BACKGROUND
aspergillus fumigatus is associated with many human health conditions  <cit> .
it is the most common cause of invasive aspergillosis, an opportunistic infection whose incidence increases in immunocompromised patients  <cit> .
aspergillosis is also prevalent in animals, such as dogs and birds  <cit> .
a. fumigatus is responsible for more human and animal diseases than other moulds even though their conidia are usually outnumbered by spores of other mould species in inhaled air, suggesting that a. fumigatus has special properties that enable it to colonize the lungs of its hosts  <cit> .
most studies with a. fumigatus have focused on the interactions of the fungus with alveolar macrophages  <cit>  or with cell line a <dig> from pneumocytes  <cit> .
however, the first tissue usually encountered by the inhaled conidia is the airway epithelium, and it is frequently damaged in patients at risk for invasive aspergillosis  <cit> .
we previously studied the interactions between a. fumigatus and the respiratory epithelium using human nasal epithelium cell  cultures  <cit> .
after one week of culture, the hnec are organized in a pseudostratified epithelium with mucus and ciliated cells, very similar to the in vivo airway epithelium  <cit> .
we found that a filtrate from a. fumigatus cultures has specific electrophysiological effects on hnec compared with one from fungi not responsible for invasive infection  <cit> .
effects included a decrease in transepithelial electrical resistance and polarization of the epithelium.
these changes to the epithelium properties could play a role in the ability of a. fumigatus to colonize the respiratory tract.
in the present study, in order to identify which compounds were responsible for these changes, we used high-performance liquid chromatography with diode-array detection  to fractionate the filtrate of a. fumigatus culture.
thus, we identified verruculogen as the toxin responsible for the toxic effects observed in the epithelial cell model.
to determine whether verruculogen was associated with conidia upon its first contact with epithelial cells, the presence of verruculogen was specifically monitored in conidial extracts by mass spectrometry analysis.
moreover, since every isolate of a. fumigatus is potentially responsible for invasive aspergillosis , the ability of every isolate to produce verruculogen was a prerequisite for involving verruculogen as a pathogenic factor.
the production of verruculogen was therefore verified in a collection of  <dig> human, animal, plant and environmental a. fumigatus isolates.
RESULTS
we first fractionated the metabolites and analyzed the organic phase profile obtained with 25°c fungi cultures, to obtain enough metabolites to be seen in hplc analysis.
for 25°c fungi cultures, we detected and identified fumigaclavine a and c, fumitremorgin c, verruculogen, and fumagillin by comparison with authentic standards .
for 37°c culture, other metabolites were identified on the basis of their uv spectrum: fumigaclavine a, gliotoxin, fumiquinazoline f, fumitremorgin c, fumagillin, and helvolic acid.
when testing the different hplc fractions and commercial products, the results obtained after  <dig> min or  <dig> hours of exposure were similar.
therefore, only the  <dig> min results were retained.
at 37°c, only the 20– <dig> min fraction  <dig>  increased the vt .
as this fraction was known to contain the secondary metabolites helvolic acid, fumagillin and verruculogen, the effects of different concentrations of these commercially available compounds were tested.
helvolic acid had no significant effect .
the effects of fumagillin are summarized in figure  <dig> and were opposite to the effects of f <dig>  only verruculogen showed effects similar to those of f <dig>  in figure  <dig>  the effects of different concentrations of verruculogen are presented, ranging from 10- <dig> to 10- <dig> m. the effects on rt and vt were slightly dissociated, as 10- <dig> and 10- <dig> m verruculogen decreased rt and increased vt, whereas 10- <dig> and 10- <dig> m verruculogen only increased vt with no significant effect on rt.
to verify that verruculogen was responsible for the effects of fraction  <dig>  f <dig> was further fractionated into five two-minute fractions .
in these hplc conditions, verruculogen was recovered in f' <dig>  while helvolic acid and fumagillin were eluted together in f' <dig> .
among the five tested fractions, only f' <dig> displayed results similar to those of f <dig>  since verruculogen is produced in greater amounts at 25°c than at 37°c  <cit> , we performed similar experiments with the organic phase obtained at 25°c to look for a concentration effect.
for 25°c culture, f' <dig> revealed an increase in vt and a decrease in rt within the same range as 10- <dig> m standard verruculogen, whereas the effects observed with f' <dig> of 37°c culture were similar to those obtained with lower concentrations of verruculogen .
fraction f' <dig> was analyzed by mass spectrometry and verruculogen was identified on the basis of electron ionization ms and uv spectral analysis, after comparison with a reference standard.
in the analysis of fraction f' <dig>  the mass spectrum of verruculogen showed two major ions at m/z  <dig> and  <dig>  corresponding to loss of water and sodium adduct, respectively .
the collision-induced dissociation ms-ms spectrum of the  <dig> ion led to fragment at m/z  <dig>  identical to that of authentic verruculogen  <cit> .
the amounts of fumagillin and verruculogen were estimated by hplc-dad using standard curves.
f' <dig> contained  <dig>  μg of fumagillin for 37°c culture and  <dig>  μg for 25°c culture.
therefore, the concentration of fumagillin in culture filtrate used to challenge the hnec was  <dig>  10- <dig> m, more than  <dig> times lower than the lowest concentration,  <dig>  10- <dig> m, of purified fumagillin resulting in a significant effect , thus definitively excluding fumagillin as the toxin involved in our observations.
for verruculogen, f' <dig> of 37°c culture contained less than  <dig> ng of verruculogen and fraction f' <dig> of 25°c culture contained  <dig>  μg of verruculogen.
the concentrations obtained after resolubilization in dmso and dilution to challenge hnec corresponded to  <dig>  10- <dig> m for 25°c culture and  <dig>  10- <dig> m of verruculogen for 37°c culture.
compared with the results obtained with the serial dilutions of standard verruculogen , fraction f' <dig> of 25°c culture showed an increase in vt and rt, as did the 10- <dig> m concentration, whereas f' <dig> of 37°c culture presented a decrease in vt in the same way as the 10- <dig> concentration.
verruculogen was detected by hplc-dad in the  <dig> a. fumigatus hyphal and conidial extracts tested, from  <dig>  to  <dig>  μg/g of wheat.
one extract of conidial fractions was also analyzed by mass spectrometry to verify the presence of verruculogen.
in positive mode, verruculogen  was detected in conidial extract.
the presence of verruculogen was confirmed after  <dig>  min of elution, at which time its mass spectrum corresponded to that of standard verruculogen.
in these extracts, seven other compounds were also specifically detected .
discussion
the production of toxins by a. fumigatus may help the fungus to colonize and invade the respiratory epithelium by modifying the natural clearance of the respiratory tract.
previous research has shown that a. fumigatus culture filtrate modifies the transepithelial resistance  and transepithelial potential differences  of hnec, an in vitro model of the air-liquid interface of airway epithelium  <cit> .
the aim of this study was to use hplc and ms-ms to identify which toxins produced by a. fumigatus are responsible for these modifications.
our data suggest that verruculogen, which has never been implicated in invasive aspergillosis, is one of the probable candidates.
the fact that a. fumigatus produces a number of biologically active substances that slow ciliary beating, damage epithelium, and that may affect colonization of the airways has already been reported using culture explants  <cit> .
among these substances, such toxins as gliotoxin, fumagillin, and helvolic acid have been implicated in the pathogenesis of aspergillosis  <cit> .
our results did not suggest the involvement of any of these toxins in the effects observed on our in vitro model of respiratory epithelium.
the hplc fraction known to contain gliotoxin had no detectable effect, suggesting that the concentration of gliotoxin in the fungal filtrate was too low to play a role in the observed effects.
for fumagillin, the effects were different from those of the whole a. fumigatus culture filtrate and were only observed with concentrations above those found in the culture filtrate.
with the helvolic acid, we did not observe any effects at the tested concentrations based on the residual extract hplc analysis.
therefore, we concluded that none of these metabolites were responsible for the electrophysiological modifications of hnec.
nevertheless, their role, especially in association, cannot be completely ruled out.
for instance, gliotoxin is known to be produced by a. fumigatus during the exponential growth phase at 37°c  <cit> .
therefore, the role of gliotoxin may be minimal in the first days of colonization, after the seeding of airway epithelium, but become crucial at a later stage of infection.
gliotoxin has been detected in patients suffering from aspergillosis  <cit>  and in bovine udder infected with a. fumigatus  <cit> .
our results strongly suggest that verruculogen is one of the candidate toxins, if not the sole toxin, responsible for the changes of hnec observed with culture filtrate.
our arguments are based on the fact that:  only the fraction containing verruculogen modified vt and rt,  the concentration of verruculogen in the organic extract corresponds to the concentration obtained from the standard curve produced with pure commercial toxin,  the temperature of 25°c, which fosters the production of verruculogen by a. fumigatus, led to increased effects, and that  heating the filtrate at 100°c for  <dig> min did not modify the effects on hnec  <cit>  and is known not to modify the structure of verruculogen .
verruculogen is one of the tremorgenic mycotoxins produced by fungi belonging to the genera penicillium and aspergillus that elicit intermittent or sustained tremors  in vertebrate animals  <cit> .
the clinical symptoms typically observed during mycotoxin poisoning include diminished activity and immobility, followed by hyper-excitability, convulsions, muscle tremor, ataxia, and tetanic seizures.
however, death rarely occurs and these symptoms are reversible if the animals are removed from the toxic-feed source.
the concentrations detected in our a. fumigatus filtrates are considerably lower than those reported when clinical symptoms of poisoning are observed in ruminants.
no data are yet available in relation to the concentration dependence of human susceptibility.
verruculogen is known as an inhibitor of k+ channels in synaptic in vitro models  <cit> .
since verruculogen has never been tested on hnec, additional research is needed to identify the prime action site on hnec.
the action of verruculogen could be extended to other targets such as dna  <cit> .
in order to include verruculogen as one of the virulence factors of a. fumigatus, toxin should be present in all the a. fumigatus isolates.
no specific isolates have been shown to be more pathogenic than others in humans  <cit>  or birds  <cit> .
according to some authors, 40% to 91% of the a. fumigatus isolates produce verruculogen  <cit> .
the production of mycotoxins at an easily detectable level is very dependent on culture medium and temperature.
for instance, verruculogen is produced in larger amounts at 25°c than at 37°c  <cit> .
generally, greater amounts of mycotoxins are detected in grain cultures than in standard liquid cultures.
moreover, the production of toxins on grains is more physiological for a mould than the production of toxins in microbiological medium such as pda or sabouraud medium.
using autoclaved grains as a culture support, we were able to show that  <dig> different a. fumigatus isolates are able to produce verruculogen.
as we do not observe direct cell lysis with low concentrations of verruculogen, further studies are required to test whether verruculogen is a virulent factor in the strictest sense, i.e.
a component that damages the host cells  <cit> .
however, a verruculogen-deficient mutant strain would probably not be informative, since the pathogenicity of a. fumigatus is multifactorial.
indeed, all experiments performed to prove the role of a specific molecule in the virulence of a. fumigatus have failed, with the exception of melanin-deficient mutants  <cit>  and mutants deficient in the siderophore biosynthesis  <cit> .
to take on a primary role in the colonization and/or invasion of airway epithelium, verruculogen not only has to be present in every a. fumigatus isolate, but also has to be associated with conidia and delivered directly onto the epithelial cells.
the presence of verruculogen at the cellular level could alter pulmonary clearance and thereby increase colonization and the risk of invasive aspergillosis.
some authors have found verruculogen in very few conidial extracts  <cit> .
using ms/ms, we found that verruculogen is associated with conidia .
therefore, verruculogen can act on its first contact with epithelial cells.
furthermore, the detection of verruculogen in conidial extracts presents opportunities for investigation into the potential role of inhalation in the intake of mycotoxins by humans.
the recent detection of abundant respirable ergot alkaloids associated with a. fumigatus conidia raises the possibility of the respiratory epithelium as an alternative to the digestive route for the intake of these mycotoxins  <cit> .
CONCLUSIONS
the hplc-ms and hplc-msn analysis techniques used in this study according to recent fda recommendations  <cit>  confirm the existence of verruculogen and other mycotoxins associated with conidia.
the identification of the toxins secreted by a. fumigatus and their role in humans is a long-running enterprise involving microbiology, chemistry, and mammalian cell cultures.
for the first time, we are including verruculogen as a toxin with deleterious effects on respiratory epithelial cells.
whatever the exact role of verruculogen, it is not the only factor involved in the prevalence of a. fumigatus in invasive disease compared with other moulds.
verruculogen is, for instance, secreted by penicillium verruculosum, which has never been implicated in invasive disease.
verruculogen may act first, leading to settlement of the fungus and subsequently allowing other toxins to be produced if the fungus grows.
additionally, verruculogen might be synergistic or antagonistic with other molecules or proteases produced by a. fumigatus.

 aspergillus fumigatus is associated with many human health conditions. it is the most common cause of invasive aspergillosis, an opportunistic infection whose incidence increases in immunocompromised patients. aspergillosis is also prevalent in animals, such as dogs and birds. a. fumigatus is responsible for more human and animal diseases than other moulds even though their conidia are usually outnumbered by spores of other mould species in inhaled air, suggesting that a. fumigatus has special properties that enable it to colonize the lungs of its hosts. most studies with a. fumigatus have focused on the interactions of the fungus with alveolar macrophages or with cell line a from pneumocytes. however, the first tissue usually encountered by the inhaled conidia is the airway epithelium, and it is frequently damaged in patients at risk for invasive aspergillosis. we previously studied the interactions between a. fumigatus and the respiratory epithelium using human nasal epithelium cell cultures. after one week of culture, the hnec are organized in a pseudostratified epithelium with mucus and ciliated cells, very similar to the in vivo airway epithelium. we found that a filtrate from a. fumigatus cultures has specific electrophysiological effects on hnec compared with one from fungi not responsible for invasive infection. effects included a decrease in transepithelial electrical resistance and polarization of the epithelium. these changes to the epithelium properties could play a role in the ability of a. fumigatus to colonize the respiratory tract. in the present study, in order to identify which compounds were responsible for these changes, we used high-performance liquid chromatography with diode-array detection to fractionate the filtrate of a. fumigatus culture. thus, we identified verruculogen as the toxin responsible for the toxic effects observed in the epithelial cell model. to determine whether verruculogen was associated with conidia upon its first contact with epithelial cells, the presence of verruculogen was specifically monitored in conidial extracts by mass spectrometry analysis. moreover, since every isolate of a. fumigatus is potentially responsible for invasive aspergillosis, the ability of every isolate to produce verruculogen was a prerequisite for involving verruculogen as a pathogenic factor. the production of verruculogen was therefore verified in a collection of human, animal, plant and environmental a. fumigatus isolates.  we first fractionated the metabolites and analyzed the organic phase profile obtained with 25°c fungi cultures, to obtain enough metabolites to be seen in hplc analysis. for 25°c fungi cultures, we detected and identified fumigaclavine a and c, fumitremorgin c, verruculogen, and fumagillin by comparison with authentic standards. for 37°c culture, other metabolites were identified on the basis of their uv spectrum: fumigaclavine a, gliotoxin, fumiquinazoline f, fumitremorgin c, fumagillin, and helvolic acid. when testing the different hplc fractions and commercial products, the results obtained after min or hours of exposure were similar. therefore, only the min results were retained. at 37°c, only the 20– min fraction increased the vt. as this fraction was known to contain the secondary metabolites helvolic acid, fumagillin and verruculogen, the effects of different concentrations of these commercially available compounds were tested. helvolic acid had no significant effect. the effects of fumagillin are summarized in figure and were opposite to the effects of f only verruculogen showed effects similar to those of f in figure the effects of different concentrations of verruculogen are presented, ranging from 10- to 10- m. the effects on rt and vt were slightly dissociated, as 10- and 10- m verruculogen decreased rt and increased vt, whereas 10- and 10- m verruculogen only increased vt with no significant effect on rt. to verify that verruculogen was responsible for the effects of fraction f was further fractionated into five two-minute fractions. in these hplc conditions, verruculogen was recovered in f' while helvolic acid and fumagillin were eluted together in f'. among the five tested fractions, only f' displayed results similar to those of f since verruculogen is produced in greater amounts at 25°c than at 37°c, we performed similar experiments with the organic phase obtained at 25°c to look for a concentration effect. for 25°c culture, f' revealed an increase in vt and a decrease in rt within the same range as 10- m standard verruculogen, whereas the effects observed with f' of 37°c culture were similar to those obtained with lower concentrations of verruculogen. fraction f' was analyzed by mass spectrometry and verruculogen was identified on the basis of electron ionization ms and uv spectral analysis, after comparison with a reference standard. in the analysis of fraction f' the mass spectrum of verruculogen showed two major ions at m/z and corresponding to loss of water and sodium adduct, respectively. the collision-induced dissociation ms-ms spectrum of the ion led to fragment at m/z identical to that of authentic verruculogen. the amounts of fumagillin and verruculogen were estimated by hplc-dad using standard curves. f' contained μg of fumagillin for 37°c culture and μg for 25°c culture. therefore, the concentration of fumagillin in culture filtrate used to challenge the hnec was 10- m, more than times lower than the lowest concentration, 10- m, of purified fumagillin resulting in a significant effect, thus definitively excluding fumagillin as the toxin involved in our observations. for verruculogen, f' of 37°c culture contained less than ng of verruculogen and fraction f' of 25°c culture contained μg of verruculogen. the concentrations obtained after resolubilization in dmso and dilution to challenge hnec corresponded to 10- m for 25°c culture and 10- m of verruculogen for 37°c culture. compared with the results obtained with the serial dilutions of standard verruculogen, fraction f' of 25°c culture showed an increase in vt and rt, as did the 10- m concentration, whereas f' of 37°c culture presented a decrease in vt in the same way as the 10- concentration. verruculogen was detected by hplc-dad in the a. fumigatus hyphal and conidial extracts tested, from to μg/g of wheat. one extract of conidial fractions was also analyzed by mass spectrometry to verify the presence of verruculogen. in positive mode, verruculogen was detected in conidial extract. the presence of verruculogen was confirmed after min of elution, at which time its mass spectrum corresponded to that of standard verruculogen. in these extracts, seven other compounds were also specifically detected. discussion the production of toxins by a. fumigatus may help the fungus to colonize and invade the respiratory epithelium by modifying the natural clearance of the respiratory tract. previous research has shown that a. fumigatus culture filtrate modifies the transepithelial resistance and transepithelial potential differences of hnec, an in vitro model of the air-liquid interface of airway epithelium. the aim of this study was to use hplc and ms-ms to identify which toxins produced by a. fumigatus are responsible for these modifications. our data suggest that verruculogen, which has never been implicated in invasive aspergillosis, is one of the probable candidates. the fact that a. fumigatus produces a number of biologically active substances that slow ciliary beating, damage epithelium, and that may affect colonization of the airways has already been reported using culture explants. among these substances, such toxins as gliotoxin, fumagillin, and helvolic acid have been implicated in the pathogenesis of aspergillosis. our results did not suggest the involvement of any of these toxins in the effects observed on our in vitro model of respiratory epithelium. the hplc fraction known to contain gliotoxin had no detectable effect, suggesting that the concentration of gliotoxin in the fungal filtrate was too low to play a role in the observed effects. for fumagillin, the effects were different from those of the whole a. fumigatus culture filtrate and were only observed with concentrations above those found in the culture filtrate. with the helvolic acid, we did not observe any effects at the tested concentrations based on the residual extract hplc analysis. therefore, we concluded that none of these metabolites were responsible for the electrophysiological modifications of hnec. nevertheless, their role, especially in association, cannot be completely ruled out. for instance, gliotoxin is known to be produced by a. fumigatus during the exponential growth phase at 37°c. therefore, the role of gliotoxin may be minimal in the first days of colonization, after the seeding of airway epithelium, but become crucial at a later stage of infection. gliotoxin has been detected in patients suffering from aspergillosis and in bovine udder infected with a. fumigatus. our results strongly suggest that verruculogen is one of the candidate toxins, if not the sole toxin, responsible for the changes of hnec observed with culture filtrate. our arguments are based on the fact that: only the fraction containing verruculogen modified vt and rt, the concentration of verruculogen in the organic extract corresponds to the concentration obtained from the standard curve produced with pure commercial toxin, the temperature of 25°c, which fosters the production of verruculogen by a. fumigatus, led to increased effects, and that heating the filtrate at 100°c for min did not modify the effects on hnec and is known not to modify the structure of verruculogen. verruculogen is one of the tremorgenic mycotoxins produced by fungi belonging to the genera penicillium and aspergillus that elicit intermittent or sustained tremors in vertebrate animals. the clinical symptoms typically observed during mycotoxin poisoning include diminished activity and immobility, followed by hyper-excitability, convulsions, muscle tremor, ataxia, and tetanic seizures. however, death rarely occurs and these symptoms are reversible if the animals are removed from the toxic-feed source. the concentrations detected in our a. fumigatus filtrates are considerably lower than those reported when clinical symptoms of poisoning are observed in ruminants. no data are yet available in relation to the concentration dependence of human susceptibility. verruculogen is known as an inhibitor of k+ channels in synaptic in vitro models. since verruculogen has never been tested on hnec, additional research is needed to identify the prime action site on hnec. the action of verruculogen could be extended to other targets such as dna. in order to include verruculogen as one of the virulence factors of a. fumigatus, toxin should be present in all the a. fumigatus isolates. no specific isolates have been shown to be more pathogenic than others in humans or birds. according to some authors, 40% to 91% of the a. fumigatus isolates produce verruculogen. the production of mycotoxins at an easily detectable level is very dependent on culture medium and temperature. for instance, verruculogen is produced in larger amounts at 25°c than at 37°c. generally, greater amounts of mycotoxins are detected in grain cultures than in standard liquid cultures. moreover, the production of toxins on grains is more physiological for a mould than the production of toxins in microbiological medium such as pda or sabouraud medium. using autoclaved grains as a culture support, we were able to show that different a. fumigatus isolates are able to produce verruculogen. as we do not observe direct cell lysis with low concentrations of verruculogen, further studies are required to test whether verruculogen is a virulent factor in the strictest sense, i.e. a component that damages the host cells. however, a verruculogen-deficient mutant strain would probably not be informative, since the pathogenicity of a. fumigatus is multifactorial. indeed, all experiments performed to prove the role of a specific molecule in the virulence of a. fumigatus have failed, with the exception of melanin-deficient mutants and mutants deficient in the siderophore biosynthesis. to take on a primary role in the colonization and/or invasion of airway epithelium, verruculogen not only has to be present in every a. fumigatus isolate, but also has to be associated with conidia and delivered directly onto the epithelial cells. the presence of verruculogen at the cellular level could alter pulmonary clearance and thereby increase colonization and the risk of invasive aspergillosis. some authors have found verruculogen in very few conidial extracts. using ms/ms, we found that verruculogen is associated with conidia. therefore, verruculogen can act on its first contact with epithelial cells. furthermore, the detection of verruculogen in conidial extracts presents opportunities for investigation into the potential role of inhalation in the intake of mycotoxins by humans. the recent detection of abundant respirable ergot alkaloids associated with a. fumigatus conidia raises the possibility of the respiratory epithelium as an alternative to the digestive route for the intake of these mycotoxins.  the hplc-ms and hplc-msn analysis techniques used in this study according to recent fda recommendations confirm the existence of verruculogen and other mycotoxins associated with conidia. the identification of the toxins secreted by a. fumigatus and their role in humans is a long-running enterprise involving microbiology, chemistry, and mammalian cell cultures. for the first time, we are including verruculogen as a toxin with deleterious effects on respiratory epithelial cells. whatever the exact role of verruculogen, it is not the only factor involved in the prevalence of a. fumigatus in invasive disease compared with other moulds. verruculogen is, for instance, secreted by penicillium verruculosum, which has never been implicated in invasive disease. verruculogen may act first, leading to settlement of the fungus and subsequently allowing other toxins to be produced if the fungus grows. additionally, verruculogen might be synergistic or antagonistic with other molecules or proteases produced by a. fumigatus. 
the role of aspergillus fumigatus mycotoxins in the colonization of the respiratory tract by conidia has not been studied extensively, even though patients at risk from invasive aspergillosis frequently exhibit respiratory epithelium damage. in a previous study, we found that filtrates of a. fumigatus cultures can specifically alter the electrophysiological properties of human nasal epithelial cells compared to those of non pathogenic moulds. we fractionated the organic phase of filtrate from 3-day old a. fumigatus cultures using high-performance liquid chromatography. we verified that verruculogen was produced by a collection of human, animal, plant and environmental a. fumigatus isolates. the role of these modifications in the colonization and invasion of the respiratory epithelium by a. fumigatus on first contact with the epithelium remains to be determined. decrease of transepithelial resistance and increase of potential differences, and contained secondary metabolites such as helvolic acid, fumagillin, and verruculogen.
 the role of aspergillus fumigatus mycotoxins in the colonization of the respiratory tract by conidia has not been studied extensively, even though patients at risk from invasive aspergillosis frequently exhibit respiratory epithelium damage. in a previous study, we found that filtrates of a. fumigatus cultures can specifically alter the electrophysiological properties of human nasal epithelial cells compared to those of non pathogenic moulds.  we fractionated the organic phase of filtrate from 3-day old a. fumigatus cultures using high-performance liquid chromatography. the different fractions were tested for their ability to modify the electrophysiological properties of hnec in an in vitro primary culture model. the fraction collected between and min mimicked the effects of the whole filtrate, i.e. decrease of transepithelial resistance and increase of potential differences, and contained secondary metabolites such as helvolic acid, fumagillin, and verruculogen. only verruculogen had effects similar to the whole filtrate. we verified that verruculogen was produced by a collection of human, animal, plant and environmental a. fumigatus isolates. using ms-ms analysis, we found that verruculogen was associated with both mycelium and conidia extracts.  verruculogen is a secondary metabolite that modifies the electrophysiological properties of hnec. the role of these modifications in the colonization and invasion of the respiratory epithelium by a. fumigatus on first contact with the epithelium remains to be determined. 