INFECTION AND IMMUNITY, JUlY 1994, p. 3004-3007 Vol. 62, No.

7
0019-9567/94/$04.OO+O
Copyright © 1994, American Society for Microbiology

Susceptibility of Melanized and Nonmelanized Cryptococcus

neoformans to Nitrogen- and Oxygen-Derived Oxidants
YULIN WANG' AND ARTURO CASADEVALL' 2*
Department of Microbiology and Immunology' and Division of Infectious Diseases of the Department of Medicine,2
Albert Einstein College of Medicine, Bronx, New York 10461
Received 14 February 1994/Returned for modification 26 March 1994/Accepted 8 April 1994

Melanized Cryptococcus neoformans cells were less susceptible than nonmelanized cells to the fungicidal
effects of nitrogen- and oxygen-derived oxidants. The results support the hypothesis that the phenoloxidase
enzyme system contributes to virulence by protecting C. neoformans against nitrogen- and oxygen-derived
oxidative antimicrobial molecules produced by immune effector cells.

Cryptococcus neoformans is a pathogenic fungus which
causes life-threatening infections in approximately 5 to 10% of
AIDS patients (36). In AIDS cases, cryptococcal infections are
very difficult to treat because antifungal therapy does not
eradicate the infection (31) despite the organism's in vitro
susceptibility to the drugs used (2). C. neoformans has a
marked propensity for causing central nervous system infec-
tions, and the major clinical findings are those associated with
meningoencephalitis (22). Among the virulence markers de-
scribed for C. neoformans are the polysaccharide capsule and a
phenoloxidase enzyme system (16, 17, 28). The capsule con-
tributes to virulence by being antiphagocytic (13, 14). The
mechanism by which the phenoloxidase enzyme contributes to
virulence is less well understood.
The C. neofornans phenoloxidase enzyme gene has recently
been cloned and was found to encode a 624-amino-acid
protein (34). The enzyme has broad substrate specificity and
can generate melanin-like pigments from a variety of sub-
strates, including L-dopa (4, 18, 23, 24, 32, 34). Production of
melanin-like pigments is a characteristic used in the identifi-
cation of C. neoformans (32). The ability to produce melanin-
like pigments has been associated with virulence in various
fungi, including C. neoformans (6, 16, 17, 26, 28). C. neofor-
mans cells with melanin-like pigments have been observed in
human brains (15, 29). In melanized cells the pigment is found
of philosophy at the Sue Golding Graduate Division of Med-
ical Science, Albert Einstein College of Medicine, Yeshiva
University, Bronx, N.Y.)
C. neoformans ATCC 24067 (serotype D) was obtained from
the American Type Culture Collection (Rockville, Md.). C.
neoformans was grown in a defined minimal medium (15 mM
glucose [EM Science, Gibbstown, N.J.], 10 mM MgSO4 [J. T.
Baker Inc., Phillipsburg, N.J.], 29.4 mM KH2PO4 [J. T. Baker],
13 mM glycine [United States Biochemical Corp., Cleveland,
Ohio], 3.0 ,uM vitamin B, [Sigma Chemical Co., St. Louis,
Mo.)] (pH 5.5) with or without 1.0 mM L-dopa (Sigma). The
cultures were incubated at 30°C in a rotatory shaker at
approximately 200 rpm. In minimal medium with L-dopa, C.
neofornans cultures were brown after 3 to 5 days and heavily
melanized after 5 to 8 days. Day 8 was chosen as the experi-
mental time point because by that time the cultures were
heavily melanized. Glycine was used as a nitrogen source
because this amino acid enhances the production of melanin-
like pigments (4). The plating efficiencies for 8-day-old mela-
nized and nonmelanized cells were identical, indicating equal
viabilities for C. neoformans grown in the presence and ab-
sence of L-dopa.
Nitrate and oxygen-derived oxidants were generated by
using two systems that have been previously used to study the
effect of oxidants on C. neofornans (1, 25). Nitric oxide (NO )-

in the cell wall (30). The phenoloxidase activity (10) and the
form of enzyme expressed (8) are regulated by temperature.
The phenoloxidase enzyme catalyzes the oxidation catechol
precursors to reactive intermediates which polymerize to mel-
anin-like compounds (4, 21). Although melanin-like pigments
are ubiquitous molecules found in organisms of all phyloge-
netic kingdoms, their functions are poorly understood (7).
Melanins are efficient free-radical scavengers (7, 19, 27), and in
C. neoformans the physiologic role of the phenoloxidase system
may involve protection against oxidants (9, 17, 25, 33). Re-
cently, C. neoformans melanin has been elegantly shown to
have antioxidant properties (11). To test the hypothesis that
melanin protects against oxidants, we exposed melanized and
nonmelanized cells to chemically generated nitrogen- and
oxygen-derived oxidants.
(The data in this paper are from a thesis to be submitted in
partial fulfillment of the requirements for the degree of doctor
*
Corresponding author. Mailing address: Department of Medicine,
Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx,
NY 10461. Phone: (718) 430-4260. Fax: (718) 597-5814.
3004
and reactive nitrogen intermediates were generated in a solu-
tion of 0.5 mM NaNO2 (Sigma) and 25 mM succinic acid
(Sigma), pH 4, as described by Alspaugh and Granger (1). In
acidic solutions, nitrite salts react with H+ to produce NO * (1)
and a variety of reactive nitrogen species with fungicidal
activity against C. neoformans (35). Oxygen-derived oxidants
were generated by using the epinephrine oxidative system
described by Polacheck et al. (25). In this system, epinephrine
serves as an electron donor, Fe3" is a transition metal catalyst,
and H202 serves as an electron acceptor; the reaction of these
elements results in the production of oxygen-derived free
radicals (25). Briefly, ferric ammonium sulfate, H202, and
epinephrine bitartrate (Sigma) were mixed in that order to
final concentrations of 0.5 mM, 0.00021%, and 1.0 mM,
respectively. Melanized and nonmelanized cells were washed
in 0.02 M phosphate-buffered saline (PBS) (pH 7.2), resus-
pended in PBS, and exposed to chemically generated NO * or
the oxygen-derived oxidants generated by the epinephrine
oxidative system. After various time intervals of exposure to
these reaction mixtures, C. neofornans cells were plated on
Sabouraud dextrose agar (Difco Laboratories) plates to deter-
mine viability as measured by the number of colonies. Percent
VOL. 62, 1994 NOTES 3005

100 r

E L-dopa
80 I
-j
W (D L-dopa
** p<0.01
at: 60 F
CO)
I-
z
w 40 I
w
0L I*

20

0 -7
1 2 3 4
TIME (Hours)
FIG. 1. Survival of 8-day-old melanized and nonmelanized C. neofornans after exposure to chemically generated NO Values are the averages
-

and standard deviations of five measurements. P values were calculated by using Student's t test. This experiment was done three times, with similar
results.

survival was determined by counting the number of colonies L-Dopa was used as the substrate for phenoloxidase synthe-
relative to untreated cells. Student's t test was done with sis of melanin-like pigments because it is a neurotransmitter
Primer of Statistics version 3.0 (McGraw-Hill Inc., New York, precursor in humans. L-Dopa alone does not protect against
N.Y.). oxidants (11). C. neoformans did not grow in agar plates with

100 r
**

80 I T

0 L-dopa
L-dopa
> 60
:3 ** p<0.01
co
z
t) 40
w
aL

20 1

0
1 2 3 3.5
TIME (Hours)
FIG. 2. Survival of 8-day-old melanized and nonmelanized C. neofornans after exposure to oxygen-derived oxidants generated in the
epinephrine oxidative system. Values are the averages and standard deviations of five measurements. P values were calculated by using Student's
t test. This experiment was done three times, with similar results.
3006 NOTES
50 r
+ L-dopa
40 F D] - L-dopa
-j
* p<0.05
**
p<0.01
cr 30 F
U)
z
20 I
LU
w
a-
10 F
**
*
0
3 4
FIG. 3. Survival of C. neoformans grown in the presence and absence of 1.0 mM L-dopa at various time points of culture growth after 2 h of
exposure to oxygen-derived oxidants generated in the epinephrine oxidative system. Values are the averages and standard deviations of five
measurements. P values were calculated by using Student's t test.
L-dopa as the sole carbon or nitrogen source. Similar findings
were reported by Polacheck et al. (25). Growth curves of C.
neofornans cultured in minimal media with and without L-
dopa were similar. HIowever, in the presence of L-dopa, C.
neoformans cells rapidly melanized.
NO and related products produced by murine immune
effector cells have been shown to be fungistatic and fungicidal
for C. neoformans (20, 35). Figure 1 shows the survival
percentages of 8-day-old melanized and nonmelanized C.
neoformans cells after various time intervals of exposure to
chemically generated NO- and related products. Figure 2
shows the survival percentages of 8-day-old melanized and
nonmelanized C. neoformans after various time intervals of
exposure to oxygen-derived oxidants generated in the epineph-
rine oxidative system (25). Oxygen-derived oxidants produced
in the epinephrine oxidative system (25) are similar to some
products of the oxidative burst of immune effector cells (5, 12).
Melanized cells exhibited significantly greater survival percent-
ages than did nonmelanized cells after exposure to nitrogen-
and oxygen-derived oxidants. Figure 3 shows the survival
percentages of C. neoformans grown in the presence and
absence of L-dopa at various times after exposure to the
products of the epinephrine oxidative system. At all time
points, cells grown in the presence of L-dopa were more
resistant to oxygen-derived free radicals than those that were
not. The significantly enhanced survival rate for 8-day-old cells
grown in the presence of L-dopa compared with that for
6-day-old cells grown in the presence of L-dopa is not under-
stood but may reflect increased melanization with culture age
(11) and/or other metabolic changes in late stationary phase.
The susceptibility of C. neoformans to the products of the
epinephrine oxidative system decreased with melanization.
Similar results have recently been reported with hypochlorite
(11). The difference in survival between melanized and non-
INFECL. IMMUN.
**
**
5 6 8
DAY
melanized cells exposed to the products of the epinephrine
oxidative system was smaller than that reported by Polacheck
et al. (25) for phenoloxidase-negative mutants and wild-type
strains. Presumably this difference reflects the fact that our
experiments compared the susceptibilities of the same strain in
the melanized and nonmelanized states whereas Polacheck et
al. compared strains with and without phenoloxidase activity
(25).
Our results indicate that melanized C. neoformans cells are
less susceptible than nonmelanized cells to the fungicidal
effects of chemically generated nitrogen- and oxygen-derived
oxidants. The oxidative systems are artificial and may or may
not be relevant to physiologic defense mechanisms. Neverthe-
less, our findings are consistent with and support the hypoth-
esis that the phenoloxidase system functions as a virulence
factor by catalyzing the production of melanin-like pigments
which in turn protect against oxidants generated by immune
effector cells (9, 11, 17, 25, 33). Melanin-like pigments may
contribute to virulence in C. neoformans in a manner analo-
gous to the way in which microbial glycolipids protect myco-
bacteria against oxidative damage by scavenging free radicals (3).
We thank Brian P. Currie, John Chan, and Lorraine Marsh for many
helpful discussions. A.C. was supported in part by a Pfizer Postdoctoral
Fellowship, a James S. McDonnell Scholar Award, and NIH grants
R01-AI33774 and R01-AI13342. This support is gratefully acknowl-
edged.
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