Aspergillus mycotoxins and their effect on the host

K. KAMEI & A. WATANABE

Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Japan

Aspergillus fumigatus is known to produce various immunosuppressive mycotoxins

including gliotoxin. However, none of these mycotoxins has been confirmed as

being directly related to the pathogenesis of aspergilli. Recent studies have made

substantial progress in the determination of mycotoxins as virulence factors.

Gliotoxin was found to be produced much faster than previously believed under

certain culture conditions, such as at 378C and under high oxygen content, which is

close to the environment in the host. Gliotoxin was also found to be detectable in

the sera of aspergillosis mice and of aspergillosis patients. Based on these findings,

it is becoming evident that gliotoxin is produced in the infected organs of patients

of aspergillosis at a significant level. In addition to these known mycotoxins,

A. fumigatus produces many mycotoxins apparently different from known toxins.

From the aspect of gene analysis, the deletion of laeA was found to block the

expression of metabolic gene clusters such as sterigmatocystin, and the gene is also

expected to be related to the production of gliotoxin. The significance of

mycotoxins as virulence factors will hopefully be clarified in the near future.

Keywords Aspergillus, mycotoxin, gliotoxin, virulence factor

Introduction

Mycotoxins, which are produced by many species of

fungi, are secondary metabolites that are toxic to

humans and animals. Most of them are of small

molecular sizes (MW B/300) [1]. Various investigations

of mycotoxins have been carried out over the years,

most from the viewpoint of food intoxication and its

prevention [2]. Mycotoxin food contamination causing

mycotoxicoses in animals and humans has been a

serious problem, and remains so in some developing

countries. Pathogenic Aspergillus spp. also produce

mycotoxins. These toxins are known for their strong

and varied biological activities. For example, aflatoxin,

the most well-known and well-investigated mycotoxin,

is known to carry the most potent carcinogenic activity

as a natural product. It also carries acute toxicity to

various human cells such as hepatocytes, renal cells,

lung epithelioid cells, etc., as well as various immuno-

suppressive activities [3�/5]. Many other mycotoxins

have fairly similar activities [6].

Considering the potent biological activity, it is likelythat mycotoxins work as virulence factors in the

development of aspergillosis. However, studies focusing

on the relation between mycotoxins and its pathogen-

esis have been limited, and significance of mycotoxins

in the virulence of Aspergillus fumigatus has not yet

been demonstrated [7].

Function of mycotoxins and their productionby Aspergillus spp. in vitro

Until recently, the relationship between mycotoxins

and the pathogenicity of the fungi that produce them

has received little attention. However, there are manymycotoxins with the capability to alter the defense

system of the host, and by this immunosuppressive

activity these mycotoxins may help the fungus to

invade the host tissue by working as virulence factors.

Among mycotoxins produced by Aspergillus spp., for

example, Aspergillus flavus produces aflatoxin that

suppresses the function of macrophages [8], and

Aspergillus ochraceus produces ochratoxin that isknown to be cytotoxic to lymphocytes [8], and it

suppresses many functions of lymphocytes, monocytes,

and granulocytes [9,10].

Correspondence: K. Kamei, 1-8-1 Inohana, Chuo-ku, Chiba, 260-

8673 Japan. Tel./Fax: �/81 43 226 2491; E-mail: k.kamei@

faculty.chiba-u.jp

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Although A. fumigatus is not known to produce

aflatoxin, the fungus is known to produce variousimmunosuppressive mycotoxins including gliotoxin

(MW 326 Da), fumagillin (459 Da), helvolic acid

(fumigacin) (569 Da), fumitremorgin A and Asp-

hemolysin (16 kDa) [11�/13]. Gliotoxin is an alkaloid

with a low molecular size which has been known to

possess a number of immunosuppressive activities, such

as inhibition of superoxide release, migration, micro-

bicidal activity [11,14�/17] cytokine release [18] byleukoyctes, and T-lymphocyte-mediated cytotoxicity

[19]. It is genotoxic [20] and also causes apoptosis in

macrophages [21]. Fumagillin is a cyclohexane deriva-

tive, and is known as an inhibitor of endothelial cell

proliferation and angiogenesis. Asp-hemolysin is a

hemolytic toxin and is cytotoxic to neutrophils and

macrophages. All of these mycotoxins, i.e., gliotoxin,

fumagillin, helvolic acid, and Asp-hemolysin, inhibitthe function of leukocytes in terms of migration,

superoxide production and fungicidal activity, with

the activities of gliotoxin being much stronger than

those of the others. However, none of these mycotoxins,

including gliotoxin, has been confirmed as being

directly related to the pathogenesis of aspergilli despite

their strong activities.

As a general characteristic, mycotoxins are thoughtto be produced slowly, reaching detectable levels after a

long culture period, which means that their release is

time-dependent in culture. This slow production means

that mycotoxins are unlikely virulence factors. In fact,

in order to collect fungal mycotoxins in culture filtrates,

fungi were traditionally cultured in conventional

poorly-aerated conditions at low temperature (e.g.,

room temperature), and the mycotoxins were extractedfrom the filtrates. Under this environment, some days

of culture were essential for the production of myco-

toxins.

However, the metabolism of fungi depends on the

environment, and therefore the culture condition plays

a critical role in the production of mycotoxins [22].

Recent studies have found that gliotoxin was produced

much faster (from 29 hours after the beginning ofincubation) than previously believed under a certain

culture condition such as at 378C [23].

Aeration during the culture was found to be another

important factor for the rapid production of gliotoxin.

When the culture filtrate of A. fumigatus is made in a

well-aerated culture system in a medium such as RPMI

1640 at 378C, the filtrate rapidly (within 15 hours of

culture) exhibits potent cytotoxic activity againstleukocytes such as macrophages and polymorphonuc-

lear leukocytes at a low concentration (1%). The

leukocytes demonstrated significant morphological

changes, and were destroyed by exposure to the culture

filtrate. These findings were confirmed by examiningmore than 10 clinical and environmental isolates. In

contrast, this activity was not seen or was much weaker

in the filtrates of other Aspergillus spp. such as

A. flavus, A. terreus or A. niger, or when A. fumigatus

was cultured under poorly-aerated conditions [24]. At

lower concentrations, the filtrate of A. fumigatus

inhibited the migration, phagocytosis and superoxide

generation by human polymorphonuclear leukocytes.When injected into murine peritoneal cavities with

spores of A. fumigatus, the culture filtrate dramatically

promoted the development of aspergillosis and shor-

tened the survival of mice [25].

Analysis of the filtrate by gas chromatography-mass

spectrometry disclosed that a significant amount of

gliotoxin became detectable in the culture filtrate when

the cytotoxic activity became evident. When the culturefiltrate was made in a special container with direct

control of oxygen concentration in the environment,

gliotoxin production was found to be dependent on the

oxygen concentration of the environment at B/14%

oxygen in a concentration-dependent manner [26,27].

The primary target of infection by A. fumigatus is the

lung, the most well-aerated organ. In this sense, the

high concentration of oxygen in the lung provides anoptimal condition for the production of gliotoxin by

A. fumigatus.

Furthermore, analysis of the filtrate also disclosed

some unknown cytotoxic components other than

gliotoxin that were not listed in the library search

program, meaning that these components were appar-

ently novel. The active substance was heat labile,

soluble in chloroform, and had a small molecular size(B/ 3 kDa). When the cytotoxic activity of the filtrate

was compared with authentic gliotoxin, the concentra-

tion of gliotoxin (ca. 4 mcg/ml) was found to be too low

to be solely responsible for the cytotoxic activity, and it

is assumed that the novel substances play a significant

additional role in the activity of the filtrate [25].

In addition to the novel active substance described

above, there are many studies reporting thatA. fumigatus produces many mycotoxins apparently

different from known toxins [28�/31]. The combination

effect of these mycotoxins may produce unexpected

synergistic results [32].

Aspergillus mycotoxins in vivo: productionand function

To work as virulence factors, mycotoxins should be

produced and be active in vivo. From this aspect,

studies have been even more limited. In terms of

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mycotoxins of large molecular size, ribonucleotoxin

(molecular size: 18 kDa) was found in the urine ofaspergillosis patients [33,34]. Restrictocin is produced

by A. fumigatus and also by non-pathogenic species

such as Aspergillus restrictus. It blocks protein synth-

esis and exerts toxicity to human cells, and was once

regarded as a candidate of a virulence factor of A.

fumigatus [33]. However, studies using restrictocin-

deficient A. fumigatus isolates by gene disruption

showed disappointing results, demonstrating no differ-ence in virulence between these strains and indicating

that restrictocin is not an important determinant of

virulence [35,36].

As for the smaller mycotoxins such as gliotoxin,

research had been hampered by a number of technical

problems until the recent development of more ad-

vanced techniques. Producing antibodies to these non-

proteinous small mycotoxins such as gliotoxin hadsometimes been problematic owing to their structure,

and few reports were available. Recently, Fox et al . [37]

reported a new simple method using thyroglobulin-

based immunogens, and successfully produced antibo-

dies to gliotoxin and helvolic acid. These antibodies are

expected to work as reliable tools for detecting the

presence of mycotoxins in infected organs [37].

In terms of the detection of gliotoxin in vivo, only afew cases of gliotoxin being detected in infected tissues

have been reported. Bauer et al . [38] reported the

presence of gliotoxin in a cow’s udder. Richard et al .

[39] made an animal model using turkeys and found a

significant amount of gliotoxin in the poults of infected

animals. Recently, Reeves et al . [40] detected gliotoxin

in the bodies of larvae of experimentally-infected

Galleria mellonella, and compared the gliotoxin con-tent between isolates [40]. These findings indicate that

A. fumigatus is capable of producing gliotoxin in vivo in

some animals. In addition, Lewis et al . [41], using LC-

tandem mass spectroscopy, recently showed that a

detectable level of gliotoxin was present in the sera of

aspergillosis mice. Gliotoxin was also found in the sera

of aspergillosis patients [41]. This shows that detection

of gliotoxin may be used as a diagnostic tool. It alsoshows that a significant amount of gliotoxin is present

in infected tissues, and a substantial biological effect

should be expected. From these findings, it is becoming

increasingly evident that gliotoxin is produced at a

significant level in the infected organs of patients with

aspergillosis. Its role in the pathogenesis of aspergillosis

remains to be clarified until a gliotoxin negative strain

of A. fumigatus is produced.The function of mycotoxin in vivo is of primary

importance for understanding the pathogenicity

of mycotoxins. Some studies have demonstrated

the worsening of aspergillosis by giving gliotoxin to

infected animals [25,42]. The gene manipulation tech-nique is believed to be a powerful tool for research, and

it may help to clarify the function of mycotoxins.

However, most mycotoxins are not proteins, and

therefore genetic analysis by manipulating genes that

directly code the synthesis of the toxins is difficult to

perform. Many trials have been undertaken to produce

a gliotoxin-deficient strain by this technique, but they

were usually unsuccessful. Recently, analysis of genes ofAspergillus spp. related to the production of some

secondary metabolites focused on isolated genes en-

coding the enzymes related to the biosynthesis of

aflatoxin and sterigmatocystin. Their expression was

also found to be mostly regulated by a product of the

regulatory gene aflR. The deletion of laeA (delta laeA)

was found to block the expression of metabolic gene

clusters, including sterigmatocystin (carcinogen), peni-cillin (antibiotic), and lovastatin (antihypercholestero-

lemic agent) [43,44]. It is expected that the gene might

also be related to the production of gliotoxin. However,

the gene is unlikely to be specifically related to the

synthesis of gliotoxin, and the metabolism of the

fungus may result in a change in the synthesis of

many metabolites including gliotoxin. Nonetheless, the

analysis of this cluster is expected to help our under-standing of the role of gliotoxin in the development of

aspergillosis. Gene disruption that focuses on the genes

more specific to gliotoxin synthesis is warranted.

The future of mycotoxin studies

Recent studies have made substantial progress in the

determination of mycotoxins as virulence factors.Although the pathogenicity of aspergillosis may be

multifactorial [45], mycotoxins should be examined not

just as a source of food contamination but also as

possible virulence factors. It is obvious that much

remains to be done in the investigation of mycotoxins

and their relation to virulence. Recently, treatment by

amphotericin B was found to augment the release of

gliotoxin by A. fumigatus, possibly causing immunolo-gical status deterioration of the host [46]. As the role of

mycotoxins in aspergillosis becomes clear, drugs that

can control the production of mycotoxins may be a new

and attractive target of development.

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