Extrolites of Aspergillus fumigatus and Other Pathogenic Species in Aspergillus Section Fumigati

Aspergillus fumigatus is an important opportunistic human pathogen known for its production of a large array of extrolites. Up to 63 species have been described in Aspergillus section Fumigati, some of which have also been reliably reported to be pathogenic, including A. felis, A. fischeri, A. fumigatiaffinis, A. fumisynnematus, A. hiratsukae, A. laciniosus, A. lentulus, A. novofumigatus, A. parafelis, A. pseudofelis, A. pseudoviridinutans, A. spinosus, A. thermomutatus, and A. udagawae. These species share the production of hydrophobins, melanins, and siderophores and ability to grow well at 37°C, but they only share some small molecule extrolites, that could be important factors in pathogenicity. According to the literature gliotoxin and other exometabolites can be contributing factors to pathogenicity, but these exometabolites are apparently not produced by all pathogenic species. It is our hypothesis that species unable to produce some of these metabolites can produce proxy-exometabolites that may serve the same function. We tabulate all exometabolites reported from species in Aspergillus section Fumigati and by comparing the profile of those extrolites, suggest that those producing many different kinds of exometabolites are potential opportunistic pathogens. The exometabolite data also suggest that the profile of exometabolites are highly specific and can be used for identification of these closely related species.


ASPERGILLUS TAXONOMY
Since 2011, all ascomycetous species can only have one name (Hawksworth et al., 2011;Hawksworth, 2012;McNeill et al., 2012). All species formerly included in Dichotomomyces, Cristaspora, Phialosimplex, Polypaecilum, in addition to Penicillium inflatum, have been formally combined into Aspergillus Samson et al., 2014). Furthermore, all species of Eurotium, Emericella, Chaetosartorya, Fennellia, Neocarpenteles, Neopetromyces, Neosartorya, Petromyces, Saitoa, and Stilbothamnium have also been transferred to Aspergillus . Ascoma producing species in section Fumigati were originally described under the name Neosartorya , but have now all been transferred to Aspergillus . Several of the species originally thought to produce only the asexual state have later been shown to be able to produce mature ascomata when crossed with the opposite mating type, for example A. fumigatus (O'Gorman et al., 2009) and A. lentulus (Swilaiman et al., 2013). Other opportunistically pathogenic species such as A. flavus (Horn et al., 2009), A. tubingensis (Horn et al., 2013), and A. terreus Arabatsis and Velegraki, 2013) can also produce mature ascomata when crossed with the opposite mating type. All species in Aspergillus and Penicillium have now been placed in the family Aspergillaceae (Houbraken and Samson, 2011). Species in Aspergillus section Fumigati are both phenotypically and genotypically distinct (Raper and Fennell, 1965;Geiser et al., 1998;Hong et al., 2005Hong et al., , 2006Hong et al., , 2008Katz et al., 2005;Geiser et al., 2007;Samson et al., 2007;Yaguchi et al., 2007). Aspergillus lentulus was originally claimed to be a sibling species of A. fumigatus, but was later shown to be phenotypically very different from A. fumigatus, especially concerning extrolite profiles (Larsen et al., 2007;Tamiya et al., 2015). The species A. pseudofelis, A. parafelis, and A. pseudoviridinutans have not been examined chemically, but they are very close phylogenetically and morphologically to A. felis and may be real sibling species with no phenotypic differences (Sugui et al., 2014). The 63 species listed in Table 1 are all those that have been described in Aspergillus section Fumigati and Neosartorya, but some of them are not yet available for the scientific community, so their identity and probably synonymy with other species is unknown. Samson et al. (2007) indicated that several species were synonyms of already known species in Aspergillus section Fumigati and Neosartorya. Thus the total number of species in Fumigati may be less than 63.
Aspergillus section Clavati contains species mostly associated to dung, and have not been reported to cause infections of vertebrate lungs . Species in Aspergillus section Clavati produce several bioactive extrolites, but few of these are found in Aspergillus section Fumigati. Examples of such Aspergillus section Clavati specific extrolites include patulin, cytochalasin E and K, antafumicins, expansolides, and clavatols, and these extrolites may be important for competition in a dung habitat, rather than in the compost habitats in which species of Aspergillus section Fumigati thrives. Some similar extrolites are in common between species in Aspergillus sections Fumigati and Clavati, however. Ribotoxins like the sarcins in Aspergillus section Clavati (Varga and Samson, 2008) are closely related to mitogillin and restrictocin in Aspergillus section Fumigati (Kao et al., 2001;Schwienbacher et al., 2005;Virágh et al., 2014). Furthermore, some tryptoquivalins are produced by species in both Aspergillus sections.
Like other filamentous fungi, A. fumigatus isolates produce extrolites in a species specific manner (Larsen et al., 2005;Frisvad et al., 2008), but some strains do not produce all the extrolites expected. This weaker exometabolic vigor is most pronounced in isolates directly isolated from patients Tamiya et al., 2015). These isolates are often floccose and less strongly sporulating. However, isolates from natural habitats, such as compost, always sporulate heavily and 1 | Species in Aspergillus section Fumigati and their extrolite production (species written in bold are known to be pathogenic to humans and/or other mammals).
produce most of the expected species specific extrolites Tepsic et al., 1997;Hong et al., 2010a,b). Production of small molecule extrolites is depending on the growth conditions and the growth media (Nielsen et al., 2011;Frisvad, 2012;Brakhage, 2013), and some of these extrolites may need biological / chemical stimulants of the producing fungus to be expressed (Brakhage and Schroeckh, 2011;Zuck et al., 2011;Netzker et al., 2015).
Being species specific, the difference between the extrolites profiles of different species of Aspergillus section Fumigati can be used in identification of the species in Aspergillus section Fumigati as an alternative to sequence-based or MALDI-TOF based identification (Panda et al., 2015), or used together with morphology and physiology in a polyphasic identification approach . For example A. fumigatus can be distinguished from A. lentulus by exometabolite profiling (Larsen et al., 2007), MALDI-TOF (Verwer et al., 2014), and sequencing (Balajee et al., 2005a;Samson et al., 2007), but only partially by morphology and Raman spectroscopy (Verwer et al., 2014).

EXTROLITES PRODUCED BY ASPERGILLUS FUMIGATUS AND OTHER PATHOGENIC SPECIES IN FUMIGATI
A. fumigatus has been reported to produce many different extrolites that are bioactive and may contribute to infection in humans and other animals (Amitani et al., 1995;Tomee and Kauffman, 2000;Reeves et al., 2006;Cramer et al., 2009;Abad et al., 2010;Coleman et al., 2011). Melanins are polyketide derived conidium pigments that may have an influence on the infection process (Tsai et al., 1998;Jahn et al., 2000;Tsai et al., 2001;Langfelder et al., 2003). Since all species in Aspergillus section Fumigati produce green conidia, it is expected that they all contain melanin (Perrin et al., 2007). Another more general small molecule pathogenicity factor is siderophores, of which A. fumigatus produces fusarinine C and triacetylfusarinine C extracellularly (Haas, 2014;Petrik et al., 2014). Furthermore hydrophobins are also present in all species of Aspergillus section Fumigati (Geiser et al., 1998;Pedersen et al., 2011). These proteins will protect conidia from being recognized by the immune system in mammals (Aimanianda et al., 2009). Other proteins, especially proteases also play a role in the infection process and may be expected to be produced by many pathogenic species (Tomee and Kauffman, 2000;Abad et al., 2010;Dhingra et al., 2012). Small molecule siderophores are also considered to be important pathogenicity factors, and given the general importance for fungi they can be expected to be produced by all pathogenic species of Aspergillus Abad et al., 2010;Haas, 2014), but probably also by non-pathogenic species.
However, other extrolites are not produced by all species in Aspergillus section Fumigati. Gliotoxin has long been known to be important for the infection process by inhibiting the immune response, phagocytosis and angiogenesis (Watanabe et al., 2003(Watanabe et al., , 2004Tsunawaki et al., 2004;Bok et al., 2005;Lewis et al., 2005;Stanzani et al., 2005;Coméra et al., 2007;Sugui et al., 2007;Ben-Ami et al., 2009;Abad et al., 2010). Gliotoxin has been reported from the pathogenic species A. fumigatus and A. thermomutatus, but also from A. denticulatus, A. ferenczii and A. turcosus ( Table 1) the latter three not yet known to be pathogenic. Annotation of the genomes of A. fumigatus and A. fischeri indicates that the latter species can also produce gliotoxin given the right conditions (Inglis et al., 2013). However, many other Aspergillus section Fumigati extrolites appear to be involved in pathogenesis. Verruculogen, produced by A. fumigatus and A. fischeri, modifies electrophysical properties of the human nasal epithelial cells (Khoufache et al., 2007) but is also a potent tremorgen (Land et al., 1993;Kelman et al., 2004). Verruculogen and fumitremorgin C (Rabindran et al., 2000) are produced by A. fumigatus and A. fischeri (Table 1) in section Fumigati. Fumagillin suppresses the immune response, neutrophil function and angiogenesis (Fallon et al., 2010(Fallon et al., , 2011 and is produced by the pathogenic species A. felis, A. fumigatus, and A. udagawae, but also by species in Aspergillus section Fumigati, such as A. aureolus and A. viridinutans that have not been reported as yet to be pathogenic ( Table 1). Pseurotin A is an inhibitor of immunoglobulin E and is responding to hypoxia (Schmeda-Hirschmann et al., 2008;Ishikawa et al., 2009;Vödisch et al., 2011). Pseurotins are produced by the pathogenic A. fumigatus and A. spinosus, but are also produced by A. duricalis and A. aureolus ( Table 1). Sulochrin inhibits eosinophil activation (Ohashi et al., 1997(Ohashi et al., , 1998 and is produced by four pathogenic species in section Fumigati: A. felis, A. fumigatus, A. lentulus, and A. udagawae ( Table 1). The related asterric acid is produced by the same species and this extrolite inhibits vascular endothelial growth factor induced tube formation (Lee et al., 2013). Another related extrolite is trypacidin, which is cytotoxic (Gauthier et al., 2012), but was originally isolated as an antiprotozoan metabolite (Balan et al., 1963). The fumiquinazolins are also cytotoxic (Lim et al., 2014), and are produced consistently by A. fumigatus (Frisvad et al., 2009). The fumiquinazolines (Takahashi et al., 1995) are produced by the pathogenic A. fumigatus and A. lentulus, but are also produced by A. aureolus ( Table 1). The chemically similar fiscalins and cottoquinazolins (norfumiquinazolins; Ames and Walsh, 2010;Shan et al., 2015) are produced by A. fischeri, indicating that these metabolites are of importance for the competitiveness of these fungi. The pyripyropenes have antiangiogenic activity (Hayashi et al., 2009) and are produced by nearly all the known pathogenic species in section Fumigati: A. fumigatus, A. fumigatiaffinis, A. fumisynnematus, A. lentulus, A. thermomutatus, and A. udagawae ( Table 1). In addition pyripyropens are produced by A. similanensis, a species that has not yet been tested for pathogenicity or isolated from any animal tissues.
Helvolic acid has been reported as an antibiotic and antifungal extrolite (Rementeria et al., 2005), but it also has been reported to affect human respiratory epithelium (Amitani et al., 1995) and the metabolism of macrophages (Shinohara et al., 1992). Helvolic acid has been reported from Aspergillus auratus, A. aureolus, A. felis, A. fischeri, A. fumigatiaffinis, A. fumigatus, A. multiplicatus, A. novofumigatus, A. thermomutatus, A. tsunodae, and A. udagawae. It is upregulated with gliotoxin in A. fumigatus (O'Keeffe et al., 2014). Thus helvolic acid may also be a pathogenicity factor, but of the species listed above A. auratus, A. aureolus, A. multiplicatus, and A. tsunodae have not been reported as pathogenic. Among bioactive proteins it seems that mitogillin is playing a role in the infection process (Schwienbacher et al., 2005;Abad et al., 2010), but these ribotoxins have not been screened in the other 62 species in Aspergillus section Fumigati.
Several small molecule extrolites have not yet been claimed to be involved in pathogenesis. The fumigaclavines are produced by the pathogenic species A. felis, A. fumigatus, A. fumigatiaffinis, and A. lentulus, but are also produced by A. fennelliae and A. ferenczii ( Table 1). Even though these ergot alkaloids are associated with conidiation in A. fumigatus (Coyle et al., 1981), their role in animal pathogenesis is unknown. The fumigatins have mostly been found in soil-borne strains of A. fumigatus , and may rather have a role in competitiveness in compost and soil, than in animal pathogenesis.

PREDICTION OF OTHER POTENTIAL OPPORTUNISTIC PATHOGENIC SPECIES IN ASPERGILLUS SECTION FUMIGATI BASED ON EXTROLITES
Among the 63 species described in Aspergillus section Fumigati, 17 have until now been reported to be opportunistic pathogens of vertebrate animals (in bold, Table 1). Several extrolites have been shown to have a certain role in the infection process, but these extrolites may have a different role in the natural habitats of these fungi, of which plant compost may be the primary habitat (Latgé, 1999;Abad et al., 2010). It appears that when growing on plant compost these fungi need a certain profile of extrolites (small molecule extrolites and exoproteins), while as vertebrate opportunistic pathogens they may need quite a different profile of extrolites (Abad et al., 2010). For example cellulases would be important in the compost situation (Srivastava et al., 2014;Miao et al., 2015), while hemolysins are probably only important for the vertebrate infection process (Abad et al., 2010). The same would be the case for antifungals and antibiotics, especially anti-streptomycete metabolites, as A. fumigatus and other members of Aspergillus section Fumigati are thermotolerant / thermophilic species competing with other thermotolerant and thermophilic species of fungi and bacteria (Langarica-Fuentes et al., 2014). Several species, such as A. assulatus, A. australensis, A. brevipes, A. "coreanus," A. duricaulis, A. fennelliae, A. galapagensis, A. neoglaber, A. marvanovae, A. nishimurae, A. papuensis, A. quadricinctus, A. solicola, A. spathulatus, A. tatenoi, A. unilateralis, A. viridinutans, and A. waksmanii produce few if any of the extrolites suspected to play a role in the infection process, and so would not be predicted to be potential opportunistic pathogens of vertebrates. Some species, such as A. auratus, A. denticulatus, A. similanensis, A. tsunodae, and A. turcosus only produce one of the extrolites believed to play a role in pathogenesis, and may or may not be prospective vertebrate pathogens. Finally A. aureolus, A. ferenczii, and A. siamensis produce several of the extrolites potentially involved in pathogenesis, and thus may be predicted to be potential opportunistic vertebrate pathogens.
The many extrolites that have been suspected to be pathogenicity factors and are produced by species in Aspergillus section Fumigati are biosynthetically derived from polyketides, amino acids, terpenes, shikimic acid or are of mixed biosynthetic origin. The formula of some of the most important extrolites common in Aspergillus section Fumigati are shown in Figure 1. Some of the extrolites are not produced in the same patterns in different species in Aspergillus section Fumigati. While A. fumigatus produces fumiquinazolins A-G, J, and K, A. fischeri produces the related norfumiquinazolins (Shan et al., 2015). These extrolites may have the same function, even though they are chemically somewhat different. Whether such proxyextrolites have the same function for pathogenicity in vertebrates is unknown. It is known, however, that other opportunistic pathogenic aspergilli in other sections of Aspergillus produce secondary metabolites that are biosynthetically and functionally closely related. While A. fumigatus, A. thermomutatus, and other species in section Fumigati produce gliotoxin, A. flavus in Aspergillus section Flavi can produce aspirochlorine and A. terreus in Aspergillus section Terrei can produce acetylaranotin (Frisvad and Larsen, 2015). While not identical to gliotoxin, these epidithiodioxopiperazines could be predicted to play a role in pathogenicity of A. flavus and A. terreus. The reports that A. niger, A. flavus, and A. terreus could produce gliotoxin Kupfahl et al., 2008) have not been confirmed Varga et al., 2011a,b).
Close phylogenetic relationships seem to be less suited pathogenicity predictors. For example A. viridinutans seems to be non-pathogenic, while the closely related A. felis is pathogenic ( Barrs et al., 2013;Novaková et al., 2014;Sugui et al., 2014). Good growth at 37 • C also seems to be a contributing factor to pathogenicity, and for example A. brevipes, A. duricaulis and A. viridinutans grow relatively poorly at 37 • C, and in addition are not considered potentially pathogenic Aspergillus species in section Fumigati, based on extrolite evidence and absence of reports of pathogenicity. However, while there a many data on the involvement of exometabolites for A. fumigatus (Abad et al., 2010), there are few data on production of exoproteins for other opportunistic pathogenic species such as A. thermomutatus.
Genome sequencing and systematic comparison of the genomes and transcriptomes of other members of Aspergillus section Fumigati may help in predicting which pathogenicity factors are especially important (Galaghan et al., 2005;Nierman et al., 2005;Wortman et al., 2006;Fedorova et al., 2008;McDonagh et al., 2008;Chooi et al., 2013;Inglis et al., 2013;Cerqueira et al., 2014;Kusuya et al., 2015;Lind et al., 2015). These data should be compared to phenotypic data such as profiles of large and small molecule extrolites, growth temperatures, carbon dioxide tolerance etc.
Altogether, approximately one third of the species in Aspergillus section Fumigati are common pathogenic species, one third are rare species of unknown pathogenicity and one third are predicted to be non-pathogenic, based on their production of relatively few exometabolites. Exometabolite pathogenicity factors found in the successful opportunistic pathogenic fungus A. fumigatus may have proxy-exometabolites with the same function in other species in that section, but also in less closely related pathogenic Aspergilli, especially species from sections Nigri, Terrei, Circumdati, and Flavi.