Search

Methamphetamine (METH) is a major public health and safety problem in the United States. Chronic METH abuse is associated with a 2-fold-higher risk of HIV infection and, possibly, additional infections, particularly those that enter through the respiratory tract or skin. Cryptococcus neoformans is an encapsulated opportunistic yeast-like fungus that is a relatively frequent cause of meningoencephalitis in immunocompromised patients, especially in individuals with AIDS. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. l-3,4-Dihydroxyphenylalanine (l-Dopa) is a natural catecholamine that is frequently used to induce melanization in C. neoformans. l-Dopa-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents, and heavy metals. Using a systemic mouse model of infection and in vitro assays to critically assess the impact of METH on C. neoformans melanization and pathogenesis, we demonstrated that METH-treated mice infected with melanized yeast cells showed increased fungal burdens in the blood and brain, exacerbating mortality. Interestingly, analyses of cultures of METH-exposed cryptococci supplemented with l-Dopa revealed that METH accelerates fungal melanization, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms.

Autophagy is used by eukaryotes in bulk cellular material recycling and in immunity to intracellular pathogens. We evaluated the role of macrophage autophagy in the response to Cryptococcus neoformans and Candida albicans , two important opportunistic fungal pathogens. The autophagosome marker LC3 (microtubule-associated protein 1 light chain 3 alpha) was present in most macrophage vacuoles containing C. albicans . In contrast, LC3 was found in only a few vacuoles containing C. neoformans previously opsonized with antibody but never after complement-mediated phagocytosis. Disruption of host autophagy in vitro by RNA interference against ATG5 (autophagy-related 5) decreased the phagocytosis of C. albicans and the fungistatic activity of J774.16 macrophage-like cells against both fungi, independent of the opsonin used. ATG5-knockout bone marrow-derived macrophages (BMMs) also had decreased fungistatic activity against C. neoformans when activated. In contrast, nonactivated ATG5-knockout BMMs actually restricted C. neoformans growth more efficiently, suggesting that macrophage autophagy plays different roles against C. neoformans , depending on the macrophage type and activation. Interference with autophagy in J774.16 cells also decreased nonlytic exocytosis of C. neoformans , increased interleukin-6 secretion, and decreased gamma interferon-induced protein 10 secretion. Mice with a conditionally knocked out ATG5 gene in myeloid cells showed increased susceptibility to intravenous C. albicans infection. In contrast, these mice manifested no increased susceptibility to C. neoformans , as measured by survival, but had fewer alternatively activated macrophages and less inflammation in the lungs after intratracheal infection than control mice. These results demonstrate the complex roles of macrophage autophagy in restricting intracellular parasitism by fungi and reveal connections with nonlytic exocytosis, humoral immunity, and cytokine signaling.

Unsaturated fatty acids (UFA) are essential components of cells. In Saccharomyces cerevisiae , stearoyl-coenzyme A (CoA) desaturase 1 ( OLE1 ) affects cell viability through the regulation of oleic (18:1) or palmitoleic (16:1) acid production. In this study, we used a targeted gene deletion approach to determine the impact of OLE1 on the emerging human pathogenic fungus Candida parapsilosis . We found that the deletion of OLE1 resulted in an auxotrophic yeast strain (designated OLE1 KO) that required unsaturated fatty acids for growth but not saturated fatty acids. Additionally, the production of UFA by OLE1 KO yeast cells was markedly reduced, suggesting that Ole1 is essential for UFA production. In contrast to wild-type C. parapsilosis , which produced pseudohyphal growth on UFA-supplemented medium agar, pseudohyphal formation in the OLE1 KO cells was severely impaired, suggesting that Ole1 regulates morphology. Furthermore, the OLE1 KO cells were hypersensitive to various stress-inducing factors, such as salts, SDS, and H 2 O 2 , especially at the physiological temperature. The results indicate that OLE1 is essential for the stress response, perhaps through the production of UFA for cell membrane biosynthesis. The OLE1 KO cells also were hypersensitive to human and fetal bovine serum, suggesting that targeting Ole1 could suppress the dissemination of yeast cells in the bloodstream. Murine-like macrophage J774.16 more efficiently killed the OLE1 KO yeasts, and significantly larger amounts of nitric oxide were detected in cocultures of macrophages and OLE1 KO cells than with wild-type or heterozygous strains. Moreover, the disruption of OLE1 significantly reduced fungal virulence in systemic murine infection. Taken together, these results demonstrate that Ole1 regulates the pathobiology of C. parapsilosis via UFA and that the OLE1 pathway is a promising antifungal target.

Heat shock proteins with molecular masses of ∼60 kDa (Hsp60) are widely distributed in nature and are highly conserved immunogenic molecules that can function as molecular chaperones and enhance cellular survival under physiological stress conditions. The fungusHistoplasma capsulatumdisplays an Hsp60 on its cell surface that is a key target of the cellular immune response during histoplasmosis, and immunization with this protein is protective. However, the role of humoral responses to Hsp60 has not been fully elucidated. We generated immunoglobulin G (IgG) isotype monoclonal antibodies (MAbs) toH. capsulatumHsp60. IgG1 and IgG2a MAbs significantly prolonged the survival of mice infected withH. capsulatum. An IgG2b MAb was not protective. The protective MAbs reduced intracellular fungal survival and increased phagolysosomal fusion of macrophages in vitro. Histological examination of infected mice showed that protective MAbs reduced the fungal burden and organ damage. Organs of infected animals treated with protective MAbs had significantly increased levels of interleukin-2 (IL-2), IL-12, and tumor necrosis factor alpha and decreased levels of IL-4 and IL-10. Hence, IgG1 and IgG2a MAbs to Hsp60 can modifyH. capsulatumpathogenesis in part by altering the intracellular fate of the fungus and inducing the production of Th1-associated cytokines.

Several dimorphic fungi are important human pathogens, but the origin and maintenance of virulence in these organisms is enigmatic, since an interaction with a mammalian host is not a requisite for fungal survival. Recently, Cryptococcus neoformans was shown to interact with macrophages, slime molds, and amoebae in a similar manner, suggesting that fungal pathogenic strategies may arise from environmental interactions with phagocytic microorganisms. In this study, we examined the interactions of three dimorphic fungi with the soil amoeba Acanthameobae castellanii . Yeast forms of Blastomyces dermatitidis , Sporothrix schenckii , and Histoplasma capsulatum were each ingested by amoebae and macrophages, and phagocytosis of yeast cells resulted in amoeba death and fungal growth. H. capsulatum conidia were also cytotoxic to amoebae. For each fungal species, exposure of yeast cells to amoebae resulted in an increase in hyphal cells. Exposure of an avirulent laboratory strain of H. capsulatum to A. castellanii selected for, or induced, a phenotype of H. capsulatum that caused a persistent murine lung infection. These results are consistent with the view that soil amoebae may contribute to the selection and maintenance of certain traits in pathogenic dimorphic fungi that confer on these microbes the capacity for virulence in mammals.

Melanin is made by several important pathogenic fungi and has been implicated in the pathogenesis of a number of fungal infections. This study investigated whether the thermally dimorphic fungal pathogenHistoplasma capsulatumvar. capsulatumproduced melanin or melanin-like compounds in vitro and during infection. Growth ofH. capsulatummycelia in chemically defined minimal medium produced pigmented conidia. Growth ofH. capsulatumyeast in chemically defined minimal medium withl-3,4-dihydroxyphenylalanine (DOPA) or (-)-epinephrine produced pigmented cells. Treatment of the pigmented cells with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were similar in size and shape to their respective propagules. Melanin-binding monoclonal antibodies (MAb) labeled pigmented conidia, yeast, and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that pigmented yeast cells and particles derived from pigmented cells were stable free radicals consistent with their identification as melanins. Tissues from mice infected withH. capsulatumand from biopsy specimens from a patient with histoplasmosis contained fungal cells that were labeled by melanin-binding MAb. Digestion of infected mouse tissues yielded dark particles that reacted with the melanin-binding MAb and were similar in appearance toH. capsulatumyeast cells. Additionally, sera from infected mice contained antibodies that bound melanin particles. Phenoloxidase activity capable of synthesizing melanin from L-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate thatH. capsulatumconidia and yeast can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role to play in the pathogenesis of histoplasmosis.

Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogenParacoccidioides brasiliensisproduce melanin or melanin-like compounds in vitro and during infection. Growth ofP. brasiliensismycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium withl-3,4-dihydroxyphenylalanine (l-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro whenP. brasiliensiswas grown inl-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis froml-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected withP. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology ofP. brasiliensisyeasts. These data strongly suggest thatP. brasiliensispropagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.

Cryptococcus neoformans has a laccase that catalyzes the synthesis of melanin in the presence of phenolic compounds (9, 17). Melanin synthesis by C. neoformans is associated with virulence (11, 14). Melanin can protect C. neoformans against antifungal compounds, oxidants, UV light, macrophages, and extremes in temperature (reviewed in reference 2). Since melanization confers reduced susceptibility to many insults, this trait may have originally been selected for environmental survival. Infections with C. neoformans are thought to occur by inhalation of aerosolized organisms from environmental sources, and the sizes of cryptococcal cells collected from pigeon excreta are compatible with aveolar deposition (10). Although pigeon excreta have not formally been considered to be a source of human infections, four independent DNA-typing studies have identified the same cryptococcal strains in collected pigeon excreta and clinical isolates (3–5, 18). Furthermore, there is considerable anecdotal evidence suggesting that humans were infected after exposure to avian excreta (reviewed in reference 2). In this study we present evidence that cryptococcal cells are melanized in pigeon excreta. (The data in this paper are from a thesis to be submitted by Angel Luis Rosas in partial fulfillment of the requirements for the degree of doctor of philosophy from the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y.) Samples of pigeon excreta were collected from areas adjacent to our institution. The presence of C. neoformans in the feces was initially investigated by a C. neoformans cell capture enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) to screen pigeon excreta slurries. The ELISA captures and immobilizes yeast cells and was performed as described previously (1). For IF, 50 μl of pigeon excreta supernatant was dried on poly-l-lysine-coated slides (Sigma Chemical Corp., St. Louis, Mo.). Blocking for nonspecific binding was done with 0.05% Tween 20 in 2% bovine serum albumin (BSA) for 1 h at 37°C. Fifty-microliter samples of the polysaccharide-binding immunoglobulin M (IgM) monoclonal antibody (MAb) 2D10 (1) or antimelanin IgM MAb 11B11 (13) (1:100 [vol/vol]) in 1% BSA were applied to the slides, which were incubated in a moisture chamber for 1 h at 37°C. After excess MAb was washed off, each slide was covered with 50 μl of a 1:100 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgM (Southern Biotech, Birmingham, Ala.) in 1% BSA for 1 h at room temperature. The slide was washed, a coverslip was placed over it with a mounting solution (50% glycerol, 50% phosphate-buffered saline [PBS]–0.1 M N-propyl gallate), and the slide was examined with an Olympus (Melville, N.Y.) AX70 microscope. Negative controls included slides with nonmelanized cells (ATCC 24067), NSO ascites fluid, or no primary MAb. IF with the MAb 2D10 and whole-cell capture ELISA demonstrated yeast cells that stained for cryptococcal polysaccharide in pigeon feces (Fig. ​(Fig.1A1A and B). IF with the melanin-binding MAb also stained yeast cells in the pigeon excreta supernatants (Fig. ​(Fig.1C1C and D). FIG. 1 Light and IF images of C. neoformans in pigeon excreta suspensions (magnification, ×1,000). (A and B) Images depict the binding of MAb 2D10 to a yeast cell. (C and D) Images depict the staining of the melanin-binding MAb 11B11 to a yeast cell. ... Excreta that demonstrated C. neoformans by capture ELISA and/or IF were plated with 50-μl aliquots on isolation agar (1 mM l-dopa, 29.4 mM KH2PO4, 10 mM MgSO4, 13 mM glycine, 15 mM glucose, and 6 μM thiamine [Sigma] with 0.1% biphenyl [Eastman Fine Chemicals, Eastman Kodak Company, Rochester, N.Y.], 0.025% vancomycin [Fugisawa USA, Inc., Deerfield, Ill.], 0.025% imipenem–cilastin [Merck and Co.], and 1.5% agar [Difco Laboratories, Detroit, Mich.]) (3) and incubated at 30°C. After a minimum of 6 days, darkly pigmented colonies were selected for further analysis. India ink suspensions of cells from colonies or after growth in Sabouraud dextrose medium (Difco) at 30°C were viewed with an Olympus AX70 microscope. Isolates from pigeon excreta melanized when they were grown in a defined chemical medium with l-dopa (15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, 13 mM glycine, 3 μM thiamine, 1 mM l-dopa [Sigma]) at 30 or 37°C. Urease activity was measured with BBL urea agar slants (Becton Dickinson, Cockeysville, Md.). Selected isolates that were India ink positive, stained with MAb 2D10, melanized in l-dopa medium, grew at 37°C, and utilized urea were further identified as C. neoformans with the API 20C Clinical Yeast System (BioMerieux Vitek, Inc., Hazelwood, Mo.). Pigeon excreta that tested positive for C. neoformans were treated with enzymes, denaturant, and hot acid and examined by scanning electron microscopy for particles that resembled melanin “ghosts” (16). Briefly, dried pigeon excreta (250 ml) were dissolved in 1 M sorbitol–0.1 M sodium citrate (pH 5.0) and then treated with 10 mg of Novozym 234 (Calbiochem, La Jolla, Calif.) per ml for 1 h at 30°C. The debris was collected by centrifugation, suspended in 4 M guanidinium isothiocyanate for 2 h at room temperature, and then incubated in 6 M HCl for 1 h at 100°C. The denaturant- and acid-resistant material was dialyzed exhaustively against distilled water. Samples of the isolated material were prepared as previously described (7) and viewed with a model JSM-6400 electron microscope (JEOL, Tokyo, Japan). Spherical structures very similar to the melanin ghosts isolated from melanized cells (16) were observed in pigeon excreta that had been treated with enzyme, denaturant, and boiling acid. These particles contained buds and bud scars, consistent with an origin from melanized yeast (Fig. ​(Fig.2A2A and B). One particle had a hole in the surface, suggestive of a budding site where the daughter cell was sheared off during the melanin isolation. FIG. 2 Scanning electron micrographs of particles isolated following treatment with enzyme, detergent, and hot acid of pigeon excreta (A and B) or cryptococcal cells grown on excreta agar (C) (magnification, ×15,000). The structures are remarkably similar ... To examine whether pigeon excreta had precursors for melanization, C. neoformans was grown on pigeon excreta agar plates. Pigeon feces were suspended in a minimal amount of PBS and autoclaved. Agar plates were prepared with a defined chemical medium (15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, 13 mM glycine, and 3 μM thiamine [Sigma] with 2% Bacto-Agar [Difco]) with 5, 10, or 20% excreta. Positive control plates were made with 1 mM l-dopa, and negative control plates lacked a substrate. C. neoformans ATCC 24067 or Candida albicans SC5314 (gift of M. Ghannoun) was grown overnight in Sabouraud dextrose medium at 30°C with shaking and then collected and washed three times in PBS. Ten-day-old C. neoformans cells growing on 10% excreta plates were collected, subjected to the melanin ghost isolation protocol (16), and prepared for scanning electron microscopy. C. neoformans grown in medium without phenolic substrate completely solubilize after treatment with enzyme, detergent, and hot HCl (16). Colonies of C. neoformans grown on the defined agar plates with or without 10% excreta were selected and suspended in PBS, and the cells were observed in India ink suspensions. Measurements of total cell diameter were done with an Olympus AX70 microscope (magnification, ×1,000) with a grid (number of segments, 20) with resolutions to 0.1 μm. Colonies of strain 24067 on 5, 10, or 20% excreta plates turned brown by day 5 and became progressively darker over time. Colonies on the defined agar without phenolic substrate were white, whereas those on plates with l-dopa were black. C. albicans grown on the excreta plates did not become pigmented, suggesting that colony pigmentation was not due simply to the uptake of some type of pigment from the excreta agar. Treatment of C. neoformans cells from excreta agar by the melanin isolation protocol yielded particles that were very similar to melanin ghosts (Fig. ​(Fig.2C).2C). The diameters of the ghost-like structures isolated from C. neoformans grown on 10% excreta agar were similar to those of ghosts prepared from cells grown on the same agar (3.2 ± 0.77 μm versus 2.88 ± 0.92 μm; n = 20; P > 0.05 by Student’s t test). Similarly, the sizes of the particles visualized in pigeon feces were 2.91 ± 1.07 μm (n = 20; P > 0.05 by Student’s t test). We conclude that C. neoformans in pigeon excreta is melanized from the following evidence: (i) yeast cells in pigeon excreta stain with MAbs to C. neoformans polysaccharide and melanin; (ii) structures remarkably similar in size and appearance to melanin ghosts can be isolated both from pigeon feces contaminated with C. neoformans and from cryptococcal cells grown on excreta agar after treatment with enzyme, denaturant, and hot acid; and (iii) C. neoformans colonies on pigeon excreta agar plates are darkly pigmented. Melanization of C. neoformans in the environment has important implications for our understanding of cryptococcal ecology and pathogenesis. Melanin synthesis in pigeon excreta may potentially protect C. neoformans cells against environmental stresses such as UV light (15) and extremes in temperature (12). Human C. neoformans infection is acquired from the environment, and there is extensive circumstantial evidence implicating pigeon excreta as a potential reservoir for infection (reviewed in reference 2). Our finding suggests that the initial infection may involve melanized C. neoformans cells. Since there is evidence that the immune responses to melanized and nonmelanized cells are different (6), our results suggest that it is important to study the effect of melanization in initial infection.

Cryptococcus neoformansis an encapsulated, environmental fungus that can cause life-threatening meningitis. Pathogenicity ofC. neoformansfor macrophages and vertebrate hosts may be a mechanism selected in evolution for protection against environmental predators. In this study, we investigated whetherDictyostelium discoideumcould serve as an alternate host forC. neoformans. D. discoideumhas a defined genetic system which provides significant advantages for the study of fungus-amoeba interactions. Our results show thatD. discoideumis susceptible to infection withC. neoformansand that the interactions are similar to those described previously for this fungus with macrophages andAcanthamoeba castellanii. AcapsularC. neoformanscells did not replicate when coincubated withD. discoideum. However, incubation of acapsularC. neoformanswithD. discoideummutants defective in myosin VII synthesis resulted in infection, validating the concept that avirulent organisms can be virulent in impaired hosts even at the unicellular level. Phagocytosis ofC. neoformansbyD. discoideumcould be inhibited with capsule-specific antibodies and various sugars. Passage of an encapsulatedC. neoformansstrain throughD. discoideumcultures increased virulence and was accompanied by larger capsules and faster time to melanization. These results add to the evidence implicating soil ameboid predators as important factors for the maintenance ofC. neoformansvirulence in the environment and suggest thatD. discoideumpromises to be an extremely useful system for studying the interaction ofC. neoformanswith phagocytic cells.

Melanin has been implicated in the pathogenesis of several important human fungal pathogens. Existing data suggest that the conidia of the dimorphic fungal pathogen Sporothrix schenckii produce melanin or melanin-like compounds; in this study we aimed to confirm this suggestion and to demonstrate in vitro and in vivo production of melanin by yeast cells. S. schenckii grown on Mycosel agar produced visibly pigmented conidia, although yeast cells grown in brain heart infusion and minimal medium broth appeared to be nonpigmented macroscopically. However, treatment of both conidia and yeast cells with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles similar in shape and size to the corresponding propagules, which were stable free radicals consistent with identification as melanins. Melanin particles extracted from S. schenckii yeast cells were used to produce a panel of murine monoclonal antibodies (MAbs) which labeled pigmented conidia, yeast cells, and the isolated particles. Tissue from hamster testicles infected with S. schenckii contained fungal cells that were labeled by melanin-binding MAbs, and digestion of infected hamster tissue yielded dark particles that were also reactive. Additionally, sera from humans with sporotrichosis contained antibodies that bound melanin particles. These findings indicate that S. schenckii conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role in the pathogenesis of sporotrichosis.

Passive immunization with monoclonal antibodies (MAbs) to melanin prolonged the survival of and reduced the fungal burden in Cryptococcus neoformans -infected mice in comparison to controls. MAbs to melanin reduced the growth rate of in vitro-melanized C. neoformans cells, suggesting a new mechanism of antibody-mediated protection.

The protective efficacy of antibodies (Abs) toCryptococcus neoformansglucuronoxylomannan (GXM) is dependent on Ab fine specificity. Two clonally related immunoglobulin M monoclonal Abs (MAbs) (12A1 and 13F1) differ in fine specificity and protective efficacy, presumably due to variable (V)-region sequence differences resulting from somatic mutations. MAb 12A1 is protective and produces annular immunofluorescence (IF) on serotype DC. neoformans,while MAb 13F1 is not protective and produces punctate IF. To determine the Ab molecular determinants responsible for the IF pattern, site-directed mutagenesis of the MAb 12A1 heavy-chain V region (VH) was followed by serological and functional studies of the various mutants. Changing two selected amino acids in the 12A1 VHbinding cavity to the corresponding residues in the 13F1 VHaltered the IF pattern from annular to punctate, reduced opsonic efficacy, and abolished recognition by an anti-idiotypic Ab. Analysis of the binding of the various mutants to peptide mimetics revealed that different amino acids were responsible for GXM binding and peptide specificity. The results suggest that V-region motifs associated with annular binding and opsonic activity may be predictive of Ab efficacy againstC. neoformans. This has important implications for immunotherapy and vaccine design that are reinforced by the finding that GXM and peptide reactivities are determined by different amino acid residues.

Cryptococcus neoformans is a human pathogenic fungus which is unusual in two respects: it has a polysaccharide capsule similar to that found in encapsulated bacteria and it can produce melanin. Capsular and melanization phenotypes are associated with virulence. In this study we analyzed the contributions of the capsular polysaccharide, melanization, and antibody binding to the capsule to the cellular charge of C. neoformans. Cell charge was inferred from measurements of zeta potential. The results indicate that (i) C. neoformans cells are significantly more negatively charged than Saccharomyces cerevisiae cells, (ii) the polysaccharide capsule of C. neoformans is responsible for the high negative charge of the cells, (iii) C. neoformans melanin is negatively charged, (iv) melanization in C. neoformans is associated with an increased negative charge per cell, and (v) antibody binding to the capsule of C. neoformans significantly alters the cell charge. These results suggest that alterations in cell charge attributable to polysaccharide capsule formation, melanization, and antibody binding may affect C. neoformans virulence given that macrophage phagocytosis is effected by the zeta potential of microorganisms.