[Emerging Infectious Diseases * Volume 4 * Number 1 * January - March 1998]

Synopses

What Makes Cryptococcus neoformans a Pathogen?

Kent L. Buchanan and Juneann W. Murphy
University of Oklahoma Health
Sciences Center, Oklahoma City, Oklahoma, USA
------------------------------------------------------------------------
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      Life-threatening infections caused by the encapsulated fungal
      pathogen Cryptococcus neoformans have been increasing steadily
      over the past 10 years because of the onset of AIDS and the
      expanded use of immunosuppressive drugs. Intricate
      host-organism interactions make the full understanding of
      pathogenicity and virulence of C. neoformans difficult. We
      discuss the current knowledge of the characteristics C.
      neoformans must possess to enter the host and establish
      progressive disease: basic growth requirements and virulence
      factors, such as the polysaccharide capsule; shed products of
      the organism; melanin production; mannitol secretion;
      superoxide dismutase; proteases; and phospholipases.

Cryptococcus neoformans is an encapsulated fungal organism (Figure 1) that
can cause disease in apparently immunocompetent, as well as
immunocompromised, hosts (1,2). Most susceptible to infection are patients
with T-cell deficiencies (1,2). C. neoformans var. neoformans causes most
cryptococcal infections in humans, so this review will focus on information
from the neoformans variety of this basidiomycetous fungus. C. neoformans
var. neoformans is found worldwide; its main habitats are debris around
pigeon roosts and soil contaminated with decaying pigeon or chicken
droppings (1,3). Not part of the normal microbial flora of humans, C.
neoformans is only transiently isolated from persons with no pathologic
features (2,4). It is generally accepted that the organism enters the host
by the respiratory route in the form of a dehydrated haploid yeast or as
basidiospores. After some time in the lungs, the organism hematogenously
spreads to extrapulmonary tissues; since it has a predilection for the
brain, infected persons usually contract meningoencephalitis (1). If
untreated, cryptococcal meningoencephalitis is 100% fatal, and even when
treated with the most effective antifungal drugs, cryptococcal infections
can be fatal if the host does not have adequate T-celldependent immune
function (2).

To be classified as a pathogen, an organism must be able to cause infection
under certain conditions. By this definition, C. neoformans can certainly
be classified as a pathogen. Because the immunodeficient are more
susceptible than the immunocompetent to infection with this yeast-like
organism, C. neoformans is frequently referred to as an opportunistic
pathogen. The factors that make C. neoformans a pathogen can be divided
into two major groups. The first comprises the basic characteristics needed
to establish an infection and survive in the human host; the second
 	comprises the virulence factors that affect the degree of pathogenicity.

                          [Fig]

Figure 1. Transmission electron micrograph of budding C. neoformans showing
the characteristic polysaccharide capsule.

Basic Characteristics for Pathogenicity

The Infectious Particle

To enter the alveolar spaces of the lungs and establish pulmonary
infection, an organism must produce viable forms smaller than 4 m in
diameter. The typical vegetative form of C. neoformans is the yeast form
with a cell diameter of 2.5 m to 10 m. The organism can also undergo
sexual reproduction, and since it is a basidiomycete (Filobasidiella
neoformans), it forms basidiospores. Sexual reproduction appears to occur
much less frequently in nature than asexual or vegetative reproduction. The
sexual spores (basidiospores) are approximately 1.8 m to 3 m in diameter
and result from crosses of the and  a-mating types on an
appropriate medium (1). Although the exact nature of the infectious
particles of C. neoformans is not known, they are presumed to be the
dehydrated yeast cells or basidiospores of the appropriate size range to
get into the lungs. Once inside the lungs, the yeast cells become
rehydrated and acquire the characteristic polysaccharide capsule (Figure
2). In the case of basidiospores, they would convert to encapsulated
blastoconidia.

                                 [Fig]

Figure 2. Proposed means of infection by C. neoformans.

Recently, Wickes et al. (5) reported that the [Alpha]-mating type of C.
neoformans can produce monokaryotic hyphae on a solid medium without a
nitrogen source or water. The monokaryotic hyphae contain clamp connections
and basidia with short chains of basidiospores and are similar in all
respects except in nuclei number to the dikaryotic hyphae of the sexual
forms (5). The haploid fruiting bodies formed by nitrogen-starved
[Alpha]-mating type isolates also produce abundant amounts of
blastoconidia, which on the appropriate medium can produce haploid hyphae
(5). None of the a-mating type strains tested produced the haploid fruiting
bodies. Backcross studies indicated that the ability to undergo haploid
fruiting lies within (or is tightly linked to) the MAT[Alpha] locus, which
is responsible for the [Alpha]-mating type phenotype (5). These results
suggest basidiospores from the monokaryotic hyphae may be another source of
the infectious particle in nature. Regardless of the nature of the
infectious particle (yeast cell or basidiospore), if a cryptococcal isolate
is not able to produce small infectious particles, it cannot be pathogenic
under the usual conditions for establishing the disease.

Mating Types

Kwon-Chung and Bennett (6) surveyed the mating types of C. neoformans
isolates from environmental and clinical sources and found a 30- to 40-fold
higher frequency of [Alpha]-mating type than [Alpha]-mating type. The
skewed ratio of [Alpha]-mating type to a-mating type was not thought to be
due to a genetic preponderance towards [Alpha]-mating type progeny because
crosses between two test strains resulted in a 1:1 ratio of [Alpha]-mating
and [Alpha]-mating type progeny (7). To examine the reason for the
disparate ratio, Kwon-Chung and colleagues (8) constructed a pair of
congenic strains of C. neoformans that differed only in their mating type.
Survival studies with these congenic strains demonstrated that the
[Alpha]-mating type (B-4500) was significantly more virulent than the
a-mating strain (B-4476) when injected intravenously into mice (8). Eighty
percent of mice infected with 10 [sup6] B-4500 ([Alpha]-mating type) were dead
within 36 days; whereas, it took 93 days for 80% of mice infected with 10 [sup6]
B-4476 (a-mating type) to die (8). Although the mating type locus of C.
neoformans has been cloned and partially characterized, the genes or gene
products that contribute to the increased virulence of [Alpha]-mating type
isolates are not known.

The mating type locus (MAT[Alpha]) cloned from an [Alpha]-mating type C.
neoformans isolate is 35 kb to 45 kb long (9). Moore and Edman (9)
demonstrated that introducing a 2.1-kb fragment of the MAT[Alpha] locus
into an a-mating strain isolate stimulated filament formation on starvation
medium. Basidia and basidiospores were not produced on the filaments. In
contrast, electroporation of an [Alpha]-mating strain with the same MAT
[Alpha] DNA fragment was ineffective in stimulating filament formation
under the same growth conditions (9). Sequence analysis of the 2.1-kb
fragment from the MAT[Alpha] locus identified a 114-bp open reading frame
that encodes a 38-amino acid propheromone peptide (9). The nature and the
effects of the active pheromone peptide on mating and possibly virulence of
C. neoformans are unknown; consequently, how the mating type relates to
virulence at the genetic level is also unknown. However, the observation of
haploid fruiting by [Alpha]-mating type isolates of C. neoformans on
nitrogen- and water-depleted medium (9) may help explain why the
[Alpha]-mating type isolates from clinical specimens are more frequent than
the a-mating type strains (6).

Growth In Vivo

To cause infection in humans, a C. neoformans isolate must grow at 37 degrees C in
an atmosphere of approximately 5% CO2 and at a pH of 7.3 to 7.4. To survive
at 37 degrees C, the organism must have an intact gene that encodes the C.
neoformans calcineurin A catalytic subunit (10). Calcineurin is a
serine-threonine specific phosphatase that is activated by Ca2+-calmodulin
and is involved in stress responses in yeasts (10). Although calcineurin A
mutant strains of C. neoformans can grow at 24 degrees C, they cannot survive in
vitro at 37 degrees C, in 5% CO2, or at alkaline pH (10). Since these are similar
to conditions in the host, one would predict that the calcineurin A mutant
would not survive in the human host. In support of that prediction, Odom et
al. have shown that such mutants are not pathogenic for immunosuppressed
rabbits (10). Calcineurin A appears to be a basic requirement for C.
neoformans survival in the host and consequently is a necessary factor for
the pathogenicity of the organism.

Virulence Factors

Virulence factors increase the degree of pathogenicity of a microbe. C.
neoformans has a number of virulence factors; generally, the virulence of
an isolate cannot be attributed to any single factor, but rather it is
attributed to many working in unison to cause progressive disease. As
virulence factors go, those of C. neoformans would be considered low-grade.
We will discuss each virulence factor separately; however, the severity of
the host's disease results from a combination of several virulence factors
superimposed on the host's innate and immune resistance status. The
virulence factors that will be discussed are capsule, cryptococcal
products, melanin production, mannitol production, and potential factors
such as superoxide dismutase, proteases, phospholipase B, and
lysophospholipase. The polysaccharide capsule and the soluble extracellular
constituents of C. neoformans (referred to here as cryptococcal products)
are probably the dominant virulence factors.

Capsule

C. neoformans has a capsule composed primarily of a high molecular weight
polysaccharide that has a backbone of [Alpha]-1,3-D-mannopyranose units
with single residues of -D-xylopyranosyl and -D-glucuronopyranosyl
attached (11-15). This polysaccharide is referred to as glucuronoxylomannan
(GXM) (11) and has four serotypes: A and D, produced by C. neoformans var.
neoformans, and B and C, produced by C. neoformans var. gattii. The
evidence indicates that the capsule is a key virulence factor for C.
neoformans; acapsular mutants are typically avirulent, whereas encapsulated
isolates have varying degrees of virulence (20). Similar observations have
been made with the CAP64 gene (21,22). Although their biochemical functions
have not been defined, it appears that the two genes are essential for
capsule formation and virulence.

Chemotactic Effects on Leukocytes

Some properties of the C. neoformans capsule enable the host to more
effectively clear the organism from tissues; however, others protect the
organism from host defenses. The capsules of serotype A and D isolates are
chemotactic for neutrophils (23). Moreover, complement is fixed by
cryptococcal capsules by the alternative pathway (24), and this process
produces chemotactic peptides such as C5a (23). Chemotaxis of leukocytes
induced by either of these mechanisms would be advantageous to the host.

Effects of Complement Interactions

Complement fixing by C. neoformans in tissue would result in chemotactic
factor production and attraction of leukocytes into the infection site.
Once in the infected tissue, the leukocytes would interact with and kill
the organism. As complement is fixed, C3b and C3bi are deposited on the
surface of the cryptococci (24). The capsule can mask the C3b and C3bi
deposits (24-26); however, if they are not completely masked, the deposited
complement components facilitate binding of the cryptococci to CR3
receptors on leukocytes (27). Such binding interactions are advantageous to
the host; they enhance the opportunity for the leukocytes to kill the
cryptococci either extracellularly or after phagocytosis. The organism can
also be opsonized by antibodies to GXM, but the capsule may block the Fc
portion of the antibody and prevent it from binding to Fc receptors on the
phagocytic host cells (28). Some of these functions of the capsule favor
the host; however, if the capsule is very large, the organism is protected
(24-26). The cryptococci could deplete complement in the host, creating an
environment that favors the cryptococci (29).

Effects on Phagocytosis

All considered, the capsule is more beneficial to the organism than to the
host. Encapsulated C. neoformans cells are not phagocytized or killed by
neutrophils, monocytes, or macrophages to the same degree as acapsular
mutants (25,30-33). Encapsulated C. neoformans have a stronger negatively
charged surface than acapsular cells or Saccharomyces cerevisiae cells
(34). The high negative charge could cause electrostatic repulsion between
the organism and the negatively charged host effector cells and reduce
intimate cell-cell interactions required for clearance of the cryptococci
(34).

Altered Antigen Presentation

The inability of macrophages to ingest the encapsulated organisms could
also diminish antigen presentation to T cells and consequently reduce
immune responses. This speculation has been experimentally demonstrated by
Collins and Bancroft (35). Other studies with human alveolar macrophages
have confirmed that antigen presentation is not as effective with
encapsulated cryptococci as with acapsular strains (33,36). Unlike
acapsular cells, encapsulated isolates cannot stimulate proliferative
responses in T cells because of the reduced secretion of interleukin-1
(IL-1) by the antigen-presenting cells stimulated with the encapsulated
yeasts (36).

Effects on Cytokine Production

In addition to being antiphagocytic, more resistant to killing, and less
able to stimulate T-cell proliferation (25,30-33,35,36), highly
encapsulated isolates of C. neoformans opsonized with a heat-labile serum
component, presumably complement, do not stimulate monocytes and
macrophages to produce proinflammatory cytokines such as TNF[Alpha], IL-1,
and IL-6 as effectively as similarly opsonized nonencapsulated or weakly
encapsulated isolates (37-41). The signal for induction of cytokine
production can be a direct result of the attachment of the monocyte or
macrophage to the acapsular cryptococci or can be an outcome of the
phagocytic process, induced by acapsular cryptococci. Since the capsule
blocks phagocytosis, any cytokines induced by the phagocytic process would
not be induced by the encapsulated C. neoformans cells. If the cryptococcal
cell wall materials must be exposed to induce cytokine production, the
capsule would block the direct induction of cytokine production. Regardless
of the mechanism of stimulation, the lack of production of proinflammatory
cytokines could have a bearing on protection. TNF[Alpha] is necessary for
the induction of the protective immune response against C. neoformans (42).
Consequently, the lack of or reduced production of TNF[Alpha] in infections
with highly encapsulated isolates of C. neoformans would prevent the
induction of protective immunity, resulting in progressive disease. The
roles of IL-1 and IL-6 in protecting against C. neoformans have not been
defined, but it is highly probable that the lack of these two cytokines
could compromise the protective responses of the host and give cryptococci
the advantage.

In contrast to the reduced TNF[Alpha], IL-1, and IL-6 produced by
stimulating monocytes and macrophages with highly encapsulated cryptococci,
IL-10 produced by these host cells increases after interaction with
encapsulated strains (43). Neutralization of IL-10 with anti-IL-10 in
cocultures of human peripheral blood mononuclear cells and encapsulated
cryptococci increased the amounts of TNF[Alpha] and IL-1 produced, which
indicates that the induction of IL-10 production by stimulating macrophages
with encapsulated C. neoformans downregulates the production of the
proinflammatory cytokines TNF[Alpha] and IL-1 (43). One might predict that
the induction of high levels of IL-10 would also preferentially stimulate
the induction of a T-helper 2 (Th2) response rather than a Th1 response in
the T cells (44). Since the Th1 response is associated with protection
against C. neoformans (45), increased levels of IL-10 would dampen
induction of the protective immune response.

Encapsulated cryptococcal cells do not affect the different types of
leukocytes in the same manner. Although encapsulated isolates do not
stimulate macrophages to produce proinflammatory cytokines, they do
stimulate neutrophils to produce proinflammatory cytokines and the
chemotactic factor IL-8 more effectively than weakly encapsulated or
acapsular organisms (46). As with the stimulation of macrophages by
acapsular cryptococci to produce proinflammatory cytokines, serum is
required for the encapsulated organisms to induce neutrophils to produce
cytokines (46). In the case of cytokine production by neutrophils in
response to encapsulated C. neoformans, it appears that the opsonization
process releases a factor into the supernatant that induces the neutrophils
to produce the cytokines (46). How these opposing in vitro observations
with macrophages and neutrophils relate to the in vivo system or
pathogenicity is yet to be determined.

Cryptococcal Products

In disseminated cryptococcosis, measurable levels of cryptococcal products
are present in the body fluids of the patients (47). Although GXM is the
major cryptococcal component in body fluids, it is highly probable that the
organism also sheds galactoxylomannan (GalXM) and mannoproteins (MP) in
vivo. This speculation is based on indirect evidence from in vivo studies
and on the fact that GalXM and MP are in culture medium when the organism
is grown in vitro (15,48). Cryptococcal antigens in serum or spinal fluid
are diagnostic for cryptococcosis (47). Furthermore, if disseminated
cryptococcosis patients have high cryptococcal antigen titers at the onset
of therapy, they are less likely to respond to therapy or more likely to
die before therapy is completed than patients with low cryptococcal antigen
titers (49). The direct relationship of cryptococcal antigen levels in body
fluids and the severity of disease suggests that the cryptococcal antigens
in the host's circulatory system or spinal fluid may have adverse effects
on host defenses.

Effects on Leukocyte Migration

It has been recently demonstrated in the mouse model that intravascular
cryptococcal antigens inhibit the migration of leukocytes from the
bloodstream into an inflammatory site (50). Intravascular cryptococcal
antigens significantly reduce leukocyte infiltration into a site of acute
inflammation induced by such stimuli as cryptococcal culture filtrate
antigen, TNFa, or the chemotactic peptide FMLP (formylmethionyl leucyl
phenylalanine) or into a delayed-type hypersensitivity reaction site
induced by purified protein derivative of Mycobacterium tuberculosis or by
C. neoformans antigen(s) (50). Each of the identified cryptococcal
products, GXM, GalXM, and MP, when given intravenously to mice, can inhibit
leukocyte migration (50). Considering that GXM is the dominant cryptococcal
component in the host's circulation, one might predict that GXM is mainly
responsible for the reduced leukocyte migration into inflammatory sites
(50). These observations imply that pulmonary cryptococcosis patients, who
have low to no cryptococcal antigen concentrations in their serum, would
have a normal influx of leukocytes into pulmonary sites of infections. On
the other hand, in severe pulmonary infections or in disseminated
cryptococcosis patients who have high levels of circulating cryptococcal
antigens, minimal inflammation would be expected in the infected tissues.
In fact, for years investigators have commented on the minimal host tissue
responses observed in patients with disseminated cryptococcosis (3). These
recent data demonstrate that the circulating cryptococcal antigens are
responsible, at least in part, for the lack of host tissue response.

Cryptococcal antigen(s) can potentially prevent leukocytes from migrating
into an inflammatory site in two nonexclusive ways (51,52). First, GXM can
stimulate neutrophils to shed L-selectin, a surface molecule necessary for
the first step in neutrophil movement into tissues (51). Without L-selectin
the neutrophils do not slow down and roll along the inflamed endothelial
cells lining the blood vessels. With this first step in extravasation
blocked, the numbers of neutrophils in infected tissues would be greatly
reduced. Second, GXM and GalXM can bind to CD18, the beta chain of the
adhesion molecule LFA-1, and prevent the binding of anti-CD18 to LFA-1
(52). Consequently, this binding of GXM and GalXM could prevent LFA-1 on
the neutrophils from binding to the LFA-1 ligand, ICAM-1 on the inflamed
endothelial cell surface. If leukocytes are inhibited from entering tissues
by either or both of these mechanisms, the organism is not effectively
eliminated, and the disease is more severe.

Induction of Immunomodulatory Cells

Cryptococcal antigens injected into the bloodstream of experimental animals
can induce regulatory T cells, which dampen or ablate the anticryptococcal
humoral as well as cell-mediated immune responses (53-71). Some clinical
correlates support the concept that the antigenemia seen in disseminated
cryptococcosis downmodulates the immune responses (72,73).

A long-lasting, specific immunologic unresponsiveness has been reported in
persons cured of cryptococcal meningitis (72-74). Although they made
antipneumococcal polysaccharide antibodies to the same degree as control
volunteers after vaccination with pneumococcal polysaccharide, cured
patients could not make antibodies to cryptococcal polysaccharides after
vaccination with cryptococcal antigens (72,73). Henderson et al. (72,73)
speculated that the intense, prolonged antigenemia associated with the
cryptococcosis may account for the observed unresponsiveness. It is not
unusual for patients with disseminated cryptococcosis to have depressed
cell-mediated immune (CMI) responses to cryptococcal antigens (75,76).
However, sufficient information is not available to determine whether the
patients had a generalized defect in CMI function or had a specifically
depressed CMI response because of the cryptococcal antigenemia.

Experimental animal models demonstrate convincingly that cryptococcal
antigens given intravenously can induce immunoregulatory T cells that
downmodulate the anticryptococcal CMI response (56-71). Serum from C.
neoformans-infected mice with a cryptococcal antigen titer of 10-4 when
transferred intravenously to naive mice induces regulatory T cells that
specifically depress the anticryptococcal CMI response (67). Similar
immunoregulatory T cells are induced by simulating the antigenemia with
intravenous injection of cryptococcal culture filtrate antigen
(56,57,60-63,66-69). The immunoregulatory CD4+ T cells, which appear in the
lymph nodes of the mice 7 days after antigen injection, diminish the
induction of T cells responsible for the anticryptococcal delayed-type
hypersensitivity response and reduce the ability of the mice to clear
cryptococci from tissues (56,57,60,68). A second immunoregulatory T cell, a
CD8+ cell, has been found in spleens of mice (57). The CD4+
immunoregulatory cells do not alter the numbers or types of leukocytes that
infiltrate an anticryptococcal delayed-type hypersensitivity reaction;
however, they do have a downregulating effect on IL-2 and IFN[Alpha]
production at the site (77). In contrast, the CD8+ immunoregulatory cells
appear to affect the numbers of neutrophils that infiltrate the
delayed-type hypersensitivity reaction site (Murphy, unpub. data). The
details of the characteristics and functions of these immunoregulatory T
cells have been reviewed (45). The available data strongly support the
notion that cryptococcal products in the circulation induce an array of
immunoregulatory T cells, which depress the anticryptococcal immune
responses and protection.

More consideration should be given to the impact of high levels of
cryptococcal antigen in the cerebrospinal fluid (CSF) on progression of
disease. As suggested by Denning et al. (78), high cryptococcal antigen
concentrations could change the osmolality of the CSF, thereby affecting
its outflow and adsorption and increasing intracranial pressure. The
increased pressure may cause headaches, visual loss, and early death (78).
It is also possible that release of mannitol by C. neoformans contributes
to increased intracranial pressure in cryptococcal meningitis patients
(78).

Melanin Synthesis

One characteristic that differentiates pathogenic isolates of C. neoformans
from nonpathogenic isolates and other Cryptococcus species is the
organism's ability to form a brown to black pigment on a medium (such as
birdseed or caffeic acid agar) that contains diphenolic compounds (1). This
pigment, first described by Staib (79), is a melaninlike compound produced
by C. neoformans isolates with phenoloxidase activity (80). The importance
of melanin production in C. neoformans virulence was first demonstrated in
the early 1980s. Rhodes, Polacheck, and Kwon-Chung (81) reported that
naturally occurring C. neoformans mutants lacking melanin (Mel-) were less
virulent in mice than melanin-producing strains. Others (82-84), using
chemically induced mutants or isogenic pairs of C. neoformans, have
confirmed and extended this observation.

Biochemical analyses suggest that in C. neoformans melanogenesis is
accomplished by conversion of dihydroxyphenols such as
3,4-dihydroxyphenylalanine (DOPA) to dopaquinone (Figure 3). This
conversion is catalyzed by a phenoloxidase and is the rate-limiting step,
presumably because subsequent steps in the pathway, such as dopaquinone
rearranging to dopachrome and ultimately autoxidation to melanin, are
spontaneous (85). C. neoformans lacks a tyrosinase enzyme required for
endogenous production of dihydroxyphenols (83); thus to produce melanin, a
C. neoformans isolate must be able to acquire diphenolic compounds from its
growth environment, and it must have the enzyme phenoloxidase to catalyze
conversion of these compounds into the subsequent melanin intermediates.
The brain is a tissue rich in catecholamines such as DOPA and is a favorite
target for infection by C. neoformans. However, the organism cannot use
catecholamines as a sole carbon source, which suggests that the brain is
not a preferred nutritional niche for growth of C. neoformans (86); rather,
it may serve as a survival niche as described below.

                                 [Fig]

Figure 3. Proposed pathway for melanin synthesis by C. neoformans.

Polacheck et al. (86) reported that melanin-producing isolates of C.
neoformans were resistant to damage by an in vitro epinephrine oxidative
system, whereas mutants lacking phenoloxidase activity were highly
susceptible, as evidenced by decreased survival. Jacobson and Tinnell (87)
found that melanin protected C. neoformans from damage by hypochlorite and
permanganate but not by hydrogen peroxide. In these experiments,
hypochlorite was 100 times more fungicidal than hydrogen peroxide, and C.
neoformans could produce sufficient levels of melanin to effectively
protect the organism from oxidative compounds produced by macrophages (87).
Wang and Casadevall (88) extended these findings by examining survival of
C. neoformans in the presence of nitric oxide and reactive nitrogen
intermediates as well as in the epinephrine oxidative system described by
Polacheck et al. (86). Wang and Casadevall cultured C. neoformans cells
with L-DOPA to melanize the yeast cells (88). Melanized cryptococci
survived damage by both nitrogen- and oxygen-derived oxidants significantly
better than nonmelanized organisms of the same strain. These results
support the hypothesis that C. neoformans uses catecholamines in the brain
to make melanin, thereby protecting the organism from oxidative damage by
scavenging free radicals (86).

Recently, cryptococcal diphenoloxidase has been purified, and the gene
CNLAC1 encoding this enzyme was cloned (89). The glycosylated protein has a
molecular weight of 75 kDa and contains copper. The substrate specificity
of the enzyme indicates that it is a laccase. CNLAC1 contains 14 introns, a
624 amino acid open reading frame; transcriptional activity is derepressed
in the absence of glucose (89), which confirms an earlier report of low
glucose requirements for melanin formation (90). Disruption of CNLAC1
resulted in loss of virulence of C. neoformans, whereas complementation
with CNLAC1 increased virulence of Mel- mutants in mice (84); these results
suggest that the laccase (phenoloxidase) encoded by CNLAC1 is a potential
virulence factor of C. neoformans. Furthermore, transcripts of the CNLAC1
gene have been detected by reverse transcription-polymerase chain reaction
(RT-PCR) in C. neoformans yeast cells isolated from CSF in a rabbit model
of cryptococcal meningitis (84).

Besides acting as an antioxidant, melanin production may help C. neoformans
survive in the host in other ways. Melanized yeast cells are less
susceptible to amphotericin B than nonmelanized yeast cells, and this may
contribute to the inability to effectively treat infections in
immunocompromised hosts (91). Furthermore, phagocytosis of melanized C.
neoformans by a macrophage cell line in the presence of an anti-GXM
antibody was decreased, which suggests that melanin deposition in the cell
wall may inhibit opsonization by specific antibodies (92). Recently,
Huffnagle and coinvestigators (93) reported that melanized heat-killed C.
neoformans strain 145 yeast cells stimulated less TNF[Alpha] production by
alveolar macrophages and less antigen-specific T-cell proliferation than
nonmelanized heat-killed 145 strain cells. The authors suggest that melanin
"cloaks" C. neoformans from recognition by host effector cells and inhibits
induction of a protective T-cell-mediated immune response (93); however, it
is possible that scavenging of host-produced oxidants and inhibition of
phagocytosis by melanin may contribute to the decreased TNF[Alpha]
production and lymphoproliferation observed.

Although melanin production is important in the virulence of C. neoformans,
there is very little evidence demonstrating the presence of melanin in
vivo. One report indicates that phenoloxidase activity in C. neoformans is
greatly diminished at 37 degrees C compared with activity at 25 degrees C,
which suggests that melanin production may be limited in vivo (94). Detection of melanin in vivo is hampered by the lack of specific antibodies or stains. A
modified Fontana-Masson stain has been used to detect a brown pigment in
the cell wall of C. neoformans cells in histologic brain sections; however,
the stain is not specific because Cryptococcus laurentii, which is Mel-,
also stains with Fontana-Masson (95).

In summary, accumulating evidence indicates that melanin production is an
effective survival (virulence) factor of C. neoformans, and melanin may
serve multiple roles in protecting this medically important fungus from
host defenses. Two caveats should be noted in labeling melanin as a major
virulence factor for C. neoformans. The C. neoformans isolate must be able
to internalize the melanin precursors, and the phenoloxidase enzyme must
make a sufficient amount of melanin precursor at 37 degrees C. Consequently, much
more work is needed to delineate the role of melanin production in
virulence of C. neoformans, especially in light of the apparent temperature
sensitivity of the cryptococcal phenoloxidase enzyme.

Mannitol Production

Accumulating evidence suggests that production of the hexitol D-mannitol
may contribute to survival of C. neoformans in the host. Wong et al. (96)
reported that of the 12 human isolates of C. neoformans examined, all
produced and secreted D-mannitol into growth medium. Further, by using a
rabbit cryptococcal meningitis model, they showed that C. neoformans can
produce D-mannitol in vivo. CSF from rabbits treated with cortisone and
infected intracisternally with C. neoformans contained more D-mannitol than
CSF from controls, cortisone-treated uninfected rabbits, or rabbits
infected with C. neoformans but not treated with cortisone (96). In the
latter group, cryptococcal infection was limited. The levels of D-mannitol
in infected rabbit CSF correlated well with both the numbers of culturable
C. neoformans and the cryptococcal antigen titer of the CSF, which suggests
that levels of D-mannitol in CSF may be prognostic (96); however, it is not
known whether different isolates of C. neoformans vary in mannitol
production. These authors suggested two means by which mannitol production
may contribute to C. neoformans pathogenesis. First, high concentrations of
D-mannitol in the CNS may contribute to brain edema. Second, mannitol is a
potent scavenger of hydroxyl radicals, and cryptococcal-produced D-mannitol
may help protect the organism from oxidative damage (96).

To investigate the role of mannitol in cryptococcosis, Chaturvedi and
co-workers (97) isolated a low mannitol producing mutant after UV
irradiation of C. neoformans strain H99. The mutant, mannitol low producer
(MLP), was similar to the parent strain H99 in many growth and morphology
characteristics and in known virulence factors such as capsule production
and phenoloxidase activity (97). However, the mutant MLP strain was more
susceptible to growth inhibition and killing by heat and high salt than the
parent strain. In addition, the mutant MLP strain was significantly less
virulent than the parent strain (MLP LD50 = 3.7 X 106 CFU, H99 LD50 = 6.9 X
102) (97). Further comparisons of H99 and MLP strains showed that
polymorphonuclear leukocytes (PMNL) killed MLP significantly better than
H99 at several effector-to-target ratios (98). Addition of superoxide
dismutase, mannitol, or dimethyl sulfoxide inhibited PMNL killing of both
strains, but addition of catalase did not alter killing, which suggests
that reactive oxygen intermediates such as the hydroxyl radical and
hypochlorous acid are potent effector molecules against C. neoformans and
mannitol may protect against oxidative killing by scavenging such compounds
(98).

The results above indicate that mannitol production by C. neoformans
correlates with increased resistance to heat stress, osmotic stress, and
damage by reactive oxygen intermediates, as well as increased pathogenicity
of this fungal agent. Additional studies are required to determine the role
of mannitol production in virulence of C. neoformans. Isogenic strains
lacking the enzyme required for mannitol production, namely mannitol
dehydrogenase, are not available because the gene for this enzyme has not
been found. However, a gene (Mtl) has been isolated from C. neoformans that
can induce expression of the S. cerevisiae mannitol dehydrogenase gene
(99). The 346 amino acid product encoded by the Mtl gene is thought to be
involved in regulating mannitol production in C. neoformans and may be
beneficial in future studies of mannitol production (99).

Other Potential Virulence Factors

Superoxide Dismutase

Jacobson and coinvestigators (100) examined superoxide dismutase (SOD)
production by C. neoformans to determine whether SOD levels increased at
37 degrees C to compensate for possible decreases in melanin production at this
temperature. These investigators observed an increase in SOD levels at
37 degrees C, which suggests that SOD may participate in free radical scavenging at
this higher temperature in vivo (100); however, there is no evidence that
SOD production serves as a virulence factor for C. neoformans.

Proteases

Muller and Sethi (101) reported that C. neoformans grown in culture
produced a protease that could digest human plasma proteins, and Brueske
(102) reported that C. neoformans culture supernatants contained a protease
capable of digesting casein. However, neither of these studies determined
whether the proteases were manufactured in vivo. Limited evidence
indicating in vivo production of proteases has been presented by Salkowski
and Balish (103). These investigators observed skin lesions on
T-cell-deficient mice after intravenous infections with C. neoformans
strain SLHA (103). Microscopic examination of the lesions suggested that
the cryptococcal yeast cells were degrading collagen bundles in the dermis
(103). Supernatants of SLHA strain cultures liquefied gelatin in vitro
indicating that this cryptococcal strain secreted a collagenaselike protein
(103). Thus, C. neoformans proteases possibly serve as virulence mechanisms
by initiating invasion of host tissues; however, more studies with isogenic
strains of C. neoformans are required before proteases can be listed as
virulence factors.

Phospholipases

Recently, Chen and co-workers (104,105) reported phospholipase,
lysophospholipase, and lysophospholipase-transacylase activity of C.
neoformans grown on egg yolk agar. Of 50 strains tested, 49 had
phospholipase activity (104), due to phospholipase B secreted into cultures
(105). Analysis of four strains with varying levels of phospholipase
activity indicated a correlation between phospholipase activity and
virulence in BALB/c mice (104). The authors suggest that extracellular
phospholipase activity produced by C. neoformans may disrupt mammalian cell
membranes and allow the yeast cells to penetrate into host tissues
(104,105); however, further investigations are necessary to establish the
role, if any, of these types of products in the virulence of C. neoformans.

Regulation of Virulence

Regulation of virulence factors such as capsule production and melanin
formation is not well understood. However, the gene GPA1, which encodes a
G-protein [Alpha]-subunit, is involved in the regulation of these virulence
factors as well as in C. neoformans mating (106). Disruption of GPA1
resulted in defects in mating in response to nitrogen starvation, capsule
production in response to iron limitation, and melanin synthesis in
response to glucose starvation (106). Furthermore, gpa1 mutants were much
less virulent in a rabbit model of cryptococcal meningitis (106).
Reconstitution of the gpa1 mutant with wild-type GPA1 restored mating,
capsule production, melanin synthesis, and virulence. Addition of cyclic
AMP also restored these phenotypes, which suggests that C. neoformans GPA1
regulates these factors by sensing the nutritional signals of the
environment and regulating cyclic AMP metabolism in the organism (106).
These findings along with results of other molecular studies are intriguing
and represent the initial steps in defining at the molecular level the
factors controlling virulence in C. neoformans.

Conclusions

Virulent isolates of C. neoformans must be able to produce small particles
that can get into the alveolar spaces, must be able to grow at 37 degrees C at a pH
of 7.3 to 7.4 in an atmosphere of approximately 5% CO2, must have an intact
calcineurin pathway, and (possibly) must be an [Alpha] -mating type. The
ability to produce a large capsule and shed great amounts of capsular
material into the body fluids makes the organism highly virulent. Other
factors, such as melanin, mannitol, superoxide dismutase, protease, and
phospholipase production, may enhance the pathogenicity of C. neoformans.
The effectiveness of many of these cryptococcal virulence factors depends
on the status of the host's defensive mechanisms. Although we have learned
much about the pathogenicity and virulence of C. neoformans, many gaps
still remain in our knowledge.

Acknowledgments

We thank Drs. J.A. Alspaugh, J.R. Perfect, and J. Heitman for making their
manuscript available before publication.

Dr. Buchanan is a research assistant professor in the Department of
Microbiology and Immunology, University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma, USA. Dr. Murphy is a George Lynn Cross Research
Professor of Microbiology and Immunology in the same department.

Address for correspondence: Juneann W. Murphy, University of Oklahoma
Health Sciences Center, Oklahoma City, OK, USA; fax: 405-271-3117; e-mail:
kent-buchanan@ouhsc.edu or juneann-murphy@ouhsc.edu.

References

  1. Kwon-Chung KJ. Cryptococcosis. In: Kwon-Chung KJ, Bennett JE, editors.
     Medical mycology. Philadelphia: Lea & Febiger, 1992:397-446.
  2. Mitchell TG, Perfect JR. Cryptococcosis in the era of AIDS-100 years
     after the discovery of Cryptococcus neoformans. Clin Microbiol Rev
     1995;8:515-48.
  3. Rippon JW. Cryptococcosis. In: Rippon JW, editor. Medical mycology.
     Philadelphia: W.B. Saunders Company; 1988, p. 582-609.
  4. Duperval B, Hermans PE, Brewer NS, Roberts GD. Cryptococcosis, with
     emphasis on the significance of isolation of Cryptococcus neoformans
     from the respiratory tract. Chest 1977;72:13-9.
  5. Wickes BL, Mayorga ME, Edman U, Edman JC. Dimorphism and haploid
     fruiting in Cryptococcus neoformans: association with the a-mating
     type. Proc Natl Acad Sci U S A 1996;93:7327-31.
  6. Kwon-Chung KJ, Bennett JE. Distribution of and mating types of
     Cryptococcus neoformans among natural and clinical isolates. Am J
     Epidemiol 1978;108:337-40.
  7. Kwon-Chung KJ, Hill WB. Sexuality and pathogenicity of Filobasidiella
     neoformans (Cryptococcus neoformans). In: Vanbreuseghem R, DeVroey C,
     Vanbreuseghem R, editors. Sexuality and pathogenicity of fungi. Paris:
     Masson; 181. p. 243-50.
  8. Kwon-Chung KJ, Edman JC, Wickes BL. Genetic association of mating
     types and virulence in Cryptococcus neoformans. Infect Immun
     1992;60:602-5.
  9. Moore TDE, Edman JC. The [Alpha]-mating type locus of Cryptococcus
     neoformans contains a pheromone gene. Mol Cell Biol 1993;13:1962-70.
 10. Odom A, Muir S, Lim E, Toffaletti DL, Perfect J, Heitman J.
     Calcineurin is required for virulence of Cryptococcus neoformans. EMBO
     J 1997;16:2576-89.
 11. Cherniak R, Reiss E, Slodki ME, Plattner RD, Blumer SO. Structure and
     antigenic activity of the capsular polysaccharide of Cryptococcus
     neoformans. Mol Immunol 1980;17:1025-32.
 12. Bhattacharjee AK, Kwon-Chung KJ, Glaudemans CP. Capsular
     polysaccharides from a parent strain and from a possible mutant strain
     of Cryptococcus neoformans serotype A. Carbohydr Res 1981;95:237-48.
 13. Bhattacharjee AK, Bennett JE, Glaudemans CP. Capsular polysaccharides
     of Cryptococcus neoformans. Reviews of Infectious Diseases
     1984;6:619-24.
 14. Cherniak R. Soluble polysaccharides of Cryptococcus neoformans. Cur
     Top Med Mycol 1988;2:40-54.
 15. Cherniak R, Sundstrom JB. Polysaccharide antigens of the capsule of
     Cryptococcus neoformans. Infect Immun 1994;62:1507-12.
 16. Bulmer GS, Sans MD, Gunn CM. Cryptococcus neoformans. I.
     Nonencapsulated mutants. J Bacteriol 1967;94:1475-9.
 17. Fromtling RA, Shadomy HJ, Jacobson ES. Decreased virulence in stable,
     acapsular mutants of Cryptococcus neoformans. Mycopathologia
     1982;79:23-9.
 18. Kozel TR, Cazin J Jr. Non-encapsulated variant of Cryptococcus
     neoformans. I. Virulence studies and characterization of soluble
     polysaccharide. Infect Immun 1971;3:287-94.
 19. Kwon-Chung KJ, Rhodes JC. Encapsulation and melanin formation as
     indicators of virulence in Cryptococcus neoformans. Infect Immun
     1986;51:218-23.
 20. Chang YC, Kwon-Chung KJ. Complementation of a capsule-deficient
     mutation of Cryptococcus neoformans restores its virulence. Mol Cell
     Biol 1994;14:4912-9.
 21. Chang YC, Penoyer LA, Kwon-Chung KJ. The second capsule gene of
     Cryptococcus neoformans, CAP64, is essential for virulence. Infect
     Immun 1996;64:1977-83.
 22. Chang YC, Cherniak R, Kozel TR, Granger LC, Morris LC, Weinhold LC,
     Kwon-Chung KJ. Structure and biological activities of acapsular
     Cryptococcus neoformans 602 complemented with the CAP64 gene. Infect
     Immun 1997;65:1584-92.
 23. Dong ZM, Murphy JW. Mobility of human neutrophils in response to
     Cryptococcus neoformans cells, culture filtrate antigen, and
     individual components of the antigen. Infect Immun 1993;61:5067-77.
 24. Kozel TR, Pfrommer GS. Activation of the complement system by
     Cryptococcus neoformans leads to binding of iC3b to the yeast. Infect
     Immun 1986;52:1-5.
 25. Kozel TR, Mastroianni RP. Inhibition of phagocytosis by cryptococcal
     polysaccharide: dissociation of the attachment and ingestion phases of
     phagocytosis. Infect Immun 1976;14:62-7.
 26. Kozel TR, Highison B, Stratton CJ. Localization on encapsulated
     Cryptococcus neoformans of serum components opsonic for phagocytosis
     by macrophages and neutrophils. Infect Immun 1984;43:574-9.
 27. Griffin FMJ. Roles of macrophage Fc and C3b receptors in phagocytosis
     of immunologically coated Cryptococcus neoformans. Proc Natl Acad Sci
     U S A 1981;78:3853-7.
 28. Kozel TR, Pfrommer GS, Guerlain AS, Highison BA, Highison GJ. Role of
     the capsule in phagocytosis of Cryptococcus neoformans. Reviews of
     Infectious Diseases 1988;10:S436-9.
 29. Macher AM, Bennett JE, Gadek JE, Frank MM. Complement depletion in
     cryptococcal sepsis. J Immunol 1978;120:1686-90.
 30. Bulmer GS, Sans MD. Cryptococcus neoformans II. Phagocytosis by human
     leukocytes. J Bacteriol 1967;94:1480-3.
 31. Bulmer GS, Sans MD. Cryptococcus neoformans. III. Inhibition of
     phagocytosis. J Bacteriol 1968;95:5-8.
 32. Kozel TR, Gotschlich EC. The capsule of Cryptococcus neoformans
     passively inhibits phagocytosis of the yeast by macrophages. J Immunol
     1982;129:1675-80.
 33. Vecchiarelli A, Pietrella D, Dottorini M, Monari C, Retini C, Todisco
     T, Bistoni F. Encapsulation of Cryptococcus neoformans regulates
     fungicidal activity and the antigen presentation process in human
     alveolar macrophages. Clin Exp Immunol 1994;98:217-23.
 34. Nosanchuk JD, Casadevall A. Cellular charge of Cryptococcus
     neoformans: Contributions from the capsular polysaccharide, melanin,
     and monoclonal antibody binding. Infect Immun 1997;65:1836-41.
 35. Collins HL, Bancroft GJ. Encapsulation of Cryptococcus neoformans
     impairs antigen-specific T-cell responses. Infect Immun
     1991;59:3883-8.
 36. Vecchiarelli A, Dottorini M, Pietrella D, Vecchiarelli A, Dottorini M,
     Pietrella D, et al. Role of human alveolar macrophages as
     antigen-presenting cells in Cryptococcus neoformans infection. Am J
     Respir Cell Mol Biol 1994;11:130-7.
 37. Levitz SM, Tabuni A, Kornfeld H, Reardon CC, Golenbock DT. Production
     of tumor necrosis factor alpha in human leukocytes stimulated by
     Cryptococcus neoformans. Infect Immun 1994;62:1975-81.
 38. Naslund PK, Miller WC, Granger DL. Cryptococcus neoformans fails to
     induce nitric oxide synthase in primed murine macrophage-like cells.
     Infect Immun 1995;63:1298-1304.
 39. Vecchiarelli A, Retini C, Pietrella D, Monari C, Tascini T, Beccari T,
     Kozel TR. Downregulation by cryptococcal polysaccharide of tumor
     necrosis factor alpha and interleukin-1 beta secretion from human
     monocytes. Infect Immun 1995;63:2919-23.
 40. Delfino D, Cianci L, Migliardo M, Mancuso G, Cusumano V, Corradini C,
     Teti G. Tumor necrosis factor-inducing activities of Cryptococcus
     neoformans components. Infect Immun 1996;64:5199-204.
 41. Delfino D, Cianci L, Lupis E, Celese A, Petrelli ML, Curro F, et al.
     Interleukin-6 production by human monocytes stimulated with
     Cryptococcus neoformans components. Infect Immun 1997;65:2454-6.
 42. Huffnagle GB, Toews GB, Burdick MD, Boyd MB, McAllister KS, McDonald
     RA, et al. Afferent phase production of TNF-[Alpha] required for the
     development of protective T cell immunity to Cryptococcus neoformans.
     J Immunol 1996;157:4529-36.
 43. Vecchiarelli A, Retini C, Monari C, Tascini C, Bistoni F, Kozel TR.
     Purified capsular polysaccharide of Cryptococcus neoformans induces
     interleukin-10 secretion by human monocytes. Infect Immun
     1996;64:2846-9.
 44. Moore KW, O'Garra A, de Waal Malefyt R, Vieira P, Mosmann TR.
     Interleukin-10. Annu Rev Immunol 1993;11:165-90.
 45. Murphy JW. Cell-mediated immunity. In: Howard DH, Miller JD, editors.
     The Mycota VI. Human and animal relationships. Berlin:
     Springer-Verlag; 1996. p. 67-97.
 46. Retini C, Vecchiarelli A, Monari C, Tascini C, Bistoni F, Kozel TR.
     Capsular polysaccharide of Cryptococcus neoformans induces
     proinflammatory cytokine release by human neutrophils. Infect Immun
     1996;64:2897-903.
 47. Gordon MA, Vedder DK. Serologic tests in diagnosis and prognosis of
     cryptococcosis. JAMA 1966;197:961-7.
 48. Reiss E, Cherniak R, Eby E, Kaufman L. Enzyme immunoassay detection of
     IgM to galactoxylomannan of Cryptococcus neoformans. Diagnostic
     Immunology 1984;2:109-15.
 49. Diamond RD, Bennett JE. Prognostic factors in cryptococcal meningitis.
     A study in 111 cases. Ann Intern Med 1974;80:176-81.
 50. Dong ZM, Murphy JW. Intravascular cryptococcal culture filtrate (CneF)
     and its major component glucuronoxylomannan (GXM) are potent
     inhibitors of leukocyte accumulation. Infect Immun 1995;63:770-8.
 51. Dong ZM, Murphy JW. Cryptococcal polysaccharides induce L-selectin
     shedding and tumor necrosis factor receptor loss from the surface of
     human neutrophils. J Clin Invest 1996;97:689-98.
 52. Dong ZM, Murphy JW. Cryptococcal polysaccharides bind to CD18 on human
     neutrophils. Infect Immun 1997;65:557-63.
 53. Kozel TR, Gulley WF, Cazin J Jr. Immune response to Cryptococcus
     neoformans soluble polysaccharide: immunological unresponsiveness.
     Infect Immun 1977;18:701-7.
 54. Breen JF, Lee IC, Vogel FR, Friedman H. Cryptococcal capsular
     polysaccharide-induced modulation of murine immune responses. Infect
     Immun 1982;36:47-51.
 55. Murphy JW, Moorhead JW. Regulation of cell-mediated immunity in
     cryptococcosis. I. Induction of specific afferent T suppressor cells
     by cryptococcal antigen. J Immunol 1982;128:276-83.
 56. Murphy JW, Mosley RL, Moorhead JW. Regulation of cell-mediated
     immunity in cryptococcosis. II. Characterization of first-order T
     suppressor cells (Ts1) and induction of second-order suppressor cell.
     J Immunol 1983;130:2876-81.
 57. Morgan MA, Blackstock R, Bulmer GS, Hall NK. Modification of
     macrophage phagocytosis in murine cryptococcosis. Infect Immun
     1983;40:493-500.
 58. Blackstock R, Hall NK. Nonspecific immune suppression by Cryptococcus
     neoformans infection. Mycopathologia 1984;86:35-43.
 59. Murphy JW. Effects of first-order Cryptococcus-specific T-suppressor
     cells on induction of cells responsible for delayed-type
     hypersensitivity. Infect Immun 1985;48:439-45.
 60. Murphy JW, Mosley RL. Regulation of cell-mediated immunity in
     cryptococcosis. III. Characterization of second-order T suppressor
     cells (Ts2). J Immunol 1985;134:577-84.
 61. Mosley RL, Murphy JW, Cox RA. Immunoadsorption of
     Cryptococcus-specific suppressor T-cell factors. Infect Immun
     1986;51:844-50.
 62. Khakpour FR, Murphy JW. Characterization of a third-order suppressor T
     cell (Ts3) induced by cryptococcal antigen(s). Infect Immun
     1987;55:1657-62.
 63. Blackstock R, McCormack JM, Hall NK. Induction of a
     macrophage-suppressive lymphokine by soluble cryptococcal antigens and
     its association with models of immunological tolerance. Infect Immun
     1987;55:233-9.
 64. Blackstock R, Hernandez NC. Inhibition of phagocytosis in
     cryptococcosis: phenotypic analysis of the suppressor cell. Cell
     Immunol 1988;114:174-87.
 65. Murphy JW, Cox RA. Induction of antigen-specific suppression by
     circulating Cryptococcus neoformans antigen. Clin Exp Immunol
     1988;73:174-80.
 66. Murphy JW. Influence of cryptococcal antigens on cell-mediated
     immunity (CMI). Reviews of Infectious Diseases 1988;10:5432-5.
 67. Murphy JW. Clearance of Cryptococcus neoformans from immunologically
     suppressed mice. Infect Immun 1989;57:1946-52.
 68. Murphy JW. Immunoregulation in cryptococcosis. In: Kurstak E, editor.
     Immunology of fungal diseases. New York: Marcel Dekker; 1989. p.
     319-45.
 69. Blackstock R, Hall NK, Hernandez NC. Characterization of a suppressor
     factor that regulates macrophage phagocytosis in murine
     cryptococcosis. Infect Immun 1989;57:1773-9.
 70. Blackstock R, Zembala M, Asherson GL. Functional equivalence of
     cryptococcal and haptene-specific T suppressor factor (TsF). I. Picryl
     and oxazolone-specific TsF, which inhibit transfer of contact
     sensitivity also inhibit phagocytosis by a subset of macrophages. Cell
     Immunol 1991;136:435-47.
 71. Henderson DK, Bennett JE, Huber MA. Long-lasting, specific immunologic
     unresponsiveness associated with cryptococcal meningitis. J Clin
     Invest 1982;69:1185-90.
 72. Henderson DK, Kan VL, Bennett JE. Tolerance to cryptococcal
     polysaccharide in cured cryptococcosis patients: failure of antibody
     secretion in vitro. Clin Exp Immunol 1986;65:639-46.
 73. Salvin SB, Smith RF. An antigen for detection of hypersensitivity to
     Cryptococcus neoformans. Proc Soc Exp Biol Med 1961;108:498-501.
 74. Diamond RD, Bennett JE. Disseminated cryptococcosis in man: decreased
     lymphocyte transformation in response to Cryptococcus neoformans. J
     Infect Dis 1973;127:694-7.
 75. Graybill JR, Alford RH. Cell-mediated immunity in cryptococcosis. Cell
     Immunol 1974;14:12-21.
 76. Buchanan KL, Murphy JW. Regulation of cytokine production during the
     expression phase of the anticryptococcal delayed-type hypersensitivity
     response. Infect Immun 1994;62:2930-9.
 77. Denning DW, Armstrong RW, Lewis BH, Stevens DA. Elevated cerebrospinal
     fluid pressures in patients with cryptococcal meningitis and acquired
     immunodeficiency syndrome. Am J Med 1991;91:267-72.
 78. Staib F. Cryptococcus neoformans und Guizotia abyssinica. Zeitschrift
     fr Hygiene 1962;148:466-75.
 79. Shaw CE, Kapica L. Production of diagnostic pigment by phenoloxidase
     activity of Cryptococcus neoformans. Applied Microbiology
     1972;24:824-30.
 80. Rhodes JC, Polacheck I, Kwon-Chung KJ. Phenoloxidase activity and
     virulence in isogenic strains of Cryptococcus neoformans. Infect Immun
     1982;36:1175-84.
 81. Kwon-Chung KJ, Polacheck I, Popkin TJ. Melanin-lacking mutants of
     Cryptococcus neoformans and their virulence for mice. J Bacteriol
     1982;150:1414-21.
 82. Torres-Guerrero H, Edman JC. Melanin-deficient mutants of Cryptococcus
     neoformans. J Med Vet Mycol 1994;32:303-13.
 83. Salas SD, Bennett JE, Kwon-Chung KJ, Perfect JR, Williamson PR. Effect
     of the laccase gene, CNLAC1, on virulence of Cryptococcus neoformans.
     J Exp Med 1996;184:377-86.
 84. Polacheck I, Kwon-Chung KJ. Melanogenesis in Cryptococcus neoformans.
     Journal of General Microbiology 1988;134:1037-41.
 85. Polacheck I, Platt Y, Aronovitch J. Catecholamines and virulence of
     Cryptococcus neoformans. Infect Immun 1990;58:2919-22.
 86. Jacobson ES, Tinnell SB. Antioxidant function of fungal melanin. J
     Bacteriol 1993;175:7102-4.
 87. Wang Y, Casadevall A. Susceptibility of melanized and nonmelanized
     Cryptococcus neoformans to nitrogen- and oxygen-derived oxidants.
     Infect Immun 1994;62:3004-7.
 88. Williamson PR. Biochemical and molecular characterization of the
     diphenol oxidase of Cryptococcus neoformans: identification as a
     laccase. J Bacteriol 1994;176:656-64.
 89. Polacheck I, Hearing VJ, Kwon-Chung KJ. Biochemical studies of
     phenoloxidase and utilization of catecholamines in Cryptococcus
     neoformans. J Bacteriol 1982;150:1212-20.
 90. Wang Y, Casadevall A. Growth of Cryptococcus neoformans in presence of
     L-dopa decreases its susceptibility to amphotericin B. Antimicrob
     Agents Chemother 1994;38:2648-50.
 91. Wang Y, Aisen P, Casadevall A. Cryptococcus neoformans melanin and
     virulence: mechanism of action. Infect Immun 1995;63:3131-6.
 92. Huffnagle GB, Chen GH, Curtis JL, McDonald RA, Strieter RM, Toews GB.
     Down-regulation of the afferent phase of T cell-mediated pulmonary
     inflammation and immunity by a high melanin-producing strain of
     Cryptococcus neoformans. J Immunol 1995;155:3507-16.
 93. Jacobson ES, Emery HS. Temperature regulation of the cryptococcal
     phenoloxidase. J Med Vet Mycol 1991;29:121-4.
 94. Kwon-Chung KJ, Hill WB, Bennett JE. New, special stain for
     histopathological diagnosis of cryptococcosis. J Clin Microbiol
     1981;13:383-7.
 95. Wong B, Perfect JR, Beggs S, Wright KA. Production of the hexitol
     D-mannitol by Cryptococcus neoformans in vitro and in rabbits with
     experimental meningitis. Infect Immun 1990;58:1664-70.
 96. Chaturvedi V, Flynn T, Niehaus WG, Wong B. Stress tolerance and
     pathogenic potential of a mannitol mutant of Cryptococcus neoformans.
     Microbiology 1996;142:937-43.
 97. Chaturvedi V, Wong B, Newman SL. Oxidative killing of Cryptococcus
     neoformans by human neutrophils. Evidence that fungal mannitol
     protects by scavenging reactive oxygen intermediates. J Immunol
     1996;156:3836-40.
 98. Perfect JR, Rude TH, Wong B, Flynn T, Chaturvedi V, Niehaus W.
     Identification of a Cryptococcus neoformans gene that directs
     expression of the cryptic Saccharomyces cerevisiae mannitol
     dehydrogenase gene. J Bacteriol 1996;178:5257-62.
 99. Jacobson ES, Jenkins ND, Todd JM. Relationship between superoxide
     dismutase and melanin in a pathogenic fungus. Infect Immun
     1994;62:4085-6.
100. Muller HE, Sethi KK. Proteolytic activity of Cryptococcus neoformans
     against human plasma proteins. Med Microbiol Immunol (Berl)
     1972;158:129-34.
101. Brueske CH. Proteolytic activity of a clinical isolate of Cryptococcus
     neoformans. J Clin Microbiol 1986;23:631-3.
102. Salkowski CA, Balish E. Cutaneous cryptococcosis in athymic and
     beige-athymic mice. Infect Immun 1991;59:1785-9.
103. Chen SCA, Muller M, Zhou JZ, Wright LC, Sorrell TC. Phospholipase
     activity in Cryptococcus neoformans: a new virulence factor? J Infect
     Dis 1997;175:414-20.
104. Chen SCA, Wright LC, Santangelo RT, Muller M, Moran VR, Kuchel PW,
     Sorrell TC. Identification of extracellular phospholipase B,
     lysophospholipase, and acyltransferase produced by Cryptococcus
     neoformans. Infect Immun 1997;65:405-11.
105. Alspaugh JA, Perfect JR, Heitman J. Cryptococcus neoformans mating and
     virulence are regulated by the G-protein [Alpha] subunit GPA1 and
     cAMP. Genes Dev 1997;11:3206-17.

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