André Moraes Nicola

Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins.

Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature‐dependent cell morphology change from mycelium (22° C) to yeast (36° C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3938 (Y = 1654 and M = 2274) ESTs were sequenced and clustered into 597 contigs and 1563 singlets, making up a total of 2160 genes, which possibly represent one‐quarter of the complete gene repertoire in P. brasiliensis. From this total, 1040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes—cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage‐specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis. All EST sequences have been deposited in GenBank under Accession Nos CA580326–CA584263. Copyright

Vesicle-associated melanization in Cryptococcus neoformans

Recently, several pathogenic fungi were shown to produce extracellular vesicles that contain various components associated with virulence. In the human pathogenic fungus Cryptococcus neoformans, these components included laccase, an enzyme that catalyses melanin synthesis. Spherical melanin granules have been observed in the cell wall of C. neoformans. Given that melanin granules have dimensions that are comparable to those of extracellular vesicles, and that metazoan organisms produce melanin in vesicular structures known as melanosomes, we investigated the role of vesicles in cryptococcal melanization. Extracellular vesicles melanized when incubated with the melanin precursor L-3,4-dihydroxyphenylalanine (L-DOPA). The kinetics of substrate incorporation into cells and vesicles was analysed using radiolabelled L-DOPA. The results indicated that substrate incorporation was different for cells and isolated vesicles. Acid-generated melanin ghosts stained with lipophilic dyes, implying the presence of associated lipid. A model for C. neoformans melanization is proposed that accounts for these observations and provides a mechanism for the assembly of melanin into relatively uniform spherical particles stacked in an orderly arrangement in the cell wall.

Nonlytic Exocytosis of Cryptococcus neoformans from Macrophages Occurs In Vivo and Is Influenced by Phagosomal pH

ABSTRACT A unique aspect of the interaction of the fungus Cryptococcus neoformans with macrophages is the phenomenon of nonlytic exocytosis, also referred to as “vomocytosis” or phagosome extrusion/expulsion, which involves the escape of fungal cells from the phagocyte with the survival of both cell types. This phenomenon has been observed only in vitro using subjective and time-consuming microscopic techniques. In spite of recent advances in our knowledge about its mechanisms, a major question still remaining is whether this phenomenon also occurs in vivo. In this study, we describe a novel flow cytometric method that resulted in a substantial gain in throughput for studying phagocytosis and nonlytic exocytosis in vitro and used it to explore the occurrence of this phenomenon in a mouse model of infection. Furthermore, we tested the hypothesis that host cell phagosomal pH affected nonlytic exocytosis. The addition of the weak bases ammonium chloride and chloroquine resulted in a significant increase of nonlytic exocytosis events, whereas the vacuolar ATPase inhibitor bafilomycin A1 had the opposite effect. Although all three agents are known to neutralize phagosomal acidity, their disparate effects suggest that phagosomal pH is an important and complex variable in this process. Our experiments established that nonlytic exocytosis occurred in vivo with a frequency that is possibly much higher than that observed in vitro. These results in turn suggest that nonlytic exocytosis has a potential role in the pathogenesis of cryptococcosis. IMPORTANCE Cryptococcus neoformans causes disease in people with immune deficiencies such as AIDS. Upon infection, C. neoformans cells are ingested by macrophage immune cells, which provide a niche for survival and replication. After ingestion, macrophages can expel the fungi without causing harm to either cell type, a process named nonlytic exocytosis. To dissect this phenomenon, we evaluated its dependence on the pH inside the macrophage and addressed its occurrence during infection of mice. We developed new techniques using flow cytometry to measure C. neoformans internalization by and nonlytic exocytosis from macrophages. Neutralizing the phagosome acidity changed the rate of nonlytic exocytosis: activity increased with the weak bases chloroquine and ammonium chloride, whereas the vacuolar ATPase inhibitor bafilomycin A1 caused it to decrease. Experiments in mice suggested that nonlytic exocytosis occurred during infection with C. neoformans. These results shed new light on the interaction between C. neoformans and host macrophages. Cryptococcus neoformans causes disease in people with immune deficiencies such as AIDS. Upon infection, C. neoformans cells are ingested by macrophage immune cells, which provide a niche for survival and replication. After ingestion, macrophages can expel the fungi without causing harm to either cell type, a process named nonlytic exocytosis. To dissect this phenomenon, we evaluated its dependence on the pH inside the macrophage and addressed its occurrence during infection of mice. We developed new techniques using flow cytometry to measure C. neoformans internalization by and nonlytic exocytosis from macrophages. Neutralizing the phagosome acidity changed the rate of nonlytic exocytosis: activity increased with the weak bases chloroquine and ammonium chloride, whereas the vacuolar ATPase inhibitor bafilomycin A1 caused it to decrease. Experiments in mice suggested that nonlytic exocytosis occurred during infection with C. neoformans. These results shed new light on the interaction between C. neoformans and host macrophages.

Ab binding alters gene expression in Cryptococcus neoformans and directly modulates fungal metabolism

Abs facilitate humoral immunity via the classical mechanisms of opsonization, complement activation, Ab-dependent cellular cytotoxicity, and toxin/viral neutralization. There is also evidence that some Abs mediate direct antimicrobial effects. For example, Ab binding to the polysaccharide capsule of the human pathogenic fungus Cryptococcus neoformans promotes opsonization but also inhibits polysaccharide release and biofilm formation. To investigate whether Ab binding affects C. neoformans directly, we analyzed fungal gene expression after binding of protective and nonprotective mAbs. The 2 IgM Abs and 1 IgG1 Ab tested each induced different changes in gene expression. The protective IgG1 mAb upregulated genes encoding proteins involved in fatty acid synthesis, the protective IgM mAb downregulated genes encoding proteins required for protein translation, and the nonprotective IgM mAb had modest effects on gene expression. Differences in gene expression correlated with mAb binding to different locations of the capsule. Of the 3 Abs tested, the protective IgG1 mAb bound to C. neoformans closest to the cell wall, produced specific differences in the pattern of phosphorylated proteins, caused changes in lipid metabolism, and resulted in increased susceptibility to the antifungal drug amphotericin B. These results suggest what we believe to be a new mode of action for Ab-mediated immunity and raise the possibility that immunoglobulins mediate cross talk between microbes and hosts through their effects on microbial metabolism.

Macrophage Autophagy in Immunity to Cryptococcus neoformans and Candida albicans

ABSTRACT 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.

Lipophilic Dye Staining of Cryptococcus neoformans Extracellular Vesicles and Capsule

ABSTRACT Cryptococcus neoformans is an encapsulated yeast that causes systemic mycosis in immunosuppressed individuals. Recent studies have determined that this fungus produces vesicles that are released to the extracellular environment both in vivo and in vitro. These vesicles contain assorted cargo that includes several molecules associated with virulence and implicated in host-pathogen interactions, such as capsular polysaccharides, laccase, urease, and other proteins. To date, visualization of extracellular vesicles has relied on transmission electron microscopy, a time-consuming technique. In this work we report the use of fluorescent membrane tracers to stain lipophilic structures in cryptococcal culture supernatants and capsules. Two dialkylcarbocyanine probes with different spectral characteristics were used to visualize purified vesicles by fluorescence microscopy and flow cytometry. Dual staining of vesicles with dialkylcarbocyanine and RNA-selective nucleic acid dyes suggested that a fraction of the vesicle population carried RNA. Use of these dyes to stain whole cells, however, was hampered by their possible direct binding to capsular polysaccharide. A fluorescent phospholipid was used as additional membrane tracer to stain whole cells, revealing punctate structures on the edge of the capsule which are consistent with vesicular trafficking. Lipophilic dyes provide new tools for the study of fungal extracellular vesicles and their content. The finding of hydrophobic regions in the capsule of C. neoformans adds to the growing evidence for a structurally complex structure composed of polysaccharide and nonpolysaccharide components.

Capsular Localization of the Cryptococcus neoformans Polysaccharide Component Galactoxylomannan

ABSTRACT Cryptococcus neoformans capsular polysaccharide is composed of at least two components, glucuronoxylomannan (GXM) and galactoxylomannans (GalXM). Although GXM has been extensively studied, little is known about the location of GalXM in the C. neoformans capsule, in part because there are no serological reagents specific to this antigen. To circumvent the poor immunogenicity of GalXM, this antigen was conjugated to protective antigen from Bacillus anthracis as a protein carrier. The resulting conjugate elicited antibodies that reacted with GalXM in mice and yielded an immune serum that proved useful for studying GalXM in the polysaccharide capsule. In acapsular cells, immune serum localized GalXM to the cell wall. In capsulated cells, immune serum localized GalXM to discrete pockets near the capsule edge. GalXM was abundant on the nascent capsules of budding daughter cells. The constituent sugars of GalXM were found in vesicle fractions consistent with vesicular transport for this polysaccharide. In addition, we generated a single-chain fraction variable fragment antibody with specificity to oxidized carbohydrates that also produced punctate immunofluorescence on encapsulated cells that partially colocalized with GalXM. The results are interpreted to mean that GalXM is a transient component of the polysaccharide capsule of mature cells during the process of secretion. Hence, the function of GalXM appears to be more consistent with that of an exopolysaccharide than a structural component of the cryptococcal capsule.

Quorum Sensing-Mediated, Cell Density-Dependent Regulation of Growth and Virulence in Cryptococcus neoformans

ABSTRACT Quorum sensing (QS) is a cell density-dependent mechanism of communication between microorganisms, characterized by the release of signaling molecules that affect microbial metabolism and gene expression in a synchronized way. In this study, we investigated cell density-dependent behaviors mediated by conditioned medium (CM) in the pathogenic encapsulated fungus Cryptococcus neoformans. CM produced dose-dependent increases in the growth of planktonic and biofilm cells, glucuronoxylomannan release, and melanin synthesis, important virulence attributes of this organism. Mass spectrometry revealed the presence of pantothenic acid (PA) in our samples, and commercial PA was able to increase growth and melanization, although not to the same extent as CM. Additionally, we found four mutants that were either unable to produce active CM or failed to respond with increased growth in the presence of wild-type CM, providing genetic evidence for the existence of intercellular communication in C. neoformans. C. neoformans CM also increased the growth of Cryptococcus albidus, Candida albicans, and Saccharomyces cerevisiae. Conversely, CM from Cryptococcus albidus, C. albicans, S. cerevisiae, and Sporothrix schenckii increased C. neoformans growth. In summary, we report the existence of a new QS system regulating the growth and virulence factor expression of C. neoformans in vitro and, possibly, also able to regulate growth in other fungi. IMPORTANCE Quorum sensing is a strategy of communication used by pathogenic microorganisms to coordinate the expression of attributes necessary to cause disease. In this work, we describe a quorum sensing system in Cryptococcus neoformans, a yeast that can cause severe central nervous system infections. Adding conditioned medium—culture medium in which C. neoformans has previously grown—to fresh cultures resulted in faster growth of C. neoformans both as isolated cells and in microbial communities called biofilms. The addition of conditioned medium also increased the secretion of capsule carbohydrates and the formation of melanin pigment, two tools used by this microorganism to thrive in the host. This remarkable example of microbial communication shows that C. neoformans cells can act in unison when expressing attributes necessary to survive in the host, a finding that could point the way to improvements in the treatment of cryptococcosis. Quorum sensing is a strategy of communication used by pathogenic microorganisms to coordinate the expression of attributes necessary to cause disease. In this work, we describe a quorum sensing system in Cryptococcus neoformans, a yeast that can cause severe central nervous system infections. Adding conditioned medium—culture medium in which C. neoformans has previously grown—to fresh cultures resulted in faster growth of C. neoformans both as isolated cells and in microbial communities called biofilms. The addition of conditioned medium also increased the secretion of capsule carbohydrates and the formation of melanin pigment, two tools used by this microorganism to thrive in the host. This remarkable example of microbial communication shows that C. neoformans cells can act in unison when expressing attributes necessary to survive in the host, a finding that could point the way to improvements in the treatment of cryptococcosis.

Fungal killing by mammalian phagocytic cells

Phagocytes are considered the most important effector cells in the immune response against fungal infections. To exert their role, they must recognize the invading fungi, internalise, and kill them within the phagosome. Major advances in the field have elucidated the roles of pattern-recognition receptors in the innate immunity sensing and the importance of reactive oxygen and nitrogen species in intracellular killing of fungi. Surprising exit mechanisms for intracellular pathogens and extracellular traps have also been discovered. These and several other recent breakthroughs in our understanding of the mechanisms used by phagocytes to kill fungal pathogens are reviewed in this work.