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Biomedical subjects

Oscar Zaragoza

Publications and source records attributed to Oscar Zaragoza.

At least 19 recordsLinked to original sources
PubMed2006

Radial mass density, charge, and epitope distribution in the Cryptococcus neoformans capsule.

Exposure of Cryptococcus neoformans cells to gamma radiation results in a gradual release of capsular polysaccharide, in a dose-dependent manner. This method allows the systematic exploration of different capsular regions. Using this methodology, capsule density was determined to change according to the radial distribution of glucuronoxylomannan and total polysaccharide, becoming denser at the inner regions of the capsule. Scanning electron microscopy of cells following gamma radiation treatment confirmed this finding. The zeta potential of the capsule also increased as the capsule size decreased. However, neither charge nor density differences were correlated with any change in sugar composition (xylose, mannose, and glucuronic acid) in the different capsular regions, since the proportions of these sugars remained constant throughout the capsule. Analysis of the capsular antigenic properties by monoclonal antibody binding and Scatchard analysis revealed fluctuations in the binding affinity within the capsule but not in the number of antibody binding sites, suggesting that the spatial organization of high- and low-affinity epitopes within the capsule changed according to radial position. Finally, evidence is presented that the structure of the capsule changes with capsule age, since the capsule of older cells became more resistant to gamma radiation-induced ablation. In summary, the capsule of C. neoformans is heterogeneous in its spatial distribution and changes with age. Furthermore, our results suggest several mechanisms by which the capsule may protect the fungal cell against exogenous environmental factors.

Antigens, Fungal↗
PubMed2006

Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport.

The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.

Animals↗
PubMed2006

The capsular dynamics of Cryptococcus neoformans.

Cryptococcus neoformans is a soil-dwelling fungus that causes life-threatening illness in immunocompromised individuals and latently infects many healthy individuals. C. neoformans, unlike other human pathogenic fungi, is surrounded by a polysaccharide capsule that is essential for survival and enables C. neoformans to thwart the mammalian immune system. The capsule is a dynamic structure that undergoes changes in size and rearranges during budding. Here, the latest information and unresolved questions regarding capsule synthesis, structure, assembly, growth and rearrangements are discussed along with the concept that self-assembly is important in capsular dynamics.

Cell Wall↗
PubMed2006

The volume and hydration of the Cryptococcus neoformans polysaccharide capsule.

We present a new method to measure capsule size in the human fungal pathogen Cryptococcus neoformans that avoids the limitations and biases inherent in India ink measurements. The method is based on the use of gamma-radiation, which efficiently releases the capsule from the cell. By comparing the volume of irradiated and non-irradiated cells, one can accurately estimate the relative size of the capsule per cell. This method was also used to obtain an estimate of the capsule weight and water content. The C. neoformans capsule is a highly hydrated structure in all the conditions measured. However, after capsule enlargement, the amount of capsular polysaccharide significantly increases, suggesting a that capsule growth has a high energy cost for the cell.

Cryptococcus neoformans↗
PubMed2006

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.

Antibodies, Fungal↗
PubMed2006

Characterization of a flocculation-like phenotype in Cryptococcus neoformans and its effects on pathogenesis.

We investigated the phenomenon of cell-cell aggregation (flocculation) in a serotype D strain of Cryptococcus neoformans (ATCC 24067, isolate RC-2). Cell aggregation into clumps of 5-40 cells (clump+ cells) occurred during the early log phase and disappeared in the beginning of the stationary phase (clump- cells). The cell aggregation phenomenon was medium dependent. Clump+ cells could be dispersed by either vortexing or proteinase K digestion. Most importantly, the transient change in cellular phenotype changed several important host-pathogen interactions. Adherence of clump+ cells to murine macrophage-like cells J774.16 was significantly (P < 0.001) enhanced compared with adherence of clump- cells. Furthermore, complement-mediated phagocytosis efficacy of dispersed clump+ cells was significantly higher (P < 0.001) compared with clump- cells. Similar findings were documented with an in vivo phagocytosis assay. Infection of mice with a low inoculum (10(4)) of clump+ cells resulted in lower fungal burden when compared with mice infected with clump- cells. Accordingly, mice infected with clump+ cells survived significantly longer than mice infected with clump- cells. These results indicate that the cellular phenotype undergoes significant changes that result in a transient flocculation-like phenotype. We hypothesize that this cell-cell aggregation is the result of changes in protein content in the polysaccharide capsule. We conclude from our data that the change in cellular phenotype has a dramatic effect on cell adherence, and on complement-mediated phagocytosis, both of which can affect the pathogenesis of the disease in the host. Our results underscore the complexity of studies that investigate host pathogen interactions and may explain differences and inconsistencies observed in in vitro and in vivo assays.

Animals↗
PubMed2006

Efficacy of voriconazole in experimental Cryptococcus neoformans infection.

Voriconazole is a third generation triazole with improved activity against many fungal pathogens. We examined the efficacy of voriconazole in a murine infection model and evaluated the drug's effect on cellular characteristics and serum polysaccharide levels. The antifungal reduced serum polysaccharide and significantly prolonged the survival of lethally infected animals.

Animals↗
PubMed2006

The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans enlarges by distal growth and is rearranged during budding.

The capsule of Cryptococcus neoformans can undergo dramatic enlargement, a phenomenon associated with virulence. A prior study that used Ab to the capsule as a marker for older capsular material concluded that capsule growth involved the intermixing of new and old capsular material with displacement of older capsular polysaccharide towards the surface. Here we have revisited that question using complement (C), which binds to capsular polysaccharide covalently, and cannot redistribute by dissociation and binding at different sites. The experimental approach involved binding of C to cells with small capsules, inducing capsule growth, and following the location of C relative to the cell wall as the capsule enlarged. C remained close to the cell wall during capsule growth, indicating that capsule enlargement occurred by addition of new polysaccharide near the capsule edge. This conclusion was confirmed by an independent method that employed radioactive metabolic labelling of newly synthesized capsule with 3H-mannose followed by gradual capsular stripping with gamma-radiation. Capsule growth proceeded to a certain size, which was a function of cell size, and was not degraded when the cells were transferred to a non-inducing medium. During budding, an opening appeared in the capsule of the mother cell that permitted the nascent bud to separate. Scanning EM suggested that a physical separation formed between the capsules of the mother and daughter cells during budding, which may avoid mixture between both capsules. Our results indicate that C. neoformans capsular enlargement also occurs by apical growth and that budding results in capsular rearrangements.

Animals↗
PubMed2006

Equatorial ring-like channels in the Cryptococcus neoformans polysaccharide capsule.

Under certain conditions, India ink particles can penetrate the capsule of the opportunistic pathogen Cryptococcus neoformans. India ink penetration gave two distinct patterns, one as a ring in the middle of the capsule, and another as a double spot located at opposite poles of the cells. These spots were perpendicularly orientated to the bud. This pattern suggests the existence of a localized structure deep in the capsule that can accumulate large insoluble particles. Although the mechanisms responsible for the assemblage and maintenance of ring-like channels are not understood, their existence deep within the capsule implies a new level of complexity for this enigmatic structure.

Animals↗
PubMed2005

Comparative analysis of Cryptococcus neoformans acid-resistant particles generated from pigmented cells grown in different laccase substrates.

Cryptococcus neoformans produces pigments in vitro in the presence of exogenous substrate. We characterized acid-resistant particles isolated from pigmented cells grown in L-dopa, methyl-dopa, (-)-epinephrine or (-)-norepinephrine. The goals of this study were to determine whether pigments made from each of these substrates were melanins and the consequences of pigmentation on related cell characteristics. The greatest yield of acid-resistant particles occurred with methyl-dopa followed by L-dopa. Electron microscopy indicated that L-dopa and methyl-dopa produced particles with thicker shells. The mAb 6D2 reacted with all particles, but a lower reactivity was observed with epinephrine-derived particles. ESR analysis revealed that epinephrine-derived particles failed to produce a stable free radical signal typical of melanins. Growth of C. neoformans in different substrates affected cell and capsule size but not capsule induction. Hence, the type of pigment produced by C. neoformans is dependent on the substrate and not all pigments meet the criteria for melanins.

Chromatography, High Pressure Liquid↗
PubMed2005

Structural and functional characterization of glycosylation in an immunoglobulin G1 to Cryptococcus neoformans glucuronoxylomannan.

Analysis of the N-linked oligosaccharides of the murine IgG1 monoclonal antibody (mAb) to Cryptococcus neoformans by LC/MS revealed five different core fucosylated, biantennary complex-type oligosaccharides at Asn-293, with the major species being a mono-galactosylated oligosaccharide with the glycosyl composition of Hex4HexNAc4Fuc (39% of the total glycan pool). The primary sequence predicted from nucleic acid sequencing differed from that measured by mass spectrometry at position 33 (ASN to ASP), a finding that may represent post-translational modification caused by spontaneous ASP deamination. Analysis of mAb 18B7 from three hybridoma clones revealed the same heterogenous N-glycan pattern, indicating that diversity in oligosaccharide structures originated from individual cells. The binding of native and de-glycosylated mAb 18B7 to cryptococcal Ag was comparable but the de-glycosylated 18B7 had shorter serum half-life and did not activate complement (C). De-glycosylated mAb 18B7 was opsonic for C. neoformans with murine macrophages through a mechanism that involved C-independent ingestion through the C receptor. Passive administration of de-glycosylated mAb 18B7 mediated comparable protective efficacy to the native mAb in mice with lethal infection. The results imply that the contribution of N-glycan structure to immunoglobulin function varies depending on the Ag-Ab system.

Amino Acid Sequence↗
PubMed2005

Antibody-mediated protection against Cryptococcus neoformans pulmonary infection is dependent on B cells.

The pathogenesis of pulmonary Cryptococcus neoformans infection and the efficacy of passive immunoglobulin G1 (IgG1) administration were investigated in B-cell-deficient and C57BL/6J mice. C57BL/6J mice lived longer than B-cell-deficient mice after both intratracheal and intravenous infections. Administration of IgG1 prior to infection prolonged the survival of C57BL/6J mice but had no effect on the survival or numbers of CFU in the lungs of B-cell-deficient mice. C. neoformans infection in B-cell-deficient mice resulted in significantly higher levels of gamma interferon (IFN-gamma), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha) than in C57BL/6J mice. IgG1 administration reduced IFN-gamma and MCP-1 levels in C57BL/6J mice but not in B-cell-deficient mice. In addition, compared to its effect in C57BL/6J mice, C. neoformans infection in FcRgammaIII-deficient, athymic, and SCID mice significantly increased IFN-gamma and MCP-1 levels. IgG1 administration was associated with reduced IFN-gamma levels in C57BL/6J mice but not in FcRgammaIII-deficient, athymic, and SCID mice. These observations suggest that IgG1-mediated protection in this system is a consequence of alterations in the inflammatory response that translate into less damage to the host without directly reducing the fungal burden. For hosts with impaired immunities, the ineffectiveness of passive antibody (Ab) may reflect an inability to down-regulate inflammation and avoid self-damage. The results indicate an important role for B cells in host defense against C. neoformans infection and demonstrate a surprising dependence of Ab-mediated protection on B cells in this system.

Animals↗
PubMed2004

Experimental modulation of capsule size in Cryptococcus neoformans.

Experimental modulation of capsule size is an important technique for the study of the virulence of the encapsulated pathogen Cryptococcus neoformans. In this paper, we summarize the techniques available for experimental modulation of capsule size in this yeast and describe improved methods to induce capsule size changes. The response of the yeast to the various stimuli is highly dependent on the cryptococcal strain. A high CO(2) atmosphere and a low iron concentration have been used classically to increase capsule size. Unfortunately, these stimuli are not reliable for inducing capsular enlargement in all strains. Recently we have identified new and simpler conditions for inducing capsule enlargement that consistently elicited this effect. Specifically, we noted that mammalian serum or diluted Sabouraud broth in MOPS buffer pH 7.3 efficiently induced capsule growth. Media that slowed the growth rate of the yeast correlated with an increase in capsule size. Finally, we summarize the most commonly used media that induce capsule growth in C. neoformans.

Journal Article↗
PubMed2004

Effects of voriconazole on Cryptococcus neoformans.

Voriconazole is a broad-spectrum triazole that offers extended activity against molds and yeasts that are not susceptible to earlier azole-type drugs. Recent studies indicate that melanization can severely reduce the susceptibility of certain antifungal drugs, but there is no information as to whether voriconazole is vulnerable to this effect. The activity of voriconazole on C. neoformans was assessed by MIC analysis and time-kill assays for melanized and nonmelanized cells. Cell morphology, capsule release, and phagocytosis of C. neoformans were studied in the presence or absence of subinhibitory concentrations of voriconazole. Voriconazole was fungicidal at concentrations of >/=8 microg/ml in vitro against the strains of C. neoformans examined, and its efficacy was not diminished by melanization. Cells grown in subinhibitory concentrations of voriconazole had smaller cellular and capsular volumes than cells grown in the absence of drug. The induction of the capsule by serum was not affected by voriconazole. Cells grown in subinhibitory concentrations of voriconazole released their capsule and were phagocytosed at rates comparable with yeast grown without the antifungal. The high activity of voriconazole against both melanized and nonmelanized cells results suggest that voriconazole may be a particularly valuable drug for cryptococcosis.

Amphotericin B↗
PubMed2004

Antibodies produced in response to Cryptococcus neoformans pulmonary infection in mice have characteristics of nonprotective antibodies.

Murine cryptocococcal pulmonary infection elicited serum immunoglobulin M (IgM) and IgG to the capsular polysaccharide, but only IgG stained yeast cells in alveoli. Both isotypes produced punctuate immunofluorescence patterns on yeast cells like those of nonprotective antibodies. The difficulties involved in associating humoral immunity with protection in murine cryptocococcal infection could reflect nonprotective antibody responses.

Animals↗
PubMed2004

Immunoreactivity of cryptococcal antigen is not stable under prolonged incubations in human serum.

The stability of cryptococcal antigen from Cryptococcus neoformans serotype A and D strains at different temperatures in serum and other solvents was studied. Samples stored at -20 or 4 degrees C had equivalent stabilities as measured by the Premier Cryptococcal Antigen kit and the Cryptococcal Antigen Latex Agglutination System (CALAS) kit. However, using the Premier Cryptococcal Antigen kit, there was a 91% loss of reactivity in samples incubated in human serum for 4 weeks at 37 degrees C. A loss of reactivity of more than 99% was observed after incubation at 45 degrees C for 4 weeks. The capsular antigen was not detected by the Premier Cryptococcal Antigen kit after 16 weeks at elevated temperatures. Antigen titers were also reduced in a latex agglutination assay (CALAS) after 4 weeks at 37 and 45 degrees C. The loss of antigen reactivity was a function of pH and temperature.

Antibodies, Monoclonal↗
PubMed2003

Trehalose accumulation induced during the oxidative stress response is independent of TPS1 mRNA levels in Candida albicans.

Growing cells of the Candida albicans trehalose-deficient mutant tps1/tps1 were extremely sensitive to severe oxidative stress exposure (H2O2). However, their viability was not affected after saline stress or heat-shock treatments, being roughly equivalent to that of the parental strain. In wild-type cells, these adverse conditions induced the intracellular accumulation of trehalose together with activation of trehalose-6P synthase, whereas the endogenous trehalose content and the corresponding biosynthetic activity were barely detectable in the tps1/tps1 mutant. The addition of cycloheximide did not prevent the marked induction of trehalose-6P synthase activity. Furthermore, the presence of H2O2 decreased the level of TPS1 mRNA expression. Hence, the conspicuous trehalose accumulation in response to oxidative stress is not induced by increased transcription of TPS1. Our results are consistent with a specific requirement of trehalose in order to withstand a severe oxidative stress in C. albicans, and suggest that trehalose accumulation observed under these conditions is a complex process that most probably involves post-translational modifications of the trehalose synthase complex.

Candida albicans↗