Researchers have identified how a fungal infection triggers inflammation


Scientists at St. Jude Children’s Research Hospital have identified the mechanisms behind inflammasome activation driven by infection with the fungal pathogen Aspergillus fumigatus.

Fungal infection, especially with A. fumigatus, is a leading cause of infection-associated deaths in people with compromised immune systems.

The work provides clues to a potential therapeutic approach for treating infectious and inflammatory disorders. The findings were published online today in Nature.

“Inflammasomes are important sentinels of an organism’s innate immune defense system,” said corresponding author and founding member of the inflammasome field Thirumala-Devi Kanneganti, Ph.D., of the St. Jude Immunology department.

“Our prior work showed that fungal pathogens activate the inflammasome, but the exact mechanism of action for inflammasome engagement was unknown.”

To understand these mechanisms for A. fumigatus, the scientists looked for pathogen-associated molecular patterns, which can stimulate the innate immune response by activating the inflammasome.

The scientists focused on NLRP3, the most-studied inflammasome sensor.

The research identified galactosaminogalactan (GAG), a novel fungal pathogen-associated molecular pattern.

GAG is essential for A. fumigatus-induced NLRP3 inflammasome activation. The scientists showed that A. fumigatus deficient in GAG fail to induce inflammasome activation.

Conversely, over-production of GAG by A. fumigatus increases inflammasome activation.

Additionally, inflammasome activation is critical for clearing A. fumigatus infections in animals. The A. fumigatus fungal strain that failed to produce GAG was more virulent in mice, while the strain that over-produced GAG was less virulent.

Similarly, inflammasome activation is protective during gut inflammation in a mouse model of colitis, an inflammatory disease. Treatment with purified GAG provided protection against colitis.

“We showed that protection against this inflammatory disease was dependent on the ability of GAG to induce inflammasome activation,” said first author Benoit Briard, Ph.D., formerly of St. Jude Immunology. “These findings demonstrate the mechanism for the therapeutic potential of GAG in inflammatory diseases.”

Aspergillus fumigatus is the dominating species causing fungal lung diseases in humans and animals [1-6]. Other species in Aspergillus section Fumigati and its teleo- morphic (sexual) state Neosartorya are also able to cause aspergillosis, however.

These species include A. lentulus [7], N. pseudofischeri [8,9] and N. udagawae [10]. While Neosartorya species produce both a sexual state with ascospores and an asexual state with conidiospores, the Aspergillus species only produce conidiospores.

Several new species have recently been described in section Fumigati and Neosartorya [11-14], and an overview of the 23 species of Neosartorya and 10 species in Aspergillus section Fumigati is provided by Samson et al. [14]. It is well known that isolates of A. fumigatus are able to produce many extrolites [14-17], but of high importance is gliotoxin, that has been found in lungs or other infected tissues.

Gliotoxin was found after experimental aspergillosis [18], and has also been found naturally occurring in turkey lungs infected with A. fumigatus [19]. Gliotoxin has also been found in a bovine udder infected with A. fumigatus [20] and in human tissues [21].

Gliotoxin may not be the only mycotoxin involved in mycosis [22] as several other extrolites have been reported from A. fumigatus [1,15,16,23-26]. The anti- biotic fumigacin (which was later shown to be a mixture of helvolic acid and gliotoxin) has also been found in human and animal pulmonary tissues [27-30].

Two isolates of A. fumigatus have been full-genome sequenced and arrays are being developed for this species [31-33]. The closely related species Neosartorya fischeri is also being full-genome sequenced and this species and another closely related species A. lentulus and several new species we have described, can be compared with, and used as controls, for A. fumigatus, as their extrolite profiles are also known [11].

There are still extrolites that have not been identified iN A. fumigatus and allied species [34-36], but a large number of extrolites are well characterized. Many extrolites from A. fumigatus are associated with the conidiospores, including gliotoxin, trypacidin, verruculogen and fumigaclavine A [37-41] and are thus likely to have effects in the initial lung infection process.

The global regulator gene laeA appear to have an effect on conidial morphology, including associated extrolites such as hydrophobins and other extrolites [42-44].
It is the purpose of this paper to list, update and revise the profile of extrolites associated with A. fumigatus and to analyse 40 strains of A. fumigatus to examine the chemo-consistency in this species.

Table 1 Isolates of Aspergillus fumigatus examined Isolate     Source

  • CBS 542.75 =ATCC 26606                      Man, USA
  • CBS 143.89                                              Man, France
  • CBS 144.89 =CEA10*                              Man, France
  • CBS 145.89                                              Man, France
  • CBS 146.89                                              Man, France
  • CBS 147.89                                              Man, France
  • IBT Stend1                                               Man, hospital Sundby, Denmark
  • IBT Stend2                                               Man, hospital Hvidovre, Denmark
  • Af293*                                                     Man, United Kingdom
  • CBS 133.61T=ATCC 1022                       Chicken lung, Connecticut, USA
  • IBT 16904                                                Saltern,Slovenia
  • IBT 16902                                                Saltern, Slovenia
  • IBT 16903                                                Saltern, Slovenia
  • IBT 16901                                                Saltern, Slovenia
  • IBT 24004                                                Saltern, Slovenia
  • ATCC 32722                                            Soil, Canada
  • NRRL 1979 =IBT 15720                          Soil, USA
  • CBS 113.26 =ATCC 1028                        Soil, Germany
  • CBS 132.54 =QM 6b                                Soil
  • CBS 457.75 =WB 5452                            Soil, India
  • CBS 151.89                                              Stone, Germany
  • CBS 152.89                                              Stone, Germany
  • NRRL 5587                                              ?
  • IBT D47i                                                  Soil, Indonsia
  • CCRC 32120                                            Soil, Taiwan
  • IBT 25732                                                Soil under banana tree, Kenya
  • IMI 376380=IBT 23720                          Unknown (reported ochratoxin A producer)
  • CBS 545.65 =ATCC 16913                      Unknown
  • WB 5033 =IBT 22612                              Unknown (white conidia)
  • IBT 23737                                                Unknown, Denmark
  • IBT 14904 =ATCC 32722                        Unknown, USA
  • CBS 192.65 =IHEM 4392                        Feed, Netherlands
  • CBS 148.89                                              Maize, France
  • CBS 149.89                                              Maize, France
  • CBS 150.89                                              Beetroot, France
  • IBT 21997 =A195                                    Feed, Spain (reported ochratoxin A producer)
  • IBT 22023                                                Silage, Germany
  • IBT PerHag                                              Silage, Sweden
  • IBT 22234                                                Ex tea factory, Uganda
  • IBT 21711                                                Food, Italy

*Full genome sequenced strains.

Materials and methods

Many isolates of Aspergillus section Fumigati were examined for extrolite profiles using HPLC and MS methods, and few isolates of A. fumigatus and A. lentulus were specifically screened for sphingofungins.
A. fumigatus isolates from different sources were emphasized (Table 1). All isolates were inoculated on Czapek yeast autolysate (CYA) agar, yeast extract sucrose (YES) agar, malt extract autolysate (MEA) agar, Oat meal (OAT) agar at 258C and on CYA at 378C (see Samson et al. [45] for formulae). Secondary metabolites were extracted from CYA and YES agar after 7 days of growth in darkness, using the extraction solvent ethyl acetate/dichloromethane/methanol (3:2:1, v/v/v) with 1% (v/v) formic acid.
HPLC with diode array detection and high resolu- tion mass spectrometric detection (HPLC-DAD- HRMS), was performed on an Agilent 1100 system with a Luna C18 II column (Phenomenex, Torrance, CA) and equipped with a photo diode array detector (DAD), and coupled to a LCT orthogonal time-of- flight MS (Waters-Micromass, Manchester, UK), with a Z-spray ESI source and a LockSpray probe [46]. Furthermore all isolates were analyzed by HPLC-DAD using the method of Frisvad and Thrane [47,48] as modified by Smedsgaard [49].
Samples were analyzed in positive and negative electrospray interphase (ESI+ and ESI-) using a water-acetonitrile linear gradient system starting from 15% acetonitrile which was increased to 100% in 20 min and holding 100% for 5 min [50]. In both ESI+ and ESI- two scan functions (1 s each) were used: the first with a potential difference of 14 V between the skimmers scanning m/z 100 to 900; the second with 40 V between the skimmers scanning m/z 100-2000.
Data analysis was performed as described previously [50], peaks were matched against an internal reference standard database (~730 compounds), the 33557 compounds in Antibase 2007 [51], previous data from our group [17], and the review data in this paper.

The profile of extrolites produced by A. fumigatus has to be collected from several literature sources in conjunction with actual metabolic profiling of a series of isolates of A. fumigatus.

The data obtained here were verified by comparison with authentic standards, similar UV and high resolution mass spectra and literature data. We examined the isolates of A. fumiga- tus for all main secondary metabolites that have been reported in the literature, often reported from one isolate only.

In this way we confirmed that isolates of A. fumigatus produced some secondary metabolites consistently, others by approximately half of the isolates, and some reported secondary metabolites were appar- ently not produced by A. fumigatus (Table 3).

It is well known that the production of extrolites is depending on the growth medium and environmental factors [52-54], but on the media Czapek yeast autolysate (CYA) agar and yeast extract sucrose (YES) agar a large number of these representatives of the 24 families of extrolites are detectable using HPLC with diode array detection [24].

The extrolites most consistently produced were fumi- quinazolines A/B, C/D and F/G (100%), gliotoxin (38%), fumigaclavine C (100%), fumitremorgins (100%), fumagillin (100%), helvolic acid (98%), pseurotin A (100%), fumigatins (35%), chloroanthraqui- nones (70%), melanins (100%, only verified, however, by observing that the bluegreen pigment is produced by all the isolates), and pyripyropenes (48%) (Table 3). The growth conditions and the incubation time chosen may not have been optimal for production of all extrolites by A. fumigatus.

  • Epoxysuccinic acid and difructosedianhydride (tricarboxylic acid cycle)

Epoxysuccinic acid seems to be the major organic acid produced by A. fumigatus [55,56], whereas production of citric acid appears to be weak [57]. The role of epoxysuccinic acid in the life cycle of A. fumigatus is unknown. Atypical carbohydrates, such as difructose dianhydride may also be produced by A. fumigatus [58]. We did not examine any of the cultures for these two metabolites, as the detection method was not adequate

for these particular metabolites.

  • Fumigatins (polyketides)

The fumigatins and spinulosins consist of at least 21 polyketide extrolites (Table 1) and have been thoroughly studied concerning their biosynthesis [59-69]. Fumiga- tin and spinulosin are reported to be antibiotically active against several gram-negative and gram-positive bacteria [23,28] and fumigatin was cited to be toxic against experimental animals by Austwick [1], while Cole and Cox [23] claimed that vertebrate toxicity was unknown. However, later it was shown that fumiqui-

nones A and B, in the same biosynthetic family, are toxic to other kinds of animals (nematodes) [70]. Antibacterial and antinematodal activity of the fumi- gatins and spinulosins can maybe explain their pre- valence in soil isolates of A. fumigatus.

We found fumigatin in several isolates (35%), but it was most common in soil-borne A. fumigatus (Table 3). However, one isolate from a patient produced fumigatin.

  • Trypacidins (polyketides, nitrogen in one extrolite)

Trypacidin [71-73] and monomethylsulochrin [15,71,73] were isolated and their structure elucidated by Turner [71] and Balan et al. [73]. Trypacidin is antiprotozoal and also an antimicrobial antibiotic [37,74]. Two other related extrolites, asperfumin, and the nitrogen containing asperfumoid has also been detected in A. fumigatus [15]. Trypacidin and mono- methylsulochrin has been found in all isolates examined of A. fumigatus [11].

Most isolates (75%) examined by us produced trypacidin and monomethylsulochrin (Table 3). Isolates producing these metabolites also produced the chlor- oanthraquinones (Table 3).

  • Chloro-anthraquinones or anthrones (polyketides)

Emodin, physcion [15], 2-chloro-emodin, 2-chloro-citreor- osein, 2-chloro-1,3,8-trihydroxy-6-methyl-9-anthrone, and 2-chloro-1,3,8-trihydroxy-6-hydroxymethyl-9-anthrone have been reported from A. fumigatus [75]. These polyketides have not been reported to have a role in the infection process.

We found UV spectrum evidence for production of several of the anthraquinone metabolites in many strains of A. fumigatus (Table 3).

  • Melanins (polyketides)

Aspergillus fumigatus is able to produce polyketide derived melanins via a heptaketide shortening from YWA1 to 1,3,6,8-tetrahydroxynaphthalene (1,3,6, 8-THN), which is the basis for production of 1, 8-dihydroxynaphthalene, the pentaketide compound that will polymerize to melanin [76-78], giving A. fumigatus the green conidium colour. Melanin  has been mentioned as one of several potential virulence factors in A. fumigatus [79,80].

As all isolated had blue-green conidia, they probably have the ability to produce this 1,8-dihydroxynaphtha- lene derived polymer.

  • Sphingofungins and fumifungin (polyketides+alanine)

The sphingofungins A-D from A. fumigatus ATCC 20857 (from soil in Uruguay) are antifungal agents [81,82] and are potent and specific inhibitors of serine palmitoyl transferase, an enzyme essential in the the biosynthesis of sphingolipids. Paecilomyces variotii produces sphingofungins E and F [83]. Fumifungin

[84] was isolated from what was probably A. viridinutans, as the fungus also produced viriditoxin, but sphingofungins may also be produced by A. fumigatus sensu stricto. These metabolites share a similar backbone to the carcinogenic mycotoxins the fumonisins produced by Fusarium species and an Aspergillus species, A. niger [85] and may thus be potential inhibitors of human nerve cells. Fumonisins have been shown to cause pulmonary edema in pigs [86] and down-regulates basal IL-8 expression in pig intes- tine [87] and therefore sphingofungins may be likely candidates to be involved in the lung infection process, also in humans.

We examined two isolates from section Fumigati for

production of sphingofungins: the full genome se- quenced A. fumigatus Af293 and A. lentulus IBT 27201. HPLC-MS data strongly indicated that both species are able to produce these compounds (Fig. 2). A viriditoxin producer that also produced fumifungin [84] was probably not A. fumigatus or A. lentulus, as none of these species are able to produce viriditoxin. The fumifungin producing strain could have been A. viridinutans, Neosartorya aurata, or N. denticulata as these three species produces viriditoxin [14].

  • Pseurotins (mixed biosynthetic origin: polyketide+ phenylalanine)

The pseurotins were first isolated from Pseudeurotium ovalis (pseurotins A, B, C, D and E) [88-91], but were later isolated from A. fumigatus (pseurotin A, 8-O- demethylpseurotin, pseurotin F1 and F2 and synerazol) [92,93]. These compounds are chitin synthase inhibi- tors, but only the epoxy-pseurotin, synerazol, has antifungal activity. It is not known whether the pseurotins have biological activities of relevance  for the lung infection process. The closely related com- pound azaspirene has been isolated from a Neosartorya species [94].

We found that pseurotin A was produced by all 40 strains examined of A. fumigatus, but some additional pseurotins, as identified based on UV-VIS spectra, were often produced at the same time.

  • Ergosterols (triterpenes)

Apart from ergosterol, produced by all fungi, A. fumigatus has been reported to produce ergosterolpal- mitat, ergosterolperoxide [95], ergosta-4,6,8(14),22.

-tetraen-3-one, ergosta-4,22-diene-3b-ol, 5a,8a-epi- dioxy-ergosta-6,22-diene-3b-ol [15]. Ergosterolperoxide has some antiviral properties [96].

We found ergosterol in all 40 isolates of A. fumigatus examined, but did not screen for the other ergosterol derived compounds.

  • Helvolic acids (triterpenes)

Helvolic acid [15,97-101] is an antibiotic that is active against both gram-positive and gram-negative bacteria. Other products in the biosynthetic family, such as helvolinic acid and 7-desacetoxyhelvolic acid have been isolated from Sarocladium oryzae [102,103], but not yet from A. fumigatus. The fusidic acids may also be closely related, but has not been found in A. fumigatus [[104], pp. 264-265]. The reported toxicity of helvolic acid may be due to contamination with gliotoxin [1,23].

Helvolic acid was produced by nearly all strains (98%) we examined of A. fumigatus (Table 3).

  • Fumagillins (sesquiterpenes)

Trans-fumagillin was isolated from A. fumigatus by Eble and Hanson [105], and its structure elucidated by Tarbbell [106,107] and McCorkindale and Sime [108]. Further extrolites in this biosynthetic family have been isolated later, inclusive fumagiringillin [109], demethox- yfumagillol [110], Sch528647 [111], RK-95113 [112]

and closely related metabolites [113]. Ovalicin [114,115],  FR-111142  [116],  or  FR65814  [117] may

also be produced, but have been reported from other  fungi. The fumitoxins, toxic to both animals and plants [118-122] were never structure elucidated, but based on the chemical data presented, they appear to be mem- bers of the fumagillin biosynthetic family. b-transber- gamoten is a precursor of fumagillin [123].

We detected fumagillin in all strains of A. fumigatus

examined (Table 3).

  • Fatty acids (fatty acids) and hexahydroxyprenyls (polyterpenes)

A fumigatus has been reported to produce a series of hydroxypolyprenols [124], ubiquinones, phthioic acid and other lipids [125-127]. The role of these metabo- lites in the infection process is unknown, but other lipids (oxylipins) have been shown to be involved in virulence [128].

We did not screen for these lipids in the 40 extracts of

A. fumigatus.

  • Siderochromes (N-hydroxyornithine with either three glycines or one glycine and two serins)

Fusigen [129], ferricrocin and N?,N??,N???-triacetylfu- sarinine C [130,132] are important iron-chelating extrolites from A. fumigatus, that may play a significant role in the infection process in animals [133,134].

As the production requires special substrates de- pleted for iron, we did not examine the cultures for the siderophores in our screening process.

  • Gliotoxins (phenylalanine, m-tyrosine, methionine)

Gliotoxin was isolated from a strain of A. fumigatus by Johnson et al. [135] and Menzel et al. [98] and later structure elucidated [136]. Fumigacin [27,29] has been found in animal and human tissue, but fumigacin was later found to be a mixture of gliotoxin and helvolic acid. Gliotoxin has been claimed to be involved in diseases caused by A. fumigatus [40,137,138]. The less toxic bisdethiobis(methylthio)gliotoxin has also been reported from A. fumigatus [139,140] as has gliotoxin G, the tetrasulphide analogue of gliotoxin [139]. Other gliotoxins, including gliotoxin monoacetate [141,142], and gliotoxin E and G [143,144] may also be extrolites of A. fumigatus, but have been isolated from Tricho- derma virens and Penicillium lilacinoechinulatum (Fris- vad JC and Thrane U, unpublished).

Gliotoxin is best produced on media with low C/N ratio, so the media used here for screening of A. fumigatus extrolites were not optimal for its expression. When tested on such media [17] all isolates of A. fumigatus seems to be able to produce gliotoxin.

  • Fumigaclavins (tryptophane and terpene unit (dimethylallyl))

Agroclavine, festuclavine, elymoclavine, chanoclavine I, fumigaclavine A, B, and C [15,145-149] are produced by A. fumigatus. The biosynthetic genes for the ergot alkaloids in A. fumigatus have been studied by Coyle and Panaccione [150]; Li and Unso¨ ld [151]; Unso¨ ld and Li [152], Stack et al. [153].

All 40 isolates examined of A. fumigatus produced fumigaclavine C (Table 3), the end-product in the biosynthetic family.

  • Fumitremorgins, verruculogen, tryprostatins, cyclotrypostatins and spirotryprostatins (tryptophane, proline and terpene (dimethylallyl) groups)

Brevianamide F [154,155] is  a  conceived  precursor of the diverse biosynthetic family of fumitremorgins, including verruculogen [24,156-159], cyclotrypostatins [160], tryprostatins [161-163], spirotryprostatins [164,165], fumitremorgin A & B [140,166,172], fumi- tremorgin C [15,146,159,173], TR-2 [174], TR-3

=12,13-dihydroxyfumitremorgin C and demethoxyfu- mitremorgin C [162,163], 12,13-dihydrofumitremorgin C [159,175], and 15-acetoxyverruculogen [23]. In all, this extrolite family consists of 20 known members. Tryprostatin A is an inhibitor of microtubule assembly [176], and in general the fumitremorgins are cell cycle inhibitors and tremorgenic mycotoxins [23].

We found that the fumitremorgins (A, B, C), TR-2 and verruculogen were produced by all isolates of A. fumigatus examined (Table 3), but the full genome sequenced Af293 [31] only produce brevianamide F [17,177].

  • Simple diketopioperazines (two amino acids)

Alanyl-leucyl and alanyl-isoleucyl, prolyl-phenylalanyl, prolyl-glycyl, prolyl-prolyl, prolyl-valyl, 4-hydroxypro- lyl-leucyl, 4-hydroxyprolyl-phenylalanyl, and prolyl- leucyl diketopirazines, all consisting of L-amino acids, have all been reported from A. fumigatus [15,178,179]. Several of those are antibiotically active [15].

We did not detect any of those simple diketopiper- azines in A. fumigatus.

  • Pyripyropenes (meroterpenoids and nicotinic acid)

Pyripyropenes A-R have been reported from A. fumi- gatus [180-187]. The pyripyropenes have the ability to inhibit acyl-CoA:cholesterol acyltransferase and may thus play a role in the infection process.

We found that approximately half of the isolates of

A. fumigatus produced pyripyropenes (48%, Table 3). We did not detect those metabolites in Af293, but the other full genome sequence strain of A. fumigatus (CBS

144.89 =CEA 10) did produced pyripyropenes.

  • Fumiquinazolines (anthranilic acid, tryptophane, valine)

Fumiquinazolines A-E [140,188,189] were reported from marine isolates of A. fumigatus. These quinazolins have been reported to be moderately cytotoxic.

We found that the fumiquinazolines were consis- tently produced by all 40 isolates of A. fumigatus examined (Table 3).

  • Tryptoquivalines (anthranilic acid, tryptophane, valine, terpene unit (dimethylallyl))

The tryptoquivalines and tryptoquivalones were ori- ginally isolated from A. clavatus [190,191] but trypto- quivaline A and E to N has been reported from A. fumigatus [192-195]. It was later shown that A. fumigatiaffinis is a very efficient producer of (some of) these tryptoquivalines [11,14]. However, tryptoquiva- line J was isolated from a strain of A. fumigatus by Afiyatullov [159], so it is possible that also A. fumigatus can produce at least some of these extrolites.

The tryptoquivalins were not detected in our ana- lyses of 40 isolates of A. fumigatus. Earlier reports of tryptoquivalins [25] from A. fumigatus were apparently based on the fact that the UV-VIS spectra of the tryptoquivalins and the fumiquinazolines are quite similar. HPLC-HR-MS analysis showed that the major compounds with such UV spectra were all fumiquina- zolines.

  • N-(2-cis(4-hydroxyphenyl)ethenyl)-formamide

N-(2-cis(4-hydroxyphenyl)ethenyl)-formamide is a pla- telet aggregation inhibitor that was isolated from a strain identified as A. fumigatus [196].

We were not able to detect this extrolite in any of 40 extracts of A. fumigatus.

  • Restrictocins (polypeptides)

Restrictocin, mitogillin and ‘asp F1’, small basic proteins, are cytotoxins that cleave ribosomal RNA [197-199]. The culture originally examined (ATCC 34475 =NRRL 2869) was first identified as A. restric- tus, but later reidentified as A. fumigatus [200]. A leader sequence in the gene coding for these proteins protects the producer strains from suicide [201], and these proteins have also been identified as major allergens from the conidia, mycelium and culture filtrate of A. fumigatus [198]. ‘Asp F1’ was first found in urine of patients that suffered from invasive aspergillosis [197], so these compounds may be of significance in A. fumigatus mediated aspergillosis.

We did not screen A. fumigatus for any proteins in this study.

  • Volatile extrolites (including sesquiterpenes, alcohols and ketones)

Sesquiterpenes provisionally detected from from A. fumigatus   include   10(14)-(-)-aromadendrene,   bicy- cloelemene, bicyclooctane-2-one, camphene, a-cadi- nene, 2-carene, caryophyllene, a & b-curcumene, cyclooctene, dihydroedulane I, b-elemol, a-farnesene, trans-b-farnesene,  (-)-fenchol,  germacrene  A  &  B, italicene, a-longipenene, megastigma-4,6(E),8(Z)-tri- ene, p-mentha-6,8-dien-2-ol, 2-methyl-2-bornane, 2-methylenebornanene, a-muurolene, neo-allo-ocimene, and b-phellandrene (39). Other volatile metabolites reported include 2-acetyl-5-methylfuran, anisole, 3-cycloheptane-1-one, 2,3-dimethylbutanoic acid meth- yl ester, 2,5-dimethylfuran, 4,4-dimethylpentenoic acid methyl ester, dodecane, 4-ethylbutan-4-olide, 2-ethyl- furan, 2-ethyl-5-methylfuran, 1-ethyl-2-methylbenzene, furaneol, 3-hexanone, isopropylfuran, 1-methoxy- 3-methylbenzene, 2-methylbutanoic acid and its methyl ester, 4-methyl-2-(3-methyl-3-butenyl)furan, 3-methyl- 1-heptene, 6-methyl-2-heptanone, 2-methyl-2,4-hexa- diene, 2-methylphenole, 1-(3-methylphenyl)-ethanone, 3-octanone, 1,3,6-octatriene, styrene, 3,5,7-trimethyl- 2E,4E,6E,8E-decatetraene, 2,3,5-trimethylfuran (39). The role of these volatiles in the infection process of

A. fumigatus, if any, is unknown.

  • Primary metabolites

The vitamin riboflavin has also been found in A. fumigatus [202,203], and so has several other primary widespread primary metabolites.

  • Biotransformations

A. fumigatus is also capable of converting some plant metabolites for example melitolic acid to 4-hydroxy- coumarin and o-coumaric acid to dicoumarol [204] and phenylacetic acid to 2,6-dihydroxyphenylacetic acid [205]. It is also not known whether this ability to bioconvert metabolites play a role in the animal infection process.

  • Proteins

Apart from the restrictocins, A. fumigatus also produce hydrophobins and several extracellular enzymes and these do play a role in the fungal infection process [16,206].

Fig. 1 LC-MS BPI (base peak ion) ESI (electrospray ionization)+ trace of raw extract of Aspergillus fumigatus Af293 on CYA agar, depicting major and typical extrolites from the species. Box indicates window for the sphingofungins in this isolate. Sphingofungin A is one of four possible compounds with almost identical masses to be present, based on mass traces.
  • Extrolites erroneously reported from Aspergillus fumigatus

Aspergillus fumigatus has been claimed to produce a large number of mycotoxins and other extrolites, including ochratoxin A [207-210], indications of afla- toxin [211], cyclopiazonic acid [212], kojic acid [213,214], sterigmatocystin [215] and fumifungin+vir- iditoxin [84]. The isolates producing these mycotoxins and other biologically active extrolites appear to be misidentified. For example cyclopiazonic acid is pro- duced by A. lentulus and isolates of the latter species can have been mistaken for A. fumigatus [17]. The isolate reported to produce fumifungin also produced viriditoxin [84], and the latter is a typical metabolite produced by A. viridinutans, another member of Aspergillus subgenus Fumigati section Fumigati [14,216-218]. In the case of ochratoxin A, sterigmato- cystin and aflatoxin, probably the mycotoxin itself was misidentified.

Molecules that may be artefacts, such as GERI-

BP002-A [219] that is a sterol biosynthesis inhibitor, have been reported as extrolites of A. fumigatus. This compound may or may not be a real secondary metabolite.

Expansolide, antafumicin A & B, and cytochalasin E were all reported from a strain claimed to be A. fumigatus [220]. These extrolites have only been found

in A. clavatus [221], so it is highly probable that the reported producing strain represented the latter species. Ruakuric acid has been isolated from a strain of A. fumigatus growing in conjunction with a coral lichen in hot sulfurous springs, New Zealand [222]. We have not been able to examine this culture and we have not yet detected compunds with the characteristics of ruakuric acid from any strain of A. fumigatus sensu


Aurasperone C was reported from A. fumigatus by Mitchell et al. [36], but this metabolite is a common metabolite in Aspergillus section Nigri [223] and we have not been able to detect it in any strain of A. fumigatus in this study.

Fumigatonin was also reported from A.  fumigatus [224], but the isolation of the chemically related novofumigatonin from A. novofumigatus [35] indicates that fumigatonin is produced only by A. novofumigatus. None of the isolates of A. fumigatus examined here (Table 1) produced aflatoxins, antafumicins, cyclopia- zonic acid, cytochalasin E, expansolides, kojic acid,

ochratoxins, sterigmatocystin, or viriditoxin.

  • Extrolites of A. fumigatus Af293

Af293, the full genome sequenced strain of A. fumiga- tus [31] produced, fumigaclavines, fumiquinazolines, trypacidin and mono-methylsulochrin, fumagillins, gliotoxins, pseurotins, chloroanthraquinones, fumitre- morgins, verruculogen, helvolic acids and sphingofun- gins (Fig. 1). The presence of sphingofungins A, C, D, or fumifungin (or all of those) was indicated by HPLC- MS analysis (Fig. 2). The formulae of these extrolites and the other secondary metabolites produced by A. fumigatus are shown in Fig. 1 and Fig. 3.

Fig. 2 Comparison of A. lentulus and A. fumigatus production of sphingofungins. Though very different in general extrolite profile, both of these isolates produce the same profile of what appears to be sphingofungins. From the bottom: Af293 TIC (total ion count) ESI+ (CYA agar); IBT 27201 Aspergillus lentulus TIC ESI+ (CYA agar); 432.3090.05 Da mass trace for Af293 and IBT 27201 respectively (this mass fits both sphingofungin A (C21H41N3O6) and sphingofungin C, D and fumifungin (the latter three: C22H41NO7).
Fig. 3 Collection of single extrolites presenting the different compound classes, in addition to those in Fig. 2, produced by Aspergillus fumigatus.

reference link :DOI: 10.1080/13693780802307720

More information: Briard, B., Fontaine, T., Samir, P. et al. Galactosaminogalactan activates the inflammasome to provide host protection. Nature (2020).


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