Fungal Peptaibiotics: the evolution of secondary metabolite regulatory genes responsible for peptaibiotic production.

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Fungal Peptaibiotics

Studies have shown in fungi the presence of beneficial activities for health and in many cases have identified bioactive compounds or groups of molecules responsible for beneficial activities such as antioxidant, anti-hypercholesterolemic, antibacterial and anti-hyperglycemic activities and antimicrobials that are important in the food and health industries. Research into fungal systems biology leads the studies of morphology, metabolic, regulatory routes and physiology of growth and development. The fungal kingdom contributes some 30 000 natural products of which 16000 are bioactive. Of these, around 11 250 originate from microscopic fungi. Peptide antibiotics constitute a considerable part of those metabolites, including therapeutically importantβ-lactam antibiotics (penicillins, cephalosporins), which account for more than 65% of the world antibiotics market. However, the significance of other, nonantibiotic peptide drugs of fungal origin is comparable – the immunosuppressant market is still dominated by the nonribosomal biosynthesised cyclosporine A (Rohrich et al., 2012). A plethora of natural products originally isolated from fungi are used in pharmaceutical and biotechnological industry. Many of these products have proven to be extremely useful for human health such as beta-lactam antibiotic penicillin, cholesterol lowering drugs lovastatin and immunosuppressant such as cephalosporin. These compounds are collectively termed as “secondary metabolites”. Fungi capable of producing these metabolites have been intensively investigated by many pharmaceutical companies ever since the discovery of penicillin. This project will focus on metabolomics of filamentous fungi to investigate the evolution of secondary metabolite regulatory genes, gene clusters responsible for peptaibiotic production.

Peptaibiotics are defined as peptides containing Aib and exerting a variety of bioactivities. They are linear or cyclic peptide antibiotics characterized by the presence of the non-proteinogenic amino acid alpha-aminoisobutyric acid. They belong to the constantly expanding group of fungal secondary metabolites with more than 1000 different peptaibiotics identified so far. The name peptaibiomics is terms used for for the analysis of a group of fungal peptide antibiotics (peptaibiotics) containing the characteristic amino acid Aib (a-aminoisobutyric acid). Peptaibiotics are biosynthesized by multi-domain non-ribosomal peptide synthetases and recent evidence from sequencing of fungal genomes suggests that a limited set of these enzymes is responsible for the biosynthesis of a multiplicity of products by domain skipping and processing events. Despite the general assumption that secondary metabolites are not essential for the growth, development and reproduction of the producing organism, the production of peptaibiotics is tightly regulated by conserved signaling pathways and their biosynthesis seems to be correlated with conidiation of the fungus. Based on their helical structure, peptaibols can form pores in membranes and this permeabilization in most cases is the basis for their antimicrobial and cytotoxic activities.

These unique peptides are produced by a widespread group of filamentous fungi comprising soil and aquatic species, wood decaying, plant pathogenic, fungicolous and coprophilous species (Bruckner et al., 2005). The first members of the peptaibiotics family, suzukacillin and alamethicin, were isolated from Trichoderma strains already in the late 1960s (Ooka et al., 1966; Meyer and Reusser, 1967). Since then, a constantly increasing number of peptaibiotics has been identified and described which recently have been summarized in the “Comprehensive Peptaibiotics Database” (Stoppacher et al., 2013). This freely available searchable database currently contains 1043 peptaibiotics including information on the peptide category, group name of the microheterogeneous mixture to which the peptide belongs to, amino acid sequence, sequence length, producing fungus, peptide subfamily, molecular formula and monoisotopic mass. Based on the estimation of ca. 1.5 million fungal species existing on our planet of which ca. 90,000 have been described yet, many more peptaibiotics await their discovery representing a rich source of beneficial biomolecules.

The fungal species, which contain peptaibiotic producing strains, are ubiquitous and cosmopolitan, even occurring in marine, Arctic and Antarctic regions (Bruckner et al. 2009) but genus Trichoderma (teleomorph Hypocrea), filamentous ascomycetes exhibiting a broad range of life-styles and interactions (Druzhinina et al., 2011), is generally regarded as the richest source of peptaibiotics. This is also reflected by the entries in the “Comprehensive Peptaibiotics Database”, the large majority (83%) of which can be assigned to Trichoderma/Hypocrea. T.viride, T.brevicompactum, T.virens, T. parceramosum/T. ghanense, and T. harzianum are the most intensively studied species with a total number of 468 different peptaibiotics (Stoppacher et al., 2013).

In addition to the genus Trichoderma, species of other fungal genera including Acremonium, Paecilomyces, Emericellopsis, Clomostachys, Stibella, Bionectria, Monicillium, Nectriopsis, Niesslia, Sepedonium, and Tolypocladium have been identified as producers of peptaibiotics (Brückner et al., 2009). There are also reports on peptaibiotics isolated from fruit-bodies of basidiomycetes (Lee et al., 1999); however, these have to be viewed critically as they actually might result from an infection by Sepedonium chrysospermum (Degenkolb et al., 2003). The only basidiomycete, which has been verified to produce peptaibiotics so far, is Peniophora cf. nuda (Peniophoraceae, Russulales) from which analogues of the cyclic peptide chlamydocin could be isolated (Toniolo et al., 2001).

Annotations of the N. crassa, A. fumigatus, A. nidulans and A. oryzae genomes predict gene functions based on homology to characterized genes and their products. It is easy to find genes encoding putative NRPSs and PKSs based on protein domain structures. Oxidoreductases, methylases, acetylases, esterases and transporters are not exclusive to secondary metabolism, so homology searches are more problematic for these genes. Nevertheless, if good candidate genes encoding these enzymes are found adjacent to signature secondary metabolic genes such as NRPS, PKS and DMATS homologues, there is an improved chance that they are involved in production of a secondary metabolite. Peptaibiotics typically contain high proportions of the non-proteinogenic amino acid Aib. As Aib is a strongly helix-promoting residue, all of the currently available peptaibiotics structures are primarily helical. This helical structure is the basis for the biological activity of these substances which can insert into membranes thereby forming pores and voltage-dependent ion channels. The resulting permeabilization of the plasma membrane leads to leakage of cytoplasmic material and cell death (Chugh and Wallace, 2001). The in vivo production and release into the host of peptaibiotics by a fungus growing on its natural host was proven by using Hypocrea pulvinata infecting basidiomes of the polypores Piptoporus betulinus and Fomitopsis pinicola as a model. As the identified hypopulvins showed structural analogies with other 18-, 19-, and 20-residue peptaibiotics, the formation of transmembrane ion channels by hypopulvins was suggested thereby supporting the parasitic lifestyle of H. pulvinata (Röhrich et al., 2012).

Peptaibiotics exert a broad range of biological activities depending on their chain length and particular structural features. They have been described to exhibit antibacterial activity primarily against gram-positive bacteria (Leclerc et al., 2001; Dornberger et al., 1995; Grafe et al., 1995), and display antifungal activity (Berg et al., 1996; Dornberger et al., 1995), antiviral activity (Kim et al., 2000; Yun et al., 2000), antimycoplasmic activities (Leclerc et al., 2001; Beven et al., 1998) and pigment induction in Phoma destructiva (Ritzau et al., 1997; Berg et al., 2003). Bioactivity includes also haemolysis of erythrocytes and leucocytes (Irmscher and Jung, 1977), insecticidal action on larvae (Matha et al., 1992; Bandani et al., 2001; Landreau et al., 2002), inhibition of the mitochondrial ATPase and uncoupling of oxidative phosphorylation in mitochondria (Krishna et al., 1990; Okuda et al., 1994; Gupta et al., 1991; Bullough et al., 1982). Furthermore, neuroleptic activities including induction of hypothermia in mice have been reported for ampullosporins and trichofumins (Ritzau et al., 1997; Kronen et al., 2001; Berg et al., 2003). For example, Alamethicin, is the most extensively studied peptaibiotics. Alamethicin was discovered as “antibiotic U-22324” from a culture broth of Trichoderma viride in 1967 by Meyer and Reusser (Degenkolb et al., 2008a). It consists of 20 amino acids with an acetylated N-terminus and the C-terminus being phenylalaninol (Phol) (Leitgeb et al., 2007). Recent studies on the interaction of alamethicin with oriented bicelles using electron paramagnetic resonance spectroscopy revealed an aggregation and orientation of alamethicin as a function of its concentration. Alamethicin shows broad biological activity including antibiotic properties against pro- and eukaryotic microbes, trypanosomes (unicellular protozoa), cytolytic activity in animal cells and elicitation of defense responses in plants (Leitgeb et al., 2007; Ishiyama et al., 2009).

Peptaibiotics show antibiotic activity against a range of prokaryotic and eukaryotic target organisms including Gram-negative and Gram-positive bacteria, cell wall- less bacteria (mollicutes), and fungi. In addition, cytolytic activity in mammalian cells has been reported and there is increasing support of peptaibols representing a novel class of plant defense elicitors (Szekeres et al., 2005). Several peptaibiotics such as alamethicin, atroviridins, trichorzianines, trichotoxins, and trichokonins have been shown to exert antibiotic activities against Gram-positive bacteria (e.g. Oh et al., 2002; Leitgeb et al., 2007; Chutrakul et al., 2008; Panizel et al., 2013). The emergence of microbes being resistant against standard antibiotics fosters the search for alternative antimicrobial substances. The antimicrobial effect of T. pseudokoningii trichokonin VI on the Gram-positive Bacillus subtilis was examined recently using atomic force microscopy. Trichokonin VI was found to alter the morphological and mechanical properties of the bacterial cells due its membrane-damaging activity which results in leakage of intracellular materials and subsequent changes in turgor pressure and collapse of the cell structure (Su et al., 2012). Alamethicin and trichorzin were found to have antibiotic activity against the mollicutes Acholeplasma laidlawii, Mycoplasma gallisepticum, M. genitalium, M. mycoides spp. mycoides, Spiroplasma apis, S. citri, S. floricola and S. melliferum thereby affecting the potential of the bacterial cell membrane (Béven et al., 1997 and 1998). There are several reports on the antifungal activities of peptaibiotics including trichotoxin (Hou et al., 1972), trichopolins (Fuji et al., 1978), paracelsin (Brückner et al., 1983), trichorzins, harzianins (Rebuffat et al., 1992), and trichokonins (Song et al., 2006) . The extensively studied alamethicin also displays antifungal activity and it was shown to permeabilize yeast mitochondria to NADH, other low molecular-weight hydrophilic compounds and even regulatory peptides (Leitgeb et al., 2007).

The regulation of fungal secondary metabolism for peptaibiotics production is a complex process and the genes involved in such processes can regulate the biosynthesis pathways at different levels. Global and pathway specific transcriptional regulation has been investigated and continues to be a great area of interest. It is necessary to understand why secondary metabolism is largely influenced by genes that encode global transcription factors. One of the challenges regarding these metabolites is to elucidate their true function in their native state. Fungi have produced secondary metabolites over millions of years either as elements to co-ordinate growth and development or to protect its species from the competition in the natural ecosystem. A more intricate knowledge of how different biosynthetic genes involved in the production of metabolites are expressed is required for further manipulation of such genes in silent clusters. An added layer of complexity resulting from cross talk between various fungal gene clusters implicates the possibility of simultaneous activation of different gene clusters resulting in a combinatorial synthesis of novel metabolites. A possibility which must be explored as a better understanding of these regulatory genes is required. The evidence of chromatin remodelling occurring during the expression of secondary metabolites genes has shown the importance of clustering of these genes. However, much more research is required into elucidating the mechanisms by which this chromatin remodelling is confined to certain gene clusters on the chromosomes and the signals that may be involved in such a process. Microarray analysis, genetic engineering and genomics have been the most important tools in understanding the entire regulatory process on a cellular level. More of these techniques are being employed to further investigate not only the model organisms such as Aspergilli but also other filamentous fungi and yeasts. Manipulating the genes expressed in the secondary metabolism in one organism or one species will help in understanding the biosynthetic pathways in similar or closely related species.

The most challenging goals concerning peptaibiotics are the elucidation of their true function in their native habitats and, closely associated with this, the identification of the physiological and ecological conditions that lead to the activation of secondary metabolism gene clusters. However, because the functions of these secondary metabolites are largely unknown, it is not yet possible to predict the regulatory networks that control their synthesis or, therefore, to develop strategies for the activation of their biosynthesis on a rational basis. Peptaibiomics will allow the determination of peptaibiotics biosynthesized by the multienzyme complexes (Raap et al., 2005) of a multitude of filamentous fungi grown on a single agar plate without time-consuming isolation and purification procedures. This method can be extended to analyze fungi growing in (sub)liquid media, or in natural habitats such as: soil, water, decaying organic material, dung, faeces, or as parasites of plants, mushrooms, toadstools and insects. The peptaibiome, i.e. the entire expression of peptaibiotics might be also considered a natural peptaibiotic library (Bruckner et al., 2005). Elucidating the molecular mechanisms regulating peptaibiomics will thus certainly contribute to an understanding of the significance of these compounds for the produc­ing organism and will further contribute to rational drug discovery by the activation of silent gene clusters. Why several peptaibiomics gene clusters do not contain pathway-specific regulators and seem to be exclusively regulated by global regulators remains to be determined. The observation of cross-pathway control between gene clusters adds another unexpected level of complexity. This needs to be considered in gene sequencing and activation approaches, but also when metabolites are assigned to biosynthesis gene clusters. Furthermore, this crosstalk implies the possi­bility of combinatorial biosynthesis — that is, the simul­taneous activation of more than one gene cluster by a pathway-specific regulator, leading to the production of novel compounds. It will be interesting to elucidate how genetic chromatin modification can be confined to specific gene clusters and which sig­nals are required to form a novel concept for the interaction of organisms at the molecular level. However, more development of a method is required which (i) provides sufficient and reliable diagnostic information on peptaibiotic production and (ii) enables a rapid analysis and differentiation among new and already known peptaibiotics. This is of major interest because a still growing number of peptaibiotics is discovered. In continuation of our work on screening fungi for the production of Aib-peptides (Psurek et al., 2005), we will present a chromatographic screening method which enables the rapid and sensitive detection and sequential characterization of this particular group of fungal peptides. The method is applicable directly onto filamentous fungi grown on single agar plates. The method comprises solid-phase extraction (SPE) followed by on-line reversed-phase high performance liquid chromatography (HPLC) coupled to electrospray ionization tandem mass spectrometry (ES-MS). In this project we will introduce an interdisciplinary approach which will co-relate gene sequencing, assembly, and comparative peptaibiomics with analyses of products. Gene sequencing will examine the roles of structural variation in the diversification of peptaibiotic gene clusters. Fungal strain specific differences in peptaibiomics expression will be investigated under different culture conditions and data will be summarised to develop a regulatory peptaibiotic database.