Joshua D. Nosanchuk

Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence.

ABSTRACT Cryptococcus neoformans produces vesicles containing its major virulence factor, the capsular polysaccharide glucuronoxylomannan (GXM). These vesicles cross the cell wall to reach the extracellular space, where the polysaccharide is supposedly used for capsule growth or delivered into host tissues. In the present study, we characterized vesicle morphology and protein composition by a combination of techniques including electron microscopy, proteomics, enzymatic activity, and serological reactivity. Secretory vesicles in C. neoformans appear to be correlated with exosome-like compartments derived from multivesicular bodies. Extracellular vesicles manifested various sizes and morphologies, including electron-lucid membrane bodies and electron-dense vesicles. Seventy-six proteins were identified by proteomic analysis, including several related to virulence and protection against oxidative stress. Biochemical tests indicated laccase and urease activities in vesicles. In addition, different vesicle proteins were recognized by sera from patients with cryptococcosis. These results reveal an efficient and general mechanism of secretion of pathogenesis-related molecules in C. neoformans, suggesting that extracellular vesicles function as “virulence bags” that deliver a concentrated payload of fungal products to host effector cells and tissues.

Impact of Melanin on Microbial Virulence and Clinical Resistance to Antimicrobial Compounds

Melanins are negatively charged, hydrophobic pigments of high molecular weight ([54][1], [88][2], [95][3], [139][4]) that are composed of polymerized phenolic and/or indolic compounds (Fig. [1][5]) ([45][6], [128][7]). Melanins are produced by organisms in all biological kingdoms, including a wide

Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans‐cell wall transfer of proteins and lipids in ascomycetes

Vesicular secretion of macromolecules has recently been described in the basidiomycete Cryptococcus neoformans, raising the question as to whether ascomycetes similarly utilize vesicles for transport. In the present study, we examine whether the clinically important ascomycete Histoplasma capsulatum produce vesicles and utilized these structures to secrete macromolecules. Transmission electron microscopy (TEM) shows transcellular secretion of vesicles by yeast cells. Proteomic and lipidomic analyses of vesicles isolated from culture supernatants reveal a rich collection of macromolecules involved in diverse processes, including metabolism, cell recycling, signalling and virulence. The results demonstrate that H. capsulatum can utilize a trans‐cell wall vesicular transport secretory mechanism to promote virulence. Additionally, TEM of supernatants collected from Candida albicans, Candida parapsilosis, Sporothrix schenckii and Saccharomyces cerevisiae documents that vesicles are similarly produced by additional ascomycetes. The vesicles from H. capsulatum react with immune serum from patients with histoplasmosis, providing an association of the vesicular products with pathogenesis. The findings support the proposal that vesicular secretion is a general mechanism in fungi for the transport of macromolecules related to virulence and that this process could be a target for novel therapeutics.

Melanization of Cryptococcus neoformans and Histoplasma capsulatum Reduces Their Susceptibilities to Amphotericin B and Caspofungin

ABSTRACT The fungal pathogens Cryptococcus neoformans and Histoplasma capsulatum produce melanin-like pigments in the presence of l-dopa in vitro and during mammalian infection. We investigated whether melanization affected the susceptibilities of the fungi to amphotericin B, caspofungin, fluconazole, itraconazole, or flucytosine (5FC). Using the standard macrodilution MIC protocol (the M27A protocol) of the National Committee for Clinical Laboratory Standards for yeast, we found no difference in the susceptibilities of melanized and nonmelanized C. neoformans and H. capsulatum isolates. Killing assays demonstrated that melanization reduced the susceptibilities of both fungi to amphotericin B and caspofungin. Laccase-deficient C. neoformans cells grown with l-dopa were significantly more susceptible than congenic melanin-producing yeast to killing by amphotericin B or caspofungin. Preincubation of amphotericin B or caspofungin with melanins decreased their antifungal activities. Elemental analysis of melanins incubated with amphotericin B or caspofungin revealed an alteration in the C:N ratios of the melanins, which indicated binding of these drugs by the melanins. In contrast, incubation of fluconazole, itraconazole, or 5FC with melanins did not significantly affect the antifungal efficacies of the drugs or the chemical composition of the melanins. The results suggest a potential explanation for the inefficacy of caspofungin against C. neoformans in vivo, despite activity in vitro. Furthermore, the results indicate that fungal melanins protect C. neoformans and H. capsulatum from the activities of amphotericin B and caspofungin and that this protection is not demonstrable by standard broth macrodilution assays.

Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice

Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.

Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum

A protective role for antibodies has not previously been described for host defense against the pathogenic fungus Histoplasma capsulatum (Hc). Mouse mAbs were generated from mice immunized with Hc yeast that binds the cell surface of Hc. Administration of mAbs before Hc infection reduced fungal burden, decreased pulmonary inflammation, and prolonged survival in a murine infection model. Protection mediated by mAbs was associated with enhanced levels of IL-4, IL-6, and IFN-gamma in the lungs of infected mice. The mAbs increased phagocytosis of yeast by J774.16 cells through a CR3-dependent process. Ingestion of mAb-opsonized Hc by J774.16 macrophage-like cells was associated with yeast cell growth inhibition and killing. The mAbs bound to a 17-kDa antigen expressed on the surface of Hc. The antigen was identified as a histone H2B-like protein. This study establishes that mAbs to a cell surface protein of Hc alter the intracellular fate of the fungus and mediate protection in a murine model of lethal histoplasmosis, and it suggests a new candidate antigen for vaccine development.

Synthesis of Polymerized Melanin by Cryptococcus neoformans in Infected Rodents

ABSTRACT The ability of Cryptococcus neoformans to synthesize polymerized melanin in vitro has been associated with virulence, but it is unclear whether this fungus synthesizes polymerized melanin during infection. To study this question, we used two approaches: one involved the generation of monoclonal antibodies (MAbs) to melanin for use in immunohistochemical studies of C. neoformans-infected rodents, and the other sought to isolate fungal melanin from infected tissues. Digestion of in vitro-melanized C. neoformanscells with proteases, denaturant, and hot concentrated acid yields melanin particles that retain the shape of fungal cells and are therefore called melanin ghosts. BALB/c mice were immunized with melanin ghosts, and two immunoglobulin M MAbs to melanin were generated from the spleen of one mouse. Immunofluorescence analyses of lung and brain tissues of rodents infected with wild-type melanin-producing (Mel+) C. neoformans strains demonstrated binding of the MAbs to the fungal cell wall. No binding was observed when infections were performed with mutant albino (Mel−)C. neoformans strains. Particles with striking similarity to melanin ghosts were recovered after digestion of lung and brain tissues from Mel+C. neoformans-infected rodents and were reactive with the MAbs to melanin. No particles were recovered from tissues infected with Mel−C. neoformans. A Mel+C. neoformans strain grown on lung or brain homogenate agar became lightly pigmented and also yielded particles similar to melanin ghosts upon digestion, providing additional evidence that lung and brain tissues contain substrate for C. neoformans melanization. These results demonstrate that C. neoformans synthesizes polymerized melanin during infection, which has important implications for pathogenesis and antifungal drug development.

Cryptococcus neoformans virulence is enhanced after growth in the genetically malleable host Dictyostelium discoideum.

ABSTRACT Cryptococcus neoformans is an encapsulated, environmental fungus that can cause life-threatening meningitis. Pathogenicity of C. neoformans for macrophages and vertebrate hosts may be a mechanism selected in evolution for protection against environmental predators. In this study, we investigated whether Dictyostelium discoideum could serve as an alternate host for C. neoformans. D. discoideum has a defined genetic system which provides significant advantages for the study of fungus-amoeba interactions. Our results show that D. discoideum is susceptible to infection with C. neoformans and that the interactions are similar to those described previously for this fungus with macrophages and Acanthamoeba castellanii. Acapsular C. neoformans cells did not replicate when coincubated with D. discoideum. However, incubation of acapsular C. neoformans with D. discoideum mutants 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 of C. neoformans by D. discoideum could be inhibited with capsule-specific antibodies and various sugars. Passage of an encapsulated C. neoformans strain through D. discoideum cultures 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 of C. neoformans virulence in the environment and suggest that D. discoideum promises to be an extremely useful system for studying the interaction of C. neoformans with phagocytic cells.

Extracellular Vesicles from Cryptococcus neoformans Modulate Macrophage Functions

ABSTRACT Cryptococcus neoformans and distantly related fungal species release extracellular vesicles that traverse the cell wall and contain a varied assortment of components, some of which have been associated with virulence. Previous studies have suggested that these extracellular vesicles are produced in vitro and during animal infection, but the role of vesicular secretion during the interaction of fungi with host cells remains unknown. In this report, we demonstrate by fluorescence microscopy that mammalian macrophages can incorporate extracellular vesicles produced by C. neoformans. Incubation of cryptococcal vesicles with murine macrophages resulted in increased levels of extracellular tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10), and transforming growth factor β (TGF-β). Vesicle preparations also resulted in a dose-dependent stimulation of nitric oxide production by phagocytes, suggesting that vesicle components stimulate macrophages to produce antimicrobial compounds. Treated macrophages were more effective at killing C. neoformans yeast. Our results indicate that the extracellular vesicles of C. neoformans can stimulate macrophage function, apparently activating these phagocytic cells to enhance their antimicrobial activity. These results establish that cryptococcal vesicles are biologically active.

Passive Immunization with Melanin-Binding Monoclonal Antibodies Prolongs Survival of Mice with Lethal Cryptococcus neoformans Infection

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