ELSEVIER
IMMUNOLOGY AND
MEDICAL
MICROBIOLOGY
FEMS Immunology
and Medical Microbiology
14 (1996) 95-102
Expression of the Cu,Zn superoxide dismutase of
Aspergillus fumigatus as determined by immunochemistry
and immunoelectron microscopy
Andrew John Hamilton
*, Mary Denise Holdom, Lisa Jeavons
Dermatology Unit, St .lohns Institute of Dermatology, Clinical Sciences Laboratories, 18th Floor, Guy’s Tower, Guy’s Hospital,
London SE1 9RT, UK
Received
18 December
1995; revised 25 January
1996; accepted 30 January
1996
Abstract
A polyclonal antibody against purified Cu,Zn superoxide dismutase (SOD) from the pathogen Aspergillusfumigatus
was
raised in a sheep. This antibody recognised purified A. fumigntus SOD, together with a single band of 19 kDa in A.
jikgatus
cytoplasmic antigen, by immunodevelopment
of Western blots. The polyclonal serum did not recognise either the
manganese or iron containing forms of the enzyme; however, it was reactive against putative Cu,Zn SODS in other members
of the genus Aspergillus. Immunofluorescent
staining of A. fumigatus cultures demonstrated expression of the Cu,Zn SOD
in conidia and hyphae, with the cell wall staining particularly intensely. Conidiophores were stained in an uniformly intense
pattern. Immunoelectron microscopy confirmed that the SOD was present within the hyphal cell wall, although there was
also labelling in the cytoplasm. SOD may protect Aspergillus against oxidants produced by immune effector cells and these
observations demonstrate that the enzyme is available to perform its antioxidant function within the cell wall.
Keywords: Aspergillus fumi,patus; Superoxide
dismutase;
Cellular expression
1. Introduction
Aspergillus jkmigatus is an opportunistic
fungal
of causing a range of
clinical conditions from allergic reactions through to
invasive and disseminated aspergillosis in immunocompromised
individuals.
Invasive aspergillosis
is
often a fatal condition and high risk groups include
bone marrow and renal transplant recipients and
pathogen
which
* Corresponding
(171) 407 6689.
is capable
author. Tel.: +44 (171) 955 4663; Fax: +44
0928-8244/96/$15.00
Copyright
PII SO928-8244(96)00015-6
0 1996 Federation
of European
leukaemia
[l-3].
A number of
have been assessed as potential virulence determinants in A. fumigatus, including severa1 proteinases [4,5], gliotoxins [6] and complement
__
inhibiting phospholipids 171.
However, there have been no direct attempts to
determine whether A. fumigatus can protect itself
from the killing mechanisms used by host immune
effector cells such as neutrophils and macrophages.
This is despite firstly suggestions
that in other
pathogens antioxidant enzymes such as superoxide
dismutase (SOD) may protect against toxic oxidative
burst metabolites
[8,9], and secondly the observation
patients
with
diverse
factors
Microbiological
acute
Societies. Published
by Elsevier Science B.V.
96
AndrewJoh Hamilton et al. / FEMS Immunology and Medical Microbiology 14 (1996) 95-102
that the killing of A. fumigatus
hyphae by neutrophils in an in vitro system can be extensively
reduced by the addition of SOD and catalase [IO].
Recently a Cu,Zn SOD has been purified to homogeneity from A. fumigatus [ 1I]. The enzyme occurs
in the culture filtrate of mid log cultures and is
recognised by immune human sera [12]. However,
nothing is known of the antigenic relationships of
this Cu,Zn SOD to similar enzymes in other Aspergillus sp. and to the iron (Fe) and manganese
(Mn) [13,14] classes of SOD which may also exist in
A. fumigatus. More significantly the kinetics of intracellular expression of the Cu,Zn SOD during conidial germination and hyphal growth have not been
studied. The latter will have a profound bearing on
whether the A. fumigatus SOD is capable of fulfilling a protective role against immune effector cell
oxidative bursts. In this study we detail the production of a specific polyclonal antibody against the A.
fumigatus SOD which we have used to assess these
questions.
2. Materials and methods
2.1. Production
of sheep anti-SOD polyclonal
and estimation of reactivity by ELBA
sera
Cu,Zn SOD was purified from mid log phase
hyphal cultures of A. fumigatus (NCPF 2010, National Collection of Pathogenic Fungi, Mycological
Reference
Laboratories,
Bristol, UK) grown on
Sabourauds broth using a combination of liquid isoelectric focusing and gel filtration Fast Protein Liquid Chromatography (FPLC) as previously described
[lo]. Purified SOD, at a concentration of 1 mg/ml in
0.05 mM Tris buffer pH 8.4 was then used to raise a
polyclonal antibody in sheep (The Binding Site Ltd,
Birmingham,
UK). A test bleed yielded 30 ml of
sera; this was followed by a booster inoculation
which generated a further 450 ml of sera.
Purified A. fumigatus Cu,Zn SOD made up in
0.06 M carbonate buffer (pH 9.6) was used to coat
96 well ELISA plates (Linbro) at a concentration of
1 pg/well
overnight at 4°C. Plates were washed
with PBS-Tween (0.01 M pH 7.4, containing 0.05%
Tween 20) and then incubated with serial dilutions
(beginning at 1:50) of the sheep anti-A. fumigatus
polyclonal made up in PBS-Tween for one hour at
37°C. Normal sheep sera (Sigma, Poole, Dorset, UK)
was used as a negative control.
After washing with PBS-Tween plates were incubated with peroxidase conjugated donkey anti-sheep
IgG (H + L) (Jackson Immunoresearch,
West Grove,
Penn., USA) diluted at 1 : 1000 under the same conditions. Final washes in PBS-Tween and PBS were
followed by development
with chromogenic
substrate as previously described [ 15,161.
2.2. Polyacrylamide
gel electrophoresis
(PAGE),
electroblotting, and immunoenzyme development
Hyphal homogenates of mid log cultures of A.
fumigatus, A. flavus, A. niger, A. terreus, and A.
nidulans (NCPF Nos. 2008, 2023, 2729, 2232) together with purified Cu,Zn SOD from A. fumigatus
and commercial sources of bacterial (E. coti) Fe
SOD and Mn SOD (Sigma) were subjected to sodium
dodecyl sulphate PAGE on 15% gels and Western
blotting as previously described [15,16]. Blots were
blocked overnight at 4°C in 2% casein, dried, and
incubated with the sheep anti-SOD polyclonal used
at dilutions of 1 : 50, 1 : 100, 1 : 500 and 1 : 1000 in
PBS-Tween with 0.5% casein [15,16]. After washing
in PBS-Tween, blots were incubated with peroxidase-linked
donkey anti-sheep IgG (Jackson Immunochemicals)
used at dilutions of 1 : 250 and
1: 1000 and made up in PBS-Tween
with 0.5%
casein. After further washes the blots were developed with diaminobenzamine
and 4-chloronaphthol
[ 15,161. Western
blots were also stained with
Coomassie brilliant blue R-250.
2.3. lmmunofluorescence
fumigatus cultures
detection
of SOD
in A.
Glass cover slips of 5 mm were sterilised and
placed on the bottom of 24-well Costar tissue culture
plates. Sabouraud’s broth, 1 ml, together with an
inoculum of an A. fumigatus conidial suspension
(equivalent to 1 X lo5 conidia/well),
produced as
previously described [ 111, were then added to each
well. Plates were incubated at 37°C and at 2-h
intervals (up to 12 h) cover slips were removed,
washed 3 times in PBS, and fixed for 10 min in
either absolute methanol or acetone. Cover slips
AndrewJoh Hamilton et al. /FEMS
Immunology and Medical Microbiology
were then air-dried and incubated with the sheep
anti-SOD polyclonal diluted at 1 : 50 and 1 : 100 in
PBS-Tween
for 1 h at room temperature.
After
PBS-Tween washes the cover slips were incubated
with rhodamine conjugated donkey anti-sheep IgG
(H + L) (Jackson Immunochemicals)
at a dilution of
1 : 100 in PBS-Tween for 1 h at room temperature.
After a final series (of washes cover slips were
mounted in a gelatin/PBS
solution. Normal sheep
sera or PBS-Tween were used as negative controls in
ABCDEFGHIJ
83,
32s
18,
Fig. 1. Western blot (immunoblot) and Coomassie brilliant blue
stain analysis of reactivity of sheep anti-A. fimigatus SOD polyclonal antibody. Relative molecular mass values are in kilodaltons. Track A: Coomassie brilliant blue stained Western blot of A.
fumigutus hyphai cytoplasmic antigen. Track B: hyphal cytoplasmic antigen immunodeveloped
with sheep anti-A. fumigatus Cu,Zn
SOD polyclonal
antibody. Track C: Coomassie brilliant blue
stained Western blot of purified A. fumigatus Cu,Zn SOD. Track
D: purified A. fumigatus Cu,Zn SOD irnmunodeveloped
with
sheep anti-A. fumigates SOD polyclonal antibody. Tracks E, F, G,
H: hyphal cytoplasmic antigens of A. flaws,
A. nidulans, A.
niger and A. terreus, respe’ctively, innnunodeveloped
with sheep
anti-A. fumigatus
SOD polyclonal
antibody. Tracks I and J:
commercial preparations of Ibacterial FeSOD and MnSOD, respectively, immunodeveloped
wi.th sheep anti-A. fumigatus SOD polyclonal antibody.
14 (1996) 95-102
place of the sheep anti-Aspergillus
clonal.
97
Cu,Zn SOD poly-
2.4. Immunoelectron microscopic detection of SOD
in A. fimigatus cultures
Sterile
thermanox
plastic
coverslips
(Nunc,
Naperville, IL) were placed on the bottom of Costar
24 well plates and cultures of A. fumigatus were
produced as described in the previous section. Coverslips were removed at 2-h intervals, washed in 0.1
sodium cacodylate, and fixed in either 2.5% or 4%
glutaraldehyde
in 0.1 M sodium cacodylate buffer
(pH 7.4) for 30 min at 4°C. Following further washes
in cacodylate buffer coverslips were placed for 15
n-tin at 0°C in 50% methanol containing 20% (w/v)
polyvinyl pyrollidone (PVP), followed by subsequent
dehydration in 70 and 90% methanol containing 20%
PVP for 1 h each at -25°C. The coverslips were
then transferred to successive mixtures of 50% LR
Gold monomer
(Sigma)/SO%
methanol
(with
lO%w/v PVP) and 70% LR Gold monomer/30%
methanol (with 10% PVP) for 60 min each at - 25°C.
After further infiltration in 100% LR Gold monomer
for 60 min at - 25°C the coverslips were infiltrated
with two 12 h changes of LR Gold monomer with
initiator (benzil, Sigma). Gelatin capsules were then
filled with 100% LR Gold and initiator and placed
on the surface of the coverslips and the resin was
then polymerised with a 360 mn U.V. source for 48 h
at - 20°C. The capsules were then stripped from the
coverslips, leaving the fungal elements embedded in
the resin capsules.
Sections of 60-90 nm in thickness were then cut
from the surface of the resin blocks and were transferred to formvar-coated
nickel grids. Grids were
floated on drops of 1% normal goat serum, PBS
Tween at room temperature for 30 min, washed in
PBS-Tween and then incubated with sheep anti-SOD
polyclonal antibody at dilutions of 1: 50 and 1: 100
in PBS-Tween for 60 min at room temperature. After
subsequent washes in PBS-Tween, grids were incubated at room temperature with 18 nm gold conjugated donkey anti-sheep IgG (H + L) (Jackson) at
dilutions of 1: 50 and 1: 100. Finally grids were
washed in PBS-Tween and distilled water and stained
with many1 acetate and Reynold’s lead citrate. Nega-
98
AndrewJoh Hamilton et al. / FEMS Immunology and Medical Microbiology
14 (1996) 95-102
Fig. 2. Immunofluorescent
labelling of A. fumigatus cultures with sheep anti-A. fumigafus Cu,Zn SOD polyclonal antibody. 2A: conidia
after 4 h incubation in Sabourauds’ media (bar represents 16 pm). 2B, 2C: germinating conidia after 6 h incubation (bars represent 14 pm
and 16 pm, respectively). 2D: elongating hyphae with septum arrowed, after 10 h incubation (bar represents 20 pm). 2E: conidiophore
developing from foot like appendage (arrowed) on side of hyphae, after 10 h incubation (bar represents 5 pm). 2F, G and H: subsequent
development and elongation of conidiophores in 12-h cultures (bars represent 14 pm. 14 pm and 20 pm. respectively).
Fig. 3. Immunoelectron
microscopical labelling of A. fumigatus cultures with sheep anti-A. fumigatus Cu,Zn SOD polyclonal antibody. 3A: cross-section of hypha; anti-A.
fumigatus Cu,Zn SOD antibody at 1: 50, donkey anti-sheep IgG gold conjugate at 1: 50. Bar represents 500 nm. 3B: conidiophore; anti-A. fumigatus Cu,Zn SOD antibody at
1: 100, donkey anti-sheep IgG gold conjugate at 1: 100. Bar represents 300 nm. 3C: cross-section of hypha; anti-A. fumigatus Cu,Zn SOD antibody at 1: 100, donkey anti-sheep
IgG gold conjugate at 1: 100. Bar represents 300 nm.
100
tive controls
tion.
AndrewJoh Hamilton et al. /FEMS
were as described
in the previous
Immunology and Medical Microbiology I4 (1996) 95-102
sec-
3. Results
The ELISA to determine the reactivity of the
sheep polyclonal against purified A. fimigatus Cu,Zn
SOD demonstrated
that the antibody had a titre
greater than 1: 3200 (OD value of 1.l compared to
negative control values of under 0.2 at 492 nm at this
dilution) under the conditions described (data not
shown). Immunodevelopment
of Western blots of
hyphal cytoplasmic
antigen demonstrated
that the
sheep anti-A. fumigatus
SOD antibody recognised
only a single band of 19 kDa (Fig. 1, Tracks A, B).
The antibody also recognised purified A. fumigatus
Cu,Zn SOD to give a single band of the same
relative molecular mass (Tracks C, D>. In addition
the anti-A. fumigatus SOD polyclonal demonstrated
some weaker recognition of a single band in hyphal
cytoplasmic antigens of A. jlavus, A. nidulans, A.
niger and A. terreus; the apparent relative molecular
masses of these bands were 18.5 kDa, 19.25 kDa,
18.5 kDa and 17.5 kDa, respectively (Fig. 1, Tracks
E, F, G, H). Finally the anti-A. fumigatus
SOD
polyclonal did not recognise commercial preparations of either Mn or Fe SODS by immunodevelopment of the respective Western blots (Fig. 1, Tracks
I, J).
Immunofluorescence
labelling of cultures of A.
fumigatus
with the sheep anti-A. fimigatus
SOD
antibody demonstrated a clear and reproducible pattern of staining (Fig. 2). Conidial cell walls were
clearly reactive with some less intense cytoplasmic
reactivity (Fig. 2A). This pattern was repeated in
germinating
conidia with particularly
intense labelling of the cell wall comprising the apical tip of
the developing hyphae (Fig. 2B, C). In some areas
hyphal septa also demonstrated labelling (Fig. 2D).
Particularly intense fluorescence
staining was evident within developing conidiophores in later stage
cultures, with continuing
strong reactivity at the
hyphal apical tips (Fig. 2D-G). Normal sheep sera
did not show any reactivity and there was no staining
when PBS-Tween was used in place of the sheep
anti-A. fumigatus
Cu,Zn SOD antibody (data not
shown).
Immunoelectron
microscopical
labelling of cultures of A. jkmigatus with the sheep anti-A. fumigatus Cu,Zn SOD antibody demonstrated a generally
similar pattern of reactivity as seen in the immunofluorescence
studies (Fig. 3A-C). Labelling of
conidiophore and hyphal cell walls was particularly
obvious, although there appeared to be some shrinkage of the cytoplasm away from the cell wall. Whilst
there was clear reactivity in the cytoplasm, typically
there was no specific labelling of mitochondria. Labelling was more intense, particularly in the cytoplasm (Fig. 3A), when primary and secondary antibodies were used at dilutions of 1 : 50, although this
did cause an increase in background staining. Fixation in 4% glutaraldehyde
appeared to reduce the
incidence of cell shrinkage and improve subcellular
morphology,
although it substantially
reduced immunolabelling
(data not shown). Negative controls
demonstrated no significant labelling.
4. Discussion
It is clear from the ELISA studies using sheep
polyclonal serum that the A. fimigatus
Cu,Zn SOD
is an immunogenic
molecule capable of eliciting
high titre antibodies, an observation supported by the
recognition of this enzyme by a significant number
of sera from patients with aspergillosis
[12]. The
polyclonal antibody would also appear to be absolutely specific for the A. fumigatus Cu,Zn SOD,
recognising
a single band on immunodeveloped
Western blots of mycelial cytoplasmic antigen, an
observation that indicates the efficiency of the purification protocol used. The polyclonal serum is also
capable of recognising,
though to a lesser extent,
single bands of slightly different relative molecular
weight in cytoplasmic antigens from other Aspergihs species. Since the polyclonal does not recognise either Mn or Fe SODS by immunoblot
this
indicates the presence of antigenically related Cu,Zn
SODS in other members of the genus Aspergillus.
These enzymes are being purified in our laboratory
and preliminary data suggest that the antibody does
indeed appear to specifically recognise the Cu,Zn
SODS from these other Aspergillus
species. The
absence of recognition of the Mn and Fe SODS by
the polyclonal is perhaps not surprising; although the
AndrewJoh Hamilton et al. /FEMS
Immunology and Medical Microbiology 14 (1996) 95-102
enzyme types catalyze the same reaction comparatively well, the Mn and Fe SODS are considered to
comprise a separate and unrelated class from the
Cu,Zn SODS with substantially different amino acid
sequences [13,14]. As such the Mn and Fe SODS are
likely to be unrelated in terms of antigenicity to the
Cu,Zn SODS resulting in the lack of cross reactivity
exhibited by the polyclonal raised in this study.
The sheep anti-A. fimigatus
polyclonal proved
highly effective when used in immunofluorescence
labelling of A. fumigatus cultures. Cu,Zn SOD is
clearly present in conidia, primarily in the cell wall,
although the cytoplasm also contains the enzyme.
Expression
continues
during conidial germination
with particularly intense polyclonal serum reactivity
to the apical tip cell wall; the latter continued to be
the case throughout the time course of the study.
Also of particular note is the intense expression of
Cu,Zn SOD throughout the developing conidiophore.
The pattern of polyclonal reactivity identified by
immunofluorescence
was confirmed by the immunoelecton microscopy observations. Thus clearly whilst
Cu,Zn SOD is present within the cytoplasm of hyphae, there are also substantial quantities throughout
the cell wall. These observations would suggest that
after synthesis in the cytoplasm the Cu,Zn SOD is
exported via the cell membrane to the cell wall. We
have previously demonstrated that the A. jiimigatus
SOD can be detected in culture filtrate [l l] and this
would suggest that at least some of the enzyme is
also in transit through the cell wall. Morphological
preservation using the immunoelectron
microscopy
fixation and embedding protocol described was adequate; the use of a higher concentration
of glutaraldehyde improved morphology but resulted in
substantial loss of antigenicity.
Cu,Zn SODS are usually found in the cytosol of
eukaryotic cells where they fulfil a housekeeping
function, although there is a report of an extracellular
Cu,Zn SOD in human blood plasma [ 171. In contrast,
the Mn and Fe SODS are found in mitochondria and
it is significant
that the anti-A. fumigatus
Cu,Zn
SOD polyclonal did not specifically label these organelles. As part of its housekeeping role it would be
expected that the Cu,Zn SOD would be preferentially
expressed in areas where cellular metabolism and
associated production of free radicals are highest,
such as at the apical tip of hyphae and in the
101
conidiophore.
However, the association of the Aspergillus Cu,Zn SOD with the cell wall suggests that
in addition the enzyme is in a position to play a role
in protection against externally, as well as internally,
generated free radicals. This contention is supported
by the detection of extracellular A. fumigutus SOD
[ill. Whether the enzyme is therefor capable of
significantly
ameliorating
the toxic oxidative burst
metabolites produced by the myeloperoxidase
system
of the immune effector cell remains a question that
requires much further study. However, it is clear
from this study that the Cu,Zn SOD is certainly in a
cellular position to fulfil this defensive function.
Acknowledgements
This work was funded by the David and Frederick
Barclay Foundation. We would also like to acknowledge Johnson and Johnson and the Special Trustees
of Guys hospital for associated funding. We thank
Ken Brady for technical assistance.
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