphology are a polyphyletic group of true fungi (Bärlocher 1992; may sometimes be insufficient for unambiguous iden- Belliveau and Bärlocher 2005). Most species belong to tification (Marvanová and Bärlocher 2001). Molecular Ascomycota and only about 10% to Basidiomycota techniques circumvent some of the shortcomings of (Belliveau and Bärlocher 2005; Baschien et al. 2006; morphological identification of spores, including diffi- Shearer et al. 2007; Bärlocher 2010). Aquatic hyphomy- culties in identifying mycelium and nonsporulating cetes are widely distributed across most biomes, strains (Bärlocher 2010). This has led to although some species appear to have restricted distri- a reassessment of the phylogeny of freshwater fungi butions (Bärlocher 2010; Jabiol et al. 2013; Duarte et al. (Nikolcheva and Bärlocher 2002; Baschien et al. 2006; 2016), occur on a wide range of plant material decom- Letourneau et al. 2010; Seena et al. 2010, 2012) and to posing in freshwaters, including leaves and wood, and the identification of cryptic species within accepted play a major role in the recycling of organic matter morphological complexes (Roldán et al. 1989; (Cornut et al. 2010; Krauss et al. 2011). Letourneau et al. 2010). Detailed taxonomic informa- Taxonomy of aquatic hyphomycetes has traditionally tion may be provided by cloning-sequencing been based on morphology and ontogeny of their large approaches (Bärlocher 2010, 2016). Next-generation and distinctly shaped spores with tetraradiate, sigmoid, sequencing (NGS) such as 454-pyrosequencing stands or variously branched forms (Ingold 1975; Webster and out among these new technologies as particularly CONTACT Andreas Bruder [email protected] *Julien Cornut and Sophie De Respinis contributed equally to this work. promising for large-scale and high-throughput species and/or DNA sequencing, with gene databases being identification in environmental samples and was suc- themselves still incomplete for aquatic hyphomycetes cessfully applied in studies of fungal ecology (Duarte (Clivot et al. 2014; Duarte et al. 2015). et al. 2015; Wurzbacher et al. 2015; Bärlocher 2016). Currently, the development of reference databases However, some of these established molecular techni- for reliable identification by MALDI-TOF MS is hin- ques are still cost- and labor-intensive, especially if dered by difficulties of obtaining high-quality spectra reliable identification requires sequencing of multiple from filamentous fungi (De Respinis et al. 2014). Lysis genes (Baschien et al. 2013). Furthermore, unambigu- of fungal cell walls (mainly composed of various poly- ous species identification is often hindered by incom- saccharides; Kemptner et al. 2009) before protein plete coverage of aquatic hyphomycete sequences in extraction from mycelium seems necessary to obtain genomic databases (see, e.g., Clivot et al. 2014; Duarte spectra of sufficient quality. However, this was chal- et al. 2015) and limitations of the commonly used lenged by De Carolis et al. (2012), who reported good internal transcribed spacer (ITS) barcode due to its fungal species differentiation without cell-wall lysis short length (e.g., Duarte et al. 2014). Consequently, using MALDI-TOF intact-cell mass spectrometry. the choice among these established methods may Furthermore, different cell-lysis methods applied in strongly influence estimates of community diversity earlier studies to various groups of fungi probably and composition (Fernandes et al. 2015). influence protein extraction efficiency (Hettick et al. Matrix-assisted laser desorption/ionization time-of- 2008; Marinach-Patrice et al. 2009; Chalupová et al. flight mass spectrometry (MALDI-TOF MS) may pro- 2012; De Respinis et al. 2014). To our knowledge, vide a valuable alternative to complement the estab- these methods have never been compared lished methods described above. MALDI-TOF MS is systematically. being routinely used for the identification of human The objective of our study was to address these bacterial and fungal pathogens (Li et al. 2000; Fenselau crucial knowledge gaps, by (i) developing the applica- and Demirev 2001; Bright et al. 2002; Erhard et al. tion of MALDI-TOF MS to aquatic hyphomycetes; (ii) 2008; Santos et al. 2010; Croxatto et al. 2012). The comparing protein extraction efficiency among differ- technique is based on the measurement of a large ent methods and protocols; and (iii) initiating the range of dominant constitutive proteins, such as ribo- development of a spectral database that would allow somal and housekeeping proteins and cell-surface rapid identification of aquatic hyphomycetes. We tested structures (Stackebrandt et al. 2005). Previous studies different protein extraction protocols based on previous showed the potential of MALDI-TOF MS to character- applications of MALDI-TOF MS to fungi to produce ize and identify terrestrial filamentous