Trends in Biotechnology
Volume 25, Issue 9, September 2007, Pages 395-400
Journal home page for Trends in Biotechnology

Review
Proteomics of filamentous fungi

https://doi.org/10.1016/j.tibtech.2007.07.008Get rights and content

Proteomic analysis, defined here as the global assessment of cellular proteins expressed in a particular biological state, is a powerful tool that can provide a systematic understanding of events at the molecular level. Proteomic studies of filamentous fungi have only recently begun to appear in the literature, despite the prevalence of these organisms in the biotechnology industry, and their importance as both human and plant pathogens. Here, we review recent publications that have used a proteomic approach to develop a better understanding of filamentous fungi, highlighting sample preparation methods and whole-cell cytoplasmic proteomics, as well as subproteomics of cell envelope, mitochondrial and secreted proteins.

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.

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