Laccase expression in murine pulmonary Cryptococcus neoformans infection

Cryptococcus neoformans laccase expression during murine infection was investigated in lung tissue by immunohistochemistry and immunogold electron microscopy. Laccase was detected in the fungal cell cytoplasm, cell wall, and capsule in vivo. The amount of laccase found in different sites varied as a function of the time of infection.     

Intracellular crystal formation as a mechanism of cytotoxicity in murine pulmonary Cryptococcus neoformans infection

Rod-like crystalline structures formed during eosinophilic Cryptococcus neoformans pneumonia in C57BL/6 mice. Crystals were found associated with yeast cells and free in host cell cytoplasm. The crystals apparently formed because of the interaction of a host protein with the cryptococcal polysaccharide. Crystal formation likely contributes to pathogenesis by causing cellular damage.     

Cryptococcus neoformans capsule structure evolution in vitro and during murine infection

Cryptococcus neoformans capsule structure modifications after prolonged in vitro growth or in vivo passaging have been reported previously. However, nothing is known about the dynamics of these modifications or about their environmental specificities. In this study, capsule structure modifications after mouse passaging and prolonged in vitro culturing were analyzed by flow cytometry using the glucuronoxylomannan-specific monoclonal antibody E1. The capsule structures of strains recovered after 0, 1, 8, and 35 days were compared by using the level of E1-specific epitope expression and its cell-to-cell heterogeneity within a given cell population. In vitro, according to these parameters, the diversity of the strains was higher on day 35 than it was initially, suggesting the absence of selection during in vitro culturing. In contrast, the diversity of the strains recovered from the brain tended to decrease over time, suggesting that selection of more adapted strains had occurred. The strains recovered on day 35 from the spleen and the lungs had different phenotypes than the strains isolated from the brain of the same mouse on the same day, thus strongly suggesting that there is organ specificity for C. neoformans strain selection. Fingerprinting of the strains recovered in vitro and in vivo over time confirmed that genotypes evolved very differently in vitro and in vivo, depending on the environment. Overall, our results suggest that organ-specific selection can occur during cryptococcosis.     

Determination of the pH of the Cryptococcus neoformans vacuole.

We have previously demonstrated the antifungal activity of the weak bases chloroquine and quinacrine against Cryptococcus neoformans. Quinacrine, being fluorescent, was seen to be concentrated within a complex vacuolar structure within the cryptococcal cell. Here we determined the pH of this compartment using the pH-sensitive fluorescent dye, 5-(and 6-) carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA). Carboxy-DCFDA was concentrated within the cryptococcal vacuole, giving a pattern of fluorescence similar to that previously observed with quinacrine. For each experiment, a standard curve of fluorescence ratio against pH was generated using buffers of defined pH containing a mixture of ionophores and inhibitors to equilibrate vacuolar pH with that of the medium. The pH of the cryptococcal vacuole of five strains was calculated to range from 5.3 to 5.9 with a mean of 5.6. This acidic pH is consistent with a model in which weak bases such as chloroquine and quinacrine are accumulated, by ion trapping within the fungal vacuole. Antifungal activity may result from the consequent disruption of pH-dependent processes as well as effects on other as yet undefined fungal targets.     

Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection

To produce chronic infection, microbial pathogens must escape host immune defenses. Infection with the human pathogenic fungus Cryptococcus neoformans is typically chronic. To understand the mechanism by which C. neoformans survives in tissue after the infection of immunocompetent hosts, we systematically studied the course of pulmonary infection in mice by electron microscopy. The macrophage was the primary phagocytic cell at all times of infection, but neutrophils also ingested yeast. Alveolar macrophages rapidly internalized yeast cells after intratracheal infection, and intracellular yeast cells were noted at all times of infection from 2 h through 28 days. However, the proportion of yeast cells in the intracellular and extracellular spaces varied with the time of infection. Early in infection, yeast cells were found predominantly in the intracellular compartment. A shift toward extracellular predominance occurred by 24 h that was accompanied by macrophage cytotoxicity and disruption. Later in infection, intracellular persistence in vivo was associated with replication, residence in a membrane-bound phagosome, polysaccharide accumulation inside cells, and cytotoxicity to macrophages, despite phagolysosomal fusion. Many phagocytic vacuoles with intracellular yeast had discontinuous membranes. Macrophage infection resulted in cells with a distinctive appearance characterized by large numbers of vacuoles filled with polysaccharide antigen. Similar results were observed in vitro using a macrophage-like cell line. Our results show that C. neoformans is a facultative intracellular pathogen in vivo. Furthermore, our observations suggest that C. neoformans occupies a unique niche among the intracellular pathogens whereby survival in phagocytic cells is accompanied by intracellular polysaccharide production.     

Role of the mannose receptor in a murine model of Cryptococcus neoformans infection

Cryptococcus neoformans is an encapsulated fungal pathogen with a predilection to infect persons with suppressed T-cell function. Cryptococcal mannoproteins (MP) are highly mannosylated antigens which elicit T-cell responses in infected mice and in convalescent patients. Key to the immunogenicity of MP is its capacity to bind to the conserved mannose receptor (MR), CD206, on dendritic cells (DCs). To test the role of the MR in the immune response to C. neoformans, wild-type and MR knockout (MR KO) mice were compared by using in vivo and ex vivo models of cryptococcosis. Following a pulmonary challenge with C. neoformans, MR KO mice died significantly faster than wild-type mice and had higher lung fungal burdens after 4 weeks of infection. Uptake of MP was similar when DCs obtained from wild-type and MR KO mice were compared. Additionally, MP did not upregulate the maturation markers major histocompatibility complex class II, CD86, and CD40 in either wild-type or MR KO DCs. However, MP stimulated lymphoproliferation in CD4(+) T cells obtained from the peripheral lymph nodes of infected wild-type but not MR KO mice. These studies demonstrate a nonredundant role for the MR in the development of CD4(+) T-cell responses to MP and protection from C. neoformans.     

Phagocytosis of Cryptococcus neoformans by alveolar macrophages.

Guinea pig pulmonary macrophages phagocytized but did not kill nonencapsulated cells of Cryptococcus neoformans. The phagocytic process was inhibited by cryptococcal capsular polysaccharide. Pulmonary macrophages, activated by preinjecting heat-killed bacteria into intact animals, did not kill the engulfed yeast cells. Labeled cells of C. neoformans were neither killed nor cleared from guinea pig lungs 6 h postexposure. The results of our experiments indicate that during the first few hours after the lung is exposed to the infectious particle of C. neoformans the pulmonary macrophage does not function primarily to kill engulfed yeast cells. We believe that a rapid yet transient acute inflammatory response probably plays a major role in this process during the first few hours after C. neoformans enters the lung.     

The pathology of human and murine pulmonary infection with Cryptococcus neoformans var. gattii

Human infection by Cryptococcus neoformans var. neoformans is well characterised and usually occurs in immunocompromised patients. Less is known about infection by Cryptococcus neoformans var. gattii, which usually produces disease in previously normal individuals. In two cases of human pulmonary infection by Cryptococcus neoformans var. gattii, we observed a mixed inflammatory pattern, including granulomas associated with numerous T lymphocytes and a lymphocytic interstitial pneumonitis with B lymphocytes and formation of follicles. We also established a murine model of pulmonary infection by Cryptococcus neoformans var. gattii, which reproduced most of these features. This model is likely to prove useful in studies of the pathogenesis of this infection.     

Surfactant protein D facilitates Cryptococcus neoformans infection

Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Furthermore, Cryptococcus neoformans has become a primary human pathogen, causing infection in seemingly healthy individuals. Although numerous studies have elucidated the virulence properties of C. neoformans, less is understood regarding lung host immune factors during early stages of fungal infection. Based on our previous studies documenting that pulmonary surfactant protein D (SP-D) protects C. neoformans cells against macrophage-mediated defense mechanisms in vitro (S. Geunes-Boyer et al., Infect. Immun. 77:2783–2794, 2009), we postulated that SP-D would facilitate fungal infection in vivo. To test this hypothesis, we examined the role of SP-D in response to C. neoformans using SP-D^−/− mice. Here, we demonstrate that mice lacking SP-D were partially protected during C. neoformans infection; they displayed a longer mean time to death and decreased fungal burden at several time points postinfection than wild-type mice. This effect was reversed by the administration of exogenous SP-D. Furthermore, we show that SP-D bound to the surface of the yeast cells and protected the pathogenic microbes against macrophage-mediated defense mechanisms and hydrogen peroxide (H₂O₂)-induced oxidative stress in vitro and in vivo. These findings indicate that C. neoformans is capable of coopting host SP-D to increase host susceptibility to the yeast. This study establishes a new paradigm for the role played by SP-D during host responses to C. neoformans and consequently imparts insight into potential future preventive and/or treatment strategies for cryptococcosis.     

Karyotype instability in Cryptococcus neoformans infection.

The electrophoretic karyotypes of 32 clinical and 3 environmental Cryptococcus neoformans isolates from New York City were studied by contour clamped homogeneous electrophoresis. There was extensive variation among the electrophoretic karyotypes of isolates from different patients. Sequential C. neoformans isolates from patients with chronic or relapsing infection had very similar karyotypes. However, minor differences in electrophoretic karyotypes were detected among sequential isolates from 50% of the patients studied, suggesting the occurrence of chromosomal rearrangements or deletions in vivo. This hypothesis was tested by infecting mice, recovering isolates from mouse organs, and comparing the electrophoretic karyotypes before and after passage. Three clinical and three environmental strains were studied before and after passage in mice. Karyotype differences were detected after mouse passage for one clinical and two environmental strains. Our results indicate (i) extensive karyotype variation among isolates from a small geographic regions, (ii) a high frequency of electrophoretic karyotype differences among sequential isolates from individual patients, and (iii) the occurrence of electrophoretic karyotype changes during experimental infection of mice. The implications of these observations are discussed.     

Binding and internalization of glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans, by murine peritoneal macrophages

Glucuronoxylomannan (GXM), the major component of the capsular polysaccharide of Cryptococcus neoformans, is essential to virulence of the yeast. Previous studies found that the interaction between GXM and phagocytic cells has biological consequences that may contribute to the pathogenesis of cryptococcosis. We found that GXM binds to and is taken up by murine peritoneal macrophages. Uptake is dose and time dependent. Examination of the sites of GXM accumulation by immunofluorescence microscopy showed that the pattern was discontinuous and punctate both on the surfaces of macrophages and at intracellular depots. Although resident macrophages showed appreciable accumulation of GXM, uptake was greatest with thioglycolate-elicited macrophages. A modest stimulation of GXM binding followed treatment of resident macrophages with phorbol 12-myristate 13-acetate, but treatment with lipopolysaccharide or gamma interferon alone or in combination had no effect. Accumulation of GXM was critically dependent on cytoskeleton function; a near complete blockade of accumulation followed treatment with inhibitors of actin. GXM accumulation by elicited macrophages was blocked by treatment with inhibitors of tyrosine kinase, protein kinase C, and phospholipase C, but not by inhibitors of phosphatidylinositol 3-kinase, suggesting a critical role for one or more signaling pathways in the macrophage response to GXM. Taken together, the results demonstrate that it is possible to experimentally enhance or suppress binding of GXM to macrophages, raising the possibility for regulation of the interaction between this essential virulence factor and binding sites on cells that are central to host resistance.     

Analysis of cell cycle and replication of mouse macrophages after in vivo and in vitro Cryptococcus neoformans infection using laser scanning cytometry

We investigated the outcome of the interaction of Cryptococcus neoformans with murine macrophages using laser scanning cytometry (LSC). Previous results in our lab had shown that phagocytosis of C. neoformans promoted cell cycle progression. LSC allowed us to simultaneously measure the phagocytic index, macrophage DNA content, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation such that it was possible to study host cell division as a function of phagocytosis. LSC proved to be a robust, reliable, and high-throughput method for quantifying phagocytosis. Phagocytosis of C. neoformans promoted cell cycle progression, but infected macrophages were significantly less likely to complete mitosis. Hence, we report a new cytotoxic effect associated with intracellular C. neoformans residence that manifested itself in impaired cell cycle completion as a consequence of a block in the G(2)/M stage of the mitotic cell cycle. Cell cycle arrest was not due to increased cell membrane permeability or DNA damage. We investigated alveolar macrophage replication in vivo and demonstrated that these cells are capable of low levels of cell division in the presence or absence of C. neoformans infection. In summary, we simultaneously studied phagocytosis, the cell cycle state of the host cell and pathogen-mediated cytotoxicity, and our results demonstrate a new cytotoxic effect of C. neoformans infection on murine macrophages: fungus-induced cell cycle arrest. Finally, we provide evidence for alveolar macrophage proliferation in vivo.     

Disseminated infection caused by urease-negative Cryptococcus neoformans.

We report a case of fungemia and disseminated disease caused by a urease-negative strain of Cryptococcus neoformans in a patient with the acquired immune deficiency syndrome. Except for failure to hydrolyze urea, the microbiological characteristics of the isolate were typical of C. neoformans. Laboratory specialists should be aware of the occurrence of atypical strains of C. neoformans, particularly those recovered from patients with the acquired immune deficiency syndrome.     

An ultrastructural examination of the interaction between macrophages and Cryptococcus neoformans.

The interaction of mononuclear phagocytes with Cryptococcus neoformans was examined in vitro and in vivo using ultrastructural techniques. Immune serum roughens the surface of the yeast and in the first 2 hr, increases the number of organisms attaching to the macrophage surface, as well as the number of contacts between individual yeasts and the phagocyte. Contact is established by means of thin filopodia and cytoplasmic flaps. During the next few days the macrophages increase in size, and, by intimate apposition of their contiguous cell surfaces, a cellular barrier surrounds the now enclosed yeast. These events are accompanied by thinning of fungal capsule, degradation of the enclosed cryptococcus, and the formation of macrophage polykaryons. Electron cytochemical techniques for peroxidase reveal that these multinucleated cells are formed predominantly by the fusion of stimulated macrophages. Destruction of the enclosed yeast probably results from the secretion of various agents by the surrounding cells.     

Antibody-mediated protection in murine Cryptococcus neoformans infection is associated with pleotrophic effects on cytokine and leukocyte responses

Cryptococcus neoformans, an encapsulated yeast, is a common cause of life-threatening meningoencephalitis in immunosuppressed patients. We previously observed that administration of a monoclonal antibody (MAb) to the capsular polysaccharide to mice with pulmonary infection prolonged survival and enhanced granulomatous inflammation without reducing lung CFU. To understand the mechanism of MAb action, we studied leukocyte recruitment and cytokine profiles in lungs of A/JCr mice. B lymphocytes were the predominant cell type in lung infiltrates, comprising 15 to 30% of the leukocytes. Despite alterations in histological appearance, fluorescence-activated cell sorter analysis revealed no significant difference in total numbers of lung leukocytes in MAb-treated mice and controls. Differences in the immune response to C. neoformans between MAb-treated mice and controls included (i) an increase in the percentage of granulocytes among lung leukocytes on day 14, (ii) higher macrophage surface expression of CD86 on day 28, (iii) larger amounts of IL-10 in lung homogenates at day 7, (iv) a trend toward smaller amounts of gamma interferon mRNA and protein on day 7, and (v) a smaller increase in the levels of interleukin-4 mRNA and protein on day 7. Hence, the immune responses to C. neoformans infection in the presence and absence of specific antibody were qualitatively similar, and antibody administration was associated with several subtle quantitative differences in immune response parameters that could translate into enhanced survival. MAb may function partly by down-regulating the inflammatory response and reducing host damage. Our findings demonstrate unexpected complexity in the interaction between specific MAb and other components of the host immune response.     

Hinge influences in murine IgG binding to Cryptococcus neoformans capsule

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One example is the 3E5 monoclonal murine IgG family, in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes despite their identical variable sequences. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1; these binding patterns have been associated with protective efficacy in murine cryptococcosis. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with an mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in antigen binding could affect antibody biology and engineering.     

Laccase protects Cryptococcus neoformans from antifungal activity of alveolar macrophages.

While laccase of Cryptococcus neoformans is implicated in the virulence of the organism, our recent studies showing absence of melanin in the infected mouse brain has led us to a search for alternative roles for laccase in cryptococcosis. We investigated the role of laccase in protection of C. neoformans against murine alveolar macrophage (AM)-mediated antifungal activity by using a pair of congenic laccase-positive (2E-TUC) and laccase-deficient (2E-TU) strains. The laccase-positive cells with laccase derepression were more resistant to the antifungal activity of AM than a laccase-deficient strain ([28.9 +/- 1.2]% versus [40.2 +/- 2.6]% killing). Addition of L-dopa to Cryptococcus to produce melanin in a laccase-positive strain resulted in a slight increase in protection of C. neoformans from the antifungal activity of macrophages ([25.4 +/- 3.4]% versus [28.9 +/- 1.2]% killing). Recombinant cryptococcal laccase exhibited iron oxidase activity in converting Fe(II) to Fe(III). Moreover, recombinant laccase inhibited killing of C. neoformans by hydroxyl radicals catalyzed by iron in a cell-free system. Addition of the hydroxyl radical scavenger mannitol or dimethyl sulfoxide to AMs prior to the introduction of cryptococcal cells decreased killing of both strains and reduced the difference in susceptibility between the laccase-positive and laccase-deficient strains. Furthermore, laccase-mediated protection from AM killing was inhibited by the addition of Fe(II), presumably by overcoming the effects of the iron oxidase activity of cryptococcal laccase. These results suggest that the iron oxidase activity of laccase may protect C. neoformans from macrophages by oxidation of phagosomal iron to Fe(III) with a resultant decrease in hydroxyl radical formation.     

Complementation of a capsule deficient Cryptococcus neoformans with CAP64 restores virulence in a murine lung infection

Cryptococcosis is a systemic infection in humans caused by the opportunistic fungal pathogen, Cryptococcus neoformans. The infection usually presents as chronic meningoencephalitis, but infects via the respiratory tract. A polysaccharide capsule is a major virulence factor, which allows the yeast to resist host defenses. However, the essential role of the capsule in allowing it to resist host defenses during the initial lung infection has not been clearly shown. A mutant acapsular C. neoformans strain 602 was complemented with the CAP64 gene to obtain an encapsulated strain, TYCC38-602. TYCC38-602 persisted in the lungs of C.B-17 mice after intratracheal inoculation and disseminated to the brain, whereas the mutant acapsular 602 and the plasmid control transformant CIP3-602 strains grew less readily in the lung and were infrequently detected in the brain. T cell-mediated immunity, developed to the encapsulated organism, was required to control growth within the lungs and had a significant impact on numbers of yeasts detected in the brain. The parent acapsular strain, but not the transformant control, also required T cells for optimal inhibition of growth within the lung, but not for maintaining control of the colony-forming units (cfu) in the brain. In summary, the cryptococcal capsule plays an important role in lung virulence and dissemination to the brain, and intact immunity is required to control lung growth of the encapsulated yeast.