Filamentous fungi are eukaryotic protein factories having greater relevance in the modern day food and pharmaceutical industries. They are known for their astounding potential towards bio-degradation and mineralization of xenobiotics and lignocellulosics. This unique Kingdom of organisms play an inevitable role in environmental cleanup and make up, adapting themselves by way of regulating their bound and secretary proteins. They make changes in the proteome composition and provide exciting opportunities in food, feed, biofuel, pharmaceutical and agro-industries. Overview of proteomic strategies in filamentous fungi with respect to extracellular proteomes, enzymes or secretomes will have greater relevance to understand the bio-based processes, such as biomass deconstruction and mycoremediation, wherein fungi are primarily involved. Scientists of "Proteogenomics Era" try to use customized protein sequence databases to identify novel peptides in filamentous fungi. Proteomics techniques can help to elucidate complex biological processes and are considered as powerful tools for the sensitive detection and rapid identification of filamentous fungi through protein expression understanding the interaction between fungal pathogen and host plant is more challenging. The expression of protein profiles in such cases is much complicated due to the presence of proteomes of two different species simultaneously, either at the infection court or at a remote place inside the partnering organisms. When the plant and its associated fungal biome is subjected to genomic analysis, the power of proteomics remains to be the chosen tool to unravel the molecular pathways of pathogencity and resistance. Sometimes, in a compatible partnership like in mycorrhiza, the proteomics tools and techniques help to understand the symbiotic relationship. Experimental designs have also progressed extensively towards organelle proteomics and the knowledge on localization of proteins is of overriding importance to understand functions of various proteins at sub-cellular level. Organelle proteomics has gained insights in to compartmentalization and localization that exists in a fungal cell with respect to protein secretomes (e.g. production and process control of antibiotics, organic acids, enzymes etc.,). Bioinformatics tools can help in the quantitative and qualitative estimation of such proteins to look beyond harnessing the full potential of filamentous fungi.
“Animals eat their food and then digest it; fungi digest their food and then eat it,” said Bennett (2006), illustrating the vast number of extracellular enzymes necessarily produced by fungi to assimilate their food.
The Fungal Kingdom includes organisms ranging from single-celled yeasts to highly evolved moulds and mushrooms. It is the largest group of organisms, with more than 500,000 described species, and it is estimated that one million species might exist (Hawksworth, 1991).
Over 8,000 species of fungi are known to cause plant diseases, sometimes leading to severe agricultural devastation. Notable examples include:
The Irish famine in Europe (1870s) and the Bengal famine in India (1945) were catastrophic events, leading to mass human suffering and large-scale migrations (Ellis et al., 2008). These instances highlight the global economic impact of fungi and their interactions with plants and animals, emphasizing the importance of fungal proteomics research.
Fungi have an ancient evolutionary history dating back 550 million years, during which three major phyla—Zygomycota, Ascomycota, and Basidiomycota—are known to have diverged from Chytridiomycota. This evolution is believed to have occurred before plants colonized land.
Despite their long history, only a few fungal species have undergone complete genome sequencing. The first complete fungal genome was of yeast (Saccharomyces cerevisiae), released in 1996. Later, in 2005, the edible fungus (Pleurotus sapidus) was subjected to secretome analysis, supporting methodological advancements in:
Fungi can be easily cultured and mass-multiplied through solid or liquid-state fermentation. Their ability to yield precise protein identity information is well established. Additionally, studies on protein localization, post-translational modifications, and in silico gene modeling have been highly successful.
The Fungal Genomics Group at the Broad Institute has sequenced and analyzed a diverse range of fungal organisms relevant to medicine, agriculture, and industry. As of April 2017, over 100 fungal genomes, including human and plant pathogens, have been sequenced and made publicly available (http://1000.fungalgenomes.org/home/).
These sequencing efforts have led to a significant increase in proteomics research on filamentous fungi. The Central Dogma of Life explains that DNA contains specific gene sequences, which are transcribed into mRNA by RNA polymerase and then translated into proteins by ribosomes. Thus, a protein is the final functional product of a gene, playing a crucial role in the organization and characterization of any organism.
The study of all proteins within a genome is called proteomics. It has become an essential component of large-scale “omics” studies and systems biology approaches for understanding the complexity of fungal biochemistry (Doyle, 2011).
Fungi and white biotechnology White biotechnology or industrial biotechnology refers to the use of living cells and / or their secretomes to create industrial products that are more easily degradable, requiring less energy and creating less waste during production. With the soaring energy prices, fear towards energy security and concern towards fast depletion of environment, white biotechnology is more important than ever. This is the next industrial co-evolutionary step towards a sustainable and environmentally benign chemical manufacturing process, empowering with the additional knowledge gained through genomics, proteomics, molecular genetics, metabolic engineering, catalysis, enzymology, solid and liquid state fermentation technology. White biotechnology works by making living cells into micro-factories, more meaningfully mushroom fungi are regarded as protein factories addressing human, animal and plant health. The Netherlands based global leader in white biotechnology, DSM with a fermentation capacity of over 30 million cubic metres per year states that bio-succinic acid can be applied for the production of agricultural films that can be 'shredded' and ploughed back into the soil after usage. These films are biodegradable within two months (https://www.dsm.com/content/dam/dsm/cworld/en_US/documents/white-biotech.pdf). Fungi are widely used in the process of enzyme extraction and lingo-cellulosics management. Filamentous fungi also have an unbeatable ability to secrete incalculable proteins, secondary metabolites and organic acids in culture medium. Sangeetha and Krishnamoorthy (2017) have extracted and purified an antifungal metabolite, cordycepin from cell free culture filtrate and mycelial mat extract of Ophiocordyceps sinensis and O. neovolkiana. In addition to the control of Fusarium spp, the metabolite fraction also showed antinemic properties. The extracellular proteome analysis of submerged cultures of Aspergillus oryzae at different stages of growth contain impressive number of enzymes related to cell wall degradation viz., glucoamylase A, xylanase G2, glucosidase A, cellulase B, amylase, glucosidase and xylanase F3 (Machida et al., 2005 and Oda et al., 2006). Similarly, some of the secreted proteins of Penicillium chrysogenum (isoamyl alcohol oxidase, sulfydryl oxidase, dihydroxyacid dehydratase, polygalacturonases, pectate lyases, ferulic acid esterases) are especially relevant in food, beverage and nutraceutical industries (Jami et al., 2010b). Quorn™™ a kind of mycoprotein is made by the addition of oxygen, nitrogen, glucose and minerals to Fusarium venenatum in a fermenter to form a continuous supply of mycoprotein, harvested and dried before egg albumen is added for binding. Since 1967 almsot 3,000 fungi have been tested before choosing Fusarium graminearum (culture A3/5) reclassified as Fusarium venenatum (PTA 2684) and the first Quorn™™ products came to market in the 1980s. The first ever mycoprotein retail product was a vegetable pie made available in 1985. Now, there are over 90 products available in the Quorn range, including meals, grills, sausages, burgers and deli slices as well as cooking ingredients like mince, strips and pieces. The production of one tonne of QuornTM mince and pieces requires just 15% of the land required to produce one tonne of beef. Additional benefit could be lower levels of greenhouse gas emissions. Quorn ™™ products are relatively low in free sugars is also a positive effect (http://www. mycoprotein.org/index.php/faq/index.html). Due to their quality protein, alkaline ash, high potassium : sodium ratio and increased fibre, mushrooms are known to contain immnunomodulatory biological response modifiers (BRM) addressing several health related issues including anticancer effect, anti HIV, reducing hypertension, hyperacidity and constipation.Fungal proteins are also known as biomarkers of pathogenicity in human and plants, also for the control of insect pests (Carlos Barreiro et al., 2012). Secretome proteomics of phytopathogenic fungi Secretomes are complex molecules secreted by living organisms (Makridakis and Vlahou, 2010). Most of these molecules are freely released proteins, sometimes they may also be associated with outer cell wall. Many fungi secrete a huge number of proteins to facilitate their saprotrophic lifestyles. Hence, study of such molecules may help to establish the life cycles of such forms of life. Tjalsma et al. (2000) proposed even the cellular machinery involved in the protein secretion as part of secretome. Greenbaum et al., (2001) indicated secretomes as functions of gene products. The recognition plant-pathogen interaction may ay be general or host specific. For example the Botrytis cinerea has wide host range and it can infect more than 200 hosts. It is also known to be a post harvest pathogen, more
Specifically in mango and banana, infecting the fruits during storage and handling inflicting serious economic losses (Fernández-Acero et al., 2010). The infection strategy by B. cinerea is mainly facilitated by a group of pathogenicity or virulence factors, which include peculiar pathogenicity-related (PR) proteins (Shah et al., 2009 and Espino et al., 2010). These proteins were identified as pectin methyl esterases, xylanases, and proteases which had a definite role in the degradation of plant defensive barriers. This finding is important in the sense of discovering newer pathogenicity factors to unravel the mechanism of pathogen infection.
Two-dimensional (2D) electrophoresis application combined with de novo sequencing and BLAST similarity search by MALDI-TOF Analyzer led to the protein identification of virulence factors (Cobos et al., 2010). Trichoderma spp are known to be the best cellulose degraders and very often referred to as mycoparasites on several plant pathogenic fungi. Suárez et al. (2005) using proteomics approach identified more than 250 extracellular proteins by serving chitin as a carbon source. Monteiro et al. (2010) employed 2D gel electrophoresis and mass spectrometry to identify the mycelium-degrading proteins secreted by the overgrowing colony of Trichoderma harzianum on plant pathogenic fungi like Macrophomina phaseolina, Rhizoctonia solani, and Fusarium sp.
Enigmatically, the hyphae of T. harzianum was not found to coil the hyphae of Fusarium sp. revealing the fact that the secretomes related to mycoparasitism and cell wall degradation are entirely different. Metabolic enzymes like μ-mannosidase, exo-b-1,3-glucanase, heat-shock, and BiP proteins have been found to be upregulated in the virulent races compared to avirulent races of several fungal pathogens; such differences only could reflect an adaptation behavior of saprophytic existence in avirulent isolates, which may have significant applications in the biological control of plant pathogens displacing virulent ones (Cao et al., 2009). Gao et al. (2011) published the genome sequence of Metarhizium anisopliae and Metarhizium acridum, both entomopathogenic fungi used as alternatives to insecticides.
Murad et al. (2006) reported about the decretion of proteases, reductases, and acetyltransferases by M. anisopliae in the presence of the exoskeleton of Callosobruchus maculatus. Barros et al. (2010) observed clear differences in the expression of identical proteins and isoforms from conidia and mycelia of M. anisopliae. This kind of secretome analysis of entomopathogenic fungi is relatively poorly explored, which could offer prospering proteomics challenges in pest control programs.
Moonlighting Proteins in Fungi
Eukaryotic pathogens possess a number of virulence factors, but a relatively recently recognized and particularly interesting group of factors capable of enhancing virulence is the set of so-called ‘moonlighting proteins.’ These proteins include a large collection of housekeeping enzymes lacking special targeting motifs that would determine their extracellular localization, but that are often present at the cell surface of pathogens.
Several such enzymes with key metabolic functions in glycolysis, the pentose phosphate cycle, or other fundamental intracellular processes perform entirely new, non-catalytic roles often associated with adhesion to host ligands (Karkowska-Kuleta and Kozik, 2014). Several hundred moonlighting proteins thus discovered so far play important roles. For example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat-shock protein 60 (Hsp60), and tRNA synthetases play a wide range of biological roles in eukaryotic cells and some are being tested for their therapeutic potentials. While analyzing the proteomics, it becomes very important to distinguish moonlight proteins from those secreted contaminant proteins that may have a distinct role in cell lysis events.
Working with the fungus Penicillium chrysogenum, Jami et al. (2010b) offered some useful tips for the collection of secreted proteins viz., 1) careful mycelia elimination by using nylon filters, 2) rigorous centrifugation and filtration through 0.45 μm filters, or 3) the use of low temperatures to avoid mycelial degradation. Sample collection is also crucial to discard those non-natural secreted proteins.
Interestingly, Jami et al. (2010b) have observed increasing levels of protein in the broth even after the culture reached the stationary phase and established a linear correlation between the presence of proteins in the culture medium and biomass production. These data strongly suggest that some of the non-natural proteins might have arrived in the culture medium as a result of cell lysis. Consequently, sample collection at an early time point is recommended in accordance with the optimal moment of extracellular protein secretion.
Further, the knowledge of the protein abundance at the intracellular proteome helps to clarify possible cell lysis events. For example, Shah et al. (2009) recorded the abundance of intracellular proteins like malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase in the case of B. cinerea and flavohemoglobin and manganese superoxide dismutase in the case of P. chrysogenum during cell lysis. Although mycelial lysis is an undesirable event with respect to fungal perspective, more interestingly the in vitro and in planta secretome of Fusarium graminearum demonstrates the presence of 13 non-secreted proteins only under in planta conditions (Paper et al., 2007). Some of these proteins are potent immunogens, which clearly indicates that even significant levels of fungal lysis during plant pathogenesis are mandatory or a useful event for establishing the infection by the fungal population.
New bioinformatics tools are also available to characterize the classical or non-classical secreted proteins by means of prediction of secretion signal motifs (Bendtsen et al., 2004 a, b). It is well known that peroxisome abundance secreted by P. chrysogenum can be rapidly decreased through autophagic pathways, which could selectively degrade peroxisomes by fusion to lysosomes or vacuoles (Oku and Sakai, 2010). Thus, integration of peroxisomes into vacuoles may lead to the secretion of proteins located in the peroxisomal matrix by exocytosis, a mechanism that has been discussed by Martín et al. (2010) as an alternative route for the release of penicillin from peroxisomes into culture medium.
Organelles, membrane, and cell wall proteome The interface of filamentous fungi with their local habitat and inner cell contents is mediated by the entrenched proteins that may be present in the cell wall, plasma, and organelle membranes. The cell wall is referred to as the largest organelle and dynamic structure of a filamentous fungus, fulfilling many vital functions, such as physical support, selective permeability barrier, osmotic stability, immobilized enzyme support, cell-cell interactions, and morphogenesis (Pitarch et al., 2008). Mostly, the proteomic analyses of filamentous fungi target three important aspects viz., 1) the mycelial extract (whole cell), 2) the cytosolic proteins, and 3) the secretome. Even so, the membranes of organelles, like mitochondria, which include proteins of the transport systems (e.g., porins), as well as proteins involved in fusion, fission, morphology, and the inheritance of the organelle, remain unknown. Hence, mapping of organelle proteome of different cell components will be very much fascinating to reveal various aspects of fungal biology, architecture, and metabolite production (like penicillin production in the microbodies of P. chrysogenum). Organelle enrichment and purification will be the most crucial step in the organelle proteomics of fungi (Carlos Barreiro et al. 2012). Homogeneous and reproducible cell disruption using automatic grinders or French press or manual methods including bead beating and crude organelle separation and enrichment have been suggested by Ferreira de Oliveira and de Graaff (2011). On the other hand, enzymatic degradation for protoplast isolation followed by gentle lysis is also recommended. Additionally, methods like detergent fractionation, filtration, low-speed and ultracentrifugation, centrifugal elutriation, and immunomagnetic separation can help in debris elimination and organelle isolation (like mitochondria, vacuoles, microbodies, endosomes, and vesicles). However, in filamentous fungi, organelle isolation by cell disruption is cumbersome and very much limited due to their compact cell wall and protease production.
Moreover, the hydrophobicity of the membrane or cell wall-surrounded proteins hinders isolation and analysis of such proteins. Hernández-Macedo et al. (2002) used SDS–PAGE and 2D electrophoresis for plasma membrane and cell wall protein separation in cases of P. chrysosporium and Lentinulla edodes. Dongbin Lim et al. (2001) identified 32 protein spots by nano-electrospray tandem mass spectrometry and reported around 220 proteins at various locations of the cell envelope of Trichoderma reesei. The most abundant protein reported was HEX1, the major protein in Woronin body, suggesting that this structure unique to filamentous fungi is linked to the cell envelope. Glycosyl-phosphatidyl-inositol linked (GPI) proteins affixed to the plasma membrane play a role in fungal morphogenesis and cell wall organization (Bruneau et al., 2001). A total number of 10 predicted GPI-anchored proteins have been reported by de Groot et al. (2009) in the cell wall fractions of A. nidulans. Investigation of drug resistance mechanisms in fungi is yet another unexplored area. In this regard, scientists are trying to fix potential biomarkers to assess the efficacy of drugs. Caspofungin is a drug used to contain A. fumigates. Cagas et al. (2011b) investigated fungal proteomic response of A. fumigatus to drug exposure by performing 2D electrophoresis and iTRAQ method from 4 subcellular compartment fractions viz., cell wall and plasma membrane, cytoplasm, microsomes, and extracellular secretion. Microarrays were also performed and the results indicated that the cell wall proteins were often found to be highly glycosylated, posing difficulties for separation from wall-bound polysaccharides. Membrane proteins are highly hydrophobic and cannot be dissolved with routine procedures. Amusingly, the polarized growth of filamentous fungi requires a sequential supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeleton. Hence, the arrangement of the cytoskeleton is a crucial step to establish and maintain cell polarity. Norio Takeshit (2016) extensively reviewed the mechanism of polarized growth with special emphasis on the role of actin and microtubule cytoskeleton and polarity marker proteins. Rapid insertions of membranes via highly active exocytosis at hyphal tips could quickly dilute the accumulated polarity marker proteins. Transient assembly and disassembly of polarity sites at the hyphal tip were revealed through high-resolution microscopy during the process of polarization. Priyadharshini et al. (2017) investigated the function of morphogenesis-related enzymes viz., mannitol dehydrogenase (MtDH), xylanase, laccase, tyrosinase, and lipoxygenase (LOX) activities at seven different growth stages viz., pinhead, tiny button, button, elongation I and II, maturation, and complete maturation both in pileus and stipe of milky mushroom, Calocybe indica var. APK2 and CBE-TNAU-1523 wild isolate. The results indicated that the secretion level of MtDH, xylanase, laccase, tyrosinase, and LOX in the mushroom reached the maximum level at stage five (elongation II) followed by stage four (elongation I) and the rate of increase in activity was higher from stage one to stage five in pileus when compared to stipe in both APK2 variety and CBE-TNAU-1523 wild strain of Calocybe indica. Among all the enzymes assayed, the activity of xylanase was recorded maximum at all the seven stages of mushroom growth followed by lipoxygenase activity. The results of this study also depict that the lipoxygenase enzyme plays a crucial role in the production of volatile compounds which may influence the flavor and fruiting body formation of mushrooms. Thus, the proteomic approach in filamentous fungi remains challenging.
Techniques in fungal proteomics
Use of high-throughput tools for complete genomic DNA sequencing, revealing of mRNA-sequences, counting of specific peptides, profiling of metabolites and secretomes enabled the modern day biologists to unravel the complex and interactive networks made out of genes and their proteins. Proteome, in the simplest sense means a set of proteins thought to be expressed by any living organism.
These proteomes are based on the translation of a completely sequenced genome that may include the sequences derived from extra-chromosomal elements such as plasmids or cellular organelle. Some proteomes may also include protein sequences based on high quality cDNAs. These sequences are included by manual review of supporting evidences available from the nearest close relatives, because sometimes they cannot be mapped while assembling the genome sequences either due to sequencing errors or due to experimental gaps.
Proteome is otherwise known as the protein which is produced as the compliment of the genome and individual gene could produce individual protein.Bhadauria et al. (2007) detailed the techniques in proteomics, which include a) sample preparation, b) separation of proteins by 2-D gel electrophoresis, c) excision of peptide mixed protein spot from the gel, c) analysis of eluted protein spots by MALDI-TOF d) sequencing of peptide mass through Mass Spectrometry, e) search for corresponding gene in biological data bases with bioinformatics tools, f) knocking out specific gene by reverse genetics (or) if the protein or gene is found to be new, primer designing to get the specific cDNA and g) localization of the presence of protein in that particular host by CDNA labelling through green flouorescent protein (GFP) or genome sequence tags like GST, HIS-TAGS etc. Proteomic studies of plant-pathogen interactions have been revealed in several cases. Mehta et al. (2008) overviewed plant-pathogen interaction and indicated that plants possess receptors that can activate basal resistance, mediated by pathogen-associated molecular patterns (PAMPs) or cell wall-degrading enzymes (CWDEs), which may result in
A compatible or incompatible interaction. In both the cases, several defence-related and biotic stress-responsive proteins are induced. Suppression of plant defences by pathogen effectors leads to susceptibility in host plants. Some host plants express resistance (R) proteins, which guard against this interference and trigger a specific resistance, referred to as the hypersensitive response (HR).
Dongbin Lim et al., (2001) extracted and separated a total of 220 cell envelope-associated proteins from Trichoderma reesei mycelium. Altogether they examined 56 proteins by nanoelectrospray tandem mass spectrometry and obtained amino acid sequences for 32 protein spots, of which 20 protein spots were identified by advanced BLAST searches against all databases available. The most abundant protein found in association with the cell envelop in T. reesei was HEX1, the major protein known for its presence in Woronin body, a unique structure in filamentous fungi. In addition, vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta have also been reported. The tandem mass spectrometry also gave clear signals for proteins, but no match was found in the databases. This result suggests that a vast majority of proteins associated with a filamentous fungal cell wall are novel.
Bioinformatics tools for fungal secretome protein analysis
The secretome has been studied in multiple fungal genomes to elucidate the potential role of those protein collections involved in a number of metabolic processes from host infection to wood degradation. The major group of secreted fungal proteins are processed by the classical secretory pathway (Lum and Min, 2011). The software Signal P 3.0 was used by Bendtsen et al.,(2004a) for the identification of classical secretion signal motifs presents in proteins (http://www.cbs.dtu.dk/services/SignalP/). This software is integrated in different bioinformatics platforms for secreted proteins identification. They also created a prediction program of secreted proteins by means of non-classical signal motifs (Bendtsen et al., 2004b) and it was called Secretome P, which also has shown good results in the fungal protein analysis (http://www.cbs.dtu.dk/services/SecretomeP/) as referred by Shah et al.(2009) and Jami et al. (2010b). On the other hand, Phobius software (Kall et al., 2004) is used to identify signal peptides and to discriminate putative transmembrane domains (http://phobius.cgb.ki.se/index.html).
Fungal Secretome Database or the Fungal Secretome Knowledge Base (http://fsd.snu.ac.kr/) allow proper identification of secreted proteins by the compilation of different software (Choi et al., 2010; Lum and Min, 2011). SignalP 3.0, SigCleave, SigPred, RPSP, TMHMM 2.0c, TargetP 1.1b, PSort II, SecretomeP 1.0f and predictNLS are also used to predict the presence of signal peptides or nuclear localization signals, trans-membrane helixes, protein probable location and secretion by non-classical pathways. Keeping amino acid composition as key factor to recognize secretory proteins, SECRETOOL comprises a group of web tools that enable secretome predictions out of amino acid sequence profiles, up to complete fungal proteomes, in one step and is freely available at http://genomics.cicbiogune.es/SECRETOOL/Secretool.php. (Cortázar et al.,2014).
Conclusion To date, Omics sciences and scretome proteomics in particular, appear to be an outstanding way to decipher the enzymatic toolbox of filamentous fungi. The nature of fungal secretomes is closely dependent on their biotic and abiotic environment, a property that depends on the ecological spectrum of fungal species. The structure of these secretomes consists of hydrolases necessary to the fungal food supply. A better understanding of fungal biology and its industrial applications, plant-fungus interactions is needed one. It can be obtained by employing various methodological approaches, one of them being secretome proteomics. This technology allows qualitative and quantitative measurements of large numbers of proteins that directly influence cellular biochemistry and thus, provide accurate analysis of cellular state or system changes during growth, development and response to environmental factors. Secretome proteomics studies have firmly established the presence of a substantial level of secreted proteins lacking signal peptides and indicated a large degree of fungal species specificity in the composition of secreted proteins. Finally, we conclude that different secretome components reflect lifestyle-associated genomic adaptations in fungi. Information's from this study provide new insights into the genetic basis and molecular evolution of fungal lifestyles and also establish a solid foundation for future discovery and functional validation of secretome proteins.
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