Cryptococcus neoformans II. Phagocytosis by Human Leukocytes

Twenty-four per cent of the leukocytes from healthy human subjects phagocytized an encapsulated strain of Cryptococcus neoformans. Phagocytosis was approximately three times more effective with nonencapsulated mutants of C. neoformans. When the mutants reverted to the encapsulated state, the percentages of phagocytosis decreased. These data indicate that cryptococcal polysaccharide inhibits the phagocytosis of C. neoformans by human leukocytes.     

Susceptibility of congenitally immunodeficient mice to a nonencapsulated strain of Cryptococcus neoformans.

The susceptibility of congenitally immunodeficient mice to a nonencapsulated strain of Cryptococcus neoformans (strain M7) was evaluated. Gnotobiotic mice with defined congenital defects in innate immunity (beige) or cell-mediated immunity (athymic) or with combined defects in innate and cellular immunity (beige athymic) were i.v. challenged with C. neoformans M7. The nonencapsulated strain of C. neoformans produced a persistent low-grade infection in the brains of all immunodeficient and immunocompetent mice used in this study. Immunocompetent mice (nu/+;bg/+) and immunodeficient bg/bg mice readily cleared nonencapsulated cryptococci from their kidneys, liver, lungs, and spleen. In contrast to nu/+ mice, nu/nu mice had a reduced capacity to clear nonencapsulated cryptococci from their kidneys and liver after i.v. challenge. Both bg/bg-nu/nu and bg/bg-nu/+ mice developed a low-grade infection in their kidneys, liver, lungs, and spleen, which was maintained throughout the 21-day study. Persistent infections were not due to reversion to an encapsulated state. These data indicate that a capsule may not always be necessary for C. neoformans to survive, in vivo, in tissues of immunodeficient and immunocompetent mice.     

Role of serum factors in the phagocytosis of weakly or heavily encapsulated Cryptococcus neoformans strains by guinea pig peripheral blood leukocytes

We investigated the opsonic activity of the serum factors affecting phagocytosis of Cryptococcus neoformans in vitro to elucidate the role of humoral factors in the host defense mechanisms against cryptococcosis. Two strains of C. neoformans, one heavily and one weakly encapsulated, were used. Guinea pig peripheral blood leukocytes (PBLs) were used for phagocytosis. The viable weakly encapsulated cells were ingested effectively by PBLs, in the presence of guinea pig normal fresh serum, while the heavily encapsulated cells were not ingested. Neither immune serum, its IgG fraction alone, nor heated serum promoted the phagocytosis of either the weakly or heavily encapsulated strain. On the other hand, immune serum promoted adherence of PBLs to viable cells of the heavily encapsulated strain, forming rosettes in the presence of fresh serum. A substantial amount of C3b component was detected on yeast cells when weakly encapsulated cells were incubated with human fresh serum, or heavily encapsulated cells were incubated with rabbit immune serum together with human fresh serum. Serum chelation experiments also indicated that the factors involved in the alternative complement pathway are opsonins for the weakly encapsulated strain. These results suggest that the alternative pathway plays an important normal opsonic role for weakly encapsulated strains and that specific antibody plays an immune opsonic role for heavily encapsulated strains of C. neoformans via the classical pathway of complement activation.     

Cryptococcus neoformans is resistant to surfactant protein A mediated host defense mechanisms

Initiation of a protective immune response to infection by the pathogenic fungus Cryptococcus neoformans is mediated in part by host factors that promote interactions between immune cells and C. neoformans yeast. Surfactant protein A (SP-A) contributes positively to pulmonary host defenses against a variety of bacteria, viruses, and fungi in part by promoting the recognition and phagocytosis of these pathogens by alveolar macrophages. In the present study we investigated the role of SP-A as a mediator of host defense against the pulmonary pathogen, C. neoformans. Previous studies have shown that SP-A binds to acapsular and minimally encapsulated strains of C. neoformans. Using in vitro binding assays we confirmed that SP-A does not directly bind to a fully encapsulated strain of C. neoformans (H99). However, we observed that when C. neoformans was incubated in bronchoalveolar fluid, SP-A binding was detected, suggesting that another alveolar host factor may enable SP-A binding. Indeed, we discovered that SP-A binds encapsulated C. neoformans via a previously unknown IgG dependent mechanism. The consequence of this interaction was the inhibition of IgG-mediated phagocytosis of C. neoformans by alveolar macrophages. Therefore, to assess the contribution of SP-A to the pulmonary host defenses we compared in vivo infections using SP-A null mice (SP-A-/-) and wild-type mice in an intranasal infection model. We found that the immune response assessed by cellular counts, TNFalpha cytokine production, and fungal burden in lungs and bronchoalveolar lavage fluids during early stages of infection were equivalent. Furthermore, the survival outcome of C. neoformans infection was equivalent in SP-A-/- and wild-type mice. Our results suggest that unlike a variety of bacteria, viruses, and other fungi, progression of disease with an inhalational challenge of C. neoformans does not appear to be negatively or positively affected by SP-A mediated mechanisms of pulmonary host defense.     

Fungicidal activity of IFN-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast-form fungus which causes pulmonary and meningeal infections preferentially in the immunocompromised host. It is thought that cell-mediated immunity is important for acquired resistance against cryptococcosis with activated macrophages as the final effector cells. However, specific polysaccharides in the capsule of C. neoformans protect the fungus from adherence to phagocytes and from subsequent phagocytosis. We have studied extracellular killing of C. neoformans by IFN-gamma-activated macrophages and their products. Murine bone marrow-derived macrophages stimulated with rIFN-gamma for 24 h were able to effectively suppress the growth of C. neoformans and the effect of IFN-gamma was augmented by LPS. Killing of C. neoformans was also achieved by cell-free supernatants from bone marrow-derived macrophages stimulated with IFN-gamma plus LPS. Our results indicate that killing of C. neoformans by activated macrophages is independent from toxic oxygen radicals and mediated by secreted protein(s) of apparent molecular mass of 15 and 30 kDa. These findings indicate that activated macrophages play a major role in host defense, although the fungus resists phagocytosis and remains in the extracellular milieu.     

Paradoxical role of capsule in murine bronchoalveolar macrophage-mediated killing of Cryptococcus neoformans

Infections with the encapsulated fungus Cryptococcus neoformans are usually acquired via inhalation, and the presence of a capsule has been identified as a virulence factor. Therefore, we studied murine bronchoalveolar macrophage (BAM)-mediated killing and phagocytosis of encapsulated and acapsular strains of C. neoformans. After 2 h, BAM killed encapsulated strains CN52 and MP415 more readily than acapsular strains CN602 and CAP67 (54.9 and 36.2% vs 26.1 and 6.7%, respectively, p less than 0.001). Pre-incubating CN602 with purified capsular polysaccharide increased killing to 42.7% (p = 0.04). Significantly greater killing of the encapsulated strains also occurred in vivo. BAM-mediated killing of CN52 appeared to proceed by non-oxidative mechanisms, as BAM released minimal amounts of H2O2 after stimulation with CN52, and killing was not reduced by inhibitors or scavengers of the respiratory burst. The association between encapsulation and susceptibility to BAM fungicidal effects was not attributable to differences in yeast ingestion. Using the same low ratio of organisms to BAM as in the killing assay, greater than 95% of both CN52 and CN602 were phagocytosed. However, BAM phagocytosed significantly greater numbers of acapsular CN602 when incubated with a higher inoculum. Phagocytosis and killing of CN52 and CN602 required fresh serum as a source of C. Phagocytosis of CN52, but not CN602, was profoundly inhibited if BAM were plated on surfaces coated with mAb against the C3bR (CR1). mAb against the iC3b receptor (CR3) did not affect phagocytosis of either strain. These data demonstrate the innate ability of BAM to preferentially kill, by apparently non-oxidative mechanisms, an encapsulated as opposed to acapsular organism. Inasmuch as different receptors appear involved in phagocytosis of encapsulated versus acapsular C. neoformans, the disparity in killing may result from the greater ability of receptors mediating uptake of encapsulated organisms to trigger the antimicrobial armamentarium of the BAM.     

Characterization of pathogenic constituents of Cryptococcus neoformans strains

We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 X 10(5) or 1 X 10(7) viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; a cell surface structure for responsible for resistance to intra- or extracellular killing and induction of the granulomatous lesion; a growth rate suitable for interacting with phagocytic elimination.     

Antibody action after phagocytosis promotes Cryptococcus neoformans and Cryptococcus gattii macrophage exocytosis with biofilm-like microcolony formation

Antibody-mediated phagocytosis was discovered over a century ago but little is known about antibody effects in phagolysosomes. We explored the consequences of antibody-mediated phagocytosis for two closely related human pathogenic fungal species, Cryptococcus neoformans and Cryptococcus gattii , of which C. neoformans encompasses two varieties: neoformans and grubii. The interaction between C. neoformans varieties grubii and neoformans and host cells has been extensively studied, but that of C. gattii and macrophages remains largely unexplored. Like C. neoformans , antibody-mediated phagocytosis of C. gattii cells was followed by intracellular replication, host cell cytoplasmic polysaccharide accumulation and phagosomal extrusion. Both C. gattii and C. neoformans cells exited macrophages in biofilm-like microcolonies where the yeast cells were aggregated in a polysaccharide matrix that contained bound antibody. In contrast, complement-opsonized C. neoformans variety grubii cells were released from macrophages dispersed as individual cells. Hence, both antibody- and complement-mediated phagocytosis resulted in intracellular replication but the mode of opsonization affected the outcome of exocytosis. The biofilm-like microcolony exit strategy of C. neoformans and C. gattii following antibody opsonization reduced fungal cell dispersion. This finding suggests that antibody agglutination effects persist in the phagosome to entangle nascent daughter cells and this phenomenon may contribute to antibody-mediated protection.     

Cryptococcus neoformans I. Nonencapsulated Mutants

Seven nonencapsulated mutants of Cryptococcus neoformans were isolated from an encapsulated strain of human origin. Initially, the mutants were avirulent for mice. After several months of subculturing, six of the seven isolates reverted to the encapsulated state and possessed varying degrees of virulence. The results of these experiments suggest that a strong correlation exists between the presence of a capsule and the virulence of C. neoformans.     

Culture of Cryptococcus neoformans in the nonencapsulated state

Forty-one strains of Cryptococcus neoformans were examined after 3 days growth on a fresh and aged medium at p_H 5 & p_H 7 for comparison of capsule formation. Over one-half of the strains did not form visible capsules on aged medium at p_H 5. Serotypes and source of isolation did not correlate with ability or inability to form capsules. Growth of C. neoformans in the nonencapsulated state makes it possible to culture many strains of C. neoformans in the form that more closely simulates the true infectious particles.     

Physicochemical surface properties of nonencapsulated and encapsulated coagulase-negative staphylococci.

Cell surfaces of three nonencapsulated and three encapsulated coagulase-negative staphylococci were characterized by their surface free energies, zeta potentials, and elemental and molecular compositions. Surface free energies were calculated from contact angle measurements with various liquids. All six strains showed a high surface free energy (103 to 126 mJ.m-2), estimated from the concept of polar and dispersion components. However, the hydrogen-donating surface free energy parameter was zero for all nonencapsulated strains. The zeta potential profile measured as a function of pH in phosphate-buffered saline for the nonencapsulated strains was completely different from that of the encapsulated strains. X-ray photoelectron spectroscopy was used to determine the elements (O, C, N, P, and K) in the outer 2 to 5 nm of the freeze-dried cell surface and showed that the hydrophilic character of the staphylococci was related to oxygen (O/C ratio, approximately 0.52)- and phosphorus (P/C ratio, approximately 0.03)-containing groups. Both the elemental and molecular characterizations (done by infrared spectroscopy) pointed to the presence of polysaccharides and polypeptides on the cell surface of the nonencapsulated and encapsulated strains.     

Physicochemical surface properties of nonencapsulated and encapsulated coagulase-negative staphylococci

Cell surfaces of three nonencapsulated and three encapsulated coagulase-negative staphylococci were characterized by their surface free energies, zeta potentials, and elemental and molecular compositions. Surface free energies were calculated from contact angle measurements with various liquids. All six strains showed a high surface free energy (103 to 126 mJ.m-2), estimated from the concept of polar and dispersion components. However, the hydrogen-donating surface free energy parameter was zero for all nonencapsulated strains. The zeta potential profile measured as a function of pH in phosphate-buffered saline for the nonencapsulated strains was completely different from that of the encapsulated strains. X-ray photoelectron spectroscopy was used to determine the elements (O, C, N, P, and K) in the outer 2 to 5 nm of the freeze-dried cell surface and showed that the hydrophilic character of the staphylococci was related to oxygen (O/C ratio, approximately 0.52)- and phosphorus (P/C ratio, approximately 0.03)-containing groups. Both the elemental and molecular characterizations (done by infrared spectroscopy) pointed to the presence of polysaccharides and polypeptides on the cell surface of the nonencapsulated and encapsulated strains.     

Quantitative analysis of phagocytosis of Cryptococcus neoformans by adherent phagocytic cells by fluorescence multi-well plate reader

Macrophages and monocytes are adherent phagocytic cells which play an important role in host defence against the yeast-like fungus Cryptococcus neoformans. Before, phagocytosis by adherent phagocytes could only be measured by means of microscopy or by a radioactive assay, which both have obvious disadvantages. We have developed a new, rapid and objective method to measure phagocytosis of C. neoformans by adherent phagocytes (e.g. alveolar macrophages) using a fluorescence multi-well plate reader. This method allows us to discriminate accurately between adherence and internalisation of C. neoformans by macrophages during long term incubation. In addition, the method was used to study the role of the mannose receptor in phagocytosis of the acapsular yeast in the absence of serum by human monocyte-derived macrophages (MDM).     

Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival

Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H(2)O(2) that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H(2)O(2). Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to beta-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells.     

Inhibition of human endothelial cell chemokine production by the opportunistic fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated fungal pathogen commonly acquired by inhalation. Extrapulmonary dissemination can lead to infection of the bloodstream and various organs, most commonly resulting in meningoencephalitis. However, infection with C. neoformans is often characterized by a scant inflammatory response. The leukocyte response to infection depends in part upon a gradient of chemotactic factors and adhesion molecules expressed by the host vascular endothelium, yet the inflammatory response of human endothelial cells (EC) to C. neoformans has not been previously investigated. We found that incubation of primary human EC with C. neoformans did not induce chemokine synthesis, and resulted in differential inhibition of cytokine-induced IL-8, IFN-gamma-inducible protein-10, and monocyte chemoattractant protein-1. In contrast, C. neoformans had little effect on EC surface expression of the leukocyte ligand, ICAM-1, as determined by flow cytometry. Modulation of chemokine production was dependent on the chemokine under study, the inoculum of C. neoformans used, fungal viability, and cell-cell contact, but independent of cryptococcal strain or encapsulation. These observations suggest a novel mechanism whereby C. neoformans can affect EC function and interfere with the host inflammatory response.     

Characterization of<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var.<Emphasis Type="Italic">neoformans</Emphasis> serotype A and A/D in samples from Egypt

The cryptococcal polysaccharide antigen was detected in 10 cerebrospinal fluid (CSF) and 23 serum samples from cryptococcal meningitis and intestinal cryptococcosis by the cryptococcal antigen latex agglutination system (CALAS). CALAS titers in CSF and serum samples of cryptococcal meningitis ranged over 8-2048 and 32-2048, respectively, while in cases of intestinal cryptococcosis, serum titers ranged over 8-2048. The isolates of yeast Cryptococcus neoformans were determined to be of serotype A or of the A/D pair. The total leukocyte count and biochemical parameters in CSF were significantly increased as indicators of microbial infection. Furthermore, the in vitro change of the teleomorph (sexual state) to the anamorph (asexual state) was also detected and the teleomorph state changed in vivo to the encapsulated anamoph state which is more virulent during infection in vivo than the yeast-like noncapsulated form. Two primers for internal transcribed spacer (ITS) regions of ribosomal DNA were used for molecular detection of C. neoformans. After PCR amplification, a DNA band of 415 bp, visualized on agarose gel, indicated the presence of C. neoformans cells in the tested CSF and serum samples. The primer sensitivity was also characterized using purified yeast chromosomal DNA as template; it was about 20 pg or more chromosomal DNA which represents about 10 cells of C. neoformans. The primers were also specific for ITS regions of C. neoformans and gave negative results with Candida albicans and E. coli chromosomal DNA templates.     

C1q enhances the phagocytosis of Cryptococcus neoformans blastospores by human monocytes

We investigated whether C1q, a subunit of the first component of C, could modulate human peripheral blood monocyte-mediated phagocytosis of Cryptococcus neoformans (CN). Adherence of monocytes to C1q-coated surfaces induced a significant enhancement of ingestion of CN blastospores that had been opsonized with specific anticapsular IgG (IgG-CN). Additionally, C1q enhanced the monocyte-mediated phagocytosis of CN opsonized with C (CN-absorbed, nonimmune, normal human serum; C-CN). Ingestion of IgG- and C-CN by control and C1q-stimulated monocytes was maximal by 1 h of incubation. The monocyte-mediated enhancement of phagocytosis caused by C1q was paralleled by a proportionate increase in fungicidal activity, an effect which was maximal by 3 h of incubation. Human serum albumin-adherent, control monocytes exhibited only a low level of killing after 3 h of incubation. C1q enhancement was blocked by preincubation of the surfaces with a goat, polyclonal F(ab')2 anti-C1q. This study describes a new cellular function for the cell surface C1q receptor: the enhancement of phagocytosis of a pathogenic organism by monocytes.     

Monoclonal antibodies can affect complement deposition on the capsule of the pathogenic fungus Cryptococcus neoformans by both classical pathway activation and steric hindrance

The capsule of the human pathogenic fungus Cryptococcus neoformans presents the immune system with a formidable problem for phagocytosis. Capsule-mediated activation of the alternative complement (C) pathway results in component 3 (particularly, C3) binding to the capsule near the cell wall surface. Hence, for cells with large capsule, C3 cannot interact with the complement receptor (CR) and is not opsonic. However, C activation in either immune serum or in the presence of monoclonal antibody (mAb) to capsular polysaccharide localizes C3 to the capsular edge. When C. neoformans cells were coated with both C and antibody (Ab) opsonins, Ab bound first and promoted C3 deposition at the edge of the capsule. The mechanism for the Ab-mediated change in C3 localization to the capsule edge involved both classical C pathway activation and steric hindrance preventing C3 penetration into the capsule. The change in C3 localization changed the mode of phagocytosis in macrophages, such that localizing C3 at the edge of the capsule allowed phagocytosis through C3-CR3 and C3-CR4 interactions, which did not occur in serum without Ab. These findings reveal a new mechanism of Ab action whereby Abs affect the location of C3 and its interaction with its receptor in macrophages depending on the immunoglobulin concentration.     

Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella spp.

Bacteriophages UAB_Phi20, UAB_Phi78, and UAB_Phi87 were encapsulated in liposomes, and their efficacy in reducing Salmonella in poultry was then studied. The encapsulated phages had a mean diameter of 309 to 326 nm and a positive charge between +31.6 and +35.1 mV (pH 6.1). In simulated gastric fluid (pH 2.8), the titer of nonencapsulated phages decreased by 5.7 to 7.8 log units, whereas encapsulated phages were significantly more stable, with losses of 3.7 to 5.4 log units. The liposome coating also improved the retention of bacteriophages in the chicken intestinal tract. When cocktails of the encapsulated and nonencapsulated phages were administered to broilers, after 72 h the encapsulated phages were detected in 38.1% of the animals, whereas the nonencapsulated phages were present in only 9.5%. The difference was significant. In addition, in an in vitro experiment, the cecal contents of broilers promoted the release of the phages from the liposomes. In broilers experimentally infected with Salmonella, the daily administration of the two cocktails for 6 days postinfection conferred similar levels of protection against Salmonella colonization. However, once treatment was stopped, protection by the nonencapsulated phages disappeared, whereas that provided by the encapsulated phages persisted for at least 1 week, showing the enhanced efficacy of the encapsulated phages in protecting poultry against Salmonella over time. The methodology described here allows the liposome encapsulation of phages of different morphologies. The preparations can be stored for at least 3 months at 4°C and could be added to the drinking water and feed of animals.