Medical Mycology 1999, 37, 375–389
Accepted 20 May 1999
Review article
Antioxidant systems in the pathogenic fungi of man and
their role in virulence
A. J. HAMILTON & M. D. HOLDOM
Dermatology Department, St Johns Institute of Dermatology, Guys Hospital, Kings College, London, UK
In the last two decades, a variety of fungal antioxidants have attracted considerable
interest, largely arising from their hypothetical role as virulence determinants.
Melanin is a potent free radical scavenger and in Cryptococcus neoformans, there is
now good evidence that the production of melanin is a significant virulence determinant. There is also recent evidence linking melanin biosynthesis to the virulence of
Aspergillus fumigatus conidia. Superoxide dismutases are important housekeeping
antioxidants and have an additional hypothetical role in virulence; however, although these enzymes have been biochemically characterized from Aspergillus and
Cryptococcus, there is as yet no firm evidence that these enzymes are involved in
pathogenicity. Catalase production may play some role in the virulence of Candida
albicans but this enzyme has not been shown, as yet, to influence the virulence of A.
fumigatus. There are some data supporting an antioxidant function for the acyclic
hexitol mannitol in C. neoformans, but further investigations are required in this
area. Research into the putative antioxidant activities of a range of other fungal
enzymes, such as acid phosphatases, remains limited at this time.
Keywords
antioxidants, fungi, virulence
Introduction
In common with all other eukaryotic cells, fungi have
had to evolve mechanisms for dealing with the damaging
effects of oxidants arising both intracellularly (as byproducts of cellular metabolism) or extracellularly (from
diverse environmental sources). As regards fungal pathogens of man, a prime source of such environmental
oxidant attack is provided by various immune effector
cells that utilize oxidants as potent antimicrobial agents.
In various bacterial [1,2] and parasitic pathogens [3,4] it
is hypothesized that resident antioxidant systems play a
Correspondence: Dermatology Laboratory, 5th Floor Thomas Guy
House, Guys Hospital, Kings College, St Thomas’s St., London,
SE1 9RT, UK. Tel: + 44-171-9554663; fax: + 44-171-4076689;
e-mail: [email protected]
© 1999 ISHAM
role as virulence determinants by preventing or interfering with these oxidant based killing mechanisms. In the
last two decades, there has been an upsurge in interest
relating to the potential for fungal antioxidants to perform similar functions. Historically, the production of
melanin in fungi such as Cryptococcus neoformans predates research on other potential fungal antioxidant systems; however, there is now a growing literature relating
to superoxide dismutase, catalase, mannitol and other
antioxidants. Whilst demonstrating the antioxidant potency, or otherwise, of such compounds in 6itro is relatively straightforward, it is considerably more difficult to
conclusively demonstrate that they actually play a role as
fungal virulence factors in 6i6o. This is particularly pertinent since it is a reasonable working assumption that all
fungal antioxidant systems evolved originally to deal with
oxidant stress unconnected with those imposed by immune effector cells.
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Melanins
Melanins are multifunctional polymers found in representatives of all biological kingdoms [5]. In fungi, they
have attracted considerable interest as putative virulence
factors, particularly in plant pathogens [6,7]. Typically,
melanins are dark brown or black pigments of high
molecular weight formed by the oxidative polymerization
of phenolic compounds [8,9]. Historically, most interest
has been directed towards melanogenesis in C. neoformans and members of the dematiaceous fungi such as
Exophiala (Wangiella) dermatitidis, although there is
some interesting recent evidence that production of
melanins is of considerable importance to other fungi.
The initial discovery that C. neoformans could produce
melanin [10] was subsequently followed by studies on
descriptions of media suitable for the differential identification of C. neoformans [11 – 16]. Work was also centred
on the precise definition of the substrates used in melanogenesis [17,18]. In fact, C. neoformans is incapable of de
no6o melanogenesis as it lacks a tyrosinase enzyme; the
latter is responsible for the oxidation of tyrosine to
dihydroxyphenol, with the subsequent oxidation through
a series of unstable intermediates to 5,6-indoquinone,
which in turn polymerizes to form melanin [19,20]. Instead, it was established that C. neoformans relies on the
presence of dihydroxyphenolic compounds [17] in combination with the activity of an enzyme, which was initially
described as a phenoloxidase [21] (the terminology used
with reference to this enzyme in C. neoformans has varied
[22]). Phenoloxidases are responsible for the oxidization
of dihydroxyphenols to the corresponding quinone.
Quinones in turn rearrange spontaneously and in the
presence of molecular oxygen form melanin via a series
of rapid enzyme independent auto-oxidations (according
to the classical Mason – Raper scheme [20]). Two intermediates, dopachrome and 5,6-dihydroxyindole, have been
isolated from this reaction sequence and have been incorporated in a model pathway for C. neoformans melanogenesis [23]. Support for the Masson – Raper derived
scheme of melanogenesis has come from a recent study
[24]. This work also provided direct evidence for the
formation of eumelanin polymers via catecholamine oxidation by laccase, followed by oxidative coupling of
dihydroxyindole. It is of note that this pathway differs
significantly from the pentaketide pathway identified in
dematiaceous human pathogenic fungi [25,26].
Variation in the activity of the C. neoformans phenoloxidase between strains and serotypes was detected at
an early stage [27,28] and variation in virulence within
strains of a given serotype that might be related to
differences in phenoloxidase activity were also noted [29].
In addition, strain differences in melanin content and
quantitative and/or qualitative differences in the melanin
produced after growth of cells on different substrates
have become apparent [30]. It also appears that C. neoformans is unique in its genus in routinely producing
melanin from diphenolic compounds via the use of a
phenoloxidase enzyme when grown at 37 °C; reports of
melanization in other cryptococci are sporadic [17,21,31].
As C. neoformans is also the only significant pathogen in
the genus [32], with other cryptococci only rarely being
clinically isolated [33,34], this observation has traditionally underpinned the linkage between melanogenesis and
virulence. A link between virulence in C. neoformans and
the occurrence of the Mel + phenotype was also suggested by the lack of Mel − types among clinical isolates
[35]. Some extremely rare encapsulated isolates from clinical specimens that do not produce melanin on either
dopamine agar or Niger seed and that remain virulent for
mice have also been noted [36]. However, no subsequent
report on these isolates occurs in the literature.
The first extensive biochemical study on the cryptococcal phenoloxidase was produced by Polacheck et al. [37]
who found that the enzyme had an optimal temperature
of 37 °C, was not inhibited by copper chelators and was
derepressed by low levels of glucose. The latter was
thought to be related to the low levels of glucose found in
the central nervous system, coupled with the suggestion
that C. neoformans may use catecholamines more efficiently in the brain than elsewhere [37]. Subsequently, it
was discovered that a given strain of C. neoformans
produces different phenoloxidases under different conditions [38], with cultures grown at 25 °C producing an
enzyme with higher electrophoretic mobility than that
produced by cultures grown at 37 °C. Differences in the
degree of glycosylation of the phenoloxidase were also
noted at the two different growth temperatures; glucose
starvation produced an analogous effect [38]. It was also
found that the C. neoformans var. gattii phenoloxidase
had a lower electrophoretic mobility compared to that of
the equivalent enzyme from var. neoformans.
It is of note that early speculation centred on whether
two forms of phenoloxidase existed, one to act on o-phenols, the other to act on p-phenols [17]. The single
enzyme hypothesis was supported by Kwon-Chung et al.
[28] who based their view on the earlier studies on Mel +
revertants (capable of melanin production) [39]. However, more recent reports focusing on the purification and
characterization of enzymes with phenoloxidase activity
imply the involvement of more than one enzyme. Thus,
hydrophobic interaction and anion exchange chromatography have been used to purify an 80 kDa enzyme (with
a possible dimer of 180 kDa) with a pI of 4·1 [40]. This
enzyme differed from a second purified 75 kDa enzyme
© 1999 ISHAM, Medical Mycology, 37, 375–389
Antioxidant systems in the fungi of man and their role in virulence
of 624 amino-acids [41] on the basis of N-terminal amino
acid sequence [40] and predicted amino acid sequence
[41]; these enzymes would appear to be different despite
their approximate size similarity. The 75 kDa enzyme has
now been classified as a laccase (a p-diphenoloxidase)
[42] (based on the parameters of absorption spectrum and
substrate specificity), which contains copper. Support for
the involvement of copper, rather than iron in enzyme
activity has come from earlier work demonstrating inhibition of crude phenoloxidase extracts by copper chelators and studies on Mel − (lacking the ability to produce
melanin) mutants [43]. Laccase activity has been shown
to be glucose repressed [41] as has previously been
demonstrated for both diphenoloxidase and pigment production [27,37].
A marked decrease in phenoloxidase activity in cultures grown at 37 °C has been noted; this is unusual
behaviour for a putative virulence factor in a parasite of
mammals [44]. However, a compensatory mechanism for
the decrease in phenoloxidase activity involving superoxide dismutase [45] has been suggested. Earlier reports [36]
had in fact suggested the uptake of the substrate L-dopa
was the rate limiting step at 37 °C with the result that
cells grown on L-dopa, dopamine or Niger seed agar,
took 24 – 48 h longer to produce the Mel + phenotype
when grown at 37 °C as compared to 30 °C.
Early classical genetic studies suggested an association
between melanization and virulence; Rhodes et al. [39]
were able to isolate a Mel − variant of C. neoformans that
was used to demonstrate that the traits Mel + and virulence in mice cosegregated amongst the progeny of a
Mel − × Mel + cross. The basis for the Mel − phenotype
was loss of detectable phenoloxidase activity, with relative virulence in various strains correlating with relative
levels of phenoloxidase [39]. A double mutant of C.
neoformans that was both Mel − and failed to grow at
37 °C (temperature sensitive, Tem − ) was also produced
[35]. This double mutant was crossed with a wild type
strain to produce progeny with various combinations of
the two markers. It was found that the Mel − Tem + and
Mel + Tem − phenotypes were equally avirulent for mice
whereas the Mel − types neither multiplied or survived in
the organs of infected mice. Only Mel + Tem + strains
killed mice and these cells had a dark layer in the cell wall
when examined by electron microscopy, in contrast to
Mel − cells that had a hyaline wall. Two defects in
melanogenesis were demonstrated in the Mel − mutants:
the absence of phenoloxidase and the absence of an
active transport system for diphenolic compounds [35].
The inter-relationship between capsule formation in C.
neoformans, which has been clearly shown to be involved
in virulence, and melanogenesis has also been studied
© 1999 ISHAM, Medical Mycology, 37, 375 – 389
377
[36]. Acapsular (Cap − ) Mel − mutants were crossed with
wild type Cap + Mel + strains; the resulting phenotypes
Cap + Mel − , Cap − Mel + and Cap − Mel − were nonlethal for mice whereas the Cap + Mel + phenotype was
almost invariably fatal. Revertants to the wild type phenotype were subsequently shown to fully regain virulence.
In contrast, Cap − Mel + and Cap − Mel − isolates did not
show reversion and as a result failed to produce fatal
infection [36]. Perhaps surprisingly, it was subsequently
found that phenoloxidase and capsular polysaccharide
production are discordantly regulated [46].
It would seem likely that multiple genes are involved in
C. neoformans melanogenesis since seven Mel − mutant
classes have been defined by Torres-Guererro and Edman
[43]. These classes included three mutants in which the
addition of copper suppressed melanogenesis, in contrast
to wild type strain melanogenesis that was enhanced by
the addition of copper; these observations imply that
melanogenesis and copper metabolism are closely linked.
A regulatory gene required for activation or repression of
phenoloxidase gene transcription, together with a gene
involved in the post-translational modification (i.e. glycosylation) of phenoloxidase [43] may also be involved in
melanization. The former may be related to the C. neoformans G-protein alpha subunit GPA1 [47], which acts
as a regulatory element controlling melanization, along
with other virulence phenotypes. The expression of the
laccase gene (CNLAC1) itself has now been strongly
implicated in virulence [48], as site directed mutagenesis
of a putative copper binding site in the enzyme has been
shown to prevent melanization that was associated with
low virulence. Significantly, complementation of this mutant restored the Mel + phenotype and resulted in restored virulence [48].
The mechanisms by which melanization in C. neoformans may play a role in virulence have received a great
deal of attention. Studies in the 1960s demonstrated that
melaninized fungi were more resistant to enzymatic or
microbial lysis than those without melanin [49–51] and it
was suggested that melanin deposited in the cell wall may
act as a shield against host defence systems [35]. Melanins
possess a range of potentially significant biological characteristics, foremost amongst which is their ability to act
as free radical scavengers [5,52] and it has been hypothesized that in C. neoformans and other fungi the role of
melanization is to protect against oxidants [8]. The latter
may be produced either by leucocytes [53–55] or may
arise from other non-leucocyte sources (for example catecholamines can act as sources of damaging radicals under
the appropriate conditions [56]).
Some evidence for the oxidant protection hypothesis
has been accumulated. Thus mutants deficient in phe-
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noloxidase and/or in a catecholamine uptake system have
been shown to be more susceptible to an in 6itro oxidative killing system containing the catecholamine
epinephrine than are the parental wild type [57]. Cell
death resulted from damage to high molecular weight
DNA. The phenoloxidase mutants that retained the catecholamine transport system demonstrated some resistance arising, it was suggested, from the paradoxical role
of catecholamines, either as sources of damaging radicals
or as free radical scavengers that are dependent on appropriate conditions [56]. The retention of a functional
catecholamine transport system might enable the accumulation of intracellular catecholamines which in turn
increase the reductive capacity of the yeast cell [57]. In
the wild type strain, the phenoloxidase may act either to
deplete levels of catecholamines, making them unavailable for oxidative reactions in the presence of hydrogen
peroxide, or to produce melanin and in so doing give rise
to an important free radical scavenger [57].
C. neoformans mutants with varying sensitivities to
oxygen exposure were subsequently produced [58], and
three of them were found to have defects in their ability
to produce melanin. The latter was suggestive of a close
relationship between melanization and oxygen resistance
[58], particularly since catecholamine uptake mechanisms
and phenoloxidase expression and activity seemed to be
affected. These mutants were also used in attempts to
determine whether the production of complete polymeric
melanin or the build up of intracellular monomeric catechol pools was responsible for resistance to oxidant damage [59]. Exposure of wild type and mutant strains to
hypochlorite (a major oxidative product of inflammatory
cells [55]) and permanganate demonstrated that exogenous melanin was responsible for protection against oxidants. However, melanin production was non-protective
for the oxidant hydrogen peroxide, although the latter is
100 times less fungicidal than hypochlorite [59]. It is of
particular note that the physiological antioxidant capacity conferred by melanin formation was in the same range
as that of oxidant production by stimulated
macrophages. The interaction of iron with C. neoformans
melanin has also been investigated [60], and Fe (II)
appears to be able to reduce melanin and co-operatively
increase redox buffering.
Other oxygen sensitive mutants have been used to
identify two loci (oxy1 and oxy2 ) that link melanization
to hyperoxia sensitivity [61]; the oxy2 locus appears to
have a particularly close association. Studies have also
been made on the susceptibility of melanized and nonmelanized C. neoformans to both oxygen and nitrogen
derived oxidants such as nitric oxide [62] (the latter has
been shown to be both fungistatic and fungicidal [63,64]).
In the case of both oxidant types melanized cells exhibited significantly greater survival than non-melanized
cells. Electron spin resonance spectroscopy has been used
to demonstrate electron transfer to or from melanin in
fungal cells [65]; the same study also demonstrated that
melanized C. neoformans cells were more resistant to the
antifungal effects of murine macrophages than nonmelanized cells. Melaninized C. neoformans cells have
also been found to be less susceptible to in 6itro treatment
with the antifungal amphotericin B [66]. The mechanism
underlying this observation is unknown although
melanin may act by ameliorating amphotericin B-mediated oxidative damage in the same way that the addition
of catalase is hypothesized to protect Candida albicans
under similar circumstances (see Catalase section).
Although the genetic studies described in previous
paragraphs implicate laccase in virulence, there is recent
debate as to whether or not melanin is actually produced
in 6i6o. Early studies using the Masson–Fontana silver
stain for melanin demonstrated that yeasts in tissue labelled clearly [67]. It is of note, however, that unpigmented cells grown on agar were also reactive, suggesting
that there were other silver reducing compounds present
in the cell wall [67]. Electron microscopy has been used to
demonstrate electron dense cell walls in pigmented C.
neoformans cells that have been grown on L-dopa [65].
This observation is suggestive of the presence of melanin.
However, evidence has been recently produced which
shows that whilst catecholamine oxidation products
(COPs) are formed by laccase positive C. neoformans cells
in the brains of infected mice, it would appear that there
is no post-enzymatic polymerization of these products to
form melanin [68]. It is suggested that these highly reactive COPs (o-quinones) mediate laccase dependent toxicity and in so doing play a direct role in pathogenesis.
However, the absence of detectable melanin during C.
neoformans infection described in this model is in contrast to other contemporary observations. In fact, until
recently it has proved very difficult to study melanization
in 6i6o because of the absence of specific reagents that
were melanin reactive. This problem has recently been
solved by two approaches. Firstly, a phage displaying a
melanin binding peptide has been developed and used to
immunohistochemically detect melanin during experimental cryptococcal infection in mice [69,70]. Secondly,
murine monoclonal antibodies have recently been produced against C. neoformans L-dopa melanin and have
been used to demonstrate that melanin-like pigments are
formed in C. neoformans cells in several tissues, including
the brain [71]. This work built on previous studies that
demonstrated that C. neoformans melanin was immunogenic in mice [72]. Melanin is detectable as early as 2 days
© 1999 ISHAM, Medical Mycology, 37, 375–389
Antioxidant systems in the fungi of man and their role in virulence
post-infection. It is also of note that antibodies to
melanin are also made during experimental cryptococcosis in mice [69,70], suggesting the presence of the pigment
during infection.
Further support for the production of melanin in 6i6o
comes from studies on the herbicide glyphosate. The
latter can prevent C. neoformans melanization in 6itro, via
the inhibition of L-dopa autopolymerization; most interestingly, glyphosate has therapeutically beneficial effects
in a mouse model of C. neoformans [73]. This suggests
that glyphosate can prolong survival in mice by inhibiting
melanin synthesis in 6i6o. Taken together, these data now
provide compelling evidence for the in 6i6o production of
melanin in this model.
The apparent discrepancy between the work of Liu et
al. [68] and that described above may be more readily
reconcilable than first appears. The mice models of infection used in each case are rather different; thus an acute,
rapidly progressive model was used in the study of Liu et
al. [68] as the authors required large numbers of yeast
cells for biochemical studies, and melanization may not
occur under such conditions. There would seem to be
little doubt that melanization does occur in the other, less
rapidly developing model used by the Casadevall group
[69 – 73], and there appears to be an element of time
dependency in the production of the pigment. It is of
course feasible that COP-mediated toxicity is an important factor in virulence early in infection, and that subsequently oxidant protection mediated by melanin is
important in pathogenesis. As ever, the precise relationship of mouse models to infection in humans is problematic, and an obvious approach will be to use the newly
developed melanin specific reagents [69 – 71] to detect
melanin expression in clinical samples from patients.
Finally, it is important to stress a note of caution on
the study of melanization in C. neoformans; recent data
has indicated that microevolution of certain phenotypes,
including melanin production, can occur within laboratory isolates [74]. As a consequence, a degree of care
should be taken when comparing data from different
laboratories that may have used the same isolate of C.
neoformans over an extended time period.
In contrast to the situation in C. neoformans, melanization in Exophiala (Wangiella) dermatidis has been shown
to occur via the pentaketide pathway, since the relevant
melanin precursors have been detected [75], and treatment with tricyclazole causes pentaketide melanin
metabolites to accumulate [76]. In this pathway, polymerization of endogenous 1,8-dihydroxynaphthalene is the
last step in melanin production [77]. However, it is important to note that the redox functions of both the
melanin from C. neoformans and that from E. dermatidis
© 1999 ISHAM, Medical Mycology, 37, 375 – 389
379
are the same, despite their disparate synthesis [59,78].
Thus, melanized strains of E. dermatidis were shown to
neutralize more oxidant and withstood higher concentrations of permanganate and hypochlorite than albino
strains did [78]. It is of note that melanin did not protect
against hydrogen peroxide in this system.
Information on how melanin in E. dermatidis might
function in relation to immune effector cells is sparse
although a recent paper has shown that it is capable of
preventing this pathogen from being killed by the
phagolysosomal oxidative burst of human neutrophils
[79]. This protection requires the presence of the complete melanin molecule, rather than precursors such as
scytalone. Seemingly, melanin does not affect either
phagocytosis or the induction of the oxidative burst in
this model. The virulence of melanin deficient E. dermatidis mutants relative to wild type isolates in mice models
has been studied, and there appears to be an association
between absence of melanin and decreased mortality [80],
although not between melanin and the neurological
symptomology of mouse phaeohyphomycosis. However,
the apparent link between melanization and virulence is
at least partly dependent on the mouse strain used [81].
Thus the DBA/2J mouse was susceptible to infection with
both melanized and non-melanized isolates, whereas
melanin deficient mutants were less virulent than the wild
type in other mouse strains. Subsequent studies using the
non-melanized Mc3W mutant also appeared to show an
association between melanization and virulence [82].
However, it is important to note that the mutants used
in these studies were not produced by the disruption of
individual genes. Thus, the use of UV to generate mutants [81] can give rise to multiple gene defects making it
unwise to draw firm conclusions about the function of
melanin from these studies. Molecular genetic based evidence of the type demonstrating the role of the C. neoformans laccase in virulence is not yet available in E.
dermatidis. As such, the relationship between melanization and pathogenesis in this organism remains to be
definitively proved, although the data described above
would imply a role in protection against immune effector
cell oxidant-mediated damage. However, a start has now
been made in the identification in the genes involved in
melanogenesis and a putative polyketide synthase gene
has been identified [83]. Relevant work on other members
of dematiaceous fungi in relation to melanization is much
more restricted although it has been shown that albino
strains of Alternaria alternata are more susceptible to
permanganate and hypochlorite than melanized strains
[78].
There has been significant recent interest in the role of
pigments within the genus Aspergillus; in fact production
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of melanin by Aspergillus species has been known about
for some considerable time [84,85]. In A. nidulans, two
conidial pigmentation genes (wA and yA) have been
identified; the former encodes a putative polyketide synthase, and the latter encodes a laccase, and disruption of
each results in white and yellow coloured conidia, respectively [86 – 89]. No data is available on how disruption of
these genes effects virulence, which might be tested in the
chronic granulomotous disease model described subsequently in the catalase section [90]. In A. fumigatus, an
UV generated mutant strain lacking conidial pigmentation has recently been described [91]. Mutant conidia
were shown to be more susceptible to killing by oxidants
in 6itro and suffered greater monocyte induced damage
than did wild type conidia; most significantly, they were
also less virulent in a mouse model. Reversion analysis
suggested that a single mutation was responsible for the
phenotypes observed, including altered conidial surface
morphology [91]. This mutation maybe similar to that
described in the A. fumigatus alb1 gene [92], which disrupts a putative polyketide synthase and which is involved in dihydroxynaphthalene-melanin biosynthesis.
Disruption of this gene led to a loss of virulence in mice
which complementation restored. However, data was not
provided on the effect of alb1 disruption on oxidative
killing, although it was found that the mutant was ingested by neutrophils at a higher rate than the wild type
[92]. A second gene involved in the pigmentation of A.
fumigatus has also been described [93]; designated arp1,
this encodes a putative scytalone dehydratase. Disruption
of this developmentally regulated gene leads to enhanced
complement deposition, although the susceptibility of the
resulting mutant to oxidant-mediated damage was not
determined. Taken together these data strongly implicate
the formation of melanin in the pathogenesis of A. fumigatus. These findings are particularly exciting, given the
previously unsuccessful attempts to define virulence factors in this important fungal pathogen.
Superoxide dismutase
Superoxide dismutases (SODs) are metalloenzymes that
catalyse the oxidation of superoxide with the concomitant reduction of hydrogen ions to hydrogen peroxide
and molecular oxygen. Two unrelated classes of these
enzymes exist; the first contain iron (Fe SOD) or manganese (Mn SOD) at the active site, and the second class
contain copper and zinc (Cu,Zn SOD) [94]. Both classes
catalyse the same reaction comparatively well. Fe SODs
are found in prokaryotes and in some plant families and
Mn SODs are found in prokaryotes and the mitochondrial matrix. Cu,Zn SODs are usually found in the cyto-
sol of eukaryotic cells [95], though they may also be
found extracellularly [96]. They may also occur in some
plant chloroplasts and in some prokaryotes [95]. Within
the cell, the roles of SODs are closely inter-related with
the activity of other antioxidants such as catalase.
Only brief mention is made here of Mn SODs since, in
fungi, as in other eukaryotes, the primary role of this
class of enzyme would appear to be to deal with superoxide generated within mitochondria [97]. As a consequence, it is not easy to hypothesize a direct role for Mn
SODs in fungal virulence, as spatially such enzymes do
not appear to be in a position to deal with extracellular
superoxide. A number of Mn SODs from fungal pathogens have indeed been characterized, including those
from C. neoformans [98] and A. fumigatus [99–101], and
the latter has been found to be an important allergen.
Thus far, no data has been produced that associates Mn
SOD with virulence in any fungal pathogen, and compared to Cu,Zn SODs (see below), they have been the
focus of less attention.
Based on spatial distribution, the main role of fungal
Cu,Zn SODs is clearly to deal with extra-mitochondrial
superoxide generated within the cytosol. However, Cu,Zn
SOD located at or just below the cell surface could have
a hypothetical role in protecting the fungal cell against
extracellularly generated superoxide. This postulated role
would of course be most effectively performed by extracellular Cu,Zn SOD, which might or might not be associated with the fungal cell wall/capsule. It is known from
various studies that the addition of extraneous SOD to in
6itro killing assays involving fungal pathogens may abrogate fungicidal activity [102–104]. Potentially Cu,Zn
SOD produced by the invading fungus might function in
the same way and so act as a virulence determinant.
In fact the existence of cytosolic Cu,Zn SODs in a
number of fungi generally regarded as non-pathogens,
such as Neurospora [105–107] and Saccharomyces has
been known for some time [107,108]. In particular, the
literature relating to oxidative stress and Cu,Zn SOD in
S. cere6isiae has continued to expand [109–111], especially with regard to studies at the molecular level. However, it was not until 1995 [112] that a Cu,Zn SOD from
a commonly encountered fungal pathogen (A. fumigatus)
was characterized. The A. fumigatus Cu,Zn SOD demonstrated the inhibitor profile common to other such enzymes, was thermotolerant and had reduced and
non-reduced relative molecular masses of 19 and 95 kDa,
respectively; the authors suggested that the latter was
indicative of a tetrameric configuration for the intact
enzyme [112]. To date all Cu,Zn SODs described have
been multimeric in structure and the vast majority are
dimeric [113,114]; however, tetrameric forms have been
identified, at least one of which is extracellular [96].
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Antioxidant systems in the fungi of man and their role in virulence
The A. fumigatus Cu,Zn SOD was also found in supernatants [112] of mid-log cultures which implied an extracellular form of the enzyme might be present. Human
sera from patients with aspergillosis was also found to
recognize A. fumigatus Cu,Zn SOD [115], an observation
that suggested that the enzyme was readily accessible to
the humoral immune system. Subsequent studies using a
monospecific polyclonal directed against purified A. fumigatus Cu,ZnSOD [116] demonstrated that the enzyme
was present in the fungal cell wall. Expression of the
Cu,Zn SOD was also particularly associated with areas of
intense metabolic activity, such as hyphal apical tips and
conidiophores [116]; this distribution was unsurprising,
given the enzymes defined role in intracellular
detoxification.
Subsequently, Cu,Zn SODs were purified and characterized from A. fla6us, A. niger, A. nidulans and A. terreus
[117]; the latter are both very rare causative agents of
human disease. A number of interesting differences were
identified in the biochemical behaviour between these
enzymes and the previously described A. fumigatus Cu,Zn
SOD, the most significant of which were the greatly
reduced activity of the purified A. nidulans and A. terreus
enzymes at 37 °C, and their relative lack of thermotolerance. The thermotolerance of the A. fumigatus, Cu,Zn
SOD is perhaps unsurprising given the organisms ability
to survive and grow within self heating compost heaps,
but an incidental effect of this appears to be that this
enzyme is correspondingly more efficient at body temperature than the equivalent enzymes from the much more
rarely pathogenic A. nidulans and A. terreus. Irrespective
of whether or not Cu,Zn SOD directly protects Aspergillus from immune cell oxidative attack, the relative
thermotolerance of the A. fumigatus Cu,Zn SOD (and
presumably that of other fundamentally important
housekeeping enzymes) should provide A. fumigatus with
a metabolic survival advantage in the human host over
other Aspergillus species.
Similar biochemical comparison work has been performed on the Cu,Zn SODs of C. neoformans var. neoformans and C. neoformans var. gattii [118]. This revealed
interesting differences in apparent structure and behaviour which is of substantial phylogenetic interest
given the recent reclassification suggested within this species [119]. Evidence for extracellular forms of Cryptococcus Cu,Zn SOD was less convincing than in the case of
Aspergillus, although it was noted that an acapsular
mutant released more detectable Cu,Zn SOD into culture
filtrate than encapsulated isolates. This led the authors to
hypothesize that capsular retention of Cu,Zn SOD was
occurring.
© 1999 ISHAM, Medical Mycology, 37, 375 – 389
381
It should be made clear that none of the data described
above provides any direct evidence for a role for either
Aspergillus or Cryptococcus Cu,Zn SODs in virulence.
Realistically, that can only come from studies on the
virulence of mutants in which the appropriate Cu,Zn
SOD have been disrupted. In fact, the genes encoding the
respective enzymes have now been cloned from A. fumigatus, A. fla6us and A. nidulans (M.D. Holdom, unpublished results) and C. neoformans (V. Chaturvedi,
unpublished results) as a prelude to these studies. There
is a clear precedence for such work; in S. cere6isiae
disruption of the Cu,Zn SOD gene results in a number of
biochemical defects [120–122] including hypersensitivity
to oxygen toxicity. A significant offshoot of this work has
been the realization that in Cu,Zn SOD deficient mutants
compensatory antioxidant mechanisms exist [122,123],
the presence of which in fungal pathogens will make
assessing the role of Cu,Zn SODs in virulence rather
difficult.
Very limited data is available on SODs from other
fungal pathogens although it is known that Trichophyton
mentagrophytes has a Cu,Zn SOD that behaves similarly
to that described from A. fumigatus [124]. In addition, the
C. albicans Cu,Zn SOD has been subject to X-ray diffraction [125]. In neither case is there any indication as yet
that these enzymes play a role in the virulence of these
respective organisms.
Catalase
Catalases are antioxidant metalloenzymes that are found
virtually ubiquitously within aerobic organisms; their role
is to protect cells from oxidative damage arising from
exposure to hydrogen peroxide [126]. These enzymes are
typically composed of four identical subunits of between
60–90 kDa each, which contain ferriprotoporphrin
(haematin). The active site of these enzymes is generally
very highly conserved, although the molecule as a whole
may exist in a number of conformational variations. At
high concentration of hydrogen peroxide the enzyme is
catalytic, whereas it is peroxidatic when peroxide concentrations are low. There is little doubt that in fungal
pathogens, as in other aerobes, the primary function of
catalase is to remove hydrogen peroxide generated as a
normal by-product of cellular metabolism. As such catalase interacts closely with the functions of peroxidases
and superoxide dismutases, and together these enzymes
play an essential role in the intracellular oxidative detoxification process. Catalases are also known to contribute
to growth regulation and development in a variety of
eukaryotes, including S. cere6isiae [127].
382
Hamilton & Holdom
However, it is known from studies using various in
6itro killing assays that oxidative mechanisms are important in the killing of a range of fungal pathogens; the
addition of catalase to such assays may demonstrably
inhibit the fungicidal activity of such mechanisms. Thus,
catalase has been shown to inhibit the killing of A.
fumigatus by neutrophils [128], of Paracoccidioides
brasiliensis by neutrophils [129,130], of Blastomyces dermatitidis by neutrophils [131 – 133], of Histoplasma capsulatum by bronchoalveolar macrophages [134], of C.
albicans hyphae by neutrophils [135] and of C. parapsilosis by Kupffer cells [136]. Accordingly, it would seem
possible that endogenously produced fungal catalase
might abrogate the effects of such oxidative killing mechanisms, and in so doing act as a virulence determinant, in
much the same way that catalase is hypothesized to
function in various bacterial and parasite pathogens
[137]. It is also of note that in C. albicans the addition of
exogenous catalase can protect cells from amphotericin
B-mediated oxidative damage [138], which is suggestive
of another potential role for endogenously produced
catalase.
Within the pathogenic fungi as a whole, catalases have
been most extensively studied in members of the genus
Aspergillus. In A. nidulans there is evidence for at least
two catalases [139]; thus the gene encoding catA has been
analysed and found to be developmentally regulated with
expression almost totally restricted to conidia [139,140].
Perhaps as might be expected conidia from catA disrupted mutants are sensitive to hydrogen peroxide when
compared to those of the wild type [139]. A second A.
nidulans catalase (CATB), with polypeptide chains of 721
amino acids in length, is produced in the mycelial phase
[140] and appears to be closely related to the CAT1 of A.
fumigatus (see below). CATB mRNA is absent from
conidia, and the enzyme can be induced by hydrogen
peroxide. Perhaps most interestingly CATA/CATB double mutants were able to grow on substrates whose
catabolism generated hydrogen peroxide, which implicates the existence of alternative hydrogen peroxide
detoxification pathways [140]. A subsequent study using
catA disrupted mutants [90] to infect a mouse model of
chronic granulatomous disease, revealed that mutant and
wild type strains were equally virulent. Given that catA
disrupted conidia are hydrogen peroxide sensitive (see
above) this observation is disappointing since it indicates
that in this model A. nidulans catalase has no discernible
role in virulence.
In A. niger a 385 kDa glycosylated catalase composed
of four subunits, each of 97 kDa, has been identified and
characterized [141]; the relationship of this catalase to a
previously described 323 kDa catalase [142] is unclear.
Subsequently, the amino acid sequence encoded by the so
called catR gene from this fungus was deduced [143]; this
protein was composed of 730 residues with sequence
homology to pre-existing catalases. The mode of action
of A. niger catalase has also been determined [144],
together with the effects of hydrogen peroxide on this
mechanism [145]. However, there is no data available on
the ability of the A. niger catalase to ameliorate the
oxidative killing mechanisms of immune effector cells.
Research into A. fumigatus catalase has a comparatively long history with the first identification of this
enzyme in 1968 [146]. Schonheider went on to describe
the immunological importance of A. fumigatus catalase
[147,148] and its antigenic relationship to catalases from
other members of the genus [149]. Subsequently, two
classes of catalases were identified; these were designated
fast (F) and slow (S) based on their electrophoretic mobility and biochemical differences [150]. The F class catalases (240 kDa) were demonstrated to have both catalase
and peroxidase activity whilst the S class catalases (specifically S1 at 420 kDa and S2 at 502 kDa) displayed
exclusive catalase activity. One of these catalases was
shown to be immunogenic and it was suggested might be
of use in immunodiagnosis [150]; this catalase is almost
certainly synonymous with the 90 kDa immunodominant
CAT 1 antigen [151].
The CAT1 gene has since been sequenced and shown
to encode a 728 amino acid polypeptide, with a signal
peptide and a propeptide [152]. Disruption of this gene to
give rise to mutants deficient in the production of the S
catalase has revealed that it makes no discernible contribution to virulence in either in 6itro or in 6i6o models
[152]. These results are disappointing given that A. fumigatus produces so much detectable catalase. However, it
is possible that the second catalase (the F band) compensates for the loss of the S gene product and studies in
which both genes are disrupted are currently underway
(S. Paris, D. Wysong, R. Diamond, J.P. Latgé, unpublished results).
The production and function of catalase in several
Candida species has attracted a significant amount of
attention. Much of this has been focused on a catalase of
C. tropicalis which has been localized by immunoelectron
microscopy within peroxisomes [153]. The gene and
cDNA encoding this peroxisomal catalase have been
sequenced [154–157], and growth on alkanes has been
shown to cause up-regulation of the enzyme [158,159].
Candida tropicalis catalase has also been functionally
expressed in Saccharomyces cere6isiae [160]; however, no
attempts have yet been made to discern what role, if any,
this enzyme plays in pathogenesis in C. tropicalis.
© 1999 ISHAM, Medical Mycology, 37, 375–389
Antioxidant systems in the fungi of man and their role in virulence
Perhaps surprisingly there have been fewer studies on
the catalase(s) of C. albicans, numerically the most important disease causing member of the genus. An attempt
was made to characterize a catalase from this species but
this study was handicapped by what appeared to be
incomplete purification [161]. Catalase expression in C.
albicans cells exposed to methanol and various growth
temperatures has also been studied, with perhaps the
most interesting observation being the apparent up-regulation of the enzyme at 37 °C [162]. However, the most
significant study relates to the recent cloning of the gene
for C. albicans catalase (CAT1); this gene encodes a
protein of 487 residues, which has homology to other
fungal catalases [163]. CAT1 deficient mutants were
shown to be more susceptible to damage mediated by
neutrophils and by exogenous hydrogen peroxide than
the wild type. More significantly, the C. albicans catalase
deficient mutant was measurably less virulent in a mouse
model than the parent [163]. However, restoration of
virulence by reintroduction of the catalase gene into
mutants has proved unsuccessful thus far; data from the
latter experiment is necessary to provide definitive linkage between CAT1 and virulence in C. albicans.
Catalase production has also attracted some attention
in H. capsulatum. Initial studies on cell free extracts of
blastospores and conidiospores of H. capsulatum demonstrated that they contained catalase [164]. This initial
study also reported that the variation of strain sensitivity
to hydrogen peroxide could be correlated with the
amount of detectable catalase [164]. However, a subsequent attempt to correlate resistance to hydrogen peroxide with the large observed differences in catalase
expression between isolates was unsuccessful [165], although it was demonstrated that glucose and oxygen
regulated production of catalase. More recently studies
have implied the existence of at least two catalase genes
in H. capsulatum, one of which is cell associated, the
other of which is secreted [166]. One of these almost
certainly corresponds to the gene encoding the serologically important M antigen, which has been shown to
have high sequence homology to known catalases [167].
Earlier reports using cross-reactive monoclonal antibodies had indeed suggested that the M antigen was a catalase [168].
Amongst other fungal pathogens there is evidence for
the existence of catalases, but much less information
regarding their role. Thus, a diagnostically useful 61 kDa
antigen from Penicillium marneffei has been found to
have a N-terminal amino acid sequence which has high
homology with other catalases [169]. Blastomyces dermatitidis also possesses catalytic activity [170], but attempts to correlate the catalase content of particular
© 1999 ISHAM, Medical Mycology, 37, 375 – 389
383
isolates with in 6i6o virulence or in 6itro killing by neutrophils were unsuccessful. Spherules of Coccidioides immitis have catalase activity, but the low levels of this
enzyme in mature spherules were too low to account for
the low levels of hydrogen peroxide found in exposed
neutrophils [171].
Mannitol
The acyclic hexitol mannitol is produced by a number of
different fungi, and it has been suggested that it plays a
role in osmotolerance [172,173], and as a storage carbohydrate [174]. However, it also has a hypothetical role as
a free radical scavenger and it is in this potential capacity
that it is reviewed here. Most of the existing literature
relates to mannitol production in Aspergillus and Cryptococcus, and these genera are the focus of this section.
Several species of Aspergillus are known to produce large
amounts of D-mannitol in 6itro [175], and A. fumigatus
has been shown to produce this compound in 6i6o [176] at
levels at which it becomes detectable in serum. However,
mannitol production in Aspergillus has not, as yet, been
shown to have any role in protection against host cell
oxidant production.
C. neoformans has also been shown to produce mannitol both in 6itro and in 6i6o [177,178], and glucose,
fructose and mannose can be converted to mannitol by
this yeast [179]. The detection of mannitol in in 6i6o
models led to speculation that its role during infection
might be both to contribute to brain oedema and to
interfere with phagocyte killing via scavenging of hydroxyl radicals [177]. Some evidence for the latter contention now exists, based on experiments on a mutant
generated by UV irradiation that produced low levels of
mannitol [180]. In 6itro studies with this mutant have
show that it is more susceptible to killing by polymorphonuclear cells than its wild type parent, and that the
addition of extraneous mannitol could protect it from
oxidant-mediated killing in a cell free system [180].
These studies are worthy of follow up and mannitol
production in C. neoformans has now been accorded the
status of a putative virulence factor by several authors
[22,181]. Of particular importance in the future will be
the targeted disruption of the specific genes involved in
mannitol biosynthesis and measurement of the effect that
this has on virulence. In fact a gene that directs expression of mannitol dehydrogenase (Mtl-1 ) has now been
elucidated [182], although data relating to its disruption
has not yet appeared in the literature. Finally, it is worth
pointing out that the role, if any, of mannitol production
in other fungal pathogens remains to be elucidated.
384
Hamilton & Holdom
Other potential antioxidant compounds
Acid phosphatase has been implicated in the virulence of
several bacterial and parasite species [183,184]; in such
circumstances it is thought to function via suppression of
the neutrophil respiratory burst [185] and superoxide
anion production. Whilst several fungal pathogens, such
as C. neoformans [186] and P. marneffei [187] have been
shown to produce significant quantities of this enzyme,
nothing is yet known of the ability of acid phosphatases
to effect pathogenicity in these fungi. Peroxidases also
have a hypothetical role in the protection of parasites
from host generated oxidant damage [188]. Although
some non-pathogenic fungi have such enzymes [189], with
the exception of the A. fumigatus catalase/peroxidase
[152], they have not been studied in any detail in any of
the human pathogens described in the previous sections.
Consequently, their potential role in pathogenicity, if
any, remains to be ascertained. Finally, brief mention
should be made of pigments other than melanin produced by fungal pathogens. The carotenoids torulene and
torularhodine are also synthesized by E. dermatitidis
[190]. These may have a role in shielding the cell from
UV induced damage [190], although they are unable to
prevent killing by neutrophils [79].
Concluding remarks
Four putative antioxidants have attracted most interest
in the various fungal pathogens of man: melanin, SOD,
catalase and mannitol. There is now good evidence implicating melanin production in the virulence of C. neoformans and A. fumigatus. Since melanin is a
multifunctional compound it is still not absolutely clear
that its role in pathogenesis is as an antioxidant alone,
although there is certainly some in 6itro evidence from C.
neoformans that it can protect against oxidant damage.
Evidence for its antioxidant role in A. fumigatus has yet
to be published although this is an obvious area for
further study. The role of melanins and other pigments in
other fungal pathogens should also be assessed or reassessed at the earliest opportunity. The evidence linking
SODs to fungal pathogenesis is at this time purely circumstantial, although we now know something of the
biochemical characteristics of these enzymes from a number of genera. Disruption experiments on the relevant
SOD genes should be performed to provide a meaningful
assessment of their role in virulence. At least in one
pathogen (C. albicans) there appears to be some evidence
that catalase can play a protective antioxidant role, although in another (A. fumigatus) disruption of a catalase
gene has no effect. However, the existence of several
catalase genes complicates matters in this organism and
priority should be given to the study of mutants in which
all catalase genes are disrupted. As an adjunct to this
approach, disruptions of several unrelated genes (i.e.
SOD and catalase) within the same mutant in various
species should also be undertaken as antioxidant enzymes
typically act in concert. Analysis of the role of mannitol,
and for that matter of other putative antioxidants, such
as acid phosphatases, should also not be neglected. There
is clearly much that remains to be done; however, we
have at the very least established the framework for
analysing putative fungal antioxidants and progress
should be rapid in this area in the next few years.
Acknowledgements
We thank Drs A. Casadevall and J.-P. Latgé for access to
unpublished information.
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