Superoxide Dismutase Influences the Virulence of Cryptococcus neoformans by Affecting Growth within Macrophages

Abstract

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and
has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels
of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal
infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis
of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeast Cryptococcus neoformans and made mutants via targeted disruption. The sod1 mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro.
A sod1 mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by
cumulative survival in the mouse inhalational model. In vitro studies established that the sod1 strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing
reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required
for pathogenicity in C. neoformans; (ii) the decreased virulence of the sod1 strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense
systems in C. neoformans can compensate for the loss of the Cu,Zn SOD in vivo.

Full text

INFECTION AND IMMUNITY, Jan. 2003, p. 173–180 Vol. 71, No. 1
0019-9567/03/$08.00⫹0 DOI: 10.1128/IAI.71.1.173–180.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Superoxide Dismutase Influences the Virulence of Cryptococcus
neoformans by Affecting Growth within Macrophages
Gary M. Cox,
1
* Thomas S. Harrison,
2
Henry C. McDade,
1
Carlos P. Taborda,
3
Garrett Heinrich,
1
Arturo Casadevall,
3
and John R. Perfect
1
Departments of Medicine and Molecular Genetics and Microbiology, Duke University Medical Center, Durham,
North Carolina
1
; Department of Infectious Diseases, St. George’s Hospital Medical School, London, United
Kingdom
2
; and Departments of Medicine and Microbiology and Immunology, Albert Einstein
College of Medicine of Yeshiva University, Bronx, New York
3
Received 31 July 2002/Returned for modification 30 August 2002/Accepted 5 October 2002
Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and
molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria
through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been
speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been
rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned
the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeast Cryptococcus neoformans and made
mutants via targeted disruption. The sod1 mutant strains had marked decreases in SOD activity and were
strikingly more susceptible to reactive oxygen species in vitro. A sod1 mutant was significantly less virulent
than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the
mouse inhalational model. In vitro studies established that the sod1 strain had attenuated growth compared
to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of
nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required
for pathogenicity in C. neoformans; (ii) the decreased virulence of the sod1 strain may be due to increased
susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems in C.
neoformans can compensate for the loss of the Cu,Zn SOD in vivo.
Invasive fungal infections in humans are increasing in prev-
alence in parallel with the growing population of immunocom-
promised patients. There is a need for new antifungal drugs to
treat these infections since the drugs currently available are
either excessively toxic or lack broad fungicidal properties.
Studies on the pathogenesis of fungal infections should provide
insights that can help with the diagnosis and treatment of these
important human diseases. Cryptococcus neoformans is a ba-
sidiomycetous yeast that has been used successfully as a model
pathogenic fungus in a variety of molecular pathogenesis stud-
ies. We used C. neoformans to evaluate the contribution of
superoxide dismutase (SOD) to the pathogenesis of fungal
infections.
SODs are metalloenzymes that detoxify oxygen radicals
through the conversion of superoxide to hydrogen peroxide
and oxygen (20). These enzymes are present in virtually all
cells, and this very high degree of conservation is testament to
their importance in cellular homeostasis. The primary role of
SODs is to protect cells from endogenously generated super-
oxide anion, which is a by-product of normal aerobic respira-
tion. SODs can be complexed with iron, manganese, and cop-
per plus zinc. The iron and manganese SODs are genetically
similar to each other, whereas the Cu,Zn SOD exhibits no
significant homology with the other two enzymes (16, 20, 21,
32). Eukaryotic cells generally contain an Mn SOD in the
mitochondrial matrix and a Cu,Zn SOD which is located pre-
dominantly in the cytoplasm and to a lesser extent in peroxi-
somes (9, 28).
In addition to superoxide resulting from endogenous pro-
duction, human-pathogenic organisms are exposed to reactive
oxygen species generated by phagocytic cells. After phagocy-
tosis by polymorphonuclear cells or macrophages, pathogens in
the phagolysosomes are exposed to a variety of toxins, includ-
ing superoxide. Superoxide anions are generated via the oxi-
dative burst in activated immune cells by enzymes that transfer
electrons from cytosolic NADPH to molecular oxygen. For
some bacteria, SOD has been shown to play a role in virulence
when the organisms have been tested in animal models, and it
has been thought that the decreased virulence of SOD mutant
strains was due to increased susceptibility to host phagocytic
cells (32, 35). The role of SOD in the pathogenesis of fungal
infections is not clear. Biochemical characterization of the C.
neoformans Cu,Zn SOD has been done (23), and the Cu,Zn
SOD gene has been cloned from three C. neoformans varieties
(10). There has also been a suggestion that the cryptococcal
SOD has antioxidant properties (23, 24, 26). We initially iden-
tified the Cu,Zn SOD in a screening for genes differentially
regulated by temperature in C. neoformans, and we decided to
study the contribution of this gene to pathogenesis using a
molecular approach. The rationale for studing the Cu,Zn SOD
instead of the Mn SOD is that the Cu,Zn SOD is the much
more abundant form of the enzyme, and the cytoplasmic loca-
tion was thought to be more relevant for possible protection
against phagocyte-derived reactive oxygen species. We made
specific mutants using targeted gene disruption, and we show
* Corresponding author. Mailing address: Box 3281, Duke Medical
Center, Durham, NC 27710. Phone: (919) 681-5055. Fax: (919) 684-
8902. E-mail: gary.cox@duke.edu.
173

below that one of the mutants is less virulent than both the
wild-type and reconstituted strains. (Portions of this work were
presented at the 101st American Society for Microbiology
General Meeting, May 2001, and at the Fifth International
Conference on Cryptococcus and Cryptococcosis, March
2002.)
MATERIALS AND METHODS
Strains and media. C. neoformans strain H99 (serotype A, Mat␣) and strain
H99R (a spontaneous ura5 auxotroph derived from H99 by plating on 5-FOA
agar) were recovered from 15% glycerol stocks stored at ⫺80°C prior to use in
the experiments described below. The strains were maintained on YPD media
(1% yeast extract, 2% peptone, 2% dextrose) and were tested on minimal media
(YNB media without amino acids and 0.5% dextrose). Transformants were
selected on ura dropout media containing 1 M sorbitol (14, 15), and reconsti-
tuted strains were selected on YPD media supplemented with 100 ␮g of nourseo-
thricin (clonNAT; Werner Bioagents, Jena, Germany) per ml as described pre-
viously (33). Strains were tested on YNB media containing 1 to 50 mM tert-butyl
hydroperoxide, 1 to 50 mM paraquat (methyl viologen), 100 to 1,000 ␮gof
oxytetracycline per ml, 10 to 50 mM FeSO
4
, and 1 mM CuSO
4
(all obtained from
Sigma). Dopamine agar (15) and egg yolk agar (15) were made as described
previously. Urease activity was measured grossly after growth on Christensen’s
agar as described previously (14).
Isolation of the SOD gene. A subtractive cDNA library with differential PCR
amplification (PCR Select; Clontech) was used to select for cDNA preferentially
expressed at 37°C versus 25°C. Briefly, yeast strain H99 was grown in either YPD
or YNB broth for 1, 4, 8, and 24 h at either 25 or 37°C in a shaking incubator.
Total RNA from yeast grown under each type of conditions was isolated by using
Trizol reagent (Life Technologies), and the RNA from preparations incubated at
each temperature was pooled and used for differential PCR amplification per-
formed according to the manufacturer’s protocol. Clones from the pool of
cDNAs preferentially expressed at 37°C versus 25°C were screened for the
intensity of hybridization by using labeled total RNA from H99 cells grown at the
two temperatures and pulsed with [
32
P]dATP. One of the cDNA clones that
exhibited approximately threefold-greater hybridization with the labeled RNA
from the yeast grown at 37°C than with the labeled RNA from yeast grown at
25°C was sequenced, and the sequence was used to search the GenBank data-
base. The cDNA clone was found to have significant homology with Cu,Zn
isoforms of SOD and was used to probe genomic and cDNA libraries to obtain
the entire locus. A 3.1-kb KpnI genomic fragment was cloned into a plasmid and
sequenced. The sequence of the genomic fragment was compared to the se-
quences of the cloned cDNAs in order to locate the coding sequence and introns.
The gene was designated SOD1.
Disruption and reconstitution of SOD1.The 3.1-kb genomic fragment was
used to make a disruption construct by digestion with BstEII and insertion of a
1,950-bp genomic fragment containing URA5 into this single site after a fill-in
reaction with DNA polymerase (Fig. 1A). The plasmid containing the disruption
construct was used to transform ura5 strain H99R by using biolistic delivery as
described previously (14, 15). Stable prototrophic transformants were analyzed
by using colony PCR and primers flanking the URA5 insertion site (Fig. 1A).
Disruption of the native SOD1 gene was confirmed by using Southern blots
probed with a labeled SOD1 cDNA fragment. A reconstitution construct was
created by inserting the nourseothricin resistance cassette into a NotI site in the
plasmid containing the 3.1-kb SOD1 genomic fragment. The reconstitution con-
struct was used to transform one of the sod1 mutant strains by using selection
with nourseothricin. Both PCR and Southern analyses were used to confirm
restoration of the wild-type SOD1.
SOD assay. SOD activity was assayed by using a standard colorimetric assay in
which xanthine oxidase serves as a free radical generator and causes the reduc-
tion of nitro blue tetrazolium (NBT). The reduced NBT can be assayed by
absorbance at 560 nm. SOD inhibits the reduction of NBT by scavenging the free
radicals generated by the xanthine oxidase. After validation of the assay by using
purified bovine erythrocyte SOD (Sigma), protein extracts from cryptococci were
tested for SOD activity and compared to controls containing equivalent amounts
of bovine serum albumin. Approximately 10
8
yeast cells were vortexed for 5 min
in 1 ml of ice-cold 50 mM potassium phosphate (pH 7.8) containing 0.5 g of
500-␮m-diameter glass beads. The homogenates were centrifuged at 4°C, and the
supernatants were assayed for protein content by the Lowry method (Sigma); 10
and 100 ␮g of total of protein were used in the assays. The homogenates were
immediately assayed for SOD activity by mixing them with xanthine, xanthine
oxidase, and NBT in a 3-ml (total volume) reaction mixture. Absorbance at 560
nm was monitored for 30 min. The data are expressed below as percentages of
the absorbance of the control sample. All assays were repeated twice with three
independent protein homogenates, and the data were pooled for analysis with
Student’sttest.
Phenotypic assays. Quinacrine staining of the yeast was performed as de-
scribed previously (13) by pelleting logarithmically growing cells and suspending
them in YPD broth containing 50 mM NaHPO
4
and 200 ␮M quinacrine with the
pH adjusted to 7.5. The cells were incubated for 5 min at 30°C, washed in
phosphate-buffered saline (PBS), and viewed on slides with a fluorescent micro-
scope. Freeze-thaw sensitivity was analyzed as described previously (34). Briefly,
logarithmically growing cells were washed in PBS, and the cell density was
adjusted to 10
5
cells/ml in PBS. The cell suspensions were frozen at ⫺20°C for
24 h, thawed at 4°C for 40 min, and then diluted to prepare quantitative cultures
on YPD medium plates. The experiments were done in triplicate, and the results
were compared to the results for quantitative cultures obtained from the same
samples just prior to freezing. Cells were grown in the presence of 100% O
2
by
suspending them in PBS in open tubes which were then placed in an air-tight
container hooked up to a vacuum pump. After vacuum evacuation of the air,
pure oxygen was released into the container via a one-way valve from an oxygen
tank, and the cycle of vacuum evacuation and replacement with oxygen was
repeated every 3 to 4 days. The cultures were kept under these conditions for 4
weeks, as described previously (31), and then quantitated by spreading aliquots
onto YPD medium plates. Cells were tested for sensitivity to oxygen radicals with
a cell-free assay in which epinephrine was used as an electron donor (36). Cells
were grown in YPD medium overnight, washed three times in PBS, and then
FIG. 1. (A) Map of the KpnI (K) genomic fragment containing
SOD1. The URA5 gene was inserted into the BstEII site (B) of SOD1
in order to create a disruption construct. Sites of the PCR primers used
to verify disruption are indicated by solid arrowheads, and the sizes of
the relevant pieces of DNA are also indicated. (B) PCR analysis of
genomic DNA from the wild-type strain (WT), the sod1 strain (‚), and
two independent reconstituted strains (Rec1 [R1] and Rec2 [R2])
performed with primers indicated in panel A. Disruption of the native
SOD1 was indicated by the single amplicon at approximately 3 kb for
the sod1 strain, and ectopic reconstitution for the Rec1 and Rec2
strains was indicated by amplification of both the native and disrupted
versions of SOD1. (C) Southern blot of genomic DNAs from the same
four strains (in the same order as in panel B) that were digested with
KpnI and probed with a labeled SOD1 cDNA. The results show the
expected displacement of the native gene to 5,050 bp in the sod1 strain
and restoration of the wild-type loci at 3,100 bp in the two reconsti-
tuted strains. (D) Northern blot of total RNA from the wild-type
(WT), sod1 (‚), Rec1 (R1), and Rec2 (R2) strains grown in YPD
broth overnight at 30°C and total RNA from wild-type yeast after
growth in YPD broth at 24 and 37°C for 3 h. Labeled actin and SOD1
cDNA fragments were used to probe the blot. Quantitation of the
SOD1 signal with a phosphoimager and with actin hybridization as a
control confirmed that there was a 3.1-fold increase in the intensity of
the hybridization signal at 37°C compared to the intensity of the hy-
bridization signal at 24°C.
174 COX ET AL. INFECT.IMMUN.

suspended in 50 mM sodium acetate (pH 5.5)–1 mM MgSO
4
. Ferric ammonium
sulfate, hydrogen peroxide, and epinephrine bitartrate were sequentially added
to final concentrations of 0.5 mM, 0.0002%, and 1 mM, respectively, to a 1-ml
(final volume) reaction mixture. Aliquots were removed at various times for
quantitative culture on YPD agar. Superoxide levels in the cell-free assay mix-
tures were measured, as described previously (39), by adding 200 ␮lofthe
reaction mixture to 1.8 ml of HEPES-cytochrome cbuffer (17 mM HEPES [pH
7.3], 120 mM NaCl, 5 mM glucose, 5 mM KCl, 1 mM CaCl
2
, 1 mM MgCl
2
, 100
mM cytochrome c). The mixture was incubated at 37°C for 30 min, and the
absorbance at 550 nm and the absorbance at 540 nm were determined with a
spectrophotometer. The amount of superoxide anion generated was calculated
by using the following formula: (A
550
⫺A
540
)⫻1,000/19.1.
Murine model. Cryptococci were used to infect 4- to 6-week-old female A/Jcr
mice (NCI/Charles River Laboratories) by nasal inhalation. Ten mice were
infected with 5 ⫻10
4
yeast cells of the H99, sod1, Rec1, and Rec2 strains in a
50-␮l dose via nasal inhalation as described previously (14, 15). The mice were
fed ad libitum and were monitored by inspection twice daily. Mice that appeared
moribund or in pain were sacrificed by CO
2
inhalation. The protocol was ap-
proved by the Duke University Animal Use Committee. Survival data from the
mouse experiments were analyzed by a Kruskal-Wallis test.
Macrophage assays. The MH-S murine alveolar macrophage cell line (Amer-
ican Type Culture Collection, Manassas, Va.) was maintained in RPMI 1640
containing 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g
of glucose per liter, 1.5 g of bicarbonate per liter, 0.05 mM 2-mercaptoethanol,
and penicillin-streptomycin at 37°C in the presence of 5% CO
2
. Macrophages
were harvested from monolayers by using 0.25% trypsin–0.03% EDTA, and the
numbers of viable cells were determined by trypan blue exclusion and counting
with a hemacytometer. The macrophage concentration was adjusted to 10
5
cells/
ml, and in experiments in which activated macrophages were used, the cells were
primed with 100 U of murine gamma interferon (Sigma) per ml and stimulated
with 0.3 ␮g of lipopolysaccharide (LPS) per ml just prior to mixing with yeasts.
One hundred microliters of a macrophage suspension was put into each well of
96-well plates. Cryptococci that had been washed three times in PBS were
counted with a hemacytometer, the concentration was adjusted to 10
5
cells/ml by
using cell culture media, and 100 ␮l was added to the MH-S cells at a multiplicity
of infection (ratio of effectors to targets) of 1:1. Some macrophages were treated
with an irreversible inhibitor of inducible nitric oxide synthase by using 1 mM
L-N-monomethyl arginine (Cayman Chemical, Ann Arbor, Mich.). Control wells
containing only macrophages and only yeast cells were included in all experi-
ments. In all experiments 10 ␮g of 18B7 (immunoglobulin G1 anti-GXM mono-
clonal antibody) per ml was added to the yeast inocula as an opsonin. The
macrophage-yeast mixtures were incubated for 1 h before they were washed with
two changes of PBS to get rid of the extracellular yeast cells. At different times,
quantitative cultures were prepared by aspirating the medium from each well and
then lysing the remaining macrophages with two changes of 100 ␮l of 0.5%
sodium dodecyl sulfate in water. The aspirated media and sodium dodecyl sulfate
solutions were combined and cultured for quantitation on Sabouraud agar con-
taining chloramphenicol. In all experiments three duplicate wells per reading
were used, and all experiments were repeated three separate times. Fifty micro-
liters of the supernatant was immediately frozen for nitrite assays. The nitrite
assays were done in 96-well plates with equal volumes of Griess reagent and
supernatant, and absorbance at 540 nm was determined with a plate reader. The
absorbance values were compared to a standard curve obtained by using sodium
nitrite dilutions. The phagocytic index was determined as described previously by
counting 600 macrophages for each yeast strain in preparations containing stim-
ulated, unstimulated, and L-NMMA-treated macrophages (19). After4hof
incubation, the wells were washed with three changes of PBS, and the macro-
phages were stained with Giemsa stain as described previously (19). The phago-
cytic index was calculated by dividing the number of attached and ingested
cryptococci by the number of macrophages (19). The quantitative culture data
were combined, and the three groups were compared to each other by using a
one-way analysis of variance with a Bonferroni correction posttest. All other
analyses were performed with the unpaired Student ttest. Primary macrophages
were derived from peripheral blood monocytes obtained by elutriation of buffy
coat cells from normal human donors as described previously (25). After culture
for 1 week, 7 ⫻10
4
macrophages were infected with 10
4
cryptococci in the
presence of 5% pooled human serum as an opsonin. After 48 h of culture at 37°C
in the presence of 5% CO
2
, the cells were lysed, and the number of CFU was
determined by quantitative culture. The results were expressed as percentages of
growth compared to the inoculum.
Nucleotide sequence accession number. The sequence of the SOD1 gene has
been deposited in the GenBank database under accession number AF324862.
RESULTS
We used a PCR subtraction technique in a genetic screening
analysis to identify cDNA preferentially expressed at 37°C
versus 25°C and obtained a 435-bp partial cDNA fragment that
was identical to a C. neoformans Cu,Zn SOD gene (SOD1)
sequence in the GenBank database. Southern blot analysis of
genomic DNA digested with a variety of restriction enzymes
demonstrated that SOD1 existed as a single copy within the
genome. The genomic locus was cloned, and the entire Cu,Zn
SOD gene was contained in a KpnI genomic fragment (Fig.
1A). By comparison of the genomic sequence with the se-
quences of the cloned full-length cDNA fragments, SOD1 was
determined to be 884 bp long and to contain four introns. The
predicted amino acid sequence contains 154 residues that ex-
hibit 63% homology with the Cu,Zn SODs from Aspergillus
fumigatus and Neurospora crassa and 61% homology with the
Cu,Zn SOD from Candida albicans.
Northern analysis confirmed that SOD1 had threefold-in-
creased expression at 37°C compared with the expression at
25°C (Fig. 1D). No differences in expression were found when
yeasts were grown in the presence of pure oxygen and exposed
to 0.5 mM Cu
2⫹
or when they were exposed to the oxidative
stressors tert-butyl hydroperoxide and paraquat (data not
shown).
In our initial attempts to disrupt the gene, we performed a
phenotypic screening analysis with minimal media containing 5
mM paraquat, but no transformants with impaired growth
were identified among the 288 transformants tested. This re-
sult made us wary of assays that relied on phenotypic screening
to identify the mutants. We then used colony PCR performed
with primers flanking the URA5 insertion in the disruption
construct as a genotypic screening technique and found that 11
of 48 transformants (23%) had amplification of only the dis-
rupted version of the gene (Fig. 1B). Southern blotting of these
11 mutant strains demonstrated that in all of them the native
band was displaced to the expected position of the disrupted
version, and 9 of them appeared to have single insertions in the
genome (Fig. 1C). By gross inspection, all nine of these mutant
strains appeared to be equivalent to each other and to the wild
type in terms of growth at 37°C on YPD medium, melanin
production on dopamine agar, capsule size in the presence of
5% CO
2
, extracellular phospholipase activity, and urease pro-
duction. Thus, mutation of SOD1 does not affect any of these
cryptococcal phenotypes that have been associated with viru-
lence. One of the mutant strains was chosen for further anal-
ysis and designated sod1.
Reconstitution of sod1 to the wild-type phenotype was ac-
complished by ectopic integration of the KpnI genomic frag-
ment containing SOD1. Reconstituted strains were verified by
performing PCR and Southern analyses (Fig. 1B and C). The
reconstituted strains were screened for growth at 37°C, mela-
nin production on dopamine agar, and capsule size in the
presence of 5% CO
2
, and two strains that had phenotypes that
were grossly similar to the phenotypes of both the wild-type
and sod1 strains were designated Rec1 and Rec2.
A standard biochemical assay established that the sod1 mu-
tant strain had significantly lower SOD activity than either the
wild-type strain or the reconstituted strains (Fig. 2A). There
were not significant differences in the SOD activities of the
VOL. 71, 2003 SUPEROXIDE AND CRYPTOCOCCAL VIRULENCE 175

wild-type, Rec1, and Rec2 strains, demonstrating that there
was full phenotypic reconstitution in Rec1 and Rec2 (Fig. 2A).
The residual SOD activity in the sod1 strain compared to the
activities in control assay mixtures containing no SOD were
presumed to be due to the manganese isoform of SOD. The
sod1 strain was found to be much more sensitive to oxygen
radicals generated in a cell-free system than the wild-type and
Rec1 strains were (Fig. 2B). Significantly lower numbers of
yeast cells were recovered after 24 h of incubation in the
reaction mixture containing the electron donor epinephrine
and the sod1 strain (2.4 ⫾0.01 log CFU/ml) than in the reac-
tion mixtures containing epinephrine and the wild-type and
Rec1 strains (6.6 ⫾0.28 and 6.4 ⫾0.47 log CFU/ml, respec-
tively) (P⬍0.0001). In the control mixtures that contained all
the same constituents except epinephrine, there were not sig-
nificant differences in the numbers of yeast cells when the three
groups were compared.
These strains were also tested for various phenotypes that
have been observed with Cu,Zn SOD mutants of Saccharomy-
ces cerevisiae. When quantitative cultures from liquid media
were used, there were not significant differences among the
sod1, wild-type, Rec1, and Rec2 strains in terms of growth on
minimal media, prolonged stationary phase in the presence of
100% oxygen, or growth on YPD agar with either tert-butyl
hydroperoxide (1 to 50 mM), paraquat (1 to 50 mM), oxytet-
racycline (20 to 2,000 ␮g/ml), or iron (1 to 10 mM). Further-
more, there was no loss of viability of the sod1 strain compared
to the viabilities of the wild-type strains after repeated cycles of
freezing at ⫺20°C and thawing. No differences in gross vacuole
morphology were apparent among these strains after quina-
crine staining of the vacuoles. Thus, the sod1 strain did not
appear to have many of the in vitro phenotypes that have been
seen with Cu,Zn SOD mutants of S. cerevisiae.
The four strains were tested in vivo by using the mouse
inhalational model (Fig. 3). Mice infected with the sod1 strain
lived significantly longer than mice infected with the wild-type,
Rec1, and Rec2 strains. In fact, all of the mice infected with the
strains carrying a wild-type copy of SOD1 died before any of
the mice infected with the sod1 strain succumbed to infection
(Fig. 3). The average survival time for mice infected with the
sod1 strain was 27 days, compared with 20 days for the group
infected with H99 (P⫽0.001), 19 days for the group infected
with Rec1 (P⫽0.001), and 21 days for the group infected with
Rec2 (P⬍0.003). There were not significant differences in
survival between the wild-type and reconstituted groups.
Therefore, the sod1 mutant was significantly less virulent than
the wild-type strain as assessed by cumulative survival.
To investigate the mechanisms for the reduced virulence of
the sod1 strain, yeast cells were tested for growth within mac-
rophages. Phagocytic cells generate oxygen radicals to kill in-
gested microorganisms, and our data obtained with the cell-
free system that generated oxygen radicals revealed that the
sod1 strain was much more sensitive to these radicals than the
wild-type strain was. Therefore, we hypothesized that SOD1
was important for the survival of C. neoformans within macro-
phages. Both primary human macrophages and murine mac-
rophage cell lines were used in this study. In the MH-S cell
line, there were not significant differences in the phagocytic
indices among the wild-type, sod1, and Rec1 strains with stim-
ulated, unstimulated, and L-NMMA-treated macrophages
(data not shown). Thus, there were not differences in the
abilities of the three strains to be taken up by macrophages
with an anti-GXM monoclonal antibody serving as an opsonin.
However, once the cryptococci were taken up, the sod1 strain
was associated with significantly slower growth within the mac-
rophages. In the MH-S cell line, the sod1 strain exhibited
significantly slower growth within unstimulated macrophages
than the wild-type and Rec1 strains exhibited (Fig. 4A). Sig-
nificantly fewer sod1 yeast cells (8.93 ⫻10
4
CFU/ml) than
wild-type and Rec1 yeast cells (1.25 ⫻10
5
and 1.39 ⫻10
5
CFU/ml, respectively) were recovered from the unstimulated
FIG. 2. (A) SOD assay in which xanthine was used as a source of
superoxide. The superoxide reduced NBT, which could be quantified
by absorbance at 560 nm. The action of SOD decreased the amount of
NBT that was reduced, thus decreasing the absorbance. Various
amounts of whole-protein extracts from the H99 (F), sod1 (E), and
Rec1 () strains were added to reaction mixtures. Each value repre-
sents the results of three independent readings, and the results are
expressed as percentages of the control absorbance (bovine serum
albumin only); the error bars indicate standard errors. The sod1 pro-
tein extracts had significantly higher absorbance with both amounts
than the wild-type, Rec1, and Rec2 strains had. The Rec2 data were
not included for clarity. (B) Recovery of yeast cells after incubation for
24 h in a cell-free system in which oxygen radicals were generated from
the electron donor epinephrine. The results are expressed in log CFU
per milliliter (mean ⫾standard deviation) and represent three inde-
pendent readings. A total of 10
6
yeast cells of the wild-type strain (solid
bars), the sod1 strain (open bars), or the Rec1 strain (gray bars) were
inoculated into a reaction mixture containing epinephrine (⫹Epi) and
a control mixture containing all of the components except epinephrine
(Control). The number of sod1 yeast cells recovered was significantly
lower than the numbers of wild-type and Rec1 yeast cells recovered (P
⬍0.0001).
FIG. 3. Survival of mice infected with equal numbers of yeast cells
via nasal inhalation. Symbols: F, H99 (wild type); , Rec1; ƒ, Rec2; E,
sod1. The sod1-infected mice lived significantly longer than the mice in
the other three groups (P⬍0.003), and there were not significant
differences in survival among the wild-type, Rec1, and Rec2 strains.
176 COX ET AL. INFECT.IMMUN.

macrophages at 24 h (P⫽0.0008 and P⫽0.001, respectively).
The number of sod1 yeast cells recovered from macrophages
stimulated with gamma interferon and LPS (7.03 ⫻10
4
CFU/
ml) was also lower than the numbers of wild-type and Rec1
cells recovered from such macrophages (1.07 ⫻10
5
and 1.19 ⫻
10
5
CFU/ml, respectively), but the difference did not quite
reach statistical significance at the P⫽0.05 level for the wild-
type strain (P⫽0.089 and P⫽0.049, respectively). The num-
ber of sod1 yeast cells recovered from the unstimulated mac-
rophages (8.93 ⫻10
4
CFU/ml) was significantly higher than
the number recovered from the stimulated macrophages (7.03
⫻10
4
CFU/ml) (P⫽0.024). Thus, the sod1 strain was more
susceptible than the wild-type strain to growth inhibition in the
unstimulated macrophages but was not more susceptible in the
stimulated macrophages. There were not significant differ-
ences in the colony counts from the macrophages at the 4-h
time point, and there were not differences in either the number
or the viability of the macrophages from any of the groups as
assessed by trypan blue exclusion (data not shown).
We reasoned that the differences in growth of the sod1 strain
between the stimulated and unstimulated macrophages at the
24-h time point may have been due to the expanded fungistatic
repertoire of the macrophages resulting from gamma inter-
feron and LPS stimulation. Nitric oxide was considered to be
the most likely fungistatic candidate in the stimulated macro-
phages, and nitrite levels in the macrophage supernatants were
measured (Fig. 4B). As expected, the stimulated macrophages
made significantly more nitric oxide than the unstimulated
macrophages made (Fig. 4B). There were not significant dif-
ferences in the nitric oxide levels of the three strains compared
to the levels in control samples containing no yeast cells for
each type of conditions. Inhibition of the inducible nitric oxide
synthase in the stimulated macrophages with the L-arginine
analog L-NMMA not only resulted in a significant decrease in
the nitric oxide levels (Fig. 4B) but also resulted in a significant
increase in the average number of sod1 yeast cells recovered
compared to the number in stimulated macrophages not
treated with the inhibitor (8.66 ⫻10
4
and 7.03 ⫻10
4
CFU/ml,
respectively) (P⫽0.04) (Fig. 4A). The number of sod1 yeast
cells recovered from the unstimulated macrophages (8.93 ⫻
10
4
CFU/ml) was similar to the number recovered from the
stimulated macrophages treated with the inhibitor (8.66 ⫻10
4
CFU/ml) (P⫽0.23). Therefore, the reason that there were not
significant differences in the numbers of sod1 and wild-type
yeast cells recovered from the stimulated macrophages was
because of the fungistatic effects of nitric oxide.
The slower intracellular growth of the sod1 strain was also
demonstrated in human macrophages. In four independent
triplicate experiments, the percentages of growth compared to
the size of the inoculum were 39% ⫾10% and 156% ⫾42%
(means ⫾standard errors) for the sod1 and wild-type strains,
respectively (P⫽0.01).
DISCUSSION
We first identified the C. neoformans SOD1 gene in a screen-
ing for genes regulated by temperature. The regulation of
SOD1 by temperature was confirmed by a Northern analysis
that showed that there was a threefold increase in SOD1 ex-
pression at 37°C compared with SOD1 expression at 25°C. The
expression of many genes involved in resistance to oxidative
damage increases in other fungi in response to stresses such as
temperature (8), and our data are the first data which show
that there is temperature-related expression of a SOD-encod-
ing gene in a human-pathogenic fungus. The increased expres-
sion of SOD1 in C. neoformans may be part of a generalized
stress response, but it may also be a response to increased
intracellular stresses related to higher rates of oxidative me-
tabolism. Interestingly, the increased expression of SOD1 has
been independently confirmed by Jim Kronstad, who also
found higher levels of SOD1 mRNA in C. neoformans strain
JEC21 grown at 37°C than in the same strain grown at 24°C
(Jim Kronstad, personal communication).
We were able to create sod1 mutants using targeted disrup-
tion, and the sod1 mutants clearly had decreased SOD activity,
as measured by a standard assay. In vitro comparison demon-
strated that the sod1 strain was largely killed in the presence of
oxygen radicals, whereas both the wild-type and reconstituted
strains were able to survive with no appreciable cell death.
Hence, SOD1 is critically important in the yeast defense
against extracellular oxygen radicals generated by epinephrine
in a cell-free system. However, despite this striking phenotype,
we were unable to find in the sod1 mutants any of the pheno-
types thought to be due to an excess of intracellular oxygen
radicals, such as those that have been described for S. cerevisiae
SOD mutants. For example, mutation of the Cu,Zn SOD in S.
cerevisiae leads to sensitivity to oxytetracycline (3), iron (17),
FIG. 4. (A) Number of yeast cells (CFU per milliliter; mean ⫾
standard deviation) recovered from MH-S macrophages incubated
with yeast after 24 h. The wild-type (solid bars), sod1 (open bars), and
Rec1 (gray bars) strains were incubated with cells stimulated with both
gamma interferon and LPS (Stim), cells with no stimulation (Unstim),
or cells stimulated with both gamma interferon and LPS and treated
with the nitric oxide synthase inhibitor L-NMMA (⫹Inhib). A number
sign indicates that the Pvalue was ⬍0.002 compared with the results
for the wild-type and Rec1 yeasts in unstimulated macrophages. An
asterisk indicates that the Pvalue was 0.024 compared with the results
for sod1 yeast in stimulated macrophages. A plus sign indicates that the
Pvalue was ⬍0.010 compared with the results for the wild-type and
Rec1 yeasts in stimulated macrophages treated with inhibitor. There
were not significant differences for the comparisons of any other
groups. (B) Nitric oxide levels in cell culture supernatants in the
experiments shown in panel A were determined by measuring nitrite
levels. Each bar and error bar indicate the average ⫹standard devi-
ation for nine samples from three different experiments. MP, macro-
phage control (no yeast); WT, wild type; sod1,sod1 mutant strain;
Rec1, reconstituted strain. Solid bars, macrophages stimulated with
gamma interferon and LPS; open bars, unstimulated macrophages;
gray bars, stimulated macrophages treated with the nitric oxide syn-
thase inhibitor L-NMMA. Significantly higher levels of nitrite were
measured for each strain in the stimulated macrophages than under
the other two conditions. There were not significant differences be-
tween the unstimulated and inhibitor-treated conditions for any of the
groups, and there were not significant differences in the nitric oxide
levels of the three strains compared to the levels of the control con-
taining no yeast for each of the three conditions.
VOL. 71, 2003 SUPEROXIDE AND CRYPTOCOCCAL VIRULENCE 177

paraquat (21), 100% oxygen (21), freeze-thaw stress (34), age
(4), and auxotrophy for methionine and lysine (21). The dif-
ferences in such disparate phenotypes associated with the same
gene in these two fungi are striking and could be due to in-
trinsic differences between the two fungi that are unrelated to
resistance to oxidative damage. However, the differences may
also reflect the fact that C. neoformans has other redundant
systems that can detoxify superoxide radicals. We believe that
part of this redundancy can be explained by the ability of C.
neoformans to produce two powerful free radical quenchers,
mannitol and melanin. Both of these products are made by C.
neoformans but not by S. cerevisiae, and both have been pos-
tulated by other investigators to be oxygen radical scavengers
in C. neoformans (11, 12, 24, 26, 29). The presence of such
redundant scavenger systems may explain why the C. neofor-
mans sod1 strain does not have some of the phenotypes that
have been found in S. cerevisiae. However, these redundant
systems cannot fully compensate for the loss of SOD1 since the
sod1 strain is much more sensitive than the wild type to oxygen
radicals, as demonstrated in our cell-free assay, and it clearly
has a decreased ability to detoxify superoxide, as shown in the
SOD assays. Therefore, we believe that the postulated redun-
dant systems can only partially compensate for the loss of
SOD1.
There have been suggestions that the virulence of C. neo-
formans strains may be related to an individual strain’s resis-
tance to oxidative stress. One study correlated the virulence of
three different clinical isolates of C. neoformans in the murine
model with in vitro resistance to reactive oxygen and nitrogen
species (41). The most virulent strain was also the strain that
was most resistant to oxidative damage. In our studies, we
showed that the sod1 mutant was significantly less virulent than
the wild-type strain in the murine inhalational model. The fact
that in two independent reconstituted strains virulence was
restored strongly supports the claim that the virulence defect
was due to the sod1 mutation itself rather than to some un-
specified mutation that occurred during the transformation
process. The virulence defect was not due to any obvious,
known virulence phenotype, such as growth rate at 37°Cor
production of melanin, extracellular phospholipase, urease,
and the polysaccharide capsule. Therefore, we reasoned that
the decreased ability of the sod1 strain to cause infection is due
to increased susceptibility to oxygen radical attack within
phagocytic cells.
One of the mechanisms by which human phagocytic cells kill
ingested microorganisms is by selective production of oxygen
radicals, including superoxide, in phagolysosomes. The impor-
tance of superoxide in human immune defenses is illustrated
by the susceptibility of patients with chronic granulomatous
disease to a variety of bacterial and fungal infections. These
patients have defects in the NADPH oxidase system and suffer
from recurrent infections due to both bacteria and fungi (27).
The importance of superoxide in the killing of cryptococci is
reflected by the fact that neutrophils from patients with
chronic granulomatous disease exhibit decreased killing of C.
neoformans in vitro (18). In some bacteria, SODs have been
shown to be important for survival within macrophages and for
virulence in animal models. For these microbes, it has been
postulated that the mechanism for decreased virulence was
increased susceptibility of the SOD mutant strains within mac-
rophages (32, 35). We reasoned that SOD1 might play a similar
role in cryptococcosis.
C. neoformans is known to reside in macrophages during
many stages of experimental and human infections (19), and
resistance to macrophage-mediated killing may be important
for virulence in this fungus. In this study, we demonstrated that
the sod1 strain was significantly more susceptible to oxygen
radicals in a cell-free system, and consequently, we wanted to
see if this strain was more susceptible to the fungistatic mech-
anisms within macrophages. Our data established that the sod1
strain had decreased growth rates compared with the growth
rates of the wild type within macrophage cell lines and primary
macrophages. Within the cell lines, the differences in growth
between the sod1 and wild-type strains were not as marked in
the stimulated macrophages as in the unstimulated macro-
phages. We feel that the intracellular growth defect of the sod1
strain was more apparent in the unstimulated macrophages
because of the limited fungistatic repertoire in the unstimu-
lated cells. Unstimulated macrophages do not produce signif-
icant amounts of nitric oxide, and in these cells the oxygen
radicals can be expected to have a more important role in
fungistasis. Therefore, the protective effects of SOD1 against
superoxide anions in the macrophages are more obvious when
nitric oxide is not being produced. We were able to show that
this effect is specific to nitric oxide by inhibiting nitric oxide
production using L-NMMA. In stimulated macrophages
treated with this drug, nitric oxide production was almost com-
pletely eliminated, and this resulted in a significant decrease in
the number of sod1 yeast cells recovered compared to the
number of wild-type cells recovered.
The fact that the sod1 mutants still exhibited significant
growth within macrophages and were still pathogenic suggests
that there are other mechanisms in addition to SOD1 that are
important in the resistance to the oxidative stresses encoun-
tered in the host. In the acidic conditions of the phagolyso-
some, superoxide becomes protonated and reacts with itself to
form hydrogen peroxide (H
2
O
2
). Hydrogen peroxide is a more
reactive oxidant than superoxide and can cause oxidation of
proteins, DNA, membrane lipids, and components of the re-
spiratory chain. Hydrogen peroxide can interact with superox-
ide via the Haber-Weiss reaction to form hydroxyl radical
(䡠OH), which is considered to be the most destructive of the
reactive oxygen species. SOD is not thought to affect the levels
of these other reactive oxygen species. Catalase, glutathione
peroxidase, and thioredoxin peroxidase have each been shown
to break down hydrogen peroxide in S. cerevisiae (5, 22), and it
is possible that these enzymes also have a significant role in the
intracellular survival of C. neoformans. As mentioned above,
both mannitol and melanin are postulated to be scavengers of
oxygen radicals (11, 12, 24, 26, 29), and both have been shown
to have some role in the protection of cryptococci within
phagocytic cells (12, 29). Nitric oxide is another important part
of the oxidative attack directed against C. neoformans in the
macrophage phagolysosome. The important fungistatic role of
nitric oxide against C. neoformans in macrophages has been
demonstrated both in vitro and in vivo (1, 2). Mice deficient in
the inducible nitric oxide synthase also had higher burdens of
infection with C. neoformans than wild-type mice had (37, 38).
One research group has reported that C. neoformans reduces
nitric oxide activity in macrophages and astrocytes via nitric
178 COX ET AL. INFECT.IMMUN.

oxide consumption by some yeast factor (40). Two candidates
for the cryptococcal yeast factor that may consume nitric oxide
are the polysaccharide capsule and flavohemoglobin. The S.
cerevisiae flavohemoglobin encoded by the YHB1 gene has
been shown to metabolize nitric oxide and to protect the yeast
against nitrosative stress (30). Flavohemoglobins have been
proposed to be a conserved protective mechanism in all mi-
croorganisms, and a flavohemoglobin-encoding gene is present
in C. neoformans (Joseph Heitman, personal communication).
Since the sod1 strain can grow in vitro and also kill mice,
SOD does not fit the classical definition of a virulence factor as
a trait that is dispensable for growth in vitro but is required for
pathogenicity (6). However, this classical definition of a viru-
lence factor is probably too restrictive and is not considered
applicable to traits associated with virulence in many organ-
isms, including some fungi (7). The definition of virulence
factor has been recently modified to a microbial trait that
promotes host damage (7). By this modified definition SOD
qualifies as a virulence factor since its presence translated into
increased host damage, as manifested by reduced survival time
in the wild-type and complemented strains. SOD can function
as a virulence factor by promoting the survival of yeast cells,
which, in turn, translates into host damage as a consequence of
microbe-mediated effects and the host response to infection.
We concluded that the Cu,Zn SOD contributes to the viru-
lence of C. neoformans but is not required for pathogenicity.
We believe that the role of SOD1 in virulence is in resistance
to oxygen radical-mediated damage within macrophage
phagolysosomes and that there are other mechanisms by which
C. neoformans can resist oxidative damage within the phagoly-
sosomes. We also believe that in order for C. neoformans to be
a successful intracellular pathogen, it must also have mecha-
nisms for the detoxification of reactive nitrogen species. Fur-
ther study of all these mechanisms should provide important
insights into how this yeast can persist within macrophages and
cause systemic infection. Given that C. neoformans has multi-
ple mechanisms with which to resist oxidative damage, it may
be very difficult to further investigate the role of oxidative
resistance in pathogenesis by targeting one specific effector
gene, such as the SOD or catalase gene. Some options include
studying strains containing multiple gene disruptions or muta-
tion of transcription factors that may control the levels of
several different genes involved in the response to oxidative
damage.
ACKNOWLEDGMENTS
This work was done as part of the Duke University Mycology Re-
search Unit (DUMRU) and was supported by Public Health Service
grants AI22774 (to A.C.), AI13342 (to A.C.), AI01334 (to G.M.C.),
and AI28388 (to J.R.P.) from the National Institute of Allergy and
Infectious Diseases. G.M.C. was a recipient of a Burroughs Wellcome
Fund New Investigator Award in Molecular Pathogenic Mycology.
We acknowledge Joseph Heitman for his support and helpful sug-
gestions and Marisol de Jesus-Berrios, Floyd L. Wormley, Jr., and J. R.
Urso for technical assistance.
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Editor: T. R. Kozel
180 COX ET AL. INFECT.IMMUN.