FREE ESSAY ON C NEOFORMANS

C NEOFORMANS

BackgroundThe organism C neoformans is an encapsulated yeast; its environmental niche has
not been completely defined, although outbreaks of disease have been associated in
particular with pigeon roosts and other large contaminated sites. There are two varieties
of C neoformans, distinguished by antigenic differences in the outer capsule of the
organism: serotypes A and D (C neoformans var neoformans, the most common strain) and
serotypes B and C (C neoformans var gatti). Cryptococcus neoformans var neoformans is the
principal pathogen in patients with AIDS. Cryptococcus neoformans var gatti, which is
found predominantly in Australia, Asia, and Southern California, has only rarely been
implicated to cause disease in this population. 
Antigenic specificity of the capsular polysaccharides define the four different serotypes
of C. neoformans; A, B, C and D. C. neoformans variety neoformans includes serotypes A
and D while C. neoformans var. gattii are associated with serotypes B and C. Worldwide,
most Cryptococcal infections in immunocompromised patients, including HIV infected
patients, are due to C. neoformans var. neoformans, serotype A. In a smaller number of
immunocompromised patients, infecton is caused by C. neoformans var. neoformans, Serotype
D. For unknown reasons, HIV infected patients and other immunosuppressed patients are
rarely infected with the variety gattii. Patients infected with C. neoformans var. gattii
are usually immunocompetent, respond slowly to treatment, and are at risk for developing
intracerebral mass lesions (cryptococcomas). 
Cryptococcus neoformans reproduces by budding and forms round yeast-like cells 3-6
micrometers in diameter. Within the host and in certain culture media, a large
polysaccharide capsule surrounds each cell. Cryptococcus neoformans forms smooth, convex,
yellow or tan colonies on solid media at 20 to 37 degrees Celsius. The identification of
this fungus is based on its microscopic appearance, biochemical tests, and its ability to
grow at 37 degrees Celsius. Most nonpathogenic strains of Cryptococcus do not grow at 37
degree Celsius. In addition, C. neoformans does not assimilate lactose and nitrates or
produce pseudomycelia on cornmeal or rice-Tween agar. Most strains of C. neoformans can
use creatinine as a nitrogen source, which may partially explain the growth of the
organism in creatinine rich avian feces. Another useful biochemical characteristic of C.
neoformans that distinguishes it from non-pathogenic strains is its ability to produce
melanin. The fungal enzyme phenol oxidase acts on certain substrates like
dihydroxyphenylalanine and caffeic acid to produce melanin.
Cryptococcus neoformans is an encapsulated yeast that was first described in an 1894
paper presented to the Greifswald Medical Society by Busse, a pathologist. Busse isolated
the yeast from the tibia of a 31 year old woman, noted its resistance to sodium
hydroxide, and published the case that same year. The following year Buschke, a surgeon,
reported the same isolate from the same patient. Thus, the early eponym of
Busse-Buschke's disease. This one case served not only to identify a new yeast, but also
to prove its pathogenic potential. 
The exact pathogenesis of infection is still unclear. Pulmonary involvement is a common
early event, and the organism is consequently assumed to gain access to the host via the
respiratory route. In the absence of normal T-cell function, it then disseminates widely
throughout the body, but especially to the central nervous system. Thus, most AIDS
patients infected with C neoformans develop meningitis, although acute cryptococcal
infection of almost every other organ has been described. Since the initial reports, the
diverse spectrum of host responses to cryptococcal infection has become apparent. The
spectrum ranges from harmless colonization of the airways and asymptomatic infection of
laboratory workers resulting in a positive skin test to meningitis or disseminated
disease. Although virulence for animals and possibly humans varies among strains of
cryptococci, virulence probably plays a relatively small part in determining the outcome
of an infection. The crucial factor appears to be the immune status of the host. The most
serious infections usually occur in individuals with defective cell mediated immunity,
such as the Acquired Immunodeficiency Syndrome (AIDS), organ transplantation,
reticuloendothelial malignancy, corticosteroid treatment (but not neutropenia or
immunoglobulin deficiency) and sarcoidosis. With the global emergence of AIDS, the
incidence of cryptococcosis is increasing and now represents a major life threatening
fungal infection in these patients. In the United States, 7% to 15% of patients with AIDS
develop cryptococcal infectious. However, in some parts of sub-Saharan Africa coinfection
with HIV and Cryptococcus neoformans approaches 90%. 
Pathophysiology: Of the 19 species which comprise the genus Cryptococcus, only
Cryptococcus neoformans is associated with human disease. Much of our understanding of
the pathogenesis and the host defense mechanisms involved in C. neoformans infections
comes from the studies of animal models. The organism is primarily transmitted by the
respiratory route and not directly from one human to another. After being inhaled, C.
neoformans is ingested by the alveolar macrophages. Unencapsulated yeast cells are
readily phagocytosed and destroyed, whereas encapsulated organisms are more resistant to
phagocytosis. Cryptococcal polysaccharide capsule has anti-phagocytic properties and may
be immunosuppressive. The anti-phagocytic properties of the capsule block recognition of
the yeast by phagocytes and inhibit leukocyte migration into the area of fungal
replication. 
The host's response to Cryptococcal infection includes both cellular and humoral
components. Animal models have demonstrated that natural killer cells participate inthe
early killing of cryptococci and that antibody dependent cell mediated killing may be
operative as well. In vitro, monocyte derived macrophages, natural killer cells, and T
lymphocytes can inhibit or kill cryptococci. A successful host response includes an
increase in helper T cell activity, skin test conversion, and a reduction in the number
of viable organisms in the tissues. In addition to cellular mechanisms, anticryptococcal
antibodies and soluble anticryptococcal factors have been described. Antibodies to
cryptococcal antigen and complement play a critical role in enhancing the macrophage and
lymphocyte mediated immune response to the organism. Monoclonal antibodies to capsular
polysaccharide have been used to passively immunize mice against C. neoformans. 
C. neoformans can cause an asymptomatic pulmonary infection followed later by the
development of meningitis, often the first indication of the disease. If limited to the
lungs, C. neoformans may cause pneumonia, poorly defined mass lesions, pulmonary nodules,
and rarely pleural effusion. Although immune defects are common in patients with
meningitis or disseminated infection, patients with disease confined to the lungs are
usually immunocompetent. 
Purpose:
Previously thought that the capsule promotes virulence by evading phagocytosis, based on
the observation that phagocytic cells rarely ingest encapsulated cells without opsonins
in vitro and on pathological studies that frequently show extracellular yeast cells in
tissue. C. neoformans was suspected of being capable of intracellular pathogenesis in
vivo, but according to the journal and any research I found, had never been demonstrated.
This study attemted to answer the question of whether C. neoformans was a facultative
intracellular pathogen by studying pulmonary infection in mice using light microscopy and
electron microscopy. The fungus was studied over the course of the infection, and both
capsular and acapsular stains were compared.
Methods and Materials:
Mice were infected intratrachealy with serotype D strain of C. neoformans variety
neoformans which is capsulated, as well as with an acapsulated strain. The mice were then
killed after various time intervals and the lung tissues examined using electron
microscopy as well as light microscopy. This gave an accurate localization of yeast
cells, and allowed visualization of cellular components.
Results:
The study showed that the capsule did not prevent phagocytosis in vivo, since cells
survived and replicated inside macrophages, despite phagolysosomal fusion.
macrophage toxicity
Initial course of pulmonary infection in mice involves rapid phagocytosis of C.
neoformans cells, macrophage toxicity, neutrophil influx, and cellular disruption. C.
neoformans intracellularly was associated with cytotxicity to host phagocytic cells. At 2
hours after infection, macrophages with intracellular yeast appeared intact, Many
phagocytotic vacuoles with intracellular cells had discontinuous membranes by 4 hours,
and by 8 hours after infection, the cell membrane of some macrophages with intracellular
yeast was disrupted, a result consistent with cell destruction as a consequence of
cytotoxic qualities of the yeast. 
In areas of the lung in which large numbers of macrophages were present, there appeared
to be two types of cells- macrophages with large numbers of yeast cells in which the
macrophage appeared to have an increase of vacuoles in their cytoplasm. In these cells
the extensive vacuolation resulted in cytoplasmic disruption of the macrophage. 
The other type of cell present contained single yeast cells but where comparatively
intact.
No funal cell components responsibele for macrophage cytotoxicity were characterized. 
Volume and Phagolysosomal Fusion:
Phagolysosomal fusion was observed in alveolar macrophages 2 hours after infection. Acid
phospphatase acitivity at the periphery of some phagosomes demonstrated phagosysosomal
fusion.
The average phagosome volume increased with the time of infection, reflecting an increase
in the average capsule size of C. neoformans cells in the lungs.
Yeast cells were found in phagosomes, lysosomes fuse with the pahgosome, and phagosomal
acidification appeared to enhance the growth of C. neoformans. This provides a growth
advantage to yeast cells in lung tissue.. The capsule separates the phagolysosomal
membrane from the yeast cell wall, and this separation could limit the fungicidal effects
of lysosomal products. 
Intracellular replication : During the 1st 24 hours, most phagosomes contained 1
organism, after this period, the # of C. neoformans cells per phagosome and the # of
phagosomes per macrophage increased (Refer to Figure).
The location of C. neoformans cells in the llung is a function of the time of infection.
They are found primarily inside alveolar macrophages shortly after infection, but by 24
hours, the majority of cells were in the extracellular space. This is consistent with the
intracellular replication followed by phagocytic cell lysis and release of live yeast
into the extracellular space. The possibility of intracellular growth followed by cell
lysis is supported by the present observation of cell debris in close proximity to yeast
cells and also to reports that C. neoformans can replicate and lyse macrophage lineage
cells in vitro. The transition from intracellular predominance to extracellular
predominance was associeated with macrophage cytotoxicity and disruption, as indicated by
1)low cytoplasmic electron density and membrane disruption and 2)the appearance of cell
debris in the alveolar space in close proximity to extracelllular yeast cells.
Capsular Vs. Acapsular:
With the acapsular strain, almost all cells were found intracellularly in macrophages 24
hours after infection. Macrophage cytoplasm and nuclei appeared normal. After 14 days,
lungs of mice appeared normal and no organisms were seen, indicating a clearance of
pulmonary infection. 
In contrast, the capsular strain, the macrophages contained multile phagosomes with more
than one yeast cell, and the macrophage cytoplasm contained vacuoles. There appeared to
be intracellular polysaccharide synthesis, and the capsule may contribulte to
intracellular survival by providing a buffer space between the site of lysosome-phagosome
fusion and the fungal cell wall. At 24 hours after infection, the distance between the
phagosome memebrane and the cell wall for phagocytosed encapsulated strains was 8.6 X
that for the nonencapsulated strain.
In vitro studies were also done to confirm that encapsulated, but not acapsular, yeast
cells replicated intracellularly and were toxic to phagocytic cells. With increased
incubation times, the number of yeast cells per phagocytic cell increased for the
encapsulated, but not the acapsular strain.
Conclusion:
intracellular localization has been associated with both control and persistence of
infection
The results demonstrated that intracellular residence was associated with multiple yeast
cells per phagosome, a higher budding index, and the emergence of a yeast cellpopulation
that was heterogeneous in size. This is indicative of intracellular replication in vivo.