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Chapter 18
Fungal metabolic diversity Andrei Stecca Steindorff1, Gabriela F. Persinoti2, Valdirene Neves Monteiro3 and Roberto Nascimento Silva4 Departamento de Biologia Celular, Universidade de Brasília, Brasília, Distrito Federal, Brazil Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil 3 Universidade Estadual de Goiás, Unidade Universitária de Ciências Exatas e Tecnológicas da Universidade Estadual de Goiás‐UnUCET, Anápolis, Goiás, Brazil 4 Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil 1
2
18.1 Introduction The way in which fungi recognize their substrate and activate the transcription of genes encoding transporters and transcriptional factors and culminate with the production of enzymes for metabolism is a subject of studies for decades. In natural environments, fungi are continuously challenged by fast changes in the environmental conditions, such as oxygen limitation and variation on nutrient abundance. These changes have a considerable impact in their lifestyles. Investigation on molecular biology using genomic and postgenomic techniques revealed that fungi are endowed with a large percentage of genes dedicated to the sensing of environmental signals and to the coordination of gene expression in response to such conditions. This diversity on metabolic pathways is quite important for the fungi to make the final decision on expression of particular set of genes in response to different external/internal signals, in order to promote their survival at low energetic cost. In the next sections, we will discuss the carbohydrate metabolism, molecule transport, energetic and secondary metabolism and some aspects of transcription
in four wide studies of fungi models: Trichoderma reesei, Neurospora crassa, Aspergillus niger and Fusarium graminearum. T. reesei is an industrially important cellulolytic filamentous fungus. T. reesei have the capacity to secrete large amounts of cellulases and hemicellulases, so it is used as a host to produce low‐cost enzymes for the conversion of plant biomass materials into industrially useful bioproducts such as sugars and bioethanol. T. reesei has a genome size of 33 Mb, organized into seven chromosomes; it has become a system for studying genomics, since it has many advantages: EST and cDNA collections and BAC libraries are available to academic researchers from the Fungal Genomics Laboratory at NCSU. DNA‐mediated transformation is a routine procedure, gene knockout protocols have been developed, and there is an active academic community of researchers worldwide (Martinez et al., 2008). N. crassa has a central role as a model organism, contributing to the fundamental understanding of genome defence systems, DNA methylation, mitochondrial protein import, circadian rhythms, post‐ transcriptional gene silencing and DNA repair. Being
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N. crassa a multicellular filamentous fungus, it has also provided a system to study cellular differentiation and development as well as other aspects of eukaryotic biology. The legacy of over 70 years of research, in addition to the availability of molecular and genetic tools, offers enormous potential for continued discovery (Galagan et al., 2003). A. niger, a member of the black aspergilli, is widely used in biotechnology for the production of food ingredients, pharmaceuticals and industrial enzymes. In their natural habitat, A. niger strains secrete large amounts of a wide variety of enzymes needed to release nutrients from biopolymers. This high secretory capacity is exploited by industry in both solid‐state and submerged fermentations (Pandey et al., 1999). A. niger has a long tradition of safe use in the production of enzymes and organic acids. Many of these products have obtained generally regarded as safe (GRAS) status (Schuster et al., 2002). Aspergillus enzymes are used in starch processing, baking, brewing and beverage industries; in animal feed; and in paper and pulping industry. Furthermore, A. niger is used as host for the production of heterologous proteins and as cell factory for the production of citric acid and gluconic acid (Archer et al., 2006). A. niger exhibits a remarkably versatile metabolism, enabling growth on a wide range of substrates and under various environmental conditions. Its ability to degrade a range of xenobiotics through various oxidative, hydroxylation and demethylation reactions provides potential for use in bioremediation (Pel et al., 2007). The genus Fusarium represents the most important group of fungal plant pathogens, causing various diseases on nearly every economically important plant species. Of equal concern is the hazard in human health and in livestock by the plethora of Fusarium mycotoxins. Besides their economic importance, species of Fusarium also serve as key model organisms for biological and evolutionary research. Fusarium graminearum is the causal agent of head blight (scab) of wheat and barley, a plant disease that constituted great impact on US agriculture and society during the 1990s. Approximately $3 billion were lost to US agriculture during wheat scab epidemics in the 1990s, resulting in devastating effects on farm communities in the upper Midwest and elsewhere. Moreover, the disease is becoming a threat to the world’s food supply due to recent head blight outbreaks in Asia, Canada, Europe and South America (Windels, 2000).
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Although fungi have important roles in biotechnology, they also cause important economic losses, since they are the etiological agents of plant and animal diseases. In humans, these microorganisms can be the cause of several infections that can be life‐threatening and/or diminish patients’ quality of life. The incidence of fungal infections is increasing worldwide, affecting both healthy and immunocompromised individuals. Due to that, we will discuss the metabolic diversity of human pathogenic fungi using the dermatophyte fungus Trichophyton rubrum as a model in sensing of nutrients and environmental changes.
18.2 Carbohydrate metabolism Fungi grow in a wide range of living or non‐living organic substrates. Among the species cited in this chapter, they have to confront a great variety of environments and different selective pressures. Thus, the capacity of an organism in being able to utilize a wide number of compounds could determine its chance of survival. Simple sugars, disaccharides, trisaccharides and glycosides of hexoses with other types of compounds are met in nature, but the carbon source in greatest abundance is the polymeric forms of simple sugars. These polysaccharides must first be hydrolysed to simple sugars before further metabolism occurs. Most hydrolytic enzymes in fungi are constitutive; but sometimes, they are adaptive, being synthesized only in the presence of the substrate or other inductive compounds. Among the proteins involved in the carbohydrate metabolism, glycoside hydrolases (GHs) are the most well‐studied group. GHs constitute an enzyme group that can catalyse the hydrolysis of the glycosidic linkage to release smaller sugars. They are extremely common enzymes with roles in nature (including degradation of biomass such as cellulose and hemicellulose), in antibacterial defence strategies (e.g. lysozyme), in pathogenesis mechanisms (e.g. mycoparasitism) and in normal cellular function (e.g. trimming mannosidases involved in N‐linked glycoprotein biosynthesis). Together with glycosyltransferases, GHs form the major catalytic machinery for the synthesis and breakage of glycosidic bonds. Carbohydrate‐active enzymes (CAZymes) are categorized into different classes and families in the
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Table 18.1 CAZyme families in Trichoderma reesei, Neurospora crassa, Aspergillus niger and Fusarium graminearum genomes CAZy
T. reesei
N. crassa
A. niger
F. graminearum
Glycosylhydrolases
200
171
250
243
Glycosyltransferases
103
76
117
110
Carbohydrate‐binding module
36
39
44
61
Carbohydrate esterase
16
21
26
42
Polysaccharide lyase
3
3
8
20
Source: Martinez et al. (2008).
CAZy database (http://www.cazy.org). CAZymes that cleave, build and rearrange oligo‐ and polysaccharides play a central role in the biology of the analysed fungi, and they are key to optimize biomass degradation by these species, especially T. reesei. Given the relative importance of this protein family to the biotechnology community, we compared the CAZome with the corresponding gene subsets from the four fungi analysed in this section (Table 18.1). Although one might expect that T. reesei, a polysaccharide‐efficient degrader plant and an important model of the degradation system, contains expansions of genes whose products are involved in digesting cell wall compounds, it has, surprisingly, few genes encoding GHs. With 200 GH‐encoding genes, it has fewer GHs than the phytopathogen F. graminearum and A. niger (Table 18.1). Less variability occurred in the glycosyltransferases, except for N. crassa, that showed the lowest value among them, suggesting that glycosyltransferases possess basal intracellular activities and that variations in composition might reflect species drift, rather than environmental pressure. The enzymes involved in plant polysaccharide depolymerization frequently carry a carbohydrate‐binding module (CBM) appended to the catalytic domain. Unexpectedly, the T. reesei genome has the smallest number of CBM‐ containing proteins among them (Table 18.1). This class (CBM) is frequently enriched in phytopathogens like F. graminearum. Similarly, T. reesei has the lowest number (16) of carbohydrate esterases among the fungi that we analysed. The Sordariomycetes (T. reesei, N. crassa and F. graminearum) showed a relative paucity of polysaccharide lyase genes, a category that typically contains 3–4 genes, except for F. graminearum, which has an expansion of 20 genes. Such a high number of polysaccharide lyases are found only in the Eurotiomycetes (in which A. niger is included), which
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have an average of 18 polysaccharide lyases. In conclusion, the T. reesei and N. crassa genome encodes a number of CAZymes that is slightly below than the other fungi analysed. Many genes encoding CAZymes are non‐randomly distributed within the genome (Diener et al., 2004). Martinez et al. (2008) perform this comparison with other fungi to analyse the non‐random distribution. They found that the concentration of CAZyme genes (primarily GH genes) in syntenic gaps in with F. graminearum and N. crassa further supports the notion that selective pressure can maintain the clustering of genes encoding proteins involved in biomass degradation. In comparison, previous studies (Galagan et al., 2005; Machida et al., 2005; Nierman et al., 2005) indicate that syntenic gaps in other genomes are enriched in genes that are important for species‐specific attributes. Although duplications may play a role in the loss of synteny, the CAZyme clusters in T. reesei show little evidence of expansion when compared to the other fungi analysed. The majority of the breaks in synteny, at which CAZyme genes are clustered, arise from movement of CAZyme genes into these regions, followed by pressure to fix the genomic rearrangements in the population (Martinez et al., 2008).
18.3
Transport metabolism
Transport systems play essential roles in cellular metabolism, such as nutrient uptake, excretion of toxic compounds and secondary metabolites and maintenance of ion homeostasis, but they also play a role in sensory processes (Saier, 2000). Insight into the distribution of transport protein classes is vital for the understanding of the metabolic capability of the analysed organisms (Ren and Paulsen, 2005). Transport
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Table 18.2 Distribution of transporter genes in Trichoderma reesei, Neurospora crassa, Aspergillus niger and Fusarium graminearum genomes according to gene ontology GO term
Number of gene models T. reesei
N. crassa
A. niger
F. graminearum
GO:0006810 transport
642
619
885
855
GO:0015837 amine transport
44
18
60
50
GO:0008643 carbohydrate transport
61
40
92
97
GO:0015893 drug transport
17
15
28
22
GO:0006818 hydrogen transport
53
29
27
31
GO:0046907 intracellular transport
115
141
116
127
GO:0006811 ion transport
122
112
102
122
GO:0006869 lipid transport
0
7
7
7
GO:0015931 nucleobase, nucleoside, nucleotide and nucleic acid transport
7
6
14
10
GO:0015849 organic acid transport
44
18
61
52
GO:0015833 peptide transport
3
1
4
4
GO:0015031 protein transport
109
141
117
122
GO:0045045 secretory pathway
19
30
24
25
GO:0016192 vesicle‐mediated transport
21
68
54
59
Total of gene models
9129
10,785
14,097
13,332
Source: JGI (http://genome.jgi.doe.gov/programs/fungi/index.jsf).
systems differ in their membrane topology and subunit composition, energy coupling mechanisms and substrate specificities. Mostly, ATP and the electrochemical transmembrane gradient of sodium ions or protons are used to drive the transport processes (Pel et al., 2007). A total of 642, 619, 885 and 855 transport proteins were predicted by the T. reesei, N. crassa, A. niger and F. graminearum genome analysis, respectively (Table 18.2). These were classified into amine, carbohydrate, drug, hydrogen, intracellular, ion, lipids, nucleic acids, peptides and protein transporters. In addition, secretory pathways and vesicle‐mediated transport were used in categorization. The number of gene models of T. reesei is relatively close to the number of gene models in N. crassa but is roughly 2500 fewer than the number of total predicted genes in F. graminearum. It is a surprising difference, given that F. graminearum and T. reesei share a relatively recent common ancestor (Martinez et al., 2008). Probably, horizontal transfer events can be the cause of this genome size difference. Between the Sordariomycetes, F. graminearum contains many genome differences. It could be reflected by the environment‐directed
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evolution and the complexity of the Fusarium genus. As expected, A. niger shows a high number of gene models (14,097) like the other species of the Aspergillus genus. In Table 18.2, we see that the number of gene models that correspond to transporters is smaller in the cellulose degraders N. crassa (619) and T. reesei (642) when compared with the others, but relative to the total gene models, they have similar transporter density. This smaller number apparently does not affect the efficiency in consumption of substrates and secretion of proteins (Galagan et al., 2003; Martinez et al., 2008). The T. reesei and N. crassa genomes have a reduced number of duplicated genes. This could explain why the genome sizes of T. reesei and N. crassa are similar and why both genomes contain few intact repeats (Martinez et al., 2008). The number of gene models in the secretory pathway and vesicle‐mediated transport in T. reesei represents this efficiency, known by the industry in recent decades. In particular, the major facilitator superfamily (MFS, IPR007114) with multiple transporter gene paralogues appears exceptionally large in both A. niger (461) and F. graminearum (292). Lower numbers were
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Chapter 18 Fungal metabolic diversity
found in T. reesei (192) and N. crassa (121). MFS proteins have been shown to facilitate the transport of a diverse range of molecules including the following: di‐ and monosaccharides, polyols, quinate, inorganic phosphate, siderophores, drugs such as anti‐fungals, mono‐ and dicarboxylates and various other organic acids (Pel et al., 2007). As biodegrading organisms, fungi not only must have the ability to degrade complex substrates but also to efficiently take up resulting small molecules. Amino acid polyamine transporter genes (IPR002293) and genes for sugar transporters (IPR003663) are abundant in F. graminearum compared to other fungi. Although most fungi can absorb organic nitrogen sources, in plants, such transporters would be beneficial for uptake of reduced nitrogen, perhaps the nutrient source most limiting to growth in plant tissue (Cuomo et al., 2007). Major facilitator transporters (IPR007114), membrane‐associated proteins that promote translocation of various small molecules across membranes in response to chemiosmotic gradients, are the second largest gene family in F. graminearum, with 294 members representing approximately 2% of its predicted genes (Cuomo et al., 2007). F. graminearum contains more predicted genes for major facilitator transporters than T. reesei and N. crassa, except for A. niger. An explanation for this high number of MFS transporter in A. niger could be the higher genome and consequently the number of gene models. A. niger is known as the more effective natural secretor of proteins (Pel et al., 2007), and certainly, the transporters are the reason for this feature. Major facilitators also have been associated with toxin efflux and reside in nearly every fungal gene cluster involved in biosynthesis of toxins active on plants, such as the trichothecene efflux transporter encoded by Tri12 (Alexander et al., 1999). The disruption of this gene (Tri12) caused a reduced growth on complex media and reduced levels of trichothecene production. Thus, the large number of major facilitator genes in fungus may reflect not only in their importance to absorption of nutrients but also in the delivery of bioactive molecules to the environment.
18.4
Energy metabolism
Metabolism serves two general functions: (1) anabolic function to synthesize structural and functional components and (2) catabolic function to extract chemical energy from nutrients.
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243
Neurospora, Trichoderma, Aspergillus and Fusarium are commonly saprophytes and obtain nutrition and energy from insoluble polymers. To the assimilation of these resources, polymers must first be broken into soluble and small molecules. Most of polymers when degraded by enzymes release glucose as final product. Glucose is then oxidized to CO2 using oxygen as the terminal electron acceptor. The role process is exergonic and forms the ATP further used in the biosynthesis of cellular constituents. Although a highly conserved pathway metabolizes glucose, the regulation of glucose utilization has been subjected to selection pressures during evolution (Chambergo et al., 2002). The glycolysis/gluconeogenesis and the tricarboxylic acid (TCA) cycle pathway during glucose metabolism are shown in Figure 18.1. The first step of glucose metabolism is its transport into the cell. This transport in fungi is mediated by sensors and by an active transport system that requires proton symport (Kubicek et al., 1993), high‐affinity glucose transporter pH‐regulated gtt1 (Delgado‐ Jarana et al., 2003) and glucose‐dependent glucose transporter regulated by O2 (Ramos et al., 2006). After uptake by fungi cells, glucose is phosphorylated at the C‐6 position by hexokinases and glucokinases. The filamentous fungi possess many copies of hexokinases (Table 18.3), but glucokinases are limited. Hexokinase is related mostly in sugar utilization for mycelium vegetative growth and probably regulates extracellular proteases during carbon starvation in Neurospora (Katz et al., 2000). On the other hand, glucokinase is associated with glucose activation from storage sugars, such as trehalose, and in Fusarium, glucokinase contributes to glucose phosphorylation under conditions of low glucose concentrations in the environment (Fleck and Brock, 2010). Phosphofructokinase 2 (PFK‐2) is a key enzyme in the glycolytic pathway. It catalyses the phosphor ylation of fructose‐6‐phosphate to fructose‐1,6‐bisphosphate by consuming of ATP molecule. PFK‐2 has been purified from T. reesei, and the enzyme is not regulated by cyclic AMP‐dependent phosphorylation but only by substrate availability, unlike in yeasts (Abrahao Neto, 1993). Except T. reesei, A. niger, N. crassa and F. graminearum contain one gene to fructose‐bisphosphate aldolase and triose‐phosphate isomerase to form glyceraldehyde‐3‐phosphate. The next step in this pathway is the conversion of glyceraldehyde‐3‐phosphate into 1,3‐diphosphoglycerate and concomitant reduction of
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Fungal biomolecules Glucose 2.7.1.1
3.1.3.9
2.7.1.2 G6-P 5.3.1.9 F6-P 2.7.1.1
3.1.3.1
FRU-1,6-P DHA-P
4.1.2.13 5.3.1.1
GA-3-P 1.2.1.12
Glycolysis/ gluconeogenesis
2.7.2.3 5.4.2.1 4.2.1.11
Ethanol
PEP 2.7.1.40
1.1.1.1 4.1.1.1
Pyruvate 1.2.4.1
4.1.1.49
Acetyl-CoA
Oxaloacetate
Acetaldehyde 1.2.1.3
Pyruvate bypass
2.3.3.1
1.1.1.37
6.2.11
Acetate
Citrate 4.2.1.3
Malate
Isocitrate
TCA cycle
4.2.1.2
Fumarate
1.1.1.42
a-Ketoglutarate
1.3.5.1 Succinate
1.2.4.2
6.2.1.4
Succinyl-CoA
Figure 18.1 Representation of glycolysis, gluconeogenesis, TCA cycle and pyruvate bypass in filamentous fungi. The numbers represent the Enzyme Commission classification
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Table 18.3 Glycolysis and gluconeogenesis orthologous genes in Aspergillus niger, Fusarium graminearum, Neurospora crassa and Trichoderma reesei genomes annotated according to KEGG database EC number
Description
A. niger
F. graminearum
N. crassa
T. reesei
1.1.1.1
Alcohol dehydrogenase
15
10
3
16
1.2.1.12
Glyceraldehyde‐3‐phosphate dehydrogenase (phosphorylating)
2
1
1
1
1.2.1.3
Aldehyde dehydrogenase (NAD+)
9
13
4
6
1.2.4.1
Pyruvate dehydrogenase (acetyl‐transferring)
3
2
2
2
2.7.1.1
Hexokinase
5
5
5
3
2.7.1.11
6‐Phosphofructokinase
1
1
1
1
2.7.1.2
Glucokinase
0
1
0
0
2.7.1.40
Pyruvate kinase
1
1
1
1
3.1.3.9
Glucose‐6‐phosphatase
0
0
0
0
3.1.3.11
Fructose‐bisphosphatase
1
1
1
0
4.1.1.1
Pyruvate decarboxylase
4
4
3
2
4.1.2.13
Fructose‐bisphosphate aldolase
1
1
1
1
4.1.1.49
Phosphoenolpyruvate carboxykinase (ATP)
1
1
1
1
5.3.1.1
Triose‐phosphate isomerase
1
1
1
2
5.3.1.9
Glucose‐6‐phosphate isomerase
1
1
1
1
6.2.1.1
Acetate–CoA ligase
2
3
2
2
NAD+ to NADH and H+ by glyceraldehyde‐3‐ phosphate dehydrogenase. A. niger possesses two genes for this reaction, while the other fungi, only one (Table 18.3). On the other hand, in N. crassa, it has been shown that glyceraldehyde‐3‐phosphate dehydrogenase is allelic to ccg‐7, a clock‐controlled gene (Bell‐Pedersen et al., 1996). The ATP‐generating steps in glycolysis occur by action of phosphoglycerate kinase and pyruvate kinase. The genes encoding 3‐phosphoglycerate kinase and pyruvate kinase from T. reesei and Trichoderma viride have been cloned (Vanhanen et al., 1989; Goldman et al., 1992; Schindler et al., 1993). The promoter region of 3‐phosphoglycerate kinase has consensus‐ binding sites for a cyclic AMP‐responsive element and the carbon catabolite repressor Cre1 (Vanhanen et al., 1991). On the other hand, the promoter region of pyruvate kinase contains consensus sequences for the binding of the glycolytic regulator genes RAP1 and GCR1, in accordance with the suggestion that the expression of glycolytic genes in Trichoderma occurs by similar pathways as to those characterized in yeast. However, detailed studies published by Chambergo et al. (2002) and Chovanec et al. (2005) showed that Trichoderma behaviour differs from that of yeast, at
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least in the presence of high glucose concentrations and O2 limitations. While in Saccharomyces cerevisiae genes related to aerobic metabolism are repressed in the presence of a high level of glucose, in T. reesei, these genes remain upregulated, thus favouring aerobic metabolism (Chambergo et al., 2002). However, at a low O2 concentration, T. viride can shift its metabolism to fermentative metabolism, suggesting that, under selection pressure imposed by O2 limitation, Trichoderma can change their lifestyle in order to guarantee energy production and survive under these conditions (Chovanec et al., 2005). The pyruvate produced at the end of glycolytic pathway can be oxidized to CO2 over the citric acid cycle (TCA) action or converted to ethanol by fermentation pathway (Figure 18.1). Fermentation in fungi starts with the decarboxylation of pyruvate to acetaldehyde and CO2 by the action of pyruvate decarboxylase (PDC). The genome of the fungi listed in Table 18.3 shows a diverse number for genes that encode this enzyme. To complete fermentation, acetaldehyde is reduced to ethanol by alcohol dehydrogenase (ADH) using NADH + H+. Except for Neurospora, fungi show several other orthologues for the gene that encode to ADH, while the yeast S. cerevisiae possesses
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A. niger is the most efficient fungi in the production of citric acid, having a great industrial importance. This ability could be explained by the fact that A. niger possesses extra copies of genes that encode for oxidoreductase (AOX, EC 1.9.3.), which may have a critical role in NADH recycling and also six isoforms of citrate synthases (Sun et al., 2007). Although citric acid production has been studied extensively in the past, there are still many questions that need to be answered to fully understand the citric acid formation process (Karaffa and Kubicek, 2003). Although many conclusions can be inferred from the available genome data, new tools in functional genomics (Schuster et al., 2012) will open new insights in fungi energy metabolism. Comparative metabolic genomics can reveal how fungi keep their versatile metabolic capacities and their robustness to adapt to different environmental conditions.
only four copies. The question here would be, why this difference between this fungi? The answer can be found looking for the environment and the abundance of oxygen and glucose. For instance, acetaldehyde formed by the decarboxylation of pyruvate by PDC is reduced to ethanol by ADH in S. cerevisiae, whereas in T. reesei, acetaldehyde is converted into acetate by aldehyde dehydrogenase (ADL1/ADL2). This is possible because T. reesei carries at least 6 isoforms of aldehyde dehydrogenase (Table 18.3), and one of them, ALD1, is not affected by glucose (Chambergo et al., 2002). The same behaviour is found in Aspergillus and Fusarium (Zhou et al., 2010). Furthermore, upon exhaustion of glucose, acetate replenishes the tricarboxylic by activating acetyl‐coenzyme A synthetase (ACS), and the activation of phosphoenolpyruvate carboxykinase (PCK) fuels the gluconeogenic pathway via oxaloacetate (Figure 18.1). Besides these forms to obtain energy demonstrated in this section, fungi have developed different strategies to survive in response to environmental changes. It has been observed that Fusarium and related fungi can produce energy using the reduction of nitrate (NO3−) or nitrite (NO2−) under hypoxic conditions (Zhou et al., 2010). Furthermore, it was described that when Fusarium is shifted to anoxic condition, this fungus can reduce NO3− to NH3 (ammonia fermentation), suggesting that fungi possess different metabolic mechanisms to get energy (Zhou et al., 2010). However, future researches using functional genomics can elucidate the diversity of metabolism in fungi under low oxygen and nutrient conditions. Concerning to TCA cycle, although the enzymes from this cycle are extremely conserved (Table 18.4),
18.4.1
Transcription factors
Promoters are constituted of core elements and specific target sequences for either positively or negatively modulation of genes transcribed by the RNA polymerase II. The gene transcription is regulated via these promoter regions by transcription factors. The regulated gene can be repressed or activated by a set of transcription factors. The chromatin itself has a repressive effect in the transcription. Thereby, the transcription factors that act as activators have several ways to induce transcription of the target gene. The genome sequencing and annotation of some filamentous fungi can give some insights into
Table 18.4 Tricarboxylic acid cycle orthologous genes in Aspergillus niger, Fusarium graminearum, Neurospora crassa and Trichoderma reesei genomes annotated according to KEGG database EC number
Description
A. niger
F. graminearum
N. crassa
T. reesei
1.1.1.37
Malate dehydrogenase
4
3
4
3
1.1.1.42
Isocitrate dehydrogenase (NADP+)
1
1
1
1
1.2.4.2
Oxoglutarate dehydrogenase (succinyl‐transferring)
2
1
2
1
1.3.5.1
Succinate dehydrogenase (ubiquinone)
3
2
3
3
2.3.3.1
Citrate (Si) synthase
4
3
2
2
4.2.1.2
Fumarate hydratase
1
1
1
1
4.2.1.3
Aconitate hydratase
4
3
2
2
6.2.1.4
Succinate–CoA ligase (GDP‐forming)
3
4
3
4
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Table 18.5
247
Transcription factors involved in cellulose, hemicellulose and β‐glucosidase regulation
Transcription factors
Regulatory rolea
JGI protein ID orthologues Trichoderma reesei
Neurospora crassa
Aspergillus niger
Fusarium graminearum
XlnR
+
122,208
6460
67,094
6,180
ACEII
+
78,445
7631
160,243
8,706
CLR‐1
+
27,600
5712
159,721
5,893
CLR‐2
+
26,163
6271
164,522
3,300
ClbR
+
Not found
4944
164,816
7,520
AreA
+
76,817
189
165,197
9,600
CREI
–
120,117
6580
156,906
11,247
ACEI
–
75,418
1048
167,502
722
PacC
Gene dependent
120,698
4372
157,309
7,758
HAP2
Gene dependent
124,286
2267
166,887
437
HAP3
Gene dependent
121,080
2551
155,314
8,196
HAP5
Gene dependent
AAK68863
4402
156,650
2,982
Xpp1
Not reported
122,879
9371
Not found
10,779
BglR
+
52,368
6866
159,374
2387
Plus for positive effect and minus for negative effect.
a
the regulation of gene transcription, but the transcriptional regulation is more complex and involves integration of signals from various factors that respond to environmental and developmental signals (Aro et al., 2003). It makes the study of transcription factors not a ‘straightforward’ approach, and some variables have to be analysed. Filamentous fungi have a key role in degradation of the most abundant biopolymers found in nature: cellulose and hemicellulose. The production of enzymes responsible for degrading these polymers (cellulases, hemicellulases, ligninases and pectinases) is regulated mainly at the transcriptional level in filamentous fungi (Aro et al., 2004). Some transcription factors involved in the regulation of cellulases, hemicellulases and β‐ glucosidases are shown in Table 18.5. Regulatory elements found in the promoters of genes encoding cellulases and hemicellulases include binding sites for the carbon catabolite repressor CRE, CCAAT element and binding sites for transcriptional element that modulates the gene expression. The CCAAT sequences are found in 5′ regions of approximately 30% of eukaryotic genes. A multimeric protein complex recognizes and binds in this sequence. The first binding complex described was designated as Hap complex, consisting of proteins Hap2, Hap3, Hap4 and Hap5, identified in
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S. cerevisiae (McNabb et al., 1995). Since then, homologues of Hap protein complex (except Hap4) have been found in various organisms, including filamentous fungi. The CCAAT motif has been described to modulate cellulase and hemicellulase genes in T. reesei (Zeilinger et al., 1998). The presence of easily metabolizable carbon source, such as glucose, results in the repression of various genes needed for the use of other alternative carbon sources. The mechanism that controls the preferential use of substrate is called carbon catabolite repression. In many filamentous fungi, glucose repression is mediated by the transcription factor CreA/CreI. Numerous cellulase and hemicellulase genes have been shown to be regulated by Cre proteins in T. reesei and Aspergillus species. In general, mutations of the cre gene lead to a derepression of the expression on glucose. A good example is the hypercellulolytic industrial mutant Rut‐C30 of T. reesei. This strain has the cre gene truncated and can produce cellulases and hemicellulases when grown on glucose medium. Despite of the complexity of these mechanisms, the utilization of plant biopolymers is well worth of study. This subject provides many features and possibilities in basic and applied fields to improve the knowledge in biology of these fungi.
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18.5
Secondary metabolism
Microorganisms are able to produce a wide variety of secondary metabolites that can be used in various applications, representing a major source of bioactive compounds for agrochemical and for pharmacology (Collemare et al., 2008; Craney et al., 2013). Secondary metabolites are defined as low‐molecular‐weight compounds that are not required for the organism growth. They are characterized by a great diversity of chemical structures and variations in different environmental conditions, conferring a selective advantage to the producer organism (Butler and Buss, 2006). Several secondary metabolites with structures and biological activities have been isolated from different microorganisms. Various studies have been done in order to understand and characterize the biosynthetic pathway leading to the discovery of new compounds (Collemare et al., 2008). The production of secondary metabolites varies according to the particular compound, the species and the presence of other microorganisms, the balance between the biosynthesis of elicitors and the rate of biotransformation (Degenkolb and Brückner, 2008; Vinale et al., 2009). Fungal secondary metabolites may be considered a large and heterogeneous group of small molecules not directly essential for growth but having an important role in signalling, development and interactions with other organisms (Mukherjee et al., 2012). Trichoderma species are well known to produce secondary metabolites with antibiotic activity, including volatile and non‐volatile compounds and peptaibols against pathogens, besides being involved in the defence mechanisms of plants (Iida et al., 1994; Samson, 1994; Schirmböck et al., 1994; Goulard et al., 1995; Yedidia et al., 1999). Secondary metabolites of Trichoderma spp. were classified into three classes: volatile antibiotics, that is, 6‐pentyl‐a‐pyrone (6PP) and most of the isocyanide derivates; water‐soluble compounds, that is, heptelidic acid or koningic acid; and peptaibols, which are linear oligopeptides of 12–22 amino acids rich in α‐aminoisobutyric acid, N‐acetylated at the N‐terminus and containing an amino alcohol (Pheol or Trpol) at the C‐terminus (Ghisalberti and Sivasithamparam, 1991). Several species of the genus Trichoderma are known to produce effective secondary metabolites against pathogens and alter the metabolism of the host plant by increasing the availability of nutrients in
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the soil. Harzianic acid is a secondary metabolite of Trichoderma harzianum. It is known by its anti‐fungal activity and as a plant growth promoter (Vinale et al., 2009). Recently, it was demonstrated that this compound has a role as siderophore with good affinity for the metal Fe3+, which may represent a mechanism for solubilization of iron and thus make it available to the soil and to the plants (Vinale et al., 2013). The secondary metabolites from Trichoderma species can result in either two mechanisms of action: influencing the microbial community due to the long‐distance high concentrations of volatile compounds such as 6‐ PP or operating in close proximity to the hyphae production of antibiotics as polar and peptaibols (Vinale et al., 2008). Metabolites produced by Gliocladium and Trichoderma species (Figure 18.2) can inhibit the growth of other microorganisms by releasing low‐molecular‐weight diffusible compounds or antibiotics, among them is the production of, for example, alamethicins, harzianic acid, gliotoxin, glioviridin, 6‐n‐pentyl‐6H‐pyran‐2‐one (6PP), T22azaphilone and others (Sivasithamparam and Ghisalberti, 1998; Benitez et al., 2004, Schuhmacher et al., 2007; Vinale et al., 2008). The isolation and identification of trichoderonic acids A (1) and B (2), novel terpenoids and heptelidic acid (3) from cultures of Trichoderma virens by spectroscopic analysis showed that these compounds are effective in inhibiting the families of mammalian DNA polymerases. It has been shown that the compounds 2 suppressed the growth of two human cancer cell lines, cervix carcinoma cells and breast carcinoma cells (Yamaguchi et al., 2010). Three new amino lipopeptides, designated trichoderins A (1), A1 (2) and B (3), were isolated from a culture of marine sponge‐derived fungus of Trichoderma sp. that showed potent activity against Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv (Pruksakorn et al., 2010). The compound harzianic acid isolated from Trichoderma harzianum (from composted hardwood bark in Western Australia) showed that this compound was effective in growth of canola seedlings, increasing the stem length between 42 and 52%. It has been showed that harzianic acid had antibiotic activity against Pythium irregulare, Sclerotinia sclerotiorum and Rhizoctonia solani (Vinale at al., 2009). Three compounds obtained from Trichoderma species, namely, heptadecanoic acid, 16‐ methyl‐, methyl ester; 9,12‐octadecadienoic acid; and cis‐9‐octadecenoic acid, identified by GC‐MS
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Chapter 18 Fungal metabolic diversity O
N H
O O
N NH H O N
O
N H
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H N
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249
HN
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OH
HN
O
NH
HN
NH
HN
HO
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NH2 O
Alamethicins
HO OH O
N O
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OH
S
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H
S
O OH
OCH3 S
CH2OH
Gliotoxin
S
N
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OCH3
N
H
H
N
O O
Glioviridin
O
O
6-n-pentyl-6H-pyran-2-one (6PP)
O O
H O H O
T22azaphilone
Figure 18.2
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Metabolites produced by Gliocladium and Trichoderma species
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analysis, were tested against a skin cancer protein (4,5‐ diarylisoxazole Hsp90 chaperone). The compound heptadecanoic acid, 16‐methyl‐, methyl ester has showed the best result by in silico docking method against skin cancer protein (Kandasamy et al., 2012). Peptaibols are a family of antibiotic peptides containing high content of the non‐proteinogenic α‐aminoisobutyric acid (Aib), and other non‐ proteinogenic amino acids can be found in peptaibols such as IVA (l‐ and d‐isovaline), Hyp (cis‐ and trans‐ 4‐l‐hydroxyproline), MePro (cis‐4‐l‐methylproline), Hyleu (β‐hydroxy‐l‐leucine), β‐Ala (β‐alanine), Pip (l‐pipecolic acid), Etnor (α‐ethyl‐norvaline) and AHMOD (2‐amino‐4‐methyl‐6hydroxy‐8‐oxoecanoic acid), which show antibiotic or other bioactivities, generally exhibiting antimicrobial activity against Gram‐positive bacteria and fungi (Wiest et al., 2002). The activities of peptaibols isolated from Trichoderma against bacterial and fungal plant pathogens have been demonstrated. The peptaibol trichokonin VI (TK VI), a peptaibol from Trichoderma pseudokoningii SMF2, induced extensive apoptotic cell death in plant fungal pathogens Fusarium oxysporum. Trichosporins B‐VIIa and B‐VIIb, produced by Trichoderma polysporum, showed antitrypanosomal activities against Trypanosoma brucei (Shi et al., 2010). Several studies have shown that the peptaibols from Trichoderma species have many important biological activities such as suppressing of tumour cells by inducing apoptosis and autophagy in hepatocellular carcinoma cells; having inhibitory properties of the envelope replication of viruses such as influenza A virus, vesicular stomatitis virus and HIV; as well as promoting wound healing, inhibiting the formation of peptide β‐amyloid in cultured cells of cerebral cortex of pigs, and potentially having an important role in neurodegenerative diseases and in maintaining homeostasis, presenting a mechanism of action similar to calcitonin (Katayama et al., 2001; Daniel and Filho, 2007; Degenkolb et al., 2008). Streptomyces are known to produce a wide variety of secondary metabolites with potential biological activities. Within the secondary metabolites reported, most of them are produced by Streptomyces, and the main products are antibiotics, antitumor agents, immunosuppressants, antihelminthics, anti‐fungals, herbicides and insecticides (Chaudhary et al., 2013). The secondary metabolic products from Streptomyces have diverse structures (Figure 18.3) from many standard metabolites and thus require specialized cellular
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machinery for production. Two major routes for the biosynthesis of secondary metabolites are groups of enzymes collectively known as non‐ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Polyketides are molecules with at least two carbonyl groups, though most of the polyketide secondary metabolites produced by members of the genus Streptomyces have numerous other prosthetic functional groups. Such polyketide natural products display a diverse range of therapeutic properties; they are used therapeutically as antibiotics, anti‐cancers, antiparasitics and anti‐fungals. Aromatic polyketides are an important class of medically relevant secondary metabolites: the anticancer agent daunorubicin and the tetracycline antibiotics belong to this class (Craney et al., 2013). A second class of secondary metabolites produced by Streptomycetes is non‐ ribosomal peptides (NRPs), which are synthesized by NRPSs, independent of mRNA or any other canonical translation machinery. These peptides are often less than 20 amino acids in length and have a diverse range of functions in microbial contexts (Koehn and Carter, 2005). Many mushrooms could be a source of natural antibiotics, mainly by the action of their secondary metabolites such as sesquiterpenes and other terpenes, steroids, anthraquinone, benzoic acid derivatives and quinolines (Figure 18.4). Thus, the sesquiterpenes enokipodins A, B, C and D with activity against Bacillus subtilis were isolated from mycelium Flammulina velutipes; however, enokipodins A and C showed only activity against Staphylococcus aureus. The terpenic compounds like confluentin, grifolin and neogrifolin from Albatrellus flettii showed activity against Bacillus cereus and Enterococcus faecalis, and the terpenes ganomycin A and B, isolated from Ganoderma pfeifferi, has activity against Bacillus subtilis, Micrococcus flavus and Staphylococcus aureus. A steroid, 3,11‐dioxolanosta‐8,24(Z)‐diene‐26‐oic acid, isolated from the Jahnoporus hirtus mushroom has activity against Bacillus cereus and Enterococcus faecalis. The organic acid, oxalic acid, has activity against Bacillus cereus and was isolated from Lentinus edodes (Figure 18.2). The peptide plectasin from Pseudoplectania nigrella showed activity against Bacillus cereus, Bacillus thuringiensis, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae and Streptococcus pyogenes (Mygind et al., 2005; Alves et al., 2012).
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251
OH
OH OH
OH
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OH Validoxylamine A (1)
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O
OH
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Geomisn (4)
OH OH
O
CH3
O O CH3
O
OH
Actinorhodin (4)
OH
O HO
O
Figure 18.3 Structures of secondary metabolites produced by Streptomyces coelicolor (2–5) and Streptomyces hygroscopicus var. jinggangensis 5008 (1). Adapted from Craney et al. (2013)
Endophytes are microorganisms that live inside plant tissues. They are present in all plants and are extremely abundant and can survive in plants for all or part of their life without causing any apparent damage or diseases. Besides that, endophytes are related to be reservoirs of metabolites that are effective against
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pathogens. Many of them are capable of synthesizing bioactive compounds that can be used by the plant for defence against pathogenic microorganisms (Abdel‐ Motaal et al., 2010; Mousa and Raizada, 2013). Its importance has been studied and demonstrated as potential sources as novel bioactive metabolites such
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O COOH
O 3,11-Dioxolanostra-8,24 (Z)-diene-26-oic acid(27) O O
H3CO
O
O
OH
6-Methylxanthopurpurin-3-O-methyl ether
R
O Enokipodins
OH
O Confluentin
Figure 18.4 Chemical structure of low‐molecular‐weight (LMW) compounds with antimicrobial potential found in mushrooms. Adapted from Alves et al. (2012)
as antimicrobial, anticancer and antiviral agents (Selim et al., 2012). Phomenone is produced by Xylaria sp., an endophytic fungus associated with Piper aduncum, and has been related to have anti‐fungal activity against Cladosporium cladosporioides and Cladosporium sphaerospermum. The steroid 3β,5α‐dihydroxy‐ 6β‐phenylacetyloxy‐ergosta‐7,22‐diene(27) compound was isolated from the liquid culture of a fungal endophyte Colletotrichum inhabiting the stems of Artemisia annua and can have an anti‐fungal activity against Phytophthora capsici, Gaeumannomyces graminis, Rhizoctonia cerealis and Helminthosporium sativum. This compound can also show antibacterial activity against Pseudomonas sp., B. subtilis, Sarcina lutea, S. aureus, A. niger and Candida albicans. Mellein is another compound that has inhibitory properties against the bacteria Bacillus megaterium and
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Escherichia coli, Ustilago violacea, Eurotium repens and the alga C. fusca. Diaporthein is a pimarane diterpene. It was purified from the culture broth of the fungus Diaporthe sp., and it showed strong inhibition of the growth of Mycobacterium tuberculosis. In Figure 18.5, some examples of the bioactive compounds isolated from endophytes are shown (Mousa and Raizada, 2013). Natural products seem to be promising sources of bioactive compounds by structural diversity found in nature produced by a wide variety of microorganisms. Mostly of these compounds were not yet explored for activation of metabolic pathways by manipulation of microorganisms. Recent advances in technologies for separation, characterization and structural elucidation of compounds facilitate the identification of bioactive products and can be applied in biotechnology purposes.
AQ1
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Chapter 18 Fungal metabolic diversity H
O
CH3
OH HO CH3
CH3
253
CH2
O
O
Phomenone
OH
O
(R)-Mellein
CH2 OH H3C
OH O HO
OH H3C
HO
Diaporthein
CH3
H3C
H3C CH3
CH3 CH3
CH3
O
HO
O
3β,5α-dihydroxy-6β-phenylacetylloxy-ergosta-7,22-diene (27)
Figure 18.5
Structures of secondary metabolites of endophytic fungi. Adapted from Mousa and Raizada (2013)
18.6 Metabolism of human pathogens: Dermatophytes As discussed before, fungi are important microorganisms playing significant roles in biotechnology for biomass degradation and production of antibiotics and other substances of economic interest, in nutrition serving as food, in agriculture through mycorrhizal associations and in the environment as decomposers of organic material. However, they also cause important economic losses as the etiological agents of plant and
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animal diseases. In humans, these microorganisms can be the cause of several infections that can be life‐threatening and/or diminish patients’ quality of life. The incidence of fungal infections is increasing worldwide, affecting both healthy and immunocompromised individuals. These infections may be superficial, affecting mainly the skin and mucous membranes, or systemic, when the fungus gets into the bloodstream causing more severe infections, such as pulmonary diseases. The symptoms associated with these infections vary greatly depending on the infected anatomical site, the host immune system and the
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infecting agent, ranging from asymptomatic to chronic and, sometimes, fatal diseases. The most frequent skin infections are the dermatophytoses, caused by a group of phylogenetically related filamentous fungi specialized in infecting and colonizing keratinized tissues in humans and animals (Grumbt et al., 2011b). These ascomycetous fungi are classified in three genera (Epidermophyton, Microsporum and Trichophyton) according to the morphology of their conidia, their asexual reproductive structures. Dermatophytes may also be classified as geophilic, zoophilic or anthropophilic species, based on their preferable environmental primary niches: soil, animals or human, respectively (Weitzman and Summerbell, 1995). Dermatophytoses such as athlete’s foot, ringworm and onychomycoses (nail infection) are among the most common fungal infections worldwide, causing infections in both healthy and immunocompromised individuals. Due to the high incidence of these infections, the genomes of the most common causative agents of dermatophytosis in human, the anthropophilic species Trichophyton rubrum and other six dermatophyte species (Trichophyton equinum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, Trichophyton verrucosum and Arthroderma benhamiae), have been completely sequenced and are publicly available at Broad Institute Dermatophyte Comparative Database (Broad, 2010; Burmester et al., 2011; Martinez et al., 2012) (http://www.broadinstitute.org/annotation/ genome/dermatophyte_comparative/MultiHome. html). These strains are clinically relevant, being previously associated with disease in humans. T. tonsurans is an anthropophilic species, mainly associated with tinea capitis, the scalp infection in children. T. equinum is a zoophilic species, infecting primarily horses, but infections in cats, dogs, and humans are also reported. M. canis is a zoophilic specie infecting mainly cats and dogs, but readily transmissible to humans, causing tinea capitis in children. T. verrucosum is a zoophilic species associated mainly with cattle dermatophytosis, infecting other ruminants and occasionally causing infections in humans, which are generally highly inflammatory. A. benhamiae is a teleomorph of Trichophyton mentagrophytes and is also a zoophilic species, infecting primarily guinea pigs, while M. gypseum is a geophilic species found in the soil, which might infect humans mainly in the arms (Achterman and White, 2011). Zoophilic species eventually cause infection in humans, being
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responsible for approximately 30% of clinical cases, triggering acute inflammatory responses in the host. Anthropophilic species, on the other hand, infect exclusively humans, corresponding to 70% of human cases of dermatophytosis, which is generally chronic with a slowly progression (White et al., 2008). The comparative genomic analyses of dermatophytes have found that these species present few differences among their genes and genome organization, presenting a high degree of collinearity and a core set of 6168 orthologous groups common to the 7 genomes analysed. It was also revealed that dermatophytes have several gene family expansions not present in other human pathogens. Such genes encode proteins related to secondary metabolism synthesis, including dermatophyte‐specific genes which can be responsible for the production of novel compounds, kinases, proteases and also proteins containing a LysM‐binding domain, responsible for chitin and other carbohydrate binding (Martinez et al., 2012). Despite this high similarity among dermatophyte genomes, each species is adapted to a specific host, which may be related to different regulations of virulent factors by each species.
18.6.1
Proteolytic activity
The main characteristic of T. rubrum and other dermatophytes is the ability to utilize the keratin present in the stratum corneum as nutrient source, due to their capacity to secrete several proteolytic enzymes, such as proteases and keratinases, which are important for the establishment and maintenance of infection. In order to cause infections, dermatophytes must adhere to host tissues to prevent its removal by the constant skin renewal through keratinization (Wagner and Sohnle, 1995). The initial contact between arthroconidia and stratum corneum is an essential step for the establishment of the infection and is mediated by adhesins (Baldo et al., 2012). After the adhesion, the process of invading host tissue is initiated by the emergence of germ tubes from the arthroconidia and is proposed to be mediated by the secreted proteases. It has been proposed by Martinez‐Rossi that the proteolytic enzymes released during the infection process are regulated by the environmental pH. In early stages of infection and in response to the human skin pH, slightly acid, dermatophytes induce the expression of acid proteases. The metabolization of keratin and
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Chapter 18 Fungal metabolic diversity
other protein substrates present in the stratum corneum generates peptides, and their further metabolism generates amino acids, such as glycine. The metabolism of glycine leads to the secretion of ammonia which promotes the alkalinization of the host microenvironment. This pH shift, from acid to alkaline, leads the pathogen to repress the secretion of acid proteases and to induce the secretion of alkaline proteases, which will be important for tissue damage and for the maintenance of the infection (Martinez‐ Rossi et al., 2004, 2012; Peres et al., 2010a). The proteases secreted by dermatophytes are important virulent factors and comprise both endo‐ and exoproteases. The endoproteases include two major families: the subtilisins, which are serine proteases, and the fungalysins (also known as metalloproteases) (Monod, 2008). Although these endoproteases are present in other pathogenic fungi, in dermatophytes, these families are expanded. While Aspergillus fumigatus presents only one gene encoding a metalloprotease and two subtilisin genes, T. rubrum presents 12 genes encoding subtilisins and 6 genes encoding fungalysins in its genome. The exoproteases include three classes of enzymes, such as dipeptidyl peptidases, aminopeptidases and carboxypeptidases (Monod, 2008). There are more than 150 genes that encode proteolytic enzymes identified in T. rubrum genome, reinforcing the importance of this class of proteins for its metabolism, as a human pathogen (Martinez et al., 2012). The subtilisins SUB1, SUB3, SUB4 and SUB5 and the metalloproteases MEP1, MEP2, MEP3 and MEP4 are highly expressed when T. rubrum is cultivated in keratin‐containing media (Maranhão et al., 2007; Zaugg et al., 2009; Peres et al., 2010b). Moreover, the genes encoding the exoproteases aminopeptidases LAP1 and LAP2, metallocarboxypeptidase MCPA and the DPPV are also strongly upregulated in these media (Zaugg et al., 2009). A similar expression profile of endo‐ and exoproteases was also observed when A. benhamiae was cultivated in keratin‐containing media (Staib et al., 2010). However, a different profile of proteases is observed in in vivo animal infection models or in human skin fragments compared to protein substrates. Only SUB3, SUB4 and MEP4 were highly upregulated in human skin fragments, while in guinea pig infection, SUB1, SUB2, SUB6 and MCPA were highly expressed (Leng et al., 2009). Furthermore, subtilisin SUB3 is involved in adherence of M. canis to human and animal epidermis,
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reinforcing its importance for virulence, although it is not required for the invasion of these tissues (Baldo et al., 2010; Bagut et al., 2012). To decompose hard keratin that is present in keratinized tissues, proteases by themselves are not enough. It has been hypothesized that sulphite is the reducing agent involved in this process (Kunert, 1972). When growing in keratin, dermatophytes secrete large quantities of sulphite as a reducing agent, rather than converting sulphite into sulphate. In the presence of sulphite, the disulphide bounds of keratin are cleaved to cysteine and S‐sulphocysteine. Then, the resultant proteins become accessible for the action of proteases secreted by the fungi (Lechenne et al., 2007). Hard keratin presents a compact structure due to the presence of disulphide bonds formed between the abundant cysteine residues in its composition, an amino acid that at high concentration is toxic to microbes and humans, although it can be catabolized to sulphite by the enzyme cysteine dioxygenase (Cdo1). In the hard keratin degradation model proposed by Grumbt, it was postulated that there is enough free cysteine in keratin to initiate the secretion of sulphite and thus keratin degradation (Grumbt et al., 2013). On the contrary, in proteases, as in other fungi, in T. rubrum and in other dermatophyte genomes, there is a single gene encoding a sulphite efflux pump SSU1 which is supposed to secrete sulphite, participating in sulphite detoxification and also in sulphitolysis (Lechenne et al., 2007). In A. benhamiae, functional analysis was performed for the genes encoding the cysteine dioxygenase (cdo1) and the sulphite efflux pump (ssu1). The ∆cdo1 strain was unable to grow on human nail and hair substrates, while ∆ssu1 mutant was incapable of growing on human hair, and its growth was strongly affected in human nail. Furthermore, ∆ssu1 mutant is sensitive to sulphite, supporting the hypothesis that sulphitolysis is mediated by this gene in dermatophytes (Grumbt et al., 2013). Proteases are highly conserved among dermatophytes at protein sequence, although the secretion levels seem to be variable depending on the species. Interestingly, the secreted proteins of T. equinum and T. tonsurans are different as well as those of T. rubrum, Trichophyton soudanense and Trichophyton violaceum, emphasizing that different regulation processes of these proteases could be niche specific and may be also related to different inflammatory reactions caused by each species in the host (Giddey et al., 2007). The different inflammatory reactions may also be related to
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the different cytokines produced by human keratinocytes in response to each species. Cytokine production was enhanced when keratinocytes were co‐cultured with the zoophilic species, while lower levels were produced by anthropophilic species T. rubrum and T. tonsurans (Shiraki et al., 2006; Tani et al., 2007). Furthermore, the keratinolytic activity of T. mentagrophytes, T. rubrum, M. canis and M. gypseum is different among each species and is influenced by different environmental conditions such as pH and temperature, being the peak of T. rubrum keratinolytic activity at pH 7.0 and 8.0 and 30–40 °C (Sharma et al., 2012).
18.6.2 pH metabolism Dermatophytes are able to grow in several nutrient sources, such as glucose, proteins, nail and skin. Another feature associated with the development and metabolism of these microorganisms is the extracellular pH. Dermatophytes are highly dependent on the initial acid pH for successful growth, since their in vitro growth is partially or completely abolished when the medium is buffered at pH 5.0 or 8.0 (Martinez‐Rossi et al., 2012). Gene regulation in response to ambient pH is mediated by the highly conserved PacC/Pal signalling pathway that controls the response to ambient pH and several other metabolic events in filamentous fungi. This pathway is composed of the zinc finger transcription factor PacC and the pal genes (A, B, C, F, H, I) that are well characterized in Aspergillus nidulans, and orthologues have been identified in several organisms, such as Saccharomyces cerevisiae, Candida albicans and Neurospora crassa, and also in dermatophytes. In A. nidulans, PacC undergoes two proteolytic steps at the C‐terminus in order to be activated. The first step is mediated by the products of the pal genes, while the second is proteasome mediated. The proteolysis leads to the active protein PacC27 that contains a DNA‐binding domain formed by three C2H2 zinc fingers capable of binding to the core consensus sequence 5′‐GCCARG‐3′ present in the promoter region of possible pH‐regulated genes (Tilburn et al., 1995). In T. rubrum, besides pH regulation, pacC gene is also associated with pathogenesis. The pacC‐1 mutant strain presents a reduction in the activity of keratinases and a loss of the capacity of growing in human nails (Ferreira‐Nozawa et al., 2006). Genes differentially expressed in either alkaline or acid pH were identified
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in T. rubrum, being some of them regulated by the transcription factor PacC at alkaline or acid pH, although pacC‐1 mutant presents the same media alkalinization profile as the wild‐type strain (Silveira et al., 2010), suggesting that PacC may be functional at both acid and alkaline pH (Rossi et al., 2013). A fatty acid desaturase, aconitase and a ribosomal protein are among the genes upregulated at acidic pH and modulated by PacC (Silveira et al., 2010).
18.6.3
Other virulent factors
Besides proteolytic activity, other factors have been associated with dermatophyte pathogenicity, mainly evidenced by transcriptional analysis. ABC transporters and multi‐drug resistance (MDR) proteins are a highly conserved class of proteins, among both eukaryotes and prokaryotes, associated with the transport of several compounds and also the efflux of possible toxic compounds to the cell. This class of transporters is highly abundant in dermatophyte genomes, and some of these proteins have already been associated with drug resistance and pathogenicity. T. rubrum TruMDR2 mutant showed an increased susceptibility to terbinafine, an anti‐fungal drug used in clinical treatment of dermatophytosis (Fachin et al., 2006), and a reduction in the infecting activity, characterized by reduced growth on human nails (Maranhão et al., 2009). The high expression levels of transporter genes by T. rubrum in keratin‐containing media and the reduction in virulence of the TruMDR2 mutant suggest that membrane transporters are involved in T. rubrum pathogenicity (Maranhão et al., 2009). The zinc finger transcription factor drn1 is a nitrogen regulatory gene homologue to A. nidulans areA and N. crassa nit‐1, involved in the regulation of several genes such as permeases associated with nitrogen utilization. The characterization of M. canis dnr1 mutant revealed a loss of the capability of growth in minimal media supplemented with nitrate and nitrite and presented a limited growth in the media containing ammonia as nitrogen source. Also, it presented reduced growth in keratin‐containing media, suggesting that dnr1 is involved in both nitrogen metabolism and pathogenicity (Yamada et al., 2006). The strong upregulation of genes coding for Hsp70 and the enzymes of the glyoxylate cycle, malate synthase and isocitrate lyase during T. rubrum growth
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in protein‐containing media and also during the development suggests that these genes may be important for dermatophyte pathogenicity (Zaugg et al., 2009; Peres et al., 2010b). However, in A. benhamiae, a mutant of the malate synthase gene (∆acuE) presented no deficiency in infecting guinea pigs or in vitro epidermal invasion, but has reduced growth in lipid media (Grumbt et al., 2011a). In the human pathogen Cryptococcus neoformans, melanin production is an important virulent factor. Recently, it was demonstrated that dermatophytes also produce melanin or melanin‐like compounds, which may be considered as a virulent factor (Youngchim et al., 2011). The genome sequences and the advances in the available genetic tools to study dermatophytes will surely enable further researches on this field to come up with a better understanding of the molecular basis of host–pathogen interactions and the pathogenicity of these infections in order to improve therapeutic strategies to control these fungal infections.
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