Trends in Biotechnology
ReviewProteomics of filamentous fungi
Introduction
Filamentous fungi comprise an important class of organisms of significant commercial relevance, even though they typically receive less attention than their lower eukaryotic relatives, such as yeasts. For example, in the biotechnology industry, filamentous fungi are used to produce a wide variety of products ranging from human therapeutics (e.g. antibacterial and antifungal agents) to specialty chemicals (e.g. commercial enzymes, organic acids), which together represent billions of dollars in annual sales [1]. Just one class of compounds, the cholesterol-lowering statins, represents a market of almost US$15 billion per year in the USA 2, 3. Filamentous fungi are also notorious pathogens in both humans 4, 5 and plants [6], and recently have received much public interest in the USA and in Denmark owing, respectively, to their prevalent infestation in buildings affected by Hurricane Katrina [7], and in schools affected by repeated flooding, raising health concerns for both adults and school children 8, 9.
The importance of studying fungi can also be highlighted by the increasing number of genomes that have been sequenced. To date, 18 different species have been sequenced and annotated: Aspergillus clavatus[10], A. flavus11, 12, A. fumigatus11, 13, A. nidulans[13], A. niger[14], A. oryzae12, 13, 15, A. terreus[10], Botrytis cinerea[16], Chaetomium globosum[16], Coprinus cinereus[16], Fusarium graminearum[16], F. verticillioides[17], Magnaporthe grisea[18], Neurospora crassa19, 20, Phanerochaete chrysosporium[21], Rhizopus oryzae16, 22, Sclerotinia sclerotiorum[16], and Stagonospora nodorum[16] (for reviews of fungal genomes, see 23, 24, 25 and for other on-going projects, see http://www.broad.mit.edu/annotation/fgi/). Yet despite their importance and the availability of sequenced genomes, there have been relatively few (although increasing) studies (Figure 1) on filamentous fungi compared with their simpler relatives, such as the model yeast Saccharomyces cerevisiae or the pathogen Candida albicans. This is true for both transcriptomic and proteomic analyses. We note that protein-level analysis is particularly relevant in eukaryotic systems, such as fungi because it allows location-specific analysis (i.e. subproteome, see Glossary), as well as the study of post-translational modifications (e.g. phosphorylation, glycosylation), which might impact on phenomena such as signal transduction [26].
In two previous reviews 27, 28, it was noted that efforts toward post-genomic studies were just beginning in filamentous fungi, and to harness their potential as hosts for recombinant protein expression would require an increase in both transcript and proteomic related research. The earliest post-genomic studies of filamentous fungi were published at the beginning of the twenty-first century by Lim et al.[29] on Trichoderma reesei cell envelope proteins and by Bruneau et al.[30] on A. fumigatus glycosylphosphatidylinositol-anchored proteins. Since then, a significant number of post-genomic studies have been published ([31], Table 1), and we believe the filamentous fungal research community has now moved beyond its initial stage into a posture of active research. Several reviews have addressed post-genomic fungal studies from a general perspective 27, 28, 31, 32, 33, 34, 35, 36, 37, and a review of transcript analysis studies has just appeared [31]. Our goal here is to complement these previous publications and provide a survey of recent proteomic studies in filamentous fungi. Specifically, we report on publications from the past five years that relate to whole-cell cytoplasmic proteomics, subproteomics of cell envelope proteins and of mitochondrial proteins, and the secretome of filamentous fungi. We have excluded studies of dimorphic fungi (e.g. C. albicans, Ustilago maydis) and yeast (for a recent review on S. cerevisiae proteomics, see [38]) to further narrow our scope.
Section snippets
Cell wall lysis and sample preparation
Because filamentous fungi have an exceptionally strong cell wall [39], several early studies were devoted to overcoming this challenge by providing an effective means of cell lysis for adequate release of intracellular proteins. For example, several researchers 40, 41, 42, 43 used mechanical lysis via glass beads to liberate cytoplasmic protein, and this approach has been more efficient than either chemical or enzymatic extraction methods [44]. In an alternative approach, Shimizu and Wariishi
Intracellular proteomics
One of the earliest intracellular filamentous fungal proteomic studies was performed by Hernández-Macedo et al.[46] on the wood-degrading fungi P. chrysosporium and Lentinula edodes. Using 2DE to conduct a differential comparison of cytoplasmic protein expression patterns in the presence or absence of iron, they visualized 21 proteins related to iron uptake in these ligninolytic fungi. However, the subsequent identification of these proteins was deficient and therefore Grinyer et al.[54]
Subproteomics
We use the term ‘subproteomics’ to describe proteomic analysis of a defined subset of an organism's protein complement, primarily specific organelles [60]. Hernández-Macedo et al.[46] described procedures for plasma membrane and outer membrane protein extraction of P. chrysosporium and L. edodes, although the proteins were only visualized in one-dimensional SDS–PAGE rather than 2DE. Later, Asif et al.[61] provided the first subproteome map of A. fumigatus surface proteins, with the goal of
Secretome
The secretome has been defined as the combination of native secreted proteins and the cellular machinery involved in their secretion [64]. Secretome-related studies are particularly relevant in understanding filamentous fungi because many fungi secrete a vast number of proteins to accommodate their saprotrophic lifestyle. In light of this, it has been said that unlike animals, ‘fungi digest their food and [then] ‘eat’ it’ [7], illustrating the large number of extracellular hydrolytic enzymes
Concluding remarks
It is our opinion that the field of fungal proteomics is rapidly entering an ‘exponential’ phase as evidenced by an apparent increase in the rate of relevant publications (Figure 1). This observation is supported by an increased number of presentations that appeared at recent conferences on both sides of the Atlantic, such as the 24th Fungal Genetics Conference at Asilomar (2007), the 8th European Conference on Fungal Genetics at Vienna (2006), and the Second International Fungal Proteomics
Acknowledgements
We gratefully acknowledge Motoyuki Shimizu (Kyushu University, http://www.kyushu-u.ac.jp/) for a helpful discussion on sample preparation protocol and Wilson Francisco (Arizona State University, http://www.asu.edu) for allowing us access to his pre-print manuscript. We are also indebted to the anonymous reviewers of this manuscript for their helpful comments in improving this report. This material is based upon work supported by the National Science Foundation under grant No. 0519080. Any
Glossary
- 2DE
- two-dimensional electrophoresis; gel-based separation of proteins by the orthogonal properties of isoelectric point (see isoelectric focusing) and molecular weight.
- Isoelectric focusing
- first dimension of separation in 2DE in which proteins are separated by their isoelectric point (pI); proteins are typically separated electrophoretically in gels containing an immobilized pH gradient (IPG).
- LC-MS/MS
- liquid chromatography-mass spectrometry/mass spectrometry, also called tandem mass spectrometry.
References (75)
Best-selling human medicines 2002–2004
Drug Discov. Today
(2005)- et al.
The arms race continues: Battle strategies between plants and fungal pathogens
Curr. Opin. Microbiol.
(2005) Comparative analysis of 87,000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides
Fungal Genet. Biol.
(2005)Genomics of Aspergillus fumigatus
Rev. Iberoam. Micol.
(2005)Fungal genomics beyond Saccharomyces cerevisiae?
Curr. Opin. Biotechnol.
(2003)Heterologous protein expression in filamentous fungi
Trends Biotechnol.
(2005)- et al.
From genomics to post-genomics in Aspergillus
Curr. Opin. Microbiol.
(2004) Use of genome information for the study of the pathogenesis of fungal infections and the development of diagnostic tools
Rev. Iberoam. Micol.
(2005)Development and application of proteomics technologies in Saccharomyces cerevisiae
Trends Biotechnol.
(2005)Co-cultivation of antifungal Lactobacillus plantarum MiLAB 393 and Aspergillus nidulans, evaluation of effects on fungal growth and protein expression
FEMS Microbiol. Lett.
(2005)
Development of a sample preparation method for fungal proteomics
FEMS Microbiol. Lett.
Peptide mass fingerprinting
Methods
Sexual reproduction and the evolution of microbial pathogens
Curr. Biol.
Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: Identification and characterisation of an elongation factor 1B protein with glutathione transferase activity
Biochem. Biophys. Res. Commun.
Proteomic analysis of rutin-induced secreted proteins from Aspergillus flavus
Fungal Genet. Biol.
Proteomic analysis of secreted proteins from Trichoderma harzianum. Identification of a fungal cell wall-induced aspartic protease
Fungal Genet. Biol.
The Phanerochaete chrysosporium secretome: Database predictions and initial mass spectrometry peptide identifications in cellulose-grown medium
J. Biotechnol.
Extracellular oxidative systems of the lignin-degrading basidiomycete Phanerochaete chrysosporium
Fungal Genet. Biol.
Crucial role of antioxidant proteins and hydrolytic enzymes in pathogenicity of Penicillium expansum: Analysis based on proteomic approach
Mol. Cell. Proteomics
The Fungi
Biosynthesis and biotechnological production of statins by filamentous fungi and application of these cholesterol-lowering drugs
Appl. Microbiol. Biotechnol.
Current approaches to diagnosis and treatment of invasive aspergillosis
Am. J. Respir. Crit. Care Med.
Advances in combating fungal diseases: Vaccines on the threshold
Nat. Rev. Microbiol.
Molds in floor dust and building-related symptoms in adolescent school children
Indoor Air
Molds in floor dust, building-related symptoms, and lung function among male and female schoolteachers
Indoor Air
Whole genome comparison of the A. fumigatus family
Med. Mycol.
Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus
Nature
Whole genome comparison of Aspergillus flavus and A. oryzae
Med. Mycol.
Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae
Nature
Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88
Nat. Biotechnol.
Genome sequencing and analysis of Aspergillus oryzae
Nature
The genome sequence of the rice blast fungus Magnaporthe grisea
Nature
The genome sequence of the filamentous fungus Neurospora crassa
Nature
Lessons from the genome sequence of Neurospora crassa: Tracing the path from genomic blueprint to multicellular organism
Microbiol. Mol. Biol. Rev.
Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78
Nat. Biotechnol.
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