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Sample records for neoformans capsular polysaccharide

  1. Synthesis of part structures of Cryptococcus neoformans serotype C capsular polysaccharide.

    PubMed

    Guazzelli, Lorenzo; McCabe, Orla; Oscarson, Stefan

    2016-10-04

    Cryptococcus neoformans is a fungal pathogen that can cause life-threatening infections in immunocompromised patients. The development of a vaccine based on the capsular polysaccharide of C. neoformans is still an open challenge due to the heterogeneity of the capsular polysaccharide and the difficulty of identifying protective epitopes. Therefore, construction of structurally defined part structures of the C. neoformans GXM capsule is in great demand. Herein is presented the synthesis of a 3-O-naphthalenylmethyl protected trisaccharide thioglycoside building block which is present in C. neoformans serotype C polysaccharide. Its property as a donor in a glycosylation reaction with a model acceptor has been evaluated together with its behaviour as an acceptor following removal of the temporary protecting group. The heavily branched hexasaccharide was obtained in good yields and excellent α-selectivity. The frame shifted octasaccharide structural triad motif for serotype C was also prepared following the same building block strategy. For the first time this structural motif, which is the most substituted amongst the four C. neoformans serotypes, was prepared. Three synthesized C. neoformans serotype C fragments of varying size, from penta-up to octasaccharide, were deprotected and will be included in unique glycoarrays to further investigate the possibility to develop a synthetic vaccine against this pathogen.

  2. Latex agglutination: diagnose the early cryptococcus neoformans test of capsular polysaccharide antigen.

    PubMed

    Wang, Huanrong; Yuan, Xueqian; Zhang, Lifeng

    2015-01-01

    This paper aims to discuss the early diagnosis value of latex agglutination test in Cryptococcal meningitis. The cerebrospinal fluid (CSF) of 112 patients with definite Cryptococcal meningitis and 26 patients with tubercular meningitis and virus meningitis were collected, latex agglutination test is adopted to detect Cryptococcal capsular polysaccharide antigen. Then it was compared with fungal culture and direct microscopy method for evaluating the sensitivity and specificity of the diagnosis. The sensitivity of three methods including latex agglutination test, fungal culture and direct microscopy was 91.1%,69.6% and 73.2% respectively. The specificity of latex agglutination test was 96.0%, 100% and 100% respectively. That latex agglutination test to detect Cryptococcal capsular polysaccharide antigen could be taken as the early diagnostic method of Cryptococcus neoformans meningitis.

  3. Synthesis of oligosaccharides corresponding to structures found in capsular polysaccharides of Cryptococcus neoformans--II.

    PubMed

    Garegg, P J; Olsson, L; Oscarson, S

    1996-11-01

    Formula 1 depicts a generalized structure of the capsular polysaccharides of four serotypes of the opportunistic microorganism Cryptococcus neoformans, which appears as one of the major infections in the late stages of development of AIDS. Syntheses are now described of two tetrasaccharides with corresponding structures. These are methyl O-alpha-D-mannopyranosyl-(1-->3)-[O-beta-D-xylopyranosyl-(1-->2)] -O-alpha-D-mannopyranosyl-(1-->3)-alpha-D-mannopyranoside and methyl O-alpha-D-mannopyranosyl-(1-->3)-[O-beta-D-glucopyranosyluronic acid-(1-->2)]-O-alpha-D-mannopyranosyl-(1-->3)-alpha-D-mannopyranoside.

  4. A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection.

    PubMed

    Fleuridor, R; Lees, A; Pirofski, L

    2001-01-15

    Defined Abs to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been shown to be protective against experimental cryptococcosis. This suggests that if a vaccine could induce similar Abs it might protect against infection. However, the potential use of a GXM-based vaccine has been limited by evidence that GXM is a poor immunogen that can induce nonprotective and deleterious, as well as protective, Abs, and that the nature of GXM oligosaccharide epitopes that can elicit a protective response is unknown. In this study, we investigated whether a peptide surrogate for a GXM epitope could induce an Ab response to GXM in mice. The immunogenicity of peptide-protein conjugates produced by linking a peptide mimetic of GXM, P13, to either BSA, P13-BSA, or tetanus toxoid, P13-tetanus toxoid, was examined in BALB/c and CBA/n mice that received four s.c. injections of the conjugates at 14- to 30-day intervals. All mice immunized with conjugate produced IgM and IgG to P13 and GXM. Challenge of conjugate-immunized mice with C. neoformans revealed longer survival and lower serum GXM levels than control mice. These results indicate that 1) P13 is a GXM mimotope and 2) that it induced a protective response against C. neoformans in mice. P13 is the first reported mimotope of a C. neoformans Ag. Therefore, the P13 conjugates are vaccine candidates for C. neoformans and their efficacy in this study suggests that peptide mimotopes selected by protective Abs deserve further consideration as vaccine candidates for encapsulated pathogens.

  5. Killing of cryptococcus neoformans by Staphylococcus aureus: the role of cryptococcal capsular polysaccharide in the fungal-bacteria interaction.

    PubMed

    Saito, Fumito; Ikeda, Reiko

    2005-11-01

    Microbes compete for the environmental niche which is their host. To investigate the effects of a pathogenic bacterium on invasion and colonization by a pathogenic yeast, Cryptococcus neoformans was co-cultured with Staphylococcus aureus. We found that the number of colony forming units of C. neoformans was decreased by Staphylococcus aureus. In contrast, the viability of Candida albicans was not affected. Under the microscope, wild-type C. neoformans cells were shown to be surrounded by S. aureus, while cells of a capsuleless mutant of C. neoformans were not. C. neoformans was not killed when a membrane separated it from S. aureus in co-culture. Killing was confirmed by staining with cyanoditolyl tetrazolium chloride: S. aureus stained red, indicating viability, while C. neojormans did not stain, indicating lethality. The in situ terminal deoxynucleotidyl transferase-mediated dUTR nick end labeling (TUNEL) assay indicated cell death with fragmentation of DNA of C. neoformans. Capsular polysaccharide from C. neoformans inhibited the killing. Treatment of the crude polysaccharide with protease increased the inhibition. The protective activity resided in the glucuronoxylomannan (GXM) fraction, although the concentration required for the inhibition was high. These results suggest that S. aureus kills C. neoformans by a process that involves attachment to the cryptococcal capsule.

  6. Receptor-mediated clearance of Cryptococcus neoformans capsular polysaccharide in vivo.

    PubMed

    Yauch, Lauren E; Mansour, Michael K; Levitz, Stuart M

    2005-12-01

    Cryptococcus neoformans capsular glucuronoxylomannan (GXM) is shed during cryptococcosis and taken up by macrophages. The roles of the putative GXM receptors CD14, CD18, Toll-like receptor 2 (TLR2), and TLR4 in GXM clearance from serum and deposition in the liver and spleen in receptor-deficient mice were studied. While alterations in the kinetics of GXM redistribution were seen in the mutant mice, none of the receptors was absolutely required for serum clearance or hepatosplenic accumulation.

  7. Immunogenicity and efficacy of Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan peptide mimotope-protein conjugates in human immunoglobulin transgenic mice.

    PubMed

    Maitta, Robert W; Datta, Kausik; Lees, Andrew; Belouski, Shelley Sims; Pirofski, Liise-anne

    2004-01-01

    Peptide mimotopes of capsular polysaccharides have been proposed as antigens for vaccines against encapsulated pathogens. In this study, we determined the antibody response to and efficacy of P13, a peptide mimetic of the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM), in mice that produce human antibodies. P13 was conjugated to tetanus toxoid (TT) or diphtheria toxoid (DT) and administered subcutaneously in Alhydrogel with or without CpG to mice transgenic for human immunoglobulin loci (XenoMouse mice) and expressing either immunoglobulin G2 (IgG2) (G2 mice) or IgG4 (G4 mice). Mice were vaccinated and revaccinated two or three times. The serum antibody responses of the mice to GXM and P13 and antibody idiotype expression were analyzed by an enzyme-linked immunosorbent assay. The results showed that both P13-TT and P13-DT were antigenic, inducing a mimetic response to P13 in both G2 and G4 mice, and immunogenic, inducing a mimotope response including VH3 (idiotype)-positive antibodies to GXM in G2 but not G4 mice. CpG led to higher titers of IgG to P13 and GXM in P13-TT-vaccinated G2 mice. C. neoformans challenge of P13-protein conjugate-vaccinated and control G2 mice induced anamnestic IgG- and VH3-positive responses to GXM and was associated with a significantly decreased risk of death and a prolongation of survival in P13-DT-vaccinated mice compared to phosphate-buffered saline-treated or protein carrier-vaccinated mice. These findings reveal that P13 elicited a human antibody response with VH3 expression in human immunoglobulin transgenic mice that has been observed for human antibodies to GXM and support the concept that peptide mimotope-based vaccines may hold promise for the treatment of C. neoformans infections.

  8. Binding of purified and radioiodinated capsular polysaccharides from Cryptococcus neoformans serotype A strains to capsule-free mutants

    SciTech Connect

    Small, J.M.; Mitchell, T.G.

    1986-12-01

    Strains 6, 15, 98, 110, and 145 of Cryptococcus neoformans serotype A vary in capsule size, animal virulence, and susceptibility to in vitro phagocytosis. The isolated capsular polysaccharides (CPSs) differ in monosaccharide composition ratios and molecular size, as determined by gel filtration. The purpose of this investigation was to characterize the binding of CPSs to capsule-free mutants of C. neoformans and to examine CPSs from these strains for differences in their ability to bind, to determine whether such differences might explain the variation in the pathobiology of these strains. CPSs were partially periodate oxidized, tyraminated, iodinated with /sup 125/I, and used in binding studies with two capsule-free mutants of C. neoformans, strain 602 and Cap59. Binding was specific for yeast species and for polysaccharide and was saturable, which is consistent with a receptor-mediated mechanism of attachment. Binding occurred rapidly and was only slowly reversible. Binding was also independent of pH from pH 5.5 to 8, of cation concentrations, and of competition by sugars up to 1.0 M concentrations. Only a portion of CPS was capable of binding, and strains varied in the extent to which their CPS bound. CPS-15-IV (peak IV was the major polysaccharide peak on DEAE-cellulose chromatography of CPS from strain 15) had the highest proportion of binding (40%), followed by CPS from strains 98, 6, 145, 110, and 15-III (peak III was an earlier eluting fraction of CPS from strain 15). The CPSs differed similarly in their ability to competitively inhibit binding. Treatment of CPS, but not yeast cells, with proteinase XIV abolished binding without altering the CPS gross structure. Treatment of yeast cells with proteases, heat, or formaldehyde did not alter binding, and both strain 602 and Cap59 bound CPS similarly. Binding to encapsulated yeast cells was minimal.

  9. Variable Region Identical IgA and IgE to Cryptococcus neoformans Capsular Polysaccharide Manifest Specificity Differences*

    PubMed Central

    Janda, Alena; Eryilmaz, Ertan; Nakouzi, Antonio; Pohl, Mary Ann; Bowen, Anthony; Casadevall, Arturo

    2015-01-01

    In recent years several groups have shown that isotype switching from IgM to IgG to IgA can affect the affinity and specificity of antibodies sharing identical variable (V) regions. However, whether the same applies to IgE is unknown. In this study we compared the fine specificity of V region-identical IgE and IgA to Cryptococcus neoformans capsular polysaccharide and found that these differed in specificity from each other. The IgE and IgA paratopes were probed by nuclear magnetic resonance spectroscopy with 15N-labeled peptide mimetics of cryptococcal polysaccharide antigen (Ag). IgE was found to cleave the peptide at a much faster rate than V region-identical IgG subclasses and IgA, consistent with an altered paratope. Both IgE and IgA were opsonic for C. neoformans and protected against infection in mice. In summary, V-region expression in the context of the ϵ constant (C) region results in specificity changes that are greater than observed for comparable IgG subclasses. These results raise the possibility that expression of certain V regions in the context of α and ϵ C regions affects their function and contributes to the special properties of those isotypes. PMID:25778397

  10. Method for producing capsular polysaccharides

    NASA Technical Reports Server (NTRS)

    Kern, Roger G. (Inventor); Petersen, Gene R. (Inventor); Richards, Gil F. (Inventor)

    1994-01-01

    Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

  11. Synthesis of a Glucuronic Acid‐Containing Thioglycoside Trisaccharide Building Block and Its Use in the Assembly of Cryptococcus Neoformans Capsular Polysaccharide Fragments†

    PubMed Central

    Guazzelli, Lorenzo; Ulc, Rebecca

    2015-01-01

    Abstract As part of an ongoing project aimed at identifying protective capsular polysaccharide epitopes for the development of vaccine candidates against the fungal pathogen Cryptococcus neoformans, the synthesis and glycosylation properties of a naphthalenylmethyl (NAP) orthogonally protected trisaccharide thioglycoside, a common building block for construction of serotype B and C capsular polysaccharide structures, were investigated. Ethyl (benzyl 2,3,4‐tri‐O‐benzyl‐β‐d‐glucopyranosyl‐ uronate)‐(1→2)‐[2,3,4‐tri‐O‐benzyl‐β‐d‐xylopyranosyl‐(1→4)]‐6‐O‐benzyl‐3‐O‐(2‐naphthalenylmethyl)‐1‐thio‐α‐d‐mannopyranoside was prepared and used both as a donor and an acceptor in glycosylation reactions to obtain spacer equipped hexa‐ and heptasaccharide structures suitable either for continued elongation or for deprotection and printing onto a glycan array or conjugation to a carrier protein. The glycosylation reactions proceeded with high yields and α‐selectivity, proving the viability of the building block approach also for construction of 4‐O‐xylosyl‐containing C. neoformans CPS structures. PMID:27308199

  12. Monoclonal antibodies specific for immunorecessive epitopes of glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans, reduce serotype bias in an immunoassay for cryptococcal antigen.

    PubMed

    Percival, Ann; Thorkildson, Peter; Kozel, Thomas R

    2011-08-01

    Immunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to remove O-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population.

  13. Biosynthesis of UDP-GlcA, a key metabolite for capsular polysaccharide synthesis in the pathogenic fungus Cryptococcus neoformans

    PubMed Central

    2004-01-01

    UDP-glucose dehydrogenase catalyses the conversion of UDP-glucose into UDP-GlcA, a critical precursor for glycan synthesis across evolution. We have cloned the gene encoding this important enzyme from the opportunistic pathogen Cryptococcus neoformans. In this fungus, UDP-GlcA is required for the synthesis of capsule polysaccharides, which in turn are essential for virulence. The gene was expressed in Escherichia coli and the 51.3-kDa recombinant protein from wild-type and five mutants was purified for analysis. The cryptococcal enzyme is strongly inhibited by UDP-xylose and NADH, has highest activity at pH 7.5 and demonstrates Km (app) values of 0.1 and 1.5 mM for NAD+ and UDP-glucose respectively. Its activity was significantly decreased by mutations in the putative sites of NAD+ and UDP-glucose binding. Unlike previously reported eukaryotic UDP-glucose dehydrogenases, which are hexamers, the cryptococcal enzyme is a dimer. PMID:15030319

  14. CAPSULAR POLYSACCHARIDE OF AZOTOBACTER AGILIS.

    PubMed

    COHEN, G H; JOHNSTONE, D B

    1964-12-01

    Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone. Capsular polysaccharide of Azotobacter agilis. J. Bacteriol. 88:1695-1699. 1964.-Capsular polysaccharide from Azotobacter agilis strain 132 was recovered from washed cells by alkaline digestion. The polysaccharide was purified by centrifugation, repeated alcohol precipitation, Sevag deproteinization, and treatment with ribonuclease and charcoal-cellulose. Methods of isolation and purification appeared to provide a polymer showing no evidence of heterogeneity when examined by chemical and physical methods. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed polysaccharide indicated that the polymer contained galactose and rhamnose at a molar ratio of approximately 1.0:0.7. A sialic acid-like component was also present in the polysaccharide. The study shows significant differences in the chemical composition of the extra-cellular polysaccharide of A. agilis and that of A. vinelandii. This adds further biochemical evidence for the right of these species to independent status.

  15. Structurally altered capsular polysaccharides produced by mutant bacteria

    NASA Technical Reports Server (NTRS)

    Kern, Roger G. (Inventor); Petersen, Gene R. (Inventor); Richards, Gil F. (Inventor)

    1995-01-01

    Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

  16. Self-Aggregation of Cryptococcus neoformans Capsular Glucuronoxylomannan Is Dependent on Divalent Cations▿ ‡

    PubMed Central

    Nimrichter, Leonardo; Frases, Susana; Cinelli, Leonardo P.; Viana, Nathan B.; Nakouzi, Antonio; Travassos, Luiz R.; Casadevall, Arturo; Rodrigues, Marcio L.

    2007-01-01

    The capsular components of the human pathogen Cryptococcus neoformans are transported to the extracellular space and then used for capsule enlargement by distal growth. It is not clear, however, how the glucuronoxylomannan (GXM) fibers are incorporated into the capsule. In the present study, we show that concentration of C. neoformans culture supernatants by ultrafiltration results in the formation of highly viscous films containing pure polysaccharide, providing a novel, nondenaturing, and extremely rapid method to isolate extracellular GXM. The weight-averaged molecular mass of GXM in the film, determined using multiangle laser light scattering, was ninefold smaller than that of GXM purified from culture supernatants by differential precipitation with cetyl trimethyl ammonium bromide (CTAB). Polysaccharides obtained either by ultrafiltration or by CTAB-mediated precipitation showed different reactivities with GXM-specific monoclonal antibodies. Viscosity analysis associated with inductively coupled plasma mass spectrometry and measurements of zeta potential in the presence of different ions implied that polysaccharide aggregation was a consequence of the interaction between the carboxyl groups of glucuronic acid and divalent cations. Consistent with this observation, capsule enlargement in living C. neoformans cells was influenced by Ca2+ in the culture medium. These results suggest that capsular assembly in C. neoformans results from divalent cation-mediated self-aggregation of extracellularly accumulated GXM molecules. PMID:17573547

  17. Capsule of Cryptococcus neoformans grows by enlargement of polysaccharide molecules

    PubMed Central

    Frases, Susana; Pontes, Bruno; Nimrichter, Leonardo; Viana, Nathan B.; Rodrigues, Marcio L.; Casadevall, Arturo

    2009-01-01

    The human pathogenic fungus Cryptococcus neoformans has a distinctive polysaccharide (PS) capsule that enlarges during infection. The capsule is essential for virulence, but the mechanism for capsular growth is unknown. In the present study, we used dynamic light scattering (LS) analysis of capsular PS and optical tweezers (OT) to explore the architecture of the capsule. Analysis of capsular PS from cells with small and large capsules by dynamic LS revealed a linear correlation between PS effective diameter and microscopic capsular diameter. This result implied that capsule growth was achieved by the addition of molecules with larger effective diameter, such that some molecules can span the entire diameter of the capsule. Measurement of polystyrene bead penetration of C. neoformans capsules by using OT techniques revealed that the outer regions were penetrable, but not the inner regions. Our results provide a mechanism for capsular enlargement based on the axial lengthening of PS molecules and suggest a model for the architecture of a eukaryotic microbial capsule. PMID:19164571

  18. Neisseria meningitidis serogroup A capsular polysaccharide acetyltransferase, methods and compositions

    DOEpatents

    Stephens, David S [Stone Mountain, GA; Gudlavalleti, Seshu K [Kensington, MD; Tzeng, Yih-Ling [Atlanta, GA; Datta, Anup K [San Diego, CA; Carlson, Russell W [Athens, GA

    2011-02-08

    Provided are methods for recombinant production of an O-acetyltransferase and methods for acetylating capsular polysaccharides, especially those of a Serogroup A Neisseria meningitidis using the recombinant O-acetyltransferase, and immunogenic compositions comprising the acetylated capsular polysaccharide.

  19. Cellular immunity to Bacteroides fragilis capsular polysaccharide

    PubMed Central

    1982-01-01

    The polysaccharide capsule of Bacteroides fragilis has been shown to be important in the virulence of the organism. The capsular polysaccharide (CP) of B. fragilis has been extensively purified. Using a murine model of intraabdominal abscess formation, we have been able to demonstrate cellular immunity to the capsular polysaccharide of B. fragilis. Immunization of C57BL/10J mice with the CP over 5 wk prevents abscess formation when the mice are challenged with B. fragilis intraperitoneally. This immunity can be transferred to naive mice with spleen cells from immune animals. The immune cells bear Thy-1.2 and Ly- 2.2 antigens. The immune response has been shown to be antigen specific, but not H-2 restricted. The possibility that these immune cells are suppressor T cells is discussed. The experimental system presented provides a model for the examination of the cellular interactions responsible for abscess formation and the cellular response to bacterial pathogens. PMID:6174672

  20. Immunogenic properties of Klebsiella pneumoniae type 2 capsular polysaccharide.

    PubMed Central

    Robert, A; Jouin, H; Fournier, J M

    1986-01-01

    The immunoprotective activity of Klebsiella pneumoniae K2 cell surface preparations and purified capsular polysaccharide was tested in mice. The 50% protective dose (PD50), expressed as capsular polysaccharide content, was 2 ng for cell surface preparations and 50 ng for purified capsular polysaccharide. Both preparations lost their immunoprotective activity after alkali treatment. Immune sera were raised in rabbits immunized with cell surface preparations. The precipitating and hemagglutinating capacity of these antisera was tested against either purified capsular polysaccharide or alkali-treated capsular polysaccharide. No difference was observed between the reactivity of the antisera against each antigen. The protective activity of these sera was tested on mice in passive transfer experiments, before and after absorption with either purified capsular polysaccharide or alkali-treated capsular polysaccharide. The sera lost their protective activity after absorption with purified capsular polysaccharide and after absorption with alkali-treated capsular polysaccharide. These experiments show that the difference in immunoprotective activity of cell surface preparations, purified capsular polysaccharide, and alkali-treated capsular polysaccharide is not due to a difference in their antigenic determinants. Cell surface preparations and purified capsular polysaccharide were fractionated by gel filtration on Sepharose 4B and by ultracentrifugation on cesium chloride density gradients. Three forms of capsular polysaccharide have been characterized. (i) A form of capsular polysaccharide with a very high protective activity (PD50 = 2 ng) that copurified with protein and lipopolysaccharide and was characterized by a low coefficient of distribution (Kd = 0.20) and a low density (1.5 to 1.6 g/cm3). (ii) A form of capsular polysaccharide with an intermediate protective activity (PD50 = 50 ng), contamined by less than 3% protein and 1% lipopolysaccharide, with a Kd of 0.35, and

  1. Antibody Binding to Cryptococcus neoformans Impairs Budding by Altering Capsular Mechanical Properties

    PubMed Central

    Cordero, Radames J. B.; Pontes, Bruno; Frases, Susana; Nakouzi, Antonio S.; Nimrichter, Leonardo; Rodrigues, Marcio L.; Viana, Nathan B.

    2013-01-01

    Abs to microbial capsules are critical for host defense against encapsulated pathogens, but very little is known about the effects of Ab binding on the capsule, apart from producing qualitative capsular reactions (“quellung” effects). A problem in studying Ab–capsule interactions is the lack of experimental methodology, given that capsules are fragile, highly hydrated structures. In this study, we pioneered the use of optical tweezers microscopy to study Ab–capsule interactions. Binding of protective mAbs to the capsule of the fungal pathogen Cryptococcus neoformans impaired yeast budding by trapping newly emerging buds inside the parental capsule. This effect is due to profound mAb-mediated changes in capsular mechanical properties, demonstrated by a concentration-dependent increase in capsule stiffness. This increase involved mAb-mediated cross-linking of capsular polysaccharide molecules. These results provide new insights into Ab-mediated immunity, while suggesting a new nonclassical mechanism of Ab function, which may apply to other encapsulated pathogens. Our findings add to the growing body of evidence that Abs have direct antimicrobial functions independent of other components of the immune system. PMID:23233725

  2. Masquerading microbial pathogens: Capsular polysaccharides mimic host-tissue molecules

    PubMed Central

    Cress, Brady F.; Englaender, Jacob A.; He, Wenqin; Kasper, Dennis; Linhardt, Robert J.; Koffas, Mattheos A. G.

    2014-01-01

    Summary Bacterial pathogens bearing capsular polysaccharides identical to mammalian glycans benefit from an additional level of protection from host immune response. The increasing prevalence of antibiotic resistant bacteria portends an impending post-antibiotic age, characterized by diminishing efficacy of common antibiotics and routine application of multifaceted, complementary therapeutic approaches to treat bacterial infections, particularly multidrug-resistant organisms. The first line of defense for most bacterial pathogens consists of a physical and immunological barrier known as the capsule, commonly composed of a viscous layer of carbohydrates that are covalently bound to the cell wall in Gram-positive bacteria or often to lipids of the outer membrane in many Gram-negative bacteria. Bacterial capsular polysaccharides are a diverse class of high molecular weight polysaccharides contributing to virulence of many human pathogens in the gut, respiratory tree, urinary tract, and other host tissues, by hiding cell-surface components that might otherwise elicit host immune response. This review highlights capsular polysaccharides that are structurally identical or similar to polysaccharides found in mammalian tissues, including polysialic acid and glycosaminoglycan capsules hyaluronan, heparosan, and chondroitin. Such non-immunogenic coatings render pathogens insensitive to certain immune responses, effectively increasing residence time in host tissues and enabling pathologically relevant population densities to be reached. Biosynthetic pathways and capsular involvement in immune system evasion are described providing a basis for potential therapies aimed at supplementing or replacing antibiotic treatment. PMID:24372337

  3. Evidence for Branching in Cryptococcal Capsular Polysaccharides and Consequences on its Biological Activity

    PubMed Central

    Cordero, Radames J.B.; Frases, Susana; Guimaräes, Allan J.; Rivera, Johanna; Casadevall, Arturo

    2011-01-01

    SUMMARY The encapsulated fungus Cryptococcus neoformans is a common cause of life-threatening disease in immunocompromised individuals. Its major virulence determinant is the polysaccharide (PS) capsule. An unsolved problem in cryptococcal biology is whether the PSs composing the capsule are linear or complex branched polymers, as well as the implications of this structural composition in pathogenesis. In this study we approached the problem by combining static and dynamic light scattering, viscosity analysis, and high-resolution microscopy and correlated the findings with biological properties. Analysis of the dependence of capsular PS molecular mass and the radius of gyration provided strong evidence against a simple linear PS configuration. Shape factors calculated from light scattering measurements in solution revealed values consistent with polymer branching. Furthermore, viscosity measurements provided complementary evidence for structural branching. Electron microscopy showed PS spherical-like structures similar to other branched PS. Finally, we show that the capacity of capsular PS to interfere in complement-mediated phagocytosis, inhibit nitric oxide production by macrophage-like cells, protect against reactive oxygen species, antibody reactivity and half-life in serum were influenced by the degree of branching, providing evidence for the notion that PS branching is an important parameter in determining the biological activity of C. neoformans PS. PMID:21208301

  4. Evidence for branching in cryptococcal capsular polysaccharides and consequences on its biological activity.

    PubMed

    Cordero, Radames J B; Frases, Susana; Guimaräes, Allan J; Rivera, Johanna; Casadevall, Arturo

    2011-02-01

    The encapsulated fungus Cryptococcus neoformans is a common cause of life-threatening disease in immunocompromised individuals. Its major virulence determinant is the polysaccharide (PS) capsule. An unsolved problem in cryptococcal biology is whether the PSs composing the capsule are linear or complex branched polymers, as well as the implications of this structural composition in pathogenesis. In this study we approached the problem by combining static and dynamic light scattering, viscosity analysis, and high-resolution microscopy and correlated the findings with biological properties. Analysis of the dependence of capsular PS molecular mass and the radius of gyration provided strong evidence against a simple linear PS configuration. Shape factors calculated from light scattering measurements in solution revealed values consistent with polymer branching. Furthermore, viscosity measurements provided complementary evidence for structural branching. Electron microscopy showed PS spherical-like structures similar to other branched PS. Finally, we show that the capacity of capsular PS to interfere in complement-mediated phagocytosis, inhibit nitric oxide production by macrophage-like cells, protect against reactive oxygen species, antibody reactivity and half-life in serum were influenced by the degree of branching, providing evidence for the notion that PS branching is an important parameter in determining the biological activity of C. neoformans PS.

  5. Finite-element model of interaction between fungal polysaccharide and monoclonal antibody in the capsule of Cryptococcus neoformans

    PubMed Central

    Rakesh, Vineet; Schweitzer, Andrew D.; Zaragoza, Oscar; Bryan, Ruth; Wong, Kevin; Datta, Ashim; Casadevall, Arturo; Dadachova, Ekaterina

    2008-01-01

    Many microorganisms such as bacteria and fungi possess so-called capsules made of polysaccharides which protect these microorganisms from environmental insults and host immune defenses. The polysaccharide capsule of C. neoformans, a human pathogenic yeast, is capable of self assembly, composed mostly of glucuronoxylomannan (GXM), a polysaccharide with molecular weight of approximately 2,000,000 Da and has several layers with different densities. The objective of this study was to model pore-hindered diffusion and binding of the GXM-specific antibody within the C. neoformans capsule. Using the finite element method (FEM), we created a model which represents the in vivo binding of GXM-specific antibody to a C. neoformans cell taking into account the intravenous infusion time of antibody, antibody diffusion through capsular pores, and Michaelis-Menten kinetics of antibody binding to capsular GXM. The model predicted rapid diffusion of antibody to all regions of the capsule where pore size was greater than the Stokes diameter of the antibody. Binding occurred primarily at intermediate regions of the capsule. The GXM concentration in each capsular region was the principal determinant of the steady-state antibody-GXM complex concentration, while the forward binding rate constant influenced the rate of complex formation in each region. The concentration profiles predicted by the model closely matched experimental immunofluorescence data. Inclusion of different antibody isotypes (IgG, IgA and IgM) into the modeling algorithm resulted in similar complex formation in outer capsular regions, but different depth of binding at inner regions. These results have implications for the development of new antibody-based therapies. PMID:18588334

  6. Roles of Lipooligosaccharide and Capsular Polysaccharide in Antimicrobial Resistance and Natural Transformation of Campylobacter jejuni

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives: To investigate the roles of surface polysaccharides, such as capsular polysaccharide (CPS) and lipooligosaccharide (LOS), in modulating natural transformation and antimicrobial resistance in Campylobacter jejuni. Methods: A series of C. jejuni mutants, which are defective in either CPS ...

  7. Genetics of Capsular Polysaccharides and Cell Envelope (Glyco)lipids

    PubMed Central

    Daffé, Mamadou; Crick, Dean C.; Jackson, Mary

    2014-01-01

    This chapter summarizes what is currently known of the structures, physiological roles, involvement in pathogenicity and biogenesis of a variety of non-covalently bound cell envelope lipids and glycoconjugates of Mycobacterium tuberculosis and other Mycobacterium species. Topics addressed in this chapter include phospholipids; phosphatidylinositol mannosides; triglycerides; isoprenoids and related compounds (polyprenyl phosphate, menaquinones, carotenoids, non-carotenoid cyclic isoprenoids); acyltrehaloses (lipooligosaccharides, trehalose mono- and di-mycolates, sulfolipids, di- and poly-acyltrehaloses); mannosyl-beta-1-phosphomycoketides; glycopeptidolipids; phthiocerol dimycocerosates, para-hydroxybenzoic acids and phenolic glycolipids; mycobactins; mycolactones; and capsular polysaccharides. PMID:25485178

  8. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

  9. Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

    PubMed Central

    Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

    2001-01-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  10. Vaccination with peptide mimotopes produces antibodies recognizing bacterial capsular polysaccharides.

    PubMed

    Wu, Yang; Zhang, Qibo; Sales, Debra; Bianco, Albert Edward; Craig, Alister

    2010-09-07

    A phage display peptide library was screened using a panel of antibodies to the capsular polysaccharides of Streptococcus agalactiae and Neisseria meningitidis. Mimotopes NPDHPRVPTFMA (2-8), LIPFHKHPHHRG (3-2) and EQEIFTNITDRV (G3) showing the highest binding capacity and strongest ELISA reaction were selected for immunization experiments. These mimotopes were either synthesised as oligodeoxynucleotides for DNA immunization or MAP (multiple antigen peptide) for peptide immunization. Mimotope-DNA vaccination, particularly for G3, induced antibodies recognizing a number of target bacteria. This response was seen after the second boost injection and was significantly enhanced by the 3rd boost injection with a Th1-associated profile, which was dominated by IgG2a, followed by IgG1. Mimotope-MAP immunization also produced strong humoral immune responses to the bacteria. Antibodies from G3 DNA immunization reacted with the surface molecules of S. agalactiae, N. meningitidis and Escherichia coli K5 shown by indirect immunofluorescence staining, indicating a possible localization to the bacterial capsule. Antibodies produced both from DNA/MAP immunization reacted with purified bacterial capsular polysaccharides by ELISA and were of high avidity. We have further characterized peptide G3 by a 'tiling path' study to examine the effect of changing individual residues in the peptide in raising antibodies, which showed that the EIFTN motif in G3 was important in generating antibodies to several capsulated bacteria. We conclude that mimotope immunization with DNA or MAP potentially induces strong antibody responses against encapsulated bacteria. It is suggested that the antibody targets are polysaccharides, and these antibodies may cross react at least among closely related species of bacteria.

  11. Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli

    PubMed Central

    Kay, Emily J.; Yates, Laura E.; Terra, Vanessa S.; Cuccui, Jon; Wren, Brendan W.

    2016-01-01

    Currently, Streptococcus pneumoniae is responsible for over 14 million cases of pneumonia worldwide annually, and over 1 million deaths, the majority of them children. The major determinant for pathogenesis is a polysaccharide capsule that is variable and is used to distinguish strains based on their serotype. The capsule forms the basis of the pneumococcal polysaccharide vaccine (PPV23) that contains purified capsular polysaccharide from 23 serotypes, and the pneumococcal conjugate vaccine (PCV13), containing 13 common serotypes conjugated to CRM197 (mutant diphtheria toxin). Purified capsule from S. pneumoniae is required for pneumococcal conjugate vaccine production, and costs can be prohibitively high, limiting accessibility of the vaccine in low-income countries. In this study, we demonstrate the recombinant expression of the capsule-encoding locus from four different serotypes of S. pneumoniae within Escherichia coli. Furthermore, we attempt to identify the minimum set of genes necessary to reliably and efficiently express these capsules heterologously. These E. coli strains could be used to produce a supply of S. pneumoniae serotype-specific capsules without the need to culture pathogenic bacteria. Additionally, these strains could be applied to synthetic glycobiological applications: recombinant vaccine production using E. coli outer membrane vesicles or coupling to proteins using protein glycan coupling technology. PMID:27110302

  12. A Monoclonal Antibody to Cryptococcus neoformans Glucuronoxylomannan Manifests Hydrolytic Activity for Both Peptides and Polysaccharides.

    PubMed

    Bowen, Anthony; Wear, Maggie P; Cordero, Radames J B; Oscarson, Stefan; Casadevall, Arturo

    2017-01-13

    Studies in the 1980s first showed that some natural antibodies were "catalytic" and able to hydrolyze peptide or phosphodiester bonds in antigens. Many naturally occurring catalytic antibodies have since been isolated from human sera and associated with positive and negative outcomes in autoimmune disease and infection. The function and prevalence of these antibodies, however, remain unclear. A previous study suggested that the 18B7 monoclonal antibody against glucuronoxylomannan (GXM), the major component of the Cryptococcus neoformans polysaccharide capsule, hydrolyzed a peptide antigen mimetic. Using mass spectrometry and Förster resonance energy transfer techniques, we confirm and characterize the hydrolytic activity of 18B7 against peptide mimetics and show that 18B7 is able to hydrolyze an oligosaccharide substrate, providing the first example of a naturally occurring catalytic antibody for polysaccharides. Additionally, we show that the catalytic 18B7 antibody increases release of capsular polysaccharide from fungal cells. A serine protease inhibitor blocked peptide and oligosaccharide hydrolysis by 18B7, and a putative serine protease-like active site was identified in the light chain variable region of the antibody. An algorithm was developed to detect similar sites present in unique antibody structures in the Protein Data Bank. The putative site was found in 14 of 63 (22.2%) catalytic antibody structures and 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. The ability of many antibodies to cleave antigen, albeit slowly, supports the notion that this activity is an important immunoglobulin function in host defense. The discovery of GXM hydrolytic activity suggests new therapeutic possibilities for polysaccharide-binding antibodies.

  13. Cryptococcus neoformans var. grubii: Separate Varietal Status for Cryptococcus neoformans Serotype A Isolates

    PubMed Central

    Franzot, Sarah P.; Salkin, Ira F.; Casadevall, Arturo

    1999-01-01

    Cryptococcus neoformans var. neoformans presently includes isolates which have been determined by the immunologic reactivity of their capsular polysaccharides to be serotype A and those which have been determined to be serotype D. However, recent analyses of the URA5 sequences and DNA fingerprinting patterns suggest significant genetic differences between the two serotypes. Therefore, we propose to recognize these genotypic distinctions, as well as previously reported phenotypic differences, by restricting C. neoformans var. neoformans to isolates which are serotype D and describing a new variety, C. neoformans var. grubii, for serotype A isolates. PMID:9986871

  14. Overproduction of Type 8 Capsular Polysaccharide Augments Staphylococcus aureus Virulence

    PubMed Central

    Luong, Thanh T.; Lee, Chia Y.

    2002-01-01

    Type 8 capsular polysaccharide (CP8) is the most prevalent capsule type in clinical isolates of Staphylococcus aureus. However, its role in virulence has not been clearly defined. CP8 strains such as strain Becker produce a small amount of capsule on their surface in vitro. In contrast, CP1 strains such as strain M produce a large amount of capsule, which has been shown to be an important antiphagocytic virulence factor. The cap8 and cap1 operons, required for the synthesis of CP8 and CP1, respectively, have been cloned and sequenced. To test whether CP8 contributes to the pathogenesis of S. aureus, we replaced the weak native promoter of the cap8 operon in strain Becker with the strong constitutive promoter of the cap1 operon of strain M. The resultant strain, CYL770, synthesized cap8-specific mRNA at a level about sevenfold higher than that in the parent strain. Remarkably, the CYL770 strain produced about 80-fold more CP8. In a mouse infection model of bacteremia, the CP8-overproducing strain persisted longer in the bloodstream, the liver, and the spleen in mice than the parent strain. In addition, strain CYL770 was more resistant to ospsonophagocytosis in vitro by human polymorphonuclear leukocytes. These results indicate that CP8 is an antiphagocytic virulence factor of S. aureus. PMID:12065477

  15. Therapeutic efficacy of a conjugate vaccine containing a peptide mimotope of cryptococcal capsular polysaccharide glucuronoxylomannan.

    PubMed

    Datta, Kausik; Lees, Andrew; Pirofski, Liise-anne

    2008-08-01

    Vaccination with P13, a peptide mimotope of the cryptococcal capsular polysaccharide glucuronoxylomannan (GXM), has been shown to confer protection against a subsequent lethal Cryptococcus neoformans challenge. In this study, we sought to investigate whether P13-based vaccines could be effective in an already-established infection. To address this question, we developed a systemic chronic cryptococcal infection model. We vaccinated chronically infected mice with P13-protein conjugates and monitored their survival. Compared to the controls, the conjugates prolonged the survival of chronically infected mice. The degree of protection was a function of the mouse strain (BALB/c or C57BL/6), the carrier protein (tetanus toxoid or diphtheria toxoid), and the route of infection (intraperitoneal or intravenous). Serum GXM levels were correlated with the day of death, but the correlation was driven by the carrier protein and mouse strain. The passive transfer of heat-treated sera from P13 conjugate-vaccinated mice conferred protection to naïve BALB/c mice, indicating that antibody immunity could contribute to protection. The measurement of peripheral blood cytokine (gamma interferon [IFN-gamma], interleukin-10 [IL-10], and IL-6) gene expression showed that P13 conjugate-vaccinated BALB/c and C57BL/6 mice mounted a strong Th2 (IL-10)-like response relative to the Th1 (IFN-gamma)-like response, with the degree depending on the mouse strain and carrier protein. Taken together, our data suggest that a vaccine could hold promise in the setting of chronic cryptococcosis, and that vaccine efficacy could depend on immunomodulation and augmentation of the natural immune response of the host.

  16. Nonencapsulated Variant of Cryptococcus neoformans I. Virulence Studies and Characterization of Soluble Polysaccharide

    PubMed Central

    Kozel, Thomas R.; Cazin, John

    1971-01-01

    A weakly virulent nonencapsulated variant of Cryptococcus neoformans is described. The chemical structure and antigenicity of the soluble polysaccharides produced by the variant strain and a typical virulent strain were compared. The soluble polysaccharides produced by both strains were composed of the same constituent monosaccharides; however, the virulent strain produced a polysaccharide having a greater uronic acid content and a larger molecular size than that of the variant strain. Soluble polysaccharides from the two strains are not closely related immunologically. Soluble polysaccharide obtained from the virulent strain did not affect persistence of the variant strain in mice. PMID:16557967

  17. Maternal group B streptococcal immunization: capsular polysaccharide (CPS)-based vaccines and their implications on prevention.

    PubMed

    Palmeiro, Jussara K; De Carvalho, Newton S; Botelho, Ana C N; Fracalanzza, Sérgio E L; Madeira, Humberto M F; Dalla-Costa, Libera M

    2011-05-12

    Group B streptococcal (GBS) capsular polysaccharide (CPS)-based conjugate vaccine, which includes types Ia, Ib, II, III, and V, could potentially prevent neonatal, pediatric, adult, and pregnancy-associated diseases. However, since GBS CPS types included in that vaccine are prevalent serotypes found in North America and Europe, it may not provide the necessary protection for individuals in countries in which other capsular types have been found.

  18. Pneumococcus with the "6E" cps Locus Produces Serotype 6B Capsular Polysaccharide.

    PubMed

    Burton, Robert L; Geno, K Aaron; Saad, Jamil S; Nahm, Moon H

    2016-04-01

    Genetic studies of serogroup 6 isolates ofStreptococcus pneumoniaeidentified putative serotype 6E. Although its capsular polysaccharide structure has not been elucidated, putative serotype 6E is described in an increasing number of studies as a potentially new serotype. We show here that SPEC6B, which is widely used as a target strain for serotype 6B opsonophagocytosis assays, has the genetic features of the putative serotype 6E but produces capsular polysaccharide identical to 6B capsular polysaccharide as determined by one-dimensional (1D) and 2D nuclear magnetic resonance (NMR). Thus, putative serotype 6E is a mere genetic variant of serotype 6B. Also, SPEC6B is appropriate as a target strain for serotype 6B opsonophagocytosis assays. This example illustrates the difficulties of assigning new bacterial serotypes based on genetic findings alone.

  19. Structural analysis of the capsular polysaccharide from Campylobacter jejuni RM1221

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete genome of Campylobacter jejuni strain RM1221 (Penner serotype HS:53) was reported recently and contains a novel capsular polysaccharide (CPS) biosynthesis locus. Cell surface carbohydrates such as CPS are known to be important for bacterial survival and often contribute to pathogenesis....

  20. Capsular Polysaccharide Expression in Commensal Streptococcus Species: Genetic and Antigenic Similarities to Streptococcus pneumoniae

    PubMed Central

    Skov Sørensen, Uffe B.; Yao, Kaihu; Yang, Yonghong; Tettelin, Hervé

    2016-01-01

    ABSTRACT Expression of a capsular polysaccharide is considered a hallmark of most invasive species of bacteria, including Streptococcus pneumoniae, in which the capsule is among the principal virulence factors and is the basis for successful vaccines. Consequently, it was previously assumed that capsule production distinguishes S. pneumoniae from closely related commensals of the mitis group streptococci. Based on antigenic and genetic analyses of 187 mitis group streptococci, including 90 recognized serotypes of S. pneumoniae, we demonstrated capsule production by the Wzy/Wzx pathway in 74% of 66 S. mitis strains and in virtually all tested strains of S. oralis (subspecies oralis, dentisani, and tigurinus) and S. infantis. Additional analyses of genomes of S. cristatus, S. parasanguinis, S. australis, S. sanguinis, S. gordonii, S. anginosus, S. intermedius, and S. constellatus revealed complete capsular biosynthesis (cps) loci in all strains tested. Truncated cps loci were detected in three strains of S. pseudopneumoniae, in 26% of S. mitis strains, and in a single S. oralis strain. The level of sequence identities of cps locus genes confirmed that the structural polymorphism of capsular polysaccharides in S. pneumoniae evolved by import of cps fragments from commensal Streptococcus species, resulting in a mosaic of genes of different origins. The demonstrated antigenic identity of at least eight of the numerous capsular polysaccharide structures expressed by commensal streptococci with recognized serotypes of S. pneumoniae raises concerns about potential misidentifications in addition to important questions concerning the consequences for vaccination and host-parasite relationships both for the commensals and for the pathogen. PMID:27935839

  1. Unusual galactofuranose modification of a capsule polysaccharide in the pathogenic yeast Cryptococcus neoformans.

    PubMed

    Heiss, Christian; Skowyra, Michael L; Liu, Hong; Klutts, J Stacey; Wang, Zhirui; Williams, Matthew; Srikanta, Deepa; Beverley, Stephen M; Azadi, Parastoo; Doering, Tamara L

    2013-04-19

    Galactofuranose (Galf) is the five-membered ring form of galactose. Although it is absent from mammalian glycans, it occurs as a structural and antigenic component of important cell surface molecules in a variety of microbes, ranging from bacteria to parasites and fungi. One such organism is Cryptococcus neoformans, a pathogenic yeast that causes lethal meningoencephalitis in immunocompromised individuals, particularly AIDS patients. C. neoformans is unique among fungal pathogens in bearing a complex polysaccharide capsule, a critical virulence factor reported to include Galf. Notably, how Galf modification contributes to the structure and function of the cryptococcal capsule is not known. We have determined that Galf is β1,2-linked to an unusual tetrasubstituted galactopyranose of the glucuronoxylomannogalactan (GXMGal) capsule polysaccharide. This discovery fills a longstanding gap in our understanding of a major polymer of the cryptococcal capsule. We also engineered a C. neoformans strain that lacks UDP-galactopyranose mutase; this enzyme forms UDP-Galf, the nucleotide sugar donor required for Galf addition. Mutase activity was required for the incorporation of Galf into glucuronoxylomannogalactan but was dispensable for vegetative growth, cell integrity, and virulence in a mouse model.

  2. Intracellular Fate of Cryptococcus neoformans

    PubMed Central

    Tacker, J. R.; Farhi, F.; Bulmer, G. S.

    1972-01-01

    Human peripheral leukocytes were found to engulf and kill cells of Cryptococcus neoformans. Fewer encapsulated than nonencapsulated cells met this fate, since cryptococcal capsular polysaccharide inhibited phagocytosis. During 10 to 12 hr of incubation of nonencapsulated cells in human serum, sufficient polysaccharide was produced to inhibit phagocytosis by 50%. The polysaccharide inhibitor was found in the sera of four patients with cryptococcosis, but not on the surfaces of their leukocytes. Additional experiments indicated that serum is not essential for effective phagocytosis. However, normal human serum contains anticryptococcal activity which is not inhibited by capsular material. Preliminary findings indicate that the phagocytic index of a patient with cryptococcosis may be correlated with the severity of his disease. PMID:4569916

  3. Capsular polysaccharide of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, and its modification with phosphorylcholine.

    PubMed

    Shi, Fang; Harada, Tomoyuki; Ogawa, Yohsuke; Ono, Hiroshi; Ohnishi-Kameyama, Mayumi; Miyamoto, Toru; Eguchi, Masahiro; Shimoji, Yoshihiro

    2012-11-01

    The capsule has been implicated in the virulence of the swine pathogen Erysipelothrix rhusiopathiae, a rod-shaped, intracellular Gram-positive bacterium that has a unique phylogenetic position in the phylum Firmicutes and is a close relative of Mollicutes (mycoplasma species). In this study, we analyzed the genetic locus and composition of the capsular polysaccharide (CPS) of the Fujisawa strain of E. rhusiopathiae. Genome analysis of the Fujisawa strain revealed that the genetic locus for capsular polysaccharide synthesis (cps) is located next to an lic operon, which is involved in the incorporation and expression of phosphorylcholine (PCho). Reverse transcription-PCR analysis showed that cps and lic are transcribed as a single mRNA, indicating that the loci form an operon. Using the cell surface antigen-specific monoclonal antibody (MAb) ER21 as a probe, the capsular materials were isolated from the Fujisawa strain by hot water extraction and treatment with DNase, RNase, pronase, and N-acetylmuramidase SG, followed by anion-exchange and gel filtration chromatography. The materials were then analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The CPS of E. rhusiopathiae is heterogeneous and consists of the major monosaccharides galacturonic acid, galactose, mannose, glucose, arabinose, xylose, and N-acetylglucosamine and some minor monosaccharides containing ribose, rhamnose, and N-acetylgalactosamine. In addition, the capsule is modified by PCho, which comigrates with the capsular materials, as determined by Western immunoblotting, and colocalizes on the cell surface, as determined by immunogold electron microscopy. Virulence testing of PCho-defective mutants in mice demonstrated that PCho is critical for the virulence of this organism.

  4. Capsular Polysaccharide of Erysipelothrix rhusiopathiae, the Causative Agent of Swine Erysipelas, and Its Modification with Phosphorylcholine

    PubMed Central

    Shi, Fang; Harada, Tomoyuki; Ogawa, Yohsuke; Ono, Hiroshi; Ohnishi-Kameyama, Mayumi; Miyamoto, Toru; Eguchi, Masahiro

    2012-01-01

    The capsule has been implicated in the virulence of the swine pathogen Erysipelothrix rhusiopathiae, a rod-shaped, intracellular Gram-positive bacterium that has a unique phylogenetic position in the phylum Firmicutes and is a close relative of Mollicutes (mycoplasma species). In this study, we analyzed the genetic locus and composition of the capsular polysaccharide (CPS) of the Fujisawa strain of E. rhusiopathiae. Genome analysis of the Fujisawa strain revealed that the genetic locus for capsular polysaccharide synthesis (cps) is located next to an lic operon, which is involved in the incorporation and expression of phosphorylcholine (PCho). Reverse transcription-PCR analysis showed that cps and lic are transcribed as a single mRNA, indicating that the loci form an operon. Using the cell surface antigen-specific monoclonal antibody (MAb) ER21 as a probe, the capsular materials were isolated from the Fujisawa strain by hot water extraction and treatment with DNase, RNase, pronase, and N-acetylmuramidase SG, followed by anion-exchange and gel filtration chromatography. The materials were then analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The CPS of E. rhusiopathiae is heterogeneous and consists of the major monosaccharides galacturonic acid, galactose, mannose, glucose, arabinose, xylose, and N-acetylglucosamine and some minor monosaccharides containing ribose, rhamnose, and N-acetylgalactosamine. In addition, the capsule is modified by PCho, which comigrates with the capsular materials, as determined by Western immunoblotting, and colocalizes on the cell surface, as determined by immunogold electron microscopy. Virulence testing of PCho-defective mutants in mice demonstrated that PCho is critical for the virulence of this organism. PMID:22949554

  5. CUTANEOUS REACTIONS IN RABBITS TO THE TYPE-SPECIFIC CAPSULAR POLYSACCHARIDES OF PNEUMOCOCCUS

    PubMed Central

    Francis, Thomas; Tillett, William S.

    1931-01-01

    The injection of the type-specific capsular polysaccharides of Pneumococcus Types I, II and III into the skin of rabbits, actively or passively immunized to one of these types of Pneumococcus, elicits a type-specific cutaneous reaction. The form of reaction resembles that described by Arthus. The reaction is produced only when type-specific precipitins for the homologous polysaccharide are demonstrable in the blood of the rabbit. In 84 per cent of actively immunized rabbits, the serum of which contained type-specific precipitins, a reaction was elicited. A positive result was obtained in 100 per cent of rabbits passively immunized with antipneumococcus horse serum whereas, attempts passively to transfer reactivity from immune rabbit to normal rabbit were unsuccessful. The recipients, in the latter group, possessed no demonstrable circulating type-specific precipitins. The reaction produced by specific capsular carbohydrates is always associated with a well grounded type-specific immunity. A brief summary of the relation of hypersensitiveness and immunity to pneumococcus is given. PMID:19869942

  6. High affinity mimotope of the polysaccharide capsule of Cryptococcus neoformans identified from an evolutionary phage peptide library.

    PubMed

    Beenhouwer, David O; May, Rena J; Valadon, Philippe; Scharff, Matthew D

    2002-12-15

    Cryptococcus neoformans causes a life-threatening meningoencephalitis in a significant percentage of AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid (TT) produce Abs that, based on the epitope recognized, can be either protective or nonprotective. Since nonprotective Abs block the efficacy of protective Abs, we are interested in developing a vaccine that would focus the immune response specifically to protective epitopes. Previously, we screened a phage display library with 2H1, a protective anti-GXM mAb, and isolated PA1, a representative peptide that had a K(d) of 295 nM for 2H1. Mice immunized with PA1 conjugated to keyhole limpet hemocyanin developed high anti-peptide (1/13,000), but low anti-GXM (maximum, 1/200) titers. We now report our efforts to improve this vaccine by screening a sublibrary with six random amino acids added to either end of the PA1 motif to identify higher affinity peptides. P206.1, a peptide isolated from this sublibrary, had 80-fold higher affinity for 2H1 (K(d) = 3.7 nM) than PA1. P206.1 bound protective, but not nonprotective, anti-GXM Abs. Mice immunized with P206.1 conjugated to various carriers did not mount an Ab response to GXM despite developing high anti-peptide titers. However, mice primed with GXM-TT and boosted with P206.1-TT developed significant anti-GXM titers (maximum, 1/180,000). This latter immunization scheme focused the immune response on protective epitopes, since only 2-5% of these titers were directed against nonprotective de-O-acetylated GXM epitopes compared with 20-60% in animals primed and boosted with GXM-TT.

  7. HPAEC-PAD method for the analysis of alkaline hydrolyzates of Haemophilus influenzae type b capsular polysaccharide.

    PubMed

    de Haan, Alex; van der Put, Robert M F; Beurret, Michel

    2013-09-01

    A gradient method has been devised for the rapid analysis of alkaline hydrolyzates of Haemophilus influenzae type b (Hib) capsular polysaccharide-based vaccines by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). As compared with published procedures, peak shape and sensitivity were significantly improved with this approach, analysis time was short and there was little interference from impurities. The limits of detection and quantification were established with a purified reference polysaccharide. We propose this method as a practical alternative for the analysis of minute amounts of Hib polysaccharide, which can be lower than with the conventional approaches.

  8. The Vi capsular polysaccharide enables Salmonella enterica serovar typhi to evade microbe-guided neutrophil chemotaxis.

    PubMed

    Wangdi, Tamding; Lee, Cheng-Yuk; Spees, Alanna M; Yu, Chenzhou; Kingsbury, Dawn D; Winter, Sebastian E; Hastey, Christine J; Wilson, R Paul; Heinrich, Volkmar; Bäumler, Andreas J

    2014-08-01

    Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

  9. A peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice.

    PubMed

    Buchwald, Ulrike K; Lees, Andrew; Steinitz, Michael; Pirofski, Liise-Anne

    2005-01-01

    Increasing antibiotic resistance and a rising patient population at risk for infection due to impaired immunity underscore the importance of vaccination against pneumococci. However, available capsular polysaccharide vaccines are often poorly immunogenic in patients at risk for pneumococcal disease. The goal of this study was to explore the potential of peptide mimotopes to function as alternative vaccine antigens to elicit a type-specific antibody response to pneumococci. We used a human monoclonal immunoglobulin A (IgA) antibody (NAD) to type 8 Streptococcus pneumoniae capsular polysaccharide (type 8 PS) to screen a phage display library, and the phage PUB1 displaying the peptide FHLPYNHNWFAL was selected after three rounds of biopanning. Inhibition studies with phage-displayed peptide or the peptide PUB1 and type 8 PS showed that PUB1 is a mimetic of type 8 PS. PUB1 conjugated to tetanus toxoid (PUB1-TT) induced a type 8 PS-specific antibody response in BALB/c mice, further defining it as a mimotope of type 8 PS. The administration of immune sera obtained from PUB1-TT-immunized mice earlier (days 14 and 21) and later (days 87 and 100) after primary and reimmunization resulted in a highly significant prolongation of the survival of naive mice after pneumococcal challenge compared to controls. The survival of PUB1-TT-immunized mice was also prolonged after pneumococcal challenge nearly 4 months after primary immunization. The efficacy of PUB1-TT-induced immune sera provides proof of principle that a mimotope-induced antibody response can protect against pneumococci and suggests that peptide mimotopes selected by type-specific human antibodies could hold promise as immunogens for pneumococci.

  10. Molecular Characterization of Type-Specific Capsular Polysaccharide Biosynthesis Genes of Streptococcus agalactiae Type Ia

    PubMed Central

    Yamamoto, Shin; Miyake, Katsuhide; Koike, Yoichi; Watanabe, Masaki; Machida, Yuichi; Ohta, Michio; Iijima, Shinji

    1999-01-01

    The type-specific capsular polysaccharide (CP) of a group B streptococcus, Streptococcus agalactiae type Ia, is a high-molecular-weight polymer consisting of the pentasaccharide repeating unit 4)-[α-d-NeupNAc-(2→3)-β-d-Galp-(1→4)-β-d-GlcpNAc-(1→3)]-β-d-Galp-(1→4)-β-d-Glcp-(1. Here, cloning, sequencing, and transcription of the type Ia-specific capsular polysaccharide synthesis (cps) genes and functional analysis of these gene products are described. A 26-kb DNA fragment containing 18 complete open reading frames (ORFs) was cloned. These ORFs were designated cpsIaA to cpsIaL, neu (neuraminic acid synthesis gene) A to D, orf1 and ung (uracil DNA glycosylase). The cps gene products of S. agalactiae type Ia were homologous to proteins involved in CP synthesis of S. agalactiae type III and S. pneumoniae serotype 14. Unlike the cps gene cluster of S. pneumoniae serotype 14, transcription of this operon may start from cpsIaA, cpsIaE, and orf1 because putative promoter sequences were found in front of these genes. Northern hybridization, reverse transcription-PCR, and primer extension analyses supported this hypothesis. DNA sequence analysis showed that there were two transcriptional terminators in the 3′ end of this operon (downstream of orf1 and ung). The functions of CpsIaE, CpsIaG, CpsIaI, and CpsIaJ were examined by glycosyltransferase assay by using the gene products expressed in Escherichia coli JM109 harboring plasmids containing various S. agalactiae type Ia cps gene fragments. Enzyme assays suggested that the gene products of cpsIaE, cpsIaG, cpsIaI, and cpsIaJ are putative glucosyltransferase, β-1,4-galactosyltransferase, β-1,3-N-acetylglucosaminyltransferase, and β-1,4-galactosyltransferase, respectively. PMID:10464185

  11. Isolation of a Bacteriophage Specific for a New Capsular Type of Klebsiella pneumoniae and Characterization of Its Polysaccharide Depolymerase

    PubMed Central

    Hsu, Chun-Ru; Lin, Tzu-Lung; Pan, Yi-Jiun; Hsieh, Pei-Fang; Wang, Jin-Town

    2013-01-01

    Background Klebsiella pneumoniae is one of the major pathogens causing hospital-acquired multidrug-resistant infections. The capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. With 78 capsular types discovered thus far, an association between capsular type and the pathogenicity of K. pneumoniae has been observed. Methodology/Principal Findings To investigate an initially non-typeable K. pneumoniae UTI isolate NTUH-K1790N, the cps gene region was sequenced. By NTUH-K1790N cps-PCR genotyping, serotyping and determination using a newly isolated capsular type-specific bacteriophage, we found that NTUH-K1790N and three other isolates Ca0507, Ca0421 and C1975 possessed a new capsular type, which we named KN2. Analysis of a KN2 CPS− mutant confirmed the role of capsule as the target recognized by the antiserum and the phage. A newly described lytic phage specific for KN2 K. pneumoniae, named 0507-KN2-1, was isolated and characterized using transmission electron microscopy. Whole-genome sequencing of 0507-KN2-1 revealed a 159 991 bp double-stranded DNA genome with a G+C content of 46.7% and at least 154 open reading frames. Based on its morphological and genomic characteristics, 0507-KN2-1 was classified as a member of the Myoviridae phage family. Further analysis of this phage revealed a 3738-bp gene encoding a putative polysaccharide depolymerase. A recombinant form of this protein was produced and assayed to confirm its enzymatic activity and specificity to KN2 capsular polysaccharides. KN2 K. pneumoniae strains exhibited greater sensitivity to this depolymerase than these did to the cognate phage, as determined by spot analysis. Conclusions/Significance Here we report that a group of clinical strains possess a novel Klebsiella capsular type. We identified a KN2-specific phage and its polysaccharide depolymerase, which could be used for efficient capsular typing. The lytic phage and depolymerase also have potential as alternative

  12. Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b.

    PubMed Central

    Kuo, J S; Doelling, V W; Graveline, J F; McCoy, D W

    1985-01-01

    Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP. Images PMID:3926752

  13. Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus.

    PubMed

    Miyafusa, Takamitsu; Caaveiro, Jose M M; Tanaka, Yoshikazu; Tanner, Martin E; Tsumoto, Kouhei

    2013-06-11

    Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein-protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.

  14. Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b

    SciTech Connect

    Kuo, J.S.; Doelling, V.W.; Graveline, J.F.; McCoy, D.W.

    1985-08-01

    Cells of Haemophilus influenzae type b were grown in a liquid medium containing (TH)palmitate or ( UC)ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( (TH)palmitate and ( UC)ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( (TH)palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with (TH)palmitate or (TH)palmitate and ( UC)ribose followed by chloroform-methanol extraction could most of the TH-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP.

  15. Highly Dynamic Genomic Loci Drive the Synthesis of Two Types of Capsular or Secreted Polysaccharides within the Mycoplasma mycoides Cluster

    PubMed Central

    Bertin, Clothilde; Pau-Roblot, Corinne; Courtois, Josiane; Manso-Silván, Lucía; Tardy, Florence; Poumarat, François; Citti, Christine; Sirand-Pugnet, Pascal; Gaurivaud, Patrice

    2014-01-01

    Mycoplasmas of the Mycoplasma mycoides cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for Mycoplasma mycoides subsp. mycoides polysaccharides in pathogenicity. Mycoplasma mycoides subsp. mycoides-secreted EPS was recently characterized as a β(1→6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the M. mycoides cluster: M. capricolum subsp. capripneumoniae, M. capricolum subsp. capricolum, M. leachii, and M. mycoides subsp. capri (including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of β(1→2)-glucopyranose (glucan) in M. capricolum subsp. capripneumoniae and M. leachii. Monoclonal antibodies specific for this glucan and for the Mycoplasma mycoides subsp. mycoides-secreted galactan were used to detect the two polysaccharides. While M. mycoides subsp. capri strains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains of M. capricolum subsp. capripneumoniae and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among M. capricolum subsp. capricolum strains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides. PMID:25398856

  16. Preparation of pneumococcal capsular polysaccharide-protein conjugate vaccines utilizing new fragmentation and conjugation technologies.

    PubMed

    Pawlowski, A; Källenius, G; Svenson, S B

    2000-03-17

    There is a global urgent need for a new efficient and inexpensive vaccine to combat pneumococcal disease, which should also be affordable in developing countries. In view of this need a simple low-cost technique to prepare such a vaccine was developed. The preparation of serotype 14 and 23F pneumococcal capsular polysaccharide (PnPS)-protein conjugates to be included in a forthcoming multivalent PnPS conjugate vaccine is described. Commercial lots of PnPSs produced according to Good Manufacturing Practice from Streptococcus pneumoniae serotype 14 (PS14) and 23F (PS23F) were partially depolymerized by sonication or irradiation in an electron beam accelerator. The PnPS fragments were conjugated to tetanus toxoid (TT) using a recently developed conjugation chemistry. The application of these new simple, efficient and inexpensive fragmentation and conjugation technologies allowed the synthesis of several PnPS-protein conjugates containing PnPS fragments of preselected sizes and differing in the degree of substitution. The PS14TT and PS23FTT conjugate vaccine candidates were characterized chemically and their immunogenicity was evaluated in rabbits and mice. All PnPS conjugate vaccines, unlike the corresponding plain polysaccharides, produced high IgG titres in both animal species. The PS14TT conjugates tended to be more immunogenic than the PS23FTT conjugates. The immune response to the PS14TT conjugates, but not to the PS23FTT conjugates, was related to the size of the conjugated polysaccharide hapten. Both types of conjugates elicited strong booster effects upon secondary immunizations, resulting in high IgG1, IgG2a and IgG2b titres.

  17. Immune suppression induced by Vi capsular polysaccharide is overcome by Vi-DT conjugate vaccine.

    PubMed

    An, So Jung; Yoon, Yeon Kyung; Kothari, Sudeep; Kim, Deok Ryun; Kim, Jeong Ah; Kothari, Neha; Lee, Eugene; Park, Tai Hyun; Carbis, Rodney

    2012-02-01

    The influence pre-exposure of mice to Vi capsular polysaccharide, purified from Salmonella enterica Serovar Typhi, on the subsequent immune response induced by a Vi-diphtheria toxoid (Vi-DT) conjugate was evaluated. Vi induced low anti Vi IgG titers with the dominant subclass being IgG3. The Vi-DT conjugate induced high titers of anti Vi IgG with the dominant subclass being IgG1 but with considerable quantities of IgG2a, IgG2b and IgG3. Priming of mice with Vi suppressed the response to a subsequent dose of conjugate and the suppression was overcome by a second dose of conjugate. Priming with conjugate prevented suppression of the anti Vi response and subsequent dosing with Vi raised titers back to previous levels but did not boost to new higher levels. The anti DT IgG response to one dose of conjugate was relatively strong and protracted and continued to rise for 12 weeks, compared to the response to one dose of DT which was poor and peaked at two weeks. The prolonged anti DT response was most likely due to the slow release of DT from the conjugate lattice as it degrades within the mouse resulting in a continuous stimulation of the immune response. The presence of increasing amounts of un-conjugated Vi, up to 50%, administered with the conjugate resulted in increasingly higher levels of both anti Vi and anti DT. Larger amounts of un-conjugated Vi inhibited the anti Vi response. These findings have implications for vaccine quality and a limit for un-conjugated polysaccharide should not exceed 50% and from a vaccine program perspective if the results presented here translate to humans then a Vi conjugate, once it becomes available, should replace Vi polysaccharide vaccines.

  18. Improvement in the purification process of the capsular polysaccharide from Haemophilus influenzae type b by using tangential ultrafiltration and diafiltration.

    PubMed

    Albani, Silvia Maria Ferreira; da Silva, Mateus Ribeiro; Takagi, Mickie; Cabrera-Crespo, Joaquin

    2012-08-01

    Capsular polysaccharide produced by Haemophilus influenzae b (Hib) is the main virulent agent and used as the antigen in the vaccine formulation. In this study, an improved process of polysaccharide purification was established based on tangential flow ultrafiltration using detergents (cocamidopropyl betaine and sodium deoxycholate), two selective ethanol precipitations steps, and extensive enzymatic hydrolysis as strategy. The relative purity (RP) related to protein and nucleic acids were 122~263 and 294~480, respectively, and compatible with the specifications established by the World Health Organization for Hib vaccine, RP≥100. These results make this process simple, cheaper, efficient, environmentally friendly, and prone to be scaled up.

  19. Cryptococcal xylosyltransferase 1 (Cxt1p) from Cryptococcus neoformans plays a direct role in the synthesis of capsule polysaccharides.

    PubMed

    Klutts, J Stacey; Doering, Tamara L

    2008-05-23

    The opportunistic yeast Cryptococcus neoformans causes serious disease in humans and expresses a prominent polysaccharide capsule that is required for its virulence. Little is known about how this capsule is synthesized. We previously identified a beta1,2-xylosyltransferase (Cxt1p) with in vitro enzymatic activity appropriate for involvement in capsule synthesis. Here, we investigate C. neoformans strains in which the corresponding gene has been deleted (cxt1Delta). Loss of CXT1 does not affect in vitro growth of the mutant cells or the general morphology of their capsules. However, NMR structural analysis of the two main capsule polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), showed that both were missing beta1,2-xylose residues. There was an approximately 30% reduction in the abundance of this residue in GXM in mutant compared with wild-type strains, and mutant GalXM was almost completely devoid of beta1,2-linked xylose. The GalXM from the mutant strain was also missing a beta1,3-linked xylose residue. Furthermore, deletion of CXT1 led to attenuation of cryptococcal growth in a mouse model of infection, suggesting that the affected xylose residues are important for normal host-pathogen interactions. Cxt1p is the first glycosyltransferase with a defined role in C. neoformans capsule biosynthesis, and cxt1Delta is the only strain identified to date with structural alterations of the capsule polysaccharide GalXM.

  20. Polysaccharides of the Genus Bacillus Cross-Reactive with the Capsular Polysaccharides of Diplococcus pneumoniae Type III, Haemophilus influenzae Type b, and Neisseria meningitidis Group A

    PubMed Central

    Myerowitz, Richard L.; Gordon, Ruth E.; Robbins, John B.

    1973-01-01

    We studied 174 strains of the genus Bacillus for cross-reacting antigens to the capsular polysaccharides of groups A and C meningococcus, types I and III pneumococcus, and Haemophilus influenzae type b. Cross-reactions were detected by immunodiffusion in agarose gel by using type-specific antisera and confirmed by absorption and inhibition experiments. Of 20 Bacillus pumilis strains, six had an antigen cross-reacting with group A meningococcal polysaccharide. Other cross-reactions included one strain of B. pumilis with H. influenzae type b, one of B. cereus var. mycoides with pneumococcus type III, and one of B. alvei with both type b and SIII polysaccharides. These cross-reacting antigens are polysaccharides of vegetative cells and may be extracellular in location. Because these bacilli have antigens cross-reacting with the virulence factors of pyogenic bacteria, they may, as normal flora, be an antigenic stimulus for “natural” serum anti-capsular antibodies to the type b Haemophilus and group A meningococcus polysaccharides. Images PMID:4150383

  1. Polysaccharides of the genus Bacillus cross-reactive with the capsular polysaccharides of Diplococcus pneumoniae type 3, Haemophilus influenzae type b, and Neisseria meningitidis group A.

    PubMed

    Myerowitz, R L; Gordon, R E; Robbins, J B

    1973-12-01

    We studied 174 strains of the genus Bacillus for cross-reacting antigens to the capsular polysaccharides of groups A and C meningococcus, types I and III pneumococcus, and Haemophilus influenzae type b. Cross-reactions were detected by immunodiffusion in agarose gel by using type-specific antisera and confirmed by absorption and inhibition experiments. Of 20 Bacillus pumilis strains, six had an antigen cross-reacting with group A meningococcal polysaccharide. Other cross-reactions included one strain of B. pumilis with H. influenzae type b, one of B. cereus var. mycoides with pneumococcus type III, and one of B. alvei with both type b and SIII polysaccharides. These cross-reacting antigens are polysaccharides of vegetative cells and may be extracellular in location. Because these bacilli have antigens cross-reacting with the virulence factors of pyogenic bacteria, they may, as normal flora, be an antigenic stimulus for "natural" serum anti-capsular antibodies to the type b Haemophilus and group A meningococcus polysaccharides.

  2. Anti-Biofilm Activity: A Function of Klebsiella pneumoniae Capsular Polysaccharide

    PubMed Central

    Dos Santos Goncalves, Marina; Delattre, Cédric; Balestrino, Damien; Charbonnel, Nicolas; Elboutachfaiti, Redouan; Wadouachi, Anne; Badel, Stéphanie; Bernardi, Thierry; Michaud, Philippe; Forestier, Christiane

    2014-01-01

    Competition and cooperation phenomena occur within highly interactive biofilm communities and several non-biocides molecules produced by microorganisms have been described as impairing biofilm formation. In this study, we investigated the anti-biofilm capacities of an ubiquitous and biofilm producing bacterium, Klebsiella pneumoniae. Cell-free supernatant from K. pneumoniae planktonic cultures showed anti-biofilm effects on most Gram positive bacteria tested but also encompassed some Gram negative bacilli. The anti-biofilm non-bactericidal activity was further investigated on Staphylococcus epidermidis, by determining the biofilm biomass, microscopic observations and agglutination measurement through a magnetic bead-mediated agglutination test. Cell-free extracts from K. pneumoniae biofilm (supernatant and acellular matrix) also showed an influence, although to a lesser extend. Chemical analyses indicated that the active molecule was a high molecular weight polysaccharide composed of five monosaccharides: galactose, glucose, rhamnose, glucuronic acid and glucosamine and the main following sugar linkage residues [→2)-α-l-Rhap-(1→]; [→4)-α-l-Rhap-(1→]; [α-d-Galp-(1→]; [→2,3)-α-d-Galp-(1→]; [→3)-β-d-Galp-(1→] and, [→4)-β-d-GlcAp-(1→]. Characterization of this molecule indicated that this component was more likely capsular polysaccharide (CPS) and precoating of abiotic surfaces with CPS extracts from different serotypes impaired the bacteria-surface interactions. Thus the CPS of Klebsiella would exhibit a pleiotropic activity during biofilm formation, both stimulating the initial adhesion and maturation steps as previously described, but also repelling potential competitors. PMID:24932475

  3. Two capsular polysaccharides enable Bacillus cereus G9241 to cause anthrax-like disease.

    PubMed

    Oh, So-Young; Budzik, Jonathan M; Garufi, Gabriella; Schneewind, Olaf

    2011-04-01

    Bacillus cereus G9241 causes an anthrax-like respiratory illness in humans; however, the molecular mechanisms of disease pathogenesis are not known. Genome sequencing identified two putative virulence plasmids proposed to provide for anthrax toxin (pBCXO1) and/or capsule expression (pBC218). We report here that B. cereus G9241 causes anthrax-like disease in immune-competent mice, which is dependent on each of the two virulence plasmids. pBCXO1 encodes pagA1, the homologue of anthrax protective antigen, as well as hasACB, providing for hyaluronic acid capsule formation, two traits that each contribute to disease pathogenesis. pBC218 harbours bpsX-H, B. cereus exo-polysaccharide, which produce a second capsule. During infection, B. cereus G9241 elaborates both hasACB and bpsX-H capsules, which together are essential for the establishment of anthrax-like disease and the resistance of bacilli to phagocytosis. A single nucleotide deletion causes premature termination of hasA translation in Bacillus anthracis, which is known to escape phagocytic killing by its pXO2 encoded poly-d-γ-glutamic acid (PDGA) capsule. Thus, multiple different gene clusters endow pathogenic bacilli with capsular material, provide for escape from innate host immune responses and aid in establishing the pathogenesis of anthrax-like disease.

  4. Agglutination by anti-capsular polysaccharide antibody is associated with protection against experimental human pneumococcal carriage

    PubMed Central

    Reiné, J; Zangari, T; Owugha, JT; Pennington, SH; Gritzfeld, JF; Wright, AD; Collins, AM; van Selm, S; de Jonge, MI; Gordon, SB; Weiser, JN; Ferreira, DM

    2016-01-01

    The ability of pneumococcal conjugate vaccine (PCV) to decrease transmission by blocking the acquisition of colonization has been attributed to herd immunity. We describe the role of mucosal IgG to capsular polysaccharide (CPS) in mediating protection from carriage, translating our findings from a murine model to humans. We used a flow-cytometric assay to quantify antibody-mediated agglutination demonstrating that hyperimmune sera generated against an unencapsulated mutant was poorly agglutinating. Passive immunization with this antiserum was ineffective to block acquisition of colonization compared to agglutinating antisera raised against the encapsulated parent strain. In the human challenge model samples were collected from PCV and control vaccinated adults. In PCV-vaccinated subjects IgG levels to CPS were increased in serum and nasal wash (NW). IgG to the inoculated strain CPS dropped in NW samples after inoculation suggesting its sequestration by colonizing pneumococci. In post-vaccination NW samples pneumococci were heavily agglutinated compared to pre-vaccination samples in subjects protected against carriage. Our results indicate that pneumococcal agglutination mediated by CPS specific antibodies is a key mechanism of protection against acquisition of carriage. Capsule may be the only vaccine target that can elicit strong agglutinating antibody responses, leading to protection against carriage acquisition and generation of herd immunity. PMID:27579859

  5. Two capsular polysaccharides enable Bacillus cereus G9241 to cause anthrax-like disease

    PubMed Central

    Oh, So-Young; Budzik, Jonathan M.; Garufi, Gabriella; Schneewind, Olaf

    2012-01-01

    Summary Bacillus cereus G9241 causes an anthrax-like respiratory illness in humans, however the molecular mechanisms of disease pathogenesis are not known. Genome sequencing identified two putative virulence plasmids proposed to provide for anthrax toxin (pBCXO1) and/or capsule expression (pBC218). We report here that B. cereus G9241 causes anthrax-like disease in immune-competent mice, which is dependent on each of the two virulence plasmids. pBCXO1 encodes pagA1, the homolog of anthrax protective antigen, as well as hasACB, providing for hyaluronic acid capsule formation, two traits that each contribute to disease pathogenesis. pBC218 harbors bpsX-H, Bacillus cereus exo-polysaccharide, which produce a second capsule. During infection, B. cereus G9241 elaborates both hasACB and bpsX-H capsules, which together are essential for the establishment of anthrax-like disease and the resistance of bacilli to phagocytosis. A single nucleotide deletion causes premature termination of hasA translation in B. anthracis, which is known to escape phagocytic killing by its pXO2 encoded poly-D-γ-glutamic acid (PDGA) capsule. Thus, multiple different gene clusters endow pathogenic bacilli with capsular material, provide for escape from innate host immune responses and aid in establishing the pathogenesis of anthrax-like disease. PMID:21371137

  6. O-METHYL PHOSPHORAMIDATE MODIFICATIONS ON THE CAPSULAR POLYSACCHARIDE OF CAMPYLOBACTER JEJUNI ARE INVOLVED IN SERUM RESISTANCE, INFECTION, AND INSECTICIDAL ACTIVITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter jejuni is the most commonly reported cause of bacterial foodborne illness in North America. C. jejuni decorates its surface polysaccharides with a variety of variable phosphorylated structures, including O-methyl phosphoramidate (MeOPN) modifications on the capsular polysaccharide. Alt...

  7. Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent.

    PubMed Central

    Cywes, C; Hoppe, H C; Daffé, M; Ehlers, M R

    1997-01-01

    The choice of host cell receptor and the mechanism of binding (opsonic versus nonopsonic) may influence the intracellular fate of Mycobacterium tuberculosis. We have identified two substrains of M. tuberculosis H37Rv, designated H37Rv-CC and -HH, that differed in their modes of binding to complement receptor type 3 (CR3) expressed in transfected Chinese hamster ovary (CHO-Mac-1) cells: H37Rv-CC bound nonopsonically, whereas H37Rv-HH bound only after opsonization in fresh serum. H37Rv-CC also bound nonopsonically to untransfected CHO cells, whereas H37Rv-HH binding was enhanced by serum and was mediated by the 1D1 antigen, a bacterial adhesin previously identified as a polar phosphatidylinositol mannoside. H37Rv-CC and -HH had identical IS6110 DNA fingerprint patterns. Of five M. tuberculosis clinical isolates examined, four displayed the same binding phenotype as H37Rv-CC, as did the Erdman strain, whereas one isolate, as well as Mycobacterium smegmatis, behaved like H37Rv-HH. Nonopsonic binding of H37Rv-CC to CHO cell-expressed CR3 was apparently to the beta-glucan lectin site, as it was cation independent and inhibited by laminarin (seaweed beta-glucan) and N-acetylglucosamine; laminarin also inhibited the binding of H37Rv-CC to monocyte-derived macrophages. Further, binding of H37Rv-CC to CHO-Mac-1 cells was inhibited by prior agitation of bacteria with glass beads (which strips outer capsular polysaccharides) and by preincubation with amyloglucosidase, as well as by the presence of capsular D-glucan and D-mannan from M. tuberculosis Erdman, but not by Erdman D-arabino-D-mannan, yeast mannan, or capsular components from H37Rv-HH. Analysis of capsular carbohydrates revealed that H37Rv-CC expressed 5-fold more glucose and 2.5-fold more arabinose and mannose than H37Rv-HH. Flow cytometric detection of surface epitopes indicated that H37Rv-CC displayed twofold less surface-exposed phosphatidylinositol mannoside and bound complement C3 less efficiently than H37Rv

  8. Characterization of the structure and biological functions of a capsular polysaccharide produced by Staphylococcus saprophyticus.

    PubMed

    Park, Sunny; Kelley, Kathryn A; Vinogradov, Evgeny; Solinga, Robert; Weidenmaier, Christopher; Misawa, Yoshiki; Lee, Jean C

    2010-09-01

    Staphylococcus saprophyticus is a common cause of uncomplicated urinary tract infections in women. S. saprophyticus strain ATCC 15305 carries two staphylococcal cassette chromosome genetic elements, SCC(15305RM) and SCC(15305cap). The SCC(15305cap) element carries 13 open reading frames (ORFs) involved in capsular polysaccharide (CP) biosynthesis, and its G+C content (26.7%) is lower than the average G+C content (33.2%) for the whole genome. S. saprophyticus strain ATCC 15305 capD, capL, and capK (capD(Ssp), capL(Ssp), and capK(Ssp)) are homologous to genes encoding UDP-FucNAc biosynthesis, and gtaB and capI(Ssp) show homology to genes involved in UDP-glucuronic acid synthesis. S. saprophyticus ATCC 15305 CP, visualized by immunoelectron microscopy, was extracted and purified using anionic-exchange and size exclusion chromatography. Analysis of the purified CP by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy and gas-liquid chromatography revealed two types of branched tetrasaccharide repeating units composed of the following: -4)-beta-Glc-(1-3)-Sug-(1-4)-beta-GlcA-(1- | beta-GlcNAc-(1-2) Sug represents two stereoisomers of 2-acetamido-2,6-dideoxy-hexos-4-ulose residues, one of which has an arabino configuration. The encapsulated ATCC 15305 strain was resistant to complement-mediated opsonophagocytic killing by human neutrophils, whereas the acapsular mutant C1 was susceptible. None of 14 clinical isolates reacted with antibodies to the ATCC 15305 CP. However, 11 of the 14 S. saprophyticus isolates were phenotypically encapsulated based on their resistance to complement-mediated opsonophagocytic killing and their failure to hemagglutinate when cultivated aerobically. Ten of the 14 clinical strains carried homologues of the conserved staphylococcal capD gene or the S. saprophyticus gtaB gene, or both. Our results suggest that some strains of S. saprophyticus are encapsulated and that more than one capsular serotype exists.

  9. In vivo Distribution and Clearance of Purified Capsular Polysaccharide from Burkholderia pseudomallei in a Murine Model

    PubMed Central

    Nualnoi, Teerapat; Kirosingh, Adam; Pandit, Sujata G.; Thorkildson, Peter; Brett, Paul J.; Burtnick, Mary N.; AuCoin, David P.

    2016-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection prominent in northern Australia and Southeast Asia. The “gold standard” for melioidosis diagnosis is bacterial isolation, which takes several days to complete. The resulting delay in diagnosis leads to delayed treatments, which could result in death. In an attempt to develop better methods for early diagnosis of melioidosis, B. pseudomallei capsular polysaccharide (CPS) was identified as an important diagnostic biomarker. A rapid lateral flow immunoassay utilizing CPS-specific monoclonal antibody was developed and tested in endemic regions worldwide. However, the in vivo fate and clearance of CPS has never been thoroughly investigated. Here, we injected mice with purified CPS intravenously and determined CPS concentrations in serum, urine, and major organs at various intervals. The results indicate that CPS is predominantly eliminated through urine and no CPS accumulation occurs in the major organs. Immunoblot analysis demonstrated that intact CPS was excreted through urine. To understand how a large molecule like CPS was eliminated without degradation, a 3-dimenational structure of CPS was modeled. The predicted CPS structure has a rod-like shape with a small diameter that could allow it to flow through the glomerulus of the kidney. CPS clearance was determined using exponential decay models and the corrected Akaike Information Criterion. The results show that CPS has a relatively short serum half-life of 2.9 to 4.4 hours. Therefore, the presence of CPS in the serum and/or urine suggests active melioidosis infection and provides a marker to monitor treatment of melioidosis. PMID:27941991

  10. The efficacy of pneumococcal capsular polysaccharide-specific antibodies to serotype 3 Streptococcus pneumoniae requires macrophages.

    PubMed

    Fabrizio, Kevin; Manix, Catherine; Tian, Haijun; van Rooijen, Nico; Pirofski, Liise-anne

    2010-11-03

    The efficacy of antibody immunity against Streptococcus pneumoniae stems from the ability of opsonic, serotype (ST)-specific antibodies to pneumococcal capsular polysaccharide (PPS) to facilitate killing of the homologous ST by host phagocytes. However, PPS-specific antibodies have been identified that are protective in mice, but do not promote opsonic killing in vitro, raising the question of how they mediate protection in vivo. To probe this question, we investigated the dependence of antibody efficacy against lethal systemic (intraperitoneal, i.p.) infection with Streptococcus pneumoniae serotype 3 (ST3) on macrophages and neutrophils for the following PPS3-specific monoclonal antibodies (MAbs) in survival experiments in mice using a non-opsonic human IgM (A7), a non-opsonic mouse IgG1 (1E2) and an opsonic mouse IgG1 (5F6). The survival of A7- and PPS3-specific and isotype control MAb-treated neutrophil-depleted and neutrophil-sufficient and macrophage-depleted and macrophage-sufficient mice were determined after i.p. challenge with ST3 strains 6303 and WU2. Neutrophils were dispensable for A7 and the mouse MAbs to mediate protection in this model, but macrophages were required for the efficacy of A7 and optimal mouse MAb-mediated protection. For A7-treated mice, macrophage-depleted mice had higher blood CFU, cytokines and peripheral neutrophil levels than macrophage-sufficient mice, and macrophage-sufficient mice had lower tissue bacterial burdens than control MAb-treated mice. These findings demonstrate that macrophages contribute to opsonic and non-opsonic PPS3-specific MAb-mediated protection against ST3 infection by enhancing bacterial clearance and suggest that neutrophils do not compensate for the absence of macrophages in the model used in this study.

  11. Cell growth rate regulates expression of group B Streptococcus type III capsular polysaccharide.

    PubMed Central

    Paoletti, L C; Ross, R A; Johnson, K D

    1996-01-01

    The capsular polysaccharide (CPS) of group B streptococci (GBS) is an important virulence factor that also serves to protect cells from nonspecific host defense mechanisms. Expression of CPS by GBS, as with other encapsulated bacterial pathogens, is not constitutive but varies during growth in vitro and in primary cultures isolated from different sites of infection. Despite this understanding, little is known about regulation of this surface-expressed carbohydrate antigen in GBS. Here we report that expression of type III CPS by GBS strain M781 grown in continuous culture with a modified chemically defined medium is regulated by growth rate. Cells in steady state at mass doubling times (tds) of 0.8, 1.4, and 1.6 h expressed an average of sixfold more cell-associated CPS than did cells held at tds of 2.3 and 11 h. Strain M781 grown at a td of 1.4 h repeatedly produced more type III CPS than those held at a td of 11.0 h, even when limited for glucose, pyridoxamine, or thiamine. In our studies, > or = 93% of the total CPS expressed by strain M781 was cell associated. Strain M781 grown at a td of 11.0 h (i.e., lowered CPS expression) was susceptible to in vitro complement-mediated opsonophagocytosis and killing by human peripheral blood leukocytes, whereas cells grown at a td of 1.4 h (i.e., higher CPS expression) were not killed unless type III CPS-specific antibody was present. Factors that allow GBS to asymptomatically colonize women yet cause invasive infection to both mother and infant are poorly understood. Our results shed new light on parameters that regulate the pathogenic potential of GBS and may also serve as a way to discern more fully the genetics and biochemistry of GBS capsule synthesis. PMID:8606082

  12. In vivo Distribution and Clearance of Purified Capsular Polysaccharide from Burkholderia pseudomallei in a Murine Model.

    PubMed

    Nualnoi, Teerapat; Kirosingh, Adam; Pandit, Sujata G; Thorkildson, Peter; Brett, Paul J; Burtnick, Mary N; AuCoin, David P

    2016-12-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection prominent in northern Australia and Southeast Asia. The "gold standard" for melioidosis diagnosis is bacterial isolation, which takes several days to complete. The resulting delay in diagnosis leads to delayed treatments, which could result in death. In an attempt to develop better methods for early diagnosis of melioidosis, B. pseudomallei capsular polysaccharide (CPS) was identified as an important diagnostic biomarker. A rapid lateral flow immunoassay utilizing CPS-specific monoclonal antibody was developed and tested in endemic regions worldwide. However, the in vivo fate and clearance of CPS has never been thoroughly investigated. Here, we injected mice with purified CPS intravenously and determined CPS concentrations in serum, urine, and major organs at various intervals. The results indicate that CPS is predominantly eliminated through urine and no CPS accumulation occurs in the major organs. Immunoblot analysis demonstrated that intact CPS was excreted through urine. To understand how a large molecule like CPS was eliminated without degradation, a 3-dimenational structure of CPS was modeled. The predicted CPS structure has a rod-like shape with a small diameter that could allow it to flow through the glomerulus of the kidney. CPS clearance was determined using exponential decay models and the corrected Akaike Information Criterion. The results show that CPS has a relatively short serum half-life of 2.9 to 4.4 hours. Therefore, the presence of CPS in the serum and/or urine suggests active melioidosis infection and provides a marker to monitor treatment of melioidosis.

  13. Expression of crossreactive idiotypes by human antibodies specific for the capsular polysaccharide of Hemophilus influenzae B.

    PubMed Central

    Lucas, A H

    1988-01-01

    Human antibodies specific, for polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Hemophilus influenzae b, were studied using idiotypic analysis. Antisera were prepared against purified F(ab')2 anti-PRP from two unrelated adults, H.H. and P.T. After repeated absorption with IgG myeloma proteins and with PRP-absorbed normal human Ig and donor Ig, anti-idiotypic (anti-Id) sera were obtained that specifically reacted with anti-PRP antibodies. Anti-IdHH and anti-IdPT reciprocally crossreacted with H.H. and P.T. anti-PRP antibodies and F(ab')2 fragments, and also reacted with the serum anti-PRP antibodies from three additional adults unrelated to P.T. and H.H. Both anti-Id sera partially inhibited anti-PRP paratopes but not anti-tetanus toxoid paratopes. PRP did not inhibit anti-Id recognition of shared or crossreactive idiotypic (CRI) determinants. Naturally occurring and PRP immunization-induced anti-PRP antibodies expressed CRI. While CRI titer increased after immunization, the increase was usually less than the rise in total anti-PRP antibody. Quantitative differences in CRI expression were also apparent between natural and immunization-induced H.H. and P.T. anti-PRP antibodies as shown by their differential inhibitability by anti-Id. Our data demonstrate that anti-PRP antibodies from five unrelated adults express CRI determinants that are probably distant from the PRP combining site. Naturally occurring and immunization-induced anti-PRP antibodies share CRI and therefore appear to be clonally related, although immunization apparently induces the expression CRI-negative antibodies as well. These results, taken with previous studies showing restricted and identical anti-PRP isoelectric focusing spectrotypes in unrelated adults, suggest that some PRP-specific V domains are structurally conserved and probably germ-line encoded. PMID:3257499

  14. Study of various presentation forms for a peptide mimetic of Neisseria meningitidis serogroup B capsular polysaccharide.

    PubMed

    Garay, Hilda; Menéndez, Tamara; Cruz-Leal, Yoelys; Coizeau, Edelgis; Noda, Jesus; Morera, Vivian; Guillén, Gerardo; Albericio, Fernando; Reyes, Osvaldo

    2011-01-19

    The formulation of a broadly protective vaccine to prevent the serogroup B Neisseria meningitidis (MenB) disease is still an unmet medical need. We have previously reported the induction of bactericidal and protective antibodies against MenB after immunization of mice with a phage-displayed peptide named 4 L-5. This peptide mimics a capsular polysaccharide (CPS) epitope in MenB. With the aim of developing vaccine formulations that could be used in humans, we evaluate in this study various forms of presentation to the immune system of the 4 L-5 sequence, based on synthetic peptides. We synthesized the following: (i) a linear 4 L-5 peptide, (ii) a multiple antigen peptide containing four copies of the 4 L-5 sequence (named MAP), which was then dimerized, and the product named dimeric MAP, and (iii) a second multiple antigen peptide, in this case with two copies of the 4 L-5 sequence and a copy of a T-helper cell epitope of tetanus toxoid, which was then dimerized and the product named MAP-TT. The linear peptide, the MAP, and the dimeric MAP were conjugated to the carrier protein P64K by different conjugation methods. Plain antigens and antigens coupled to P64K were used to immunize BALB/c mice. Of those variants that gave immunogenic results, MAP-TT rendered the highest levels of specific antipeptide IgG antibodies and serum bactericidal activity. These results can find application in the development of meningococcal vaccine candidates and in peptide-based vaccines strategies.

  15. Protection against Streptococcus suis Serotype 2 Infection Using a Capsular Polysaccharide Glycoconjugate Vaccine

    PubMed Central

    Calzas, Cynthia; Shiao, Tze Chieh; Neubauer, Axel; Kempker, Jennifer; Roy, René; Gottschalk, Marcelo

    2016-01-01

    Streptococcus suis serotype 2 is an encapsulated bacterium and one of the most important bacterial pathogens in the porcine industry. Despite decades of research for an efficient vaccine, none is currently available. Based on the success achieved with other encapsulated pathogens, a glycoconjugate vaccine strategy was selected to elicit opsonizing anti-capsular polysaccharide (anti-CPS) IgG antibodies. In this work, glycoconjugate prototypes were prepared by coupling S. suis type 2 CPS to tetanus toxoid, and the immunological features of the postconjugation preparations were evaluated in vivo. In mice, experiments evaluating three different adjuvants showed that CpG oligodeoxyribonucleotide (ODN) induces very low levels of anti-CPS IgM antibodies, while the emulsifying adjuvants Stimune and TiterMax Gold both induced high levels of IgGs and IgM. Dose-response trials comparing free CPS with the conjugate vaccine showed that free CPS is nonimmunogenic independently of the dose used, while 25 μg of the conjugate preparation was optimal in inducing high levels of anti-CPS IgGs postboost. With an opsonophagocytosis assay using murine whole blood, sera from immunized mice showed functional activity. Finally, the conjugate vaccine showed immunogenicity and induced protection in a swine challenge model. When conjugated and administered with emulsifying adjuvants, S. suis type 2 CPS is able to induce potent IgM and isotype-switched IgGs in mice and pigs, yielding functional activity in vitro and protection against a lethal challenge in vivo, all features of a T cell-dependent response. This study represents a proof of concept for the potential of glycoconjugate vaccines in veterinary medicine applications against invasive bacterial infections. PMID:27113360

  16. Polysaccharides, mimotopes and vaccines for fungal and encapsulated pathogens.

    PubMed

    Pirofski, L A

    2001-09-01

    Vaccination is a rational alternative to treatment for Cryptococcus neoformans infections, as these infections are currently intractable in immunocompromised (including HIV-infected) individuals. Vaccines composed of the cryptococcal capsular polysaccharide glucuronoxylomannan (GXM), the key C. neoformans virulence factor, elicit protective antibodies in mice, although deleterious antibodies can also be induced. By contrast, polysaccharides are poor immunogens in HIV-infected humans and others with B-cell defects. Peptide mimotopes of GXM can induce protective immunity to C. neoformans in mice, however, our knowledge of the mechanisms of mimotope-induced protection is incomplete and further work is needed if polysaccharide- or mimotope-based vaccines are to be used to manage C. neoformans infection.

  17. Type 5 and 8 capsular polysaccharides are expressed by Staphylococcus aureus isolates from rabbits, poultry, pigs, and horses.

    PubMed Central

    Poutrel, B; Sutra, L

    1993-01-01

    A total of 103 Staphylococcus aureus isolates from rabbits (n = 37), poultry (n = 33), pigs (n = 27), and horses (n = 6) and 14 Staphylococcus intermedius isolates from wild animals were serotyped for capsular polysaccharide types 5 and 8 by an enzyme-linked immunosorbent assay using polyclonal rabbit antibodies. About 98% of the S. aureus isolates were typeable. Type 5 was predominant in the poultry (75.8%) and pig (66.7%) isolates, whereas type 8 was more frequent among the isolates from rabbits (59.5%) and horses (83.3%). By contrast, none of the 14 S. intermedius isolates was typeable. PMID:8432841

  18. Structure of the neutral capsular polysaccharide of Acinetobacter baumannii NIPH146 that carries the KL37 capsule gene cluster.

    PubMed

    Arbatsky, Nikolay P; Shneider, Mikhail M; Kenyon, Johanna J; Shashkov, Alexander S; Popova, Anastasiya V; Miroshnikov, Konstantin A; Volozhantsev, Nikolay V; Knirel, Yuriy A

    2015-09-02

    Capsular polysaccharide (CPS) was isolated from Acinetobacter baumannii NIPH146, and the following structure of branched pentasaccharide repeating unit was established by sugar analyses along with 1D and 2D NMR spectroscopy: In comparison to most other known capsular polysaccharides of A. baumannii, the CPS studied is neutral and lacks any specific monosaccharide component. The synthesis, assembly and export of this structure could be attributed to genes in a novel capsule biosynthesis gene cluster, designated KL37, which was found in the NIPH146 genome. The CPS of A. baumannii NIPH146 shares the α-d-Galp-(1→6)-β-d-Glcp-(1→3)-d-GalpNAc-(1→ trisaccharide fragment with the CPS units of several A. baumannii strains, including ATCC 17978 and LUH 5537 that carry the KL3 and KL22 gene clusters, respectively. KL37 contains two genes for glycosyltransferases that are related to two glycosyltransferase genes present in both KL3 and KL22, and the encoded proteins could be tentatively assigned to linkages between sugars in the CPS repeat.

  19. Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a.

    PubMed Central

    Ward, C K; Inzana, T J

    1997-01-01

    Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens. PMID:9169799

  20. Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein

    NASA Astrophysics Data System (ADS)

    Abdelhameed, Ali Saber; Morris, Gordon A.; Almutairi, Fahad; Adams, Gary G.; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E.

    2016-10-01

    The structural integrity of meningococcal native, micro-fluidized and activated capsular polysaccharides and their glycoconjugates – in the form most relevant to their potential use as vaccines (dilute solution) - have been investigated with respect to their homogeneity, conformation and flexibility. Sedimentation velocity analysis showed that the polysaccharide size distributions were generally bimodal with some evidence for higher molar mass forms at higher concentration. Weight average molar masses Mw where lower for activated polysaccharides. Conjugation with tetanus toxoid protein however greatly increased the molar mass and polydispersity of the final conjugates. Glycoconjugates had an approximately unimodal log-normal but broad and large molar mass profiles, confirmed by sedimentation equilibrium “SEDFIT MSTAR” analysis. Conformation analysis using HYDFIT (which globally combines sedimentation and viscosity data), “Conformation Zoning” and Wales-van Holde approaches showed a high degree of flexibility – at least as great as the unconjugated polysaccharides, and very different from the tetanus toxoid (TT) protein used for the conjugation. As with the recently published finding for Hib-TT complexes, it is the carbohydrate component that dictates the solution behaviour of these glycoconjugates, although the lower intrinsic viscosities suggest some degree of compaction of the carbohydrate chains around the protein.

  1. Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein

    PubMed Central

    Abdelhameed, Ali Saber; Morris, Gordon A.; Almutairi, Fahad; Adams, Gary G.; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E.

    2016-01-01

    The structural integrity of meningococcal native, micro-fluidized and activated capsular polysaccharides and their glycoconjugates – in the form most relevant to their potential use as vaccines (dilute solution) - have been investigated with respect to their homogeneity, conformation and flexibility. Sedimentation velocity analysis showed that the polysaccharide size distributions were generally bimodal with some evidence for higher molar mass forms at higher concentration. Weight average molar masses Mw where lower for activated polysaccharides. Conjugation with tetanus toxoid protein however greatly increased the molar mass and polydispersity of the final conjugates. Glycoconjugates had an approximately unimodal log-normal but broad and large molar mass profiles, confirmed by sedimentation equilibrium “SEDFIT MSTAR” analysis. Conformation analysis using HYDFIT (which globally combines sedimentation and viscosity data), “Conformation Zoning” and Wales-van Holde approaches showed a high degree of flexibility – at least as great as the unconjugated polysaccharides, and very different from the tetanus toxoid (TT) protein used for the conjugation. As with the recently published finding for Hib-TT complexes, it is the carbohydrate component that dictates the solution behaviour of these glycoconjugates, although the lower intrinsic viscosities suggest some degree of compaction of the carbohydrate chains around the protein. PMID:27782149

  2. Genotyping and investigating capsular polysaccharide synthesis gene loci of non-serotypeable Streptococcus suis isolated from diseased pigs in Canada.

    PubMed

    Zheng, Han; Qiu, Xiaotong; Roy, David; Segura, Mariela; Du, Pengchen; Xu, Jianguo; Gottschalk, Marcelo

    2017-02-20

    Streptococcus suis (S. suis) is an important swine pathogen and an emerging zoonotic agent. Most clinical S. suis strains express capsular polysaccharides (CPS), which can be typed by antisera using the coagglutination test. In this study, 79 S. suis strains recovered from diseased pigs in Canada and which could not be typed using antisera were further characterized by capsular gene typing and sequencing. Four patterns of cps locus were observed: (1) fifteen strains were grouped into previously reported serotypes but presented several mutations in their cps loci, when compared to available data from reference strains; (2) seven strains presented a complete deletion of the cps locus, which would result in an inability to synthesize capsule; (3) forty-seven strains were classified in recently described novel cps loci (NCLs); and (4) ten strains carried novel NCLs not previously described. Different virulence gene profiles (based on the presence of mrp, epf, and/or sly) were observed in these non-serotypeable strains. This study provides further insight in understanding the genetic characteristics of cps loci in non-serotypeable S. suis strains recovered from diseased animals. When using a combination of the previously described 35 serotypes and the complete NCL system, the number of untypeable strains recovered from diseased animals in Canada would be significantly reduced.

  3. Recombinant Escherichia coli K5 strain with the deletion of waaR gene decreases the molecular weight of the heparosan capsular polysaccharide.

    PubMed

    Huang, Haichan; Liu, Xiaobo; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

    2016-09-01

    Heparosan, the capsular polysaccharide of Escherichia coli K5 having a carbohydrate backbone similar to that of heparin, has become a potential precursor for bioengineering heparin. In the heparosan biosynthesis pathway, the gene waaR encoding α-1-, 2- glycosyltransferase catalyze s the third glucosyl residues linking to the oligosaccharide chain. In the present study, a waaR deletion mutant of E. coli K5 was constructed. The mutant showed improvement of capsule polysaccharide yield. It is interesting that the heparosan molecular weight of the mutant is reduced and may become more suitable as a precursor for the production of low molecular weight heparin derived from the wild-type K5 capsular polysaccharide.

  4. Structural determination of the K14 capsular polysaccharide from an ST25 Acinetobacter baumannii isolate, D46.

    PubMed

    Kenyon, Johanna J; Hall, Ruth M; De Castro, Cristina

    2015-11-19

    The structure of the capsular polysaccharide (CPS) recovered from D46, an extensively antibiotic resistant ST25 Acinetobacter baumannii clinical isolate, was elucidated. The structure was resolved on the basis of NMR spectroscopy and chemical analyses, and was found to contain a branched neutral pentasaccharide with a backbone composed of GalpNAc and Galp residues, all d configured, and a d-Glcp side group. The KL14 gene cluster found in the D46 genome includes genes for four glycosyltransferases but no modules for synthesis of complex sugars, and this is consistent with the structure of K14. The K14 structure and KL14 sequence clarify the relationship between the structure and K locus sequence for A. nosocomialis isolate LUH5541. The identity of the first sugar of the K14 repeat unit (K unit), and the functions of the four encoded glycosyltransferases and Wzy polymerase were predicted.

  5. Antibodies to Staphylococcus aureus capsular polysaccharides 5 and 8 perform similarly in vitro but are functionally distinct in vivo.

    PubMed

    Liu, Bo; Park, Saeyoung; Thompson, Christopher D; Li, Xue; Lee, Jean C

    2016-12-09

    The capsular polysaccharide (CP) produced by Staphylococcus aureus is a virulence factor that allows the organism to evade uptake and killing by host neutrophils. Polyclonal antibodies to the serotype 5 (CP5) and type 8 (CP8) capsular polysaccharides are opsonic and protect mice against experimental bacteremia provoked by encapsulated staphylococci. Thus, passive immunotherapy using CP antibodies has been considered for the prevention or treatment of invasive antibiotic-resistant S. aureus infections. In this report, we generated monoclonal antibodies (mAbs) against S. aureus CP5 or CP8. Backbone specific mAbs reacted with native and O-deacetylated CPs, whereas O-acetyl specific mAbs reacted only with native CPs. Reference strains of S. aureus and a selection of clinical isolates reacted by colony immunoblot with the CP5 and CP8 mAbs in a serotype-specific manner. The mAbs mediated in vitro CP type-specific opsonophagocytic killing of S. aureus strains, and mice passively immunized with CP5 mAbs were protected against S. aureus bacteremia. Neither CP8-specific mAbs or polyclonal antibodies protected mice against bacteremia provoked by serotype 8 S. aureus clinical isolates, although these same antibodies did protect against a serotype 5 S. aureus strain genetically engineered to produce CP8. We detected soluble CP8 in culture supernatants of serotype 8 clinical isolates and in the plasma of infected animals. Serotype 5 S. aureus released significantly less soluble CP5 in vitro and in vivo. The release of soluble CP8 by S. aureus may contribute to the inability of CP8 vaccines or antibodies to protect against serotype 8 staphylococcal infections.

  6. Regulated expression of polysaccharide utilization and capsular biosynthesis loci in biofilm and planktonic Bacteroides thetaiotaomicron during growth in chemostats

    PubMed Central

    TerAvest, Michaela A.; He, Zhen; Rosenbaum, Miriam A.; Martens, Eric C.; Cotta, Michael A.; Gordon, Jeffrey I.; Angenent, Largus T.

    2014-01-01

    Bacteroides thetaiotaomicron is a prominent member of the human distal gut microbiota that specializes in breaking down diet and host-derived polysaccharides. While polysaccharide utilization has been well studied in B. thetaiotaomicron, other aspects of its behavior are less well characterized, including the factors that allow it to maintain itself in the gut. Biofilm formation may be a mechanism for bacterial retention in the gut. Therefore, we used custom GeneChips to compare the transcriptomes of biofilm and planktonic B. thetaiotaomicron during growth in mono-colonized chemostats. We identified 1154 genes with a fold-change greater than 2, with confidence greater than or equal to 95%. Among the prominent changes observed in biofilm populations were: (i) greater expression of genes in polysaccharide utilization loci that are involved in foraging of O-glycans normally found in the gut mucosa; and (ii) regulated expression of capsular polysaccharide biosynthesis loci. Hierarchical clustering of the data with different datasets, which were obtained during growth under a range of conditions in minimal media and in intestinal tracts of gnotobiotic mice, revealed that within this group of differentially expressed genes, biofilm communities were more similar to the in vivo samples than to planktonic cells and exhibited features of substrate limitation. The current study also validates the use of chemostats as an in vitro ‘gnotobiotic’ model to study gene expression of attached populations of this bacterium. This is important to gut microbiota research, because bacterial attachment and the consequences of disruptions in attachment are difficult to study in vivo. PMID:23996813

  7. Serological, chemical, and structural analyses of the Escherichia coli cross-reactive capsular polysaccharides K13, K20, and K23.

    PubMed Central

    Vann, W F; Soderstrom, T; Egan, W; Tsui, F P; Schneerson, R; Orskov, I; Orskov, F

    1983-01-01

    The Escherichia coli K13, K20, and K23 capsular polysaccharide antigens are serologically related. All of these polysaccharides contain ribose and 2-keto-3-deoxyoctonate in equimolar quantities. The K13 and K20 polysaccharides are partially O-acetylated. A comparison of these polysaccharides after O-deacetylation, by nuclear magnetic resonance and permethylation analysis, showed that these polysaccharides contained the disaccharide repeat unit leads to)-beta-ribofuranosyl-(1 leads to 7)-beta-2-keto-3-deoxyoctonate. They differed in the presence and location of an acetyl moiety. The K13 polysaccharide was O-acetylated at C-4 of the 2-keto-3-deoxyoctonate. The K20 antigen was O-acetylated at C-5 of the ribose moiety. The K23 polymer was nonacetylated. The cross-reactivity of these antigens was demonstrated by tandem-crossed immunoelectrophoresis. Antibodies to K23 could be completely absorbed from OK K23 serum by K13, K20, and K23 antigenic extracts. The K13 and K20 antibodies could be completely absorbed from their respective antisera only by homologous antigenic extracts. Monoclonal antibodies were prepared against a protein conjugate of the K13 polysaccharide. Analyses of the reactions of these antibodies with the three polysaccharides suggest that the K13 polysaccharide has at least three antigenic sites, one of which is common to the K13, K20, and K23 polysaccharides. PMID:6187684

  8. NMR and molecular dynamics studies of the conformational epitope of the type III group B Streptococcus capsular polysaccharide and derivatives.

    PubMed

    Brisson, J R; Uhrinova, S; Woods, R J; van der Zwan, M; Jarrell, H C; Paoletti, L C; Kasper, D L; Jennings, H J

    1997-03-18

    The conformational epitope of the type III group B Streptococcus capsular polysaccharide (GBSP III) exhibits unique properties which can be ascribed to the presence of sialic acid in its structure and the requirement for an extended binding site. By means of NMR and molecular dynamics studies on GBSP III and its fragments, the extended epitope of GBSP III was further defined. The influence of sialic acid on the conformational properties of GBSP III was examined by performing conformational analysis on desialylated GBSP III, which is identical to the polysaccharide of Streptococcus pneumoniae type 14, and also on oxidized and reduced GBSP III. Conformational changes were gauged by 1H and 13C chemical shift analysis, NOE, 1D selective TOCSY-NOESY experiments, J(HH) and J(CH) variations, and NOE of OH resonances. Changes in mobility were examined by 13C T1 and T2 measurements. Unrestrained molecular dynamics simulations with explicit water using the AMBER force field and the GLYCAM parameter set were used to assess static and dynamic conformational models, simulate the observable NMR parameters and calculate helical parameters. GBSP III was found to be capable of forming extended helices. Hence, the length dependence of the conformational epitope could be explained by its location on extended helices within the random coil structure of GBSP III. The interaction of sialic acid with the backbone of the PS was also found to be important in defining the conformational epitope of GBSP III.

  9. Role of capsular polysaccharide (CPS) in biofilm formation and regulation of CPS production by quorum-sensing in Vibrio vulnificus.

    PubMed

    Lee, Kyung-Jo; Kim, Jeong-A; Hwang, Won; Park, Soon-Jung; Lee, Kyu-Ho

    2013-11-01

    Extracellular polysaccharides, such as lipopolysaccharide and loosely associated exopolysaccharides, are essential for Vibrio vulnificus to form biofilms. The role of another major component of the V. vulnificus extracellular matrix, capsular polysaccharide (CPS), which contributes to colony opacity, has been characterized in biofilm formation. A CPS-deficient mutant, whose wbpP gene encoding UDP-GlcNAc C4-epimerase was knocked out, formed significantly more biofilm than wild type, due to increased hydrophobicity of the cell surface, adherence to abiotic surfaces and cell aggregation. To elucidate the direct effect of CPS on biofilm structure, extracted CPS and a CPS-degrading enzyme, α-N-acetylgalactosaminidase, were added in biofilm assays, resulting in reduction and increment of biofilm sizes respectively. Therefore, it is suggested that CPS play a critical role in determining biofilm size by restricting continual growth of mature biofilms. Since CPS is required after maturation, CPS biosynthesis should be controlled in a cell density-dependent manner, e.g. by quorum-sensing (QS) regulation. Analysing transcription of the CPS gene cluster revealed that it was activated by SmcR, a QS master regulator, via binding to the upstream region of the cluster. Therefore, CPS was produced when biofilm cell density reached high enough to turn on QS regulation and limited biofilms to appropriate sizes.

  10. E. coli Group 1 Capsular Polysaccharide Exportation Nanomachinary as a Plausible Antivirulence Target in the Perspective of Emerging Antimicrobial Resistance

    PubMed Central

    Sachdeva, Shivangi; Palur, Raghuvamsi V.; Sudhakar, Karpagam U.; Rathinavelan, Thenmalarchelvi

    2017-01-01

    Bacteria evolving resistance against the action of multiple drugs and its ability to disseminate the multidrug resistance trait(s) across various strains of the same bacteria or different bacterial species impose serious threat to public health. Evolution of such multidrug resistance is due to the fact that, most of the antibiotics target bacterial survival mechanisms which exert selective pressure on the bacteria and aids them to escape from the action of antibiotics. Nonetheless, targeting bacterial virulence strategies such as bacterial surface associated polysaccharides biosynthesis and their surface accumulation mechanisms may be an attractive strategy, as they impose less selective pressure on the bacteria. Capsular polysaccharide (CPS) or K-antigen that is located on the bacterial surface armors bacteria from host immune response. Thus, unencapsulating bacteria would be a good strategy for drug design, besides CPS itself being a good vaccine target, by interfering with CPS biosynthesis and surface assembly pathway. Gram-negative Escherichia coli uses Wzy-polymerase dependent (Groups 1 and 4) and ATP dependent (Groups 1 and 3) pathways for CPS production. Considering E. coli as a case in point, this review explains the structure and functional roles of proteins involved in Group 1 Wzy dependent CPS biosynthesis, surface expression and anchorage in relevance to drug and vaccine developments. PMID:28217109

  11. A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate

    PubMed Central

    Abdelhameed, Ali Saber; Adams, Gary G.; Morris, Gordon A.; Almutairi, Fahad M.; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E.

    2016-01-01

    Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 106 g.mol−1) compared to the native (Mw ~ 1.2 × 106 g.mol−1). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 106 g.mol−1), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution. PMID:26915577

  12. Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide.

    PubMed

    Mawas, Fatme; Bolgiano, Barbara; Rigsby, Peter; Crane, Dennis; Belgrave, Danielle; Corbel, Michael J

    2007-10-01

    Haemophilus influenzae b conjugate vaccines (Hib) are almost entirely evaluated by physico-chemical methods to ensure the consistency of manufacture of batches. As different assays are employed for the quantification of Hib capsular polysaccharide PRP (polyribosyl ribitol phosphate; 5-D-ribitol-(1-->1)-beta-D-ribose-3-phosphate) in final formulations and bulk components, there was deemed a need for an International Standard of Hib PRP polysaccharide to be made available. Ten laboratories from 8 different countries participated in a collaborative study to determine the PRP content and assess the suitability of a candidate International Standard PRP preparation (02/208). The results illustrate that a reduction in between-laboratory variability could be achieved by use of a common reference preparation and data analysis showed no significant differences in the values obtained by the different assays: ribose, phosphorus, and high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), suggesting the suitability of the proposed reference for use across these assays for quantification of PRP content in Hib vaccines. On the basis of the results of this study, the First International Standard for PRP, NIBSC Code 02/208, has been established by the Expert Committee of Biological Standards of the World Health Organisation, with a content of 4.933+/-0.267mg/ampoule, as determined by the ribose assays carried out by 7 of the participating laboratories.

  13. A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate.

    PubMed

    Abdelhameed, Ali Saber; Adams, Gary G; Morris, Gordon A; Almutairi, Fahad M; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E

    2016-02-26

    Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 10(6) g.mol(-1)) compared to the native (Mw ~ 1.2 × 10(6) g.mol(-1)). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 10(6) g.mol(-1)), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution.

  14. The Elastic Properties of the Cryptococcus neoformans Capsule

    PubMed Central

    Frases, Susana; Pontes, Bruno; Nimrichter, Leonardo; Rodrigues, Marcio L.; Viana, Nathan B.; Casadevall, Arturo

    2009-01-01

    Abstract Microbial capsules are important for virulence, but their architecture and physical properties are poorly understood. The human pathogenic fungus Cryptococcus neoformans has a large polysaccharide capsule that is necessary for virulence and is the target of protective antibody responses. To study the C. neoformans capsule we developed what we believe is a new approach whereby we probed the capsular elastic properties by applying forces using polystyrene beads manipulated with optical tweezers. This method allowed us to determine the Young's modulus for the capsule in various conditions that affect capsule growth. The results indicate that the Young's modulus of the capsule decreases with its size and increases with the Ca2+ concentration in solution. Also, capsular polysaccharide manifests an unexpected affinity for polystyrene beads, a property that may function in attachment to host cells and environmental structures. Bead probing with optical tweezers provides a new, nondestructive method that may have wide applicability for studying the effects of growth conditions, immune components, and drugs on capsular properties. PMID:19686640

  15. Hydrolytic stability of pneumococcal group 6 (type 6A and 6B) capsular polysaccharides.

    PubMed

    Zon, G; Szu, S C; Egan, W; Robbins, J D; Robbins, J B

    1982-07-01

    The hydrolyses of the immunologically cross-reactive and constitutionally isomeric group 6 pneumococcal polysaccharides, types 6A and 6B, were investigated by 31P nuclear magnetic resonance spectroscopy, gel filtration through Sepharose 4B, reducing-sugar analysis, and rocket immunoelectrophoresis. Phosphorus nuclear magnetic resonance spectroscopy showed that cleavage of the repeating-unit phosphodiester linkages at pH 10, 60 degrees C was considerably faster (greater than 10(3) ) for the type 6A than the type 6B polysaccharide. Under these reaction conditions, 31P nuclear magnetic resonance kinetic measurements showed that the Na+ form of the type 6A polysaccharide underwent phosphodiester-linkage hydrolysis two times slower than the corresponding Ca+2 form; a stoichiometrically excess amount of Ca+2 caused a 30-fold enhancement of the latter hydrolysis rate. The spectroscopic characterization of phosphorus-containing end groups resulting from hydrolysis of the type 6A polymer provided additional mechanistic information. Heating the type 6A and 6B polysaccharides at 56 degrees C for various times led to gel filtration coefficients of distribution (Kd values) which indicated that the type 6A material underwent size reductions considerably faster than did the type 6B antigen; these increased Kd values qualitatively correlated with the loss of immunochemical reactivity measured by rocket immunoelectrophoresis. The application of a statistical theory to the depolymerization of the type 6A and 6B polysaccharides was consistent with random bond cleavage, as evidenced by the calculated versus measured gel filtration patterns. Although the molecular changes causing the size reductions were not fully elaborated, it was established that the acetal linkages of the type 6A and 6B polysaccharides were comparatively resistant to hydrolysis and that depolymerization by hydrolysis of the phosphodiester linkage was a major factor only in the type 6A structure. It was concluded

  16. Hydrolytic stability of pneumococcal group 6 (type 6A and 6B) capsular polysaccharides.

    PubMed Central

    Zon, G; Szu, S C; Egan, W; Robbins, J D; Robbins, J B

    1982-01-01

    The hydrolyses of the immunologically cross-reactive and constitutionally isomeric group 6 pneumococcal polysaccharides, types 6A and 6B, were investigated by 31P nuclear magnetic resonance spectroscopy, gel filtration through Sepharose 4B, reducing-sugar analysis, and rocket immunoelectrophoresis. Phosphorus nuclear magnetic resonance spectroscopy showed that cleavage of the repeating-unit phosphodiester linkages at pH 10, 60 degrees C was considerably faster (greater than 10(3) ) for the type 6A than the type 6B polysaccharide. Under these reaction conditions, 31P nuclear magnetic resonance kinetic measurements showed that the Na+ form of the type 6A polysaccharide underwent phosphodiester-linkage hydrolysis two times slower than the corresponding Ca+2 form; a stoichiometrically excess amount of Ca+2 caused a 30-fold enhancement of the latter hydrolysis rate. The spectroscopic characterization of phosphorus-containing end groups resulting from hydrolysis of the type 6A polymer provided additional mechanistic information. Heating the type 6A and 6B polysaccharides at 56 degrees C for various times led to gel filtration coefficients of distribution (Kd values) which indicated that the type 6A material underwent size reductions considerably faster than did the type 6B antigen; these increased Kd values qualitatively correlated with the loss of immunochemical reactivity measured by rocket immunoelectrophoresis. The application of a statistical theory to the depolymerization of the type 6A and 6B polysaccharides was consistent with random bond cleavage, as evidenced by the calculated versus measured gel filtration patterns. Although the molecular changes causing the size reductions were not fully elaborated, it was established that the acetal linkages of the type 6A and 6B polysaccharides were comparatively resistant to hydrolysis and that depolymerization by hydrolysis of the phosphodiester linkage was a major factor only in the type 6A structure. It was concluded

  17. Capsular Polysaccharide (CPS) Release by Serotype 3 Pneumococcal Strains Reduces the Protective Effect of Anti-Type 3 CPS Antibodies.

    PubMed

    Choi, Eun Hwa; Zhang, Fan; Lu, Ying-Jie; Malley, Richard

    2015-12-16

    The efficacy of the serotype 3 (ST3) pneumococcal conjugate vaccine (PCV) remains unclear. While the synthesis of capsular polysaccharide (CPS) of most serotypes is wzy dependent, the strains of two serotypes, 3 and 37, synthesize CPS by the synthase-dependent pathway, resulting in a polysaccharide that is not covalently linked to peptidoglycan and can be released during growth. We hypothesized that the release of CPS during growth reduces anti-type 3 CPS antibody-mediated protection and may explain the lower efficacy of the type 3 component of PCV than that of other PCVs. The in vitro-released CPS concentrations per 10(7) CFU of ST3 and ST37 strains were significantly higher than those for the ST1, ST4, ST6B, and ST14 strains. Following intraperitoneal (i.p.) injection in mice, blood concentrations of CPS were significantly higher for the ST3 than for the ST4/5 strains. The opsonophagocytic killing assay (OPKA) titer of anti-type 3 CPS antibody was significantly reduced by type 3 CPS, culture supernatant, or serum from Streptococcus pneumoniae ST3 strain WU2-infected mice. Mice were injected with capsule-specific antibodies and challenged i.p. with or without the addition of sterile culture supernatant containing type-specific CPS. The addition of 0.2 μl of culture supernatant from WU2 inhibited passive protection, whereas 100-fold-more culture supernatant from S. pneumoniae ST4 strain TIGR4 was required for the inhibition of protection. We conclude that released type 3 CPS interferes with antibody-mediated killing and protection by anti-CPS antibodies. The relative failure of ST3 PCV may be due to CPS release, suggesting that alternative immunization approaches for ST3 may be necessary.

  18. Capsular Polysaccharide (CPS) Release by Serotype 3 Pneumococcal Strains Reduces the Protective Effect of Anti-Type 3 CPS Antibodies

    PubMed Central

    Choi, Eun Hwa; Zhang, Fan; Lu, Ying-Jie

    2015-01-01

    The efficacy of the serotype 3 (ST3) pneumococcal conjugate vaccine (PCV) remains unclear. While the synthesis of capsular polysaccharide (CPS) of most serotypes is wzy dependent, the strains of two serotypes, 3 and 37, synthesize CPS by the synthase-dependent pathway, resulting in a polysaccharide that is not covalently linked to peptidoglycan and can be released during growth. We hypothesized that the release of CPS during growth reduces anti-type 3 CPS antibody-mediated protection and may explain the lower efficacy of the type 3 component of PCV than that of other PCVs. The in vitro-released CPS concentrations per 107 CFU of ST3 and ST37 strains were significantly higher than those for the ST1, ST4, ST6B, and ST14 strains. Following intraperitoneal (i.p.) injection in mice, blood concentrations of CPS were significantly higher for the ST3 than for the ST4/5 strains. The opsonophagocytic killing assay (OPKA) titer of anti-type 3 CPS antibody was significantly reduced by type 3 CPS, culture supernatant, or serum from Streptococcus pneumoniae ST3 strain WU2-infected mice. Mice were injected with capsule-specific antibodies and challenged i.p. with or without the addition of sterile culture supernatant containing type-specific CPS. The addition of 0.2 μl of culture supernatant from WU2 inhibited passive protection, whereas 100-fold-more culture supernatant from S. pneumoniae ST4 strain TIGR4 was required for the inhibition of protection. We conclude that released type 3 CPS interferes with antibody-mediated killing and protection by anti-CPS antibodies. The relative failure of ST3 PCV may be due to CPS release, suggesting that alternative immunization approaches for ST3 may be necessary. PMID:26677201

  19. Structure of the capsular polysaccharide of Vibrio cholerae O139 synonym Bengal containing D-galactose 4,6-cyclophosphate.

    PubMed

    Knirel, Y A; Paredes, L; Jansson, P E; Weintraub, A; Widmalm, G; Albert, M J

    1995-09-01

    The capsular polysaccharide (CPS) of Vibrio cholerae O139 synonym Bengal, which is thought to carry determinants of O-specificity, was isolated by phenol/water extraction followed by delipidation of the contaminating lipopolysaccharide at pH 4.2 and gel-permeation chromatography. The CPS contained D-galactose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), D-galacturonic acid (D-GalA), and phosphate. The CPS was studied by NMR spectroscopy, methylation analysis, and selective degradations, including partial acid hydrolysis at pH 3.1 and dephosphorylation with aqueous 48% hydrofluoric acid, which both resulted in complete cleavage of Col. It was concluded that the CPS is built up of hexasaccharide repeating units containing inter alia D-galactose 4,6-cyclophosphate and having the following structure [structure: see text] These data basically confirm the structure of the V. cholerae CPS proposed on the basis of an NMR study [L. M. Preston et al. (1995) J. Bacteriol. 177, 835-838] and specify exactly the absolute configurations of the constituent monosaccharides and the position of the cyclic phosphate.

  20. Fur regulation of the capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43.

    PubMed

    Lin, Ching-Ting; Wu, Chien-Chen; Chen, Yu-Sheng; Lai, Yi-Chyi; Chi, Chia; Lin, Jing-Ciao; Chen, Yeh; Peng, Hwei-Ling

    2011-02-01

    The ferric uptake regulator Fur has been reported to repress the expression of rmpA, a regulatory gene for the mucoid phenotype, leading to decreased capsular polysaccharide (CPS) biosynthesis in Klebsiella pneumoniae CG43. Here, quantitative real-time PCR (qRT-PCR) analyses and electrophoretic mobility shift assays showed that Fur also repressed the expression of the CPS regulatory genes rmpA2 and rcsA. Interestingly, deletion of rmpA or rcsA but not rmpA2 from the Δfur strain was able to suppress the deletion effect of Fur. The availability of extracellular iron affected the amount of CPS, suggesting that Fur regulates CPS biosynthesis in an Fe(II)-dependent manner. Increased production of siderophores was observed in the Δfur strain, suggesting that uptake of extracellular iron in K. pneumoniae is regulated by Fur. Fur titration assays and qRT-PCR analyses demonstrated that at least six of the eight putative iron-acquisition systems, identified by a blast search in the contig database of K. pneumoniae CG43, were directly repressed by Fur. We conclude that Fur has a dual role in the regulation of CPS biosynthesis and iron acquisition in K. pneumoniae.

  1. Capsular polysaccharides facilitate enhanced iron acquisition by the colonial cyanobacterium Microcystis sp. isolated from a freshwater lake.

    PubMed

    Li, Zheng-Ke; Dai, Guo-Zheng; Juneau, Philippe; Qiu, Bao-Sheng

    2016-02-01

    Microcystis sp., especially in its colonial form, is a common dominant species during cyanobacterial blooms in many iron-deficient water bodies. It is still not entirely clear, however, how the colonial forms of Microcystis acclimate to iron-deficient habitats, and the responses of unicellular and colonial forms to iron-replete and iron-deficient conditions were examined here. Growth rates and levels of photosynthetic pigments declined to a greater extent in cultures of unicellular Microcystis than in cultures of the colonial form in response to decreasing iron concentrations, resulting in the impaired photosynthetic performance of unicellular Microcystis as compared to colonial forms as measured by variable fluorescence and photosynthetic oxygen evolution. These results indicate that the light-harvesting ability and photosynthetic capacity of colonial Microcystis was less affected by iron deficiency than the unicellular form. The carotenoid contents and nonphotochemical quenching of colonial Microcystis were less reduced than those of the unicellular form under decreasing iron concentrations, indicating that the colonial morphology enhanced photoprotection and acclimation to iron-deficient conditions. Furthermore, large amounts of iron were detected in the capsular polysaccharides (CPS) of the colonies, and more iron was found to be attached to the colonial Microcystis CPS under decreasing iron conditions as compared to unicellular cultures. These results demonstrated that colonial Microcystis can acclimate to iron deficiencies better than the unicellular form, and that CPS plays an important role in their acclimation advantage in iron-deficient waters.

  2. Humoral immune response of a pneumococcal conjugate vaccine: capsular polysaccharide serotype 14-Lysine modified PspA.

    PubMed

    Santamaria, Raquel; Goulart, Cibelly; Perciani, Catia T; Barazzone, Giovana C; Carvalho, Rimenys; Gonçalves, Viviane M; Leite, Luciana C C; Tanizaki, Martha M

    2011-11-03

    Polysaccharide-protein conjugates are so far the current antigens used for pneumococcal vaccines for children under 2 years of age. In this study, pneumococcal surface protein A (PspA) was used as a carrier protein for pneumococcal capsular polysaccharide serotype 14 as an alternative to broaden the vaccine coverage. PspA was modified by reductive amination with formaldehyde in order to improve the specificity of the reaction between protein and polysaccharide, inhibiting polymerization and the gel formation reaction. In the synthesis process, the currently used activator, 1-[3-(dimethylamine)propyl]-3-ethylcarbodiimide hydrochloride (EDAC) was substituted for 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). BALB/c mice were immunized with either the PS14-mPspA conjugate or the co-administered components in a three dose regimen and sera from the immunized animals were assayed for immunity induced against both antigens: PS14 and mPspA. Modification of more than 70% of lysine residues from PspA (mPspA) did not interfere in the immune response as evaluated by the anti-PspA titer and C3 complement deposition assay. Sera of mice immunized with conjugated PS14-mPspA showed similar IgG titers, avidity and isotype profile as compared to controls immunized with PspA or mPspA alone. The complement deposition was higher in the sera of mice immunized with the conjugate vaccine and the opsonophagocytic activity was similar for both sera. Conjugation improved the immune response against PS14. The anti PS14 IgG titer was higher in sera of mice immunized with the conjugate than with co-administered antigens and presented an increased avidity index, induction of a predominant IgG1 isotype and increased complement deposition on a bacteria with a surface serotype 14. These results strongly support the use of PspA as carrier in a conjugate vaccine where both components act as antigens.

  3. Evidence for a LOS and a capsular polysaccharide in Capnocytophaga canimorsus

    PubMed Central

    Renzi, Francesco; Ittig, Simon J.; Sadovskaya, Irina; Hess, Estelle; Lauber, Frederic; Dol, Melanie; Shin, Hwain; Mally, Manuela; Fiechter, Chantal; Sauder, Ursula; Chami, Mohamed; Cornelis, Guy R.

    2016-01-01

    Capnocytophaga canimorsus is a dog’s and cat’s oral commensal which can cause fatal human infections upon bites or scratches. Infections mainly start with flu-like symptoms but can rapidly evolve in fatal septicaemia with a mortality as high as 40%. Here we present the discovery of a polysaccharide capsule (CPS) at the surface of C. canimorsus 5 (Cc5), a strain isolated from a fulminant septicaemia. We provide genetic and chemical data showing that this capsule is related to the lipooligosaccharide (LOS) and probably composed of the same polysaccharide units. A CPS was also found in nine out of nine other strains of C. canimorsus. In addition, the genomes of three of these strains, sequenced previously, contain genes similar to those encoding CPS biosynthesis in Cc5. Thus, the presence of a CPS is likely to be a common property of C. canimorsus. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages. An antiserum raised against the capsule increased the killing of C. canimorsus by human serum thus showing that anti-capsule antibodies have a protective role. These findings provide a new major element in the understanding of the pathogenesis of C. canimorsus. PMID:27974829

  4. New insight into chondroitin and heparosan-like capsular polysaccharide synthesis by profiling of the nucleotide sugar precursors

    PubMed Central

    di Lauro, Irene; Di Nuzzo, Rosaria; De Rosa, Mario; Schiraldi, Chiara

    2017-01-01

    Escherichia coli K4 and K5 capsular polysaccharides (K4 and K5 CPSs) have been used as starting material for the biotechnological production of chondroitin sulfate (CS) and heparin (HP) respectively. The CPS covers the outer cell wall but in late exponential or stationary growth phase it is released in the surrounding medium. The released CPS concentration was used, so far, as the only marker to connect the strain production ability to the different cultivation conditions employed. Determining also the intracellular UDP-sugar precursor concentration variations, during the bacterial growth, and correlating it with the total CPS production (as sum of the inner and the released ones), could help to better understand the chain biosynthetic mechanism and its bottlenecks. In the present study, for the first time, a new capillary electrophoresis method was set up to simultaneously analyse the UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) and the inner CPS portion, extracted at the same time from the bacterial biomasses; separation was performed at 18°C and 18 kV with a borate-based buffer and detection at 200 nm. The E. coli K4 and K5 UDP-sugar pools were profiled, for the first time, at different time points of shake flask growths on a glycerol-containing medium and on the same medium supplemented with the monosaccharide precursors of the CPSs: their concentrations varied from 0.25 to 11 μM·gcdw−1, according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced. PMID:28104792

  5. Sub-MICs of Azithromycin Decrease Biofilm Formation of Streptococcus suis and Increase Capsular Polysaccharide Content of S. suis

    PubMed Central

    Yang, Yan-Bei; Chen, Jian-Qing; Zhao, Yu-Lin; Bai, Jing-Wen; Ding, Wen-Ya; Zhou, Yong-Hui; Chen, Xue-Ying; Liu, Di; Li, Yan-Hua

    2016-01-01

    Streptococcus suis (S. suis) caused serious disease symptoms in humans and pigs. S. suis is able to form thick biofilms and this increases the difficulty of treatment. After growth with 1/2 minimal inhibitory concentration (MIC) of azithromycin, 1/4 MIC of azithromycin, or 1/8 MIC of azithromycin, biofilm formation of S. suis dose-dependently decreased in the present study. Furthermore, scanning electron microscopy analysis revealed the obvious effect of azithromycin against biofilm formation of S. suis. Especially, at two different conditions (1/2 MIC of azithromycin non-treated cells and treated cells), we carried out comparative proteomic analyses of cells by using iTRAQ technology. Finally, the results revealed the existence of 19 proteins of varying amounts. Interestingly, several cell surface proteins (such as ATP-binding cassette superfamily ATP-binding cassette transporter (G7SD52), CpsR (K0FG35), Cps1/2H (G8DTL7), CPS16F (E9NQ13), putative uncharacterized protein (G7SER0), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (G5L259), putative uncharacterized protein (G7S2D6), amino acid permease (B0M0G6), and NsuB (G5L351)) were found to be implicated in biofilm formation. More importantly, we also found that azithromycin affected expression of the genes cps1/2H, cpsR and cps16F. Especially, after growth with 1/2 MIC of azithromycin and 1/4 MIC of azithromycin, the capsular polysaccharide content of S. suis was significantly higher. PMID:27812354

  6. Group B Streptococcus Capsular Polysaccharide-Cholera Toxin B Subunit Conjugate Vaccines Prepared by Different Methods for Intranasal Immunization

    PubMed Central

    Shen, Xuzhuang; Lagergård, Teresa; Yang, Yonghong; Lindblad, Marianne; Fredriksson, Margareta; Holmgren, Jan

    2001-01-01

    Group B Streptococcus (GBS) type III capsular polysaccharide (CPS III) was conjugated to recombinant cholera toxin B subunit (rCTB) using three different methods which employed (i) cystamine and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), (ii) carbodiimide with adipic acid dihydrazide (ADH) as a spacer, or (iii) reductive amination (RA). The CPS III-rCTB conjugates were divided into large- and small-molecular-weight (Mr) fractions, and the immunogenicities of the different preparations after intranasal (i.n.) immunization were studied in mice. Both large- and small-Mr conjugates of CPS III-rCTBRA or CPS III-rCTBADH induced high, almost comparable levels of CPS-specific immunoglobulin G (IgG) in serum, lungs, and vagina that were generally superior to those obtained with CPS III-rCTBSPDP conjugates or a CPS III and rCTB mixture. However, the smaller-Mr conjugates of CPS III-rCTBRA or CPS III-rCTBADH in most cases elicited a lower anti-CPS IgA immune response than the large-Mr conjugates, and the highest anti-CPS IgA titers in both tissues and serum were obtained with the large-Mr CPS III-rCTBRA conjugate. Serum IgG anti-CPS titers induced by the CPS III-rCTBRA conjugate had high levels of specific IgG1, IgG2a, IgG2b, and IgG3 antibodies. Based on the effectiveness of RA for coupling CPS III to rCTB, RA was also tested for conjugating GBS CPS Ia with rCTB. As for the CPS III-rCTB conjugates, the immunogenicity of CPS Ia was greatly increased by conjugation to rCTB. Intranasal immunization with a combination of CPS Ia-rCTB and CPS III-rCTB conjugates was shown to induce anti-CPS Ia and III immune responses in serum and lungs that were fully comparable with the responses to immunization with the monovalent CPS Ia-rCTB or CPS III-rCTB conjugates. These results suggest that the GBS CPS III-rCTB and CPS Ia-rCTB conjugates prepared by the RA method may be used in bivalent and possibly also in multivalent mucosal GBS conjugate vaccines. PMID:11119518

  7. A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge.

    PubMed Central

    Fattom, A I; Sarwar, J; Ortiz, A; Naso, R

    1996-01-01

    The efficacy of capsular polysaccharide (CP)-specific antibodies elicited by active immunization with vaccines composed of Staphylococcus aureus types 5 and 8 CP linked to Pseudomonas aeruginosa exoprotein A or with immune immunoglobulin G (I-IgG) obtained from vaccinated plasma donors was tested in lethal and sublethal bacterial mouse challenge models. A dose of 2 x 10(5) CFU of S. aureus type 5 CP per mouse administered intraperitoneally (i.p.) with 5% hog mucin was found to cause 80 to 100% mortality in BALB/c mice within 2 to 5 days. Mice passively immunized i.p. 24 h earlier or subcutaneously 48 h earlier with 0.5 ml of I-IgG showed significantly higher average survival rates than animals receiving standard IgG or saline (P < 0.01) following the bacterial challenge. Animals actively immunized with the monovalent type 5 CP-P. aeruginosa exoprotein A conjugate showed a survival rate of 73% compared with 13% in phosphate-buffered saline-immunized animals. The prechallenge geometric mean titer of type 5 CP antibodies in animals that died was significantly (P < 0.05) lower than that of animals which survived the challenge (95.7 versus 223.6 micrograms/ml, respectively). The IgG was further evaluated in mice challenged i.p. with a sublethal dose of 5 x 10(4) CFU per mouse. Serial blood counts were performed on surviving animals at 6, 12, 24, and 48 h. Surviving animals were sacrificed at 72 h, and bacterial counts were performed on their kidneys, livers, and peritoneal lavage fluids. Animals receiving I-IgG had lower bacterial counts in blood samples and lower bacterial densities in kidneys, livers, and peritoneal lavage samples than mice immunized with standard IgG (P < 0.05). These data suggest that S. aureus type 5 CP antibodies induced by active immunization or administered by passive immunization confer protection against S. aureus infections. PMID:8613375

  8. The contribution of naturally occurring IgM antibodies, IgM cross-reactivity and complement dependency in murine humoral responses to pneumococcal capsular polysaccharides.

    PubMed

    Jones, Hannah E; Taylor, Philip R; McGreal, Eamon; Zamze, Susanne; Wong, Simon Y C

    2009-09-25

    Immunogenicity of 12 capsular polysaccharides (CPS) from Streptococcus pneumoniae did not correlate with pre-existing levels of natural IgM anti-CPS antibodies in mice. Immunization of mice with individual CPS, with the exception of type 14 (the only neutral CPS tested), increased serum IgM that also bound other CPS serotypes independent of structural similarity or commonly known contaminants. Surprisingly only IgM response to type 4 (which has a small immunodominant epitope) was dependent on either complement C3 or complement receptors CD35/CD21. IgG anti-CPS responses were infrequently induced, but critically dependent on complement. Our results have clarified the role of complement in the induction of IgM and IgG anti-CPS antibody responses in mice and have implications for CPS vaccine development.

  9. Cloning of type 8 capsule genes and analysis of gene clusters for the production of different capsular polysaccharides in Staphylococcus aureus.

    PubMed

    Sau, S; Lee, C Y

    1996-04-01

    Eleven serotypes of capsular polysaccharide from Staphylococcus aureus have been reported. We have previously cloned a cluster of type 1 capsule (cap1) genes responsible for type 1 capsular polysaccharide biosynthesis in S. aureus M. To clone the type 8 capsule (cap8) genes, a plasmid library of type 8 strain Becker was screened with a labelled DNA fragment containing the cap1 genes under low-stringency conditions. One recombinant plasmid containing a 14-kb insert was chosen for further study and found to complement 14 of the 18 type 8 capsule-negative (Cap8-) mutants used in the study. Additional library screening, subcloning, and complementation experiments showed that all of the 18 Cap8- mutants were complemented by DNA fragments derived from a 20.5-kb contiguous region of the Becker chromosome. The mutants were mapped into six complementation groups, indicating that the cap8 genes are clustered. By Southern hybridization analyses under high-stringency conditions, we found that DNA fragments containing the cap8 gene cluster show extensive homology with all 17 strains tested, including type 1 strains. By further Southern analyses and cloning of the cap8-related homolog from strain M, we show that strain M carries an additional capsule gene cluster different from the cap1 gene cluster. In addition, by using DNA fragments containing different regions of the cap8 gene cluster as probes to hybridize DNA from different strains, we found that the central region of the cap8 gene cluster hybridizes only to DNAs from certain strains tested whereas the flanking regions hybridize to DNAs of all strains tested. Thus, the cap8 gene clusters and its closely related homologs are likely to have organizations similar to those of the encapsulation genes of other bacterial systems.

  10. Regulated expression of polysaccharide utilization and capsular biosynthesis loci in biofilm and planktonic Bacteroides thetaiotaomicron during growth in chemostats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteroides thetaiotaomicron is a prominent member of the human distal gut microbiota that specializes in breaking down diet and host-derived polysaccharides. While polysaccharide utilization has been well studied in B. thetaiotaomicron, other aspects of its behavior are less well characterized, in...

  11. Vibrio cholerae O139 Conjugate Vaccines: Synthesis and Immunogenicity of V. cholerae O139 Capsular Polysaccharide Conjugates with Recombinant Diphtheria Toxin Mutant in Mice

    PubMed Central

    Kossaczka, Zuzana; Shiloach, Joseph; Johnson, Virginia; Taylor, David N.; Finkelstein, Richard A.; Robbins, John B.; Szu, Shousun C.

    2000-01-01

    Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to ≤50. Treatment with 2-ME reduced the titers of 17 of 20 patients to ≤50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned. PMID:10948122

  12. Comparison of radioimmunoassay and enzyme-linked immunosorbent assay in measurement of antibodies to Neisseria meningitidis group A capsular polysaccharide.

    PubMed Central

    Beuvery, E C; Kayhty, M H; Leussink, A B; Kanhai, V

    1984-01-01

    Antibodies to meningococcal group A polysaccharide were determined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) in serum samples from 16 adults vaccinated with bivalent meningococcal group A and C polysaccharide vaccine. The specific antibody levels in the serum samples were expressed as micrograms of antibody protein per milliliter of serum. For RIA the polysaccharide was radiolabeled extrinsically with 125I. Both native polysaccharide and polysaccharide labeled with 127I were used in ELISA. Because these antigens gave similar results, it can be concluded that the introduction of tyramine and iodine by the labeling procedure did not alter the antigenic activity of the polysaccharide. The reproducibility of RIA was clearly better than that of ELISA. The antibody levels detected by the methods were equal, which means that ELISA can be used satisfactorily to measure antibodies to meningococcal group A polysaccharide quantitatively. Some discrepant results were found due to an underestimation of immunoglobulin M antibodies in ELISA. This was shown by a correlation test in which a weakly significant negative correlation was found between the immunoglobulin M antibody level/immunoglobulin G antibody level ratio and the RIA antibody level/ELISA antibody level ratio. PMID:6436314

  13. Gene content and diversity of the loci encoding biosynthesis of capsular polysaccharides of the 15 serovar reference strains of Haemophilus parasuis.

    PubMed

    Howell, Kate J; Weinert, Lucy A; Luan, Shi-Lu; Peters, Sarah E; Chaudhuri, Roy R; Harris, David; Angen, Oystein; Aragon, Virginia; Parkhill, Julian; Langford, Paul R; Rycroft, Andrew N; Wren, Brendan W; Tucker, Alexander W; Maskell, Duncan J

    2013-09-01

    Haemophilus parasuis is the causative agent of Glässer's disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3' end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer's disease.

  14. Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

    PubMed Central

    Weinert, Lucy A.; Luan, Shi-Lu; Peters, Sarah E.; Chaudhuri, Roy R.; Harris, David; Angen, Øystein; Aragon, Virginia; Parkhill, Julian; Langford, Paul R.; Rycroft, Andrew N.; Wren, Brendan W.; Tucker, Alexander W.; Maskell, Duncan J.

    2013-01-01

    Haemophilus parasuis is the causative agent of Glässer's disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3′ end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer's disease. PMID:23873912

  15. Distribution of the O-acetyl groups and β-galactofuranose units in galactoxylomannans of the opportunistic fungus Cryptococcus neoformans.

    PubMed

    Previato, Jose O; Vinogradov, Evgeny; Maes, Emmanuel; Fonseca, Leonardo M; Guerardel, Yann; Oliveira, Priscila A V; Mendonça-Previato, Lucia

    2016-12-16

    Galactoxylomannans (GalXMs) are a mixture of neutral and acidic capsular polysaccharides produced by the opportunistic fungus Cryptococcus neoformans that exhibit potent suppressive effects on the host immune system. Previous studies describing the chemical structure of C. neoformans GalXMs have reported species without O-acetyl substituents. Herein we describe that C. neoformans grown in capsule-inducing medium produces highly O-acetylated GalXMs. The location of the O-acetyl groups was determined by nuclear magnetic resonance (NMR) spectroscopy. In the neutral GalXM (NGalXM), 80% of 3-linked mannose (α-Manp) residues present in side chains are acetylated at the O-2 position. In the acidic GalXM also termed glucuronoxylomannogalactan (GXMGal), 85% of the 3-linked α-Manp residues are acetylated either in the O-2 (75%) or in the O-6 (25%) position, but O-acetyl groups are not present at both positions simultaneously. In addition, NMR spectroscopy and methylation analysis showed that β-galactofuranose (β-Galf) units are linked to O-2 and O-3 positions of nonbranched α-galactopyranose (α-Galp) units present in the GalXMs backbone chain. These findings highlight new structural features of C. neoformans GalXMs. Among these features, the high degree of O-acetylation is of particular interest, since O-acetyl group-containing polysaccharides are known to possess a range of immunobiological activities.

  16. Phase II, randomized, multicenter, double-blind, placebo-controlled trial of a polyclonal anti-Staphylococcus aureus capsular polysaccharide immune globulin in treatment of Staphylococcus aureus bacteremia.

    PubMed

    Rupp, Mark E; Holley, H Preston; Lutz, Jon; Dicpinigaitis, Peter V; Woods, Christopher W; Levine, Donald P; Veney, Naomi; Fowler, Vance G

    2007-12-01

    New treatment modalities are needed for the treatment of infections due to multidrug-resistant Staphylococcus aureus. S. aureus capsular polysaccharide immune globulin (Altastaph) is a polyclonal immune globulin preparation that is being developed as adjunctive therapy for persons with S. aureus infections complicated by bacteremia. In a phase II, multicenter, randomized, double-blind, placebo-controlled trial, 40 subjects with documented S. aureus bacteremia received standard therapy plus either Altastaph at 200 mg/kg of body weight in each of two infusions 24 h apart or placebo. During the 42-day observation period, antibody pharmacokinetics and safety were the primary characteristics studied. Information regarding the resolution of bacteremia and fever was also analyzed. Anti-type-5 and anti-type-8 capsular antibody levels peaked after the second infusion at 550 mug/ml and 419 mug/ml, respectively, and remained above 100 mug/ml at day 28. A total of 316 adverse events were noted in 39 of 40 subjects. Infusion-related adverse events in Altastaph recipients were infrequent and similar to those among recipients of commercial intravenously administered immunoglobulin G products. Five of 21 (23%) subjects in the Altastaph group died, whereas 2 of 18 (11%) subjects in the placebo group died (P = 0.42). Compared to the control patients, the Altastaph recipients had a shorter median time to the resolution of fever (2 days and 7 days, respectively; P = 0.09) and a shorter length of hospital stay (9 days and 14 days, respectively; P = 0.03). However, these findings are exploratory, and there were few differences in the other variables measured. High levels of opsonizing antibodies were maintained for the initial 4 weeks. Although the study was not powered to show efficacy, these preliminary findings and safety profile suggest that Altastaph may be an effective adjunct to antibiotics and warrants further investigation (ClinicalTrials.gov number NCT00063089).

  17. Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

    PubMed

    Cai, Jian-Piao; Liu, Ling-Li; To, Kelvin K W; Lau, Candy C Y; Woo, Patrick C Y; Lau, Susanna K P; Guo, Yong-Hui; Ngan, Antonio H Y; Che, Xiao-Yan; Yuen, Kwok-Yung

    2015-01-01

    Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans.

  18. Synthesis of di- and tri-saccharide fragments of Salmonella typhi Vi capsular polysaccharide and their zwitterionic analogues.

    PubMed

    Fusari, Matteo; Fallarini, Silvia; Lombardi, Grazia; Lay, Luigi

    2015-12-01

    Zwitterionic polysaccharides (ZPS) behave like traditional T cell-dependent antigens, suggesting the design of new classes of vaccines alternative to currently used glycoconjugates and based on the artificial introduction of a zwitterionic charge motif onto the carbohydrate structure of pathogen antigens. Here we report the new synthesis and antigenic evaluation of di-/tri-saccharide fragments of Salmonella typhi Vi polysaccharide, as well as of their corresponding zwitterionic analogues. Our strategy is based on versatile intermediates enabling chain elongation either by iterative single monomer attachment or by faster and more flexible approach using disaccharide donors. The effect of structural modifications of the synthetic compounds on antigenic properties was evaluated by competitive ELISA. All the oligosaccharides were recognized by specific anti-Vi polyclonal antibodies in a concentration-dependent manner, and the introduction of a zwitterionic motif into the synthetic molecules did not prevent the binding.

  19. Recognition of riboflavin and the capsular polysaccharide of Haemophilus influenzae type b by antibodies generated to the haptenic epitope D-ribitol.

    PubMed

    Ravi, G; Venkatesh, Yeldur P

    2014-04-01

    D-Ribitol, a five-carbon sugar alcohol, is an important metabolite in the pentose phosphate pathway; it is an integral part of riboflavin (vitamin B2) and cell wall polysaccharides in most Gram-positive and a few Gram-negative bacteria. Antibodies specific to D-ribitol were generated in New Zealand white rabbits by using reductively aminated D-ribose-BSA conjugate as the immunogen. MALDI-TOF and amino group analyses of ribitol-BSA conjugate following 120 h reaction showed ~27-30 mol of ribitol conjugated per mole BSA. The presence of sugar alcohol in the conjugates was also confirmed by an increase in molecular mass and a positive periodic acid-Schiff staining in SDS-PAGE. Caprylic acid precipitation of rabbit serum followed by hapten affinity chromatography on ribitol-KLH-Sepharose CL-6B resulted in pure ribitol-specific antibodies (~45-50 μg/mL). The affinity constant of ribitol antibodies was found to be 2.9 × 10(7) M(-1) by non-competitive ELISA. Ribitol antibodies showed 100% specificity towards ribitol, ~800% cross-reactivity towards riboflavin, 10-15% cross-reactivity with sorbitol, xylitol and mannitol, and 5-7% cross-reactivity with L-arabinitol and meso-erythritol. The specificity of antibody to ribitol was further confirmed by its low cross-reactivity (0.4%) with lumichrome. Antibodies to D-ribitol recognized the purified capsular polysaccharide of Haemophilus influenzae type b, which could be specifically inhibited by ribitol. In conclusion, antibodies specific to D-ribitol have been generated and characterized, which have potential applications in the detection of free riboflavin and ribitol in biological samples, as well as identification of cell-surface macromolecules containing ribitol.

  20. Capsular Polysaccharide of Mycoplasma ovipneumoniae Induces Sheep Airway Epithelial Cell Apoptosis via ROS-Dependent JNK/P38 MAPK Pathways

    PubMed Central

    Jiang, Zhongjia; Song, Fuyang; Li, Yanan; Xue, Di; Zhao, Ning; Zhang, Jiamei; Deng, Guangcun; Li, Min

    2017-01-01

    In an attempt to better understand the pathogen-host interaction between invading Mycoplasma ovipneumoniae (M. ovipneumoniae) and sheep airway epithelial cells, biological effects and possible molecular mechanism of capsular polysaccharide of M. ovipneumoniae (CPS) in the induction of cell apoptosis were explored using sheep bronchial epithelial cells cultured in air-liquid interface (ALI). The CPS of M. ovipneumoniae was first isolated and purified. Results showed that CPS had a cytotoxic effect by disrupting the integrity of mitochondrial membrane, accompanied with an increase of reactive oxygen species and decrease of mitochondrial membrane potential (ΔΨm). Of importance, the CPS exhibited an ability to induce caspase-dependent cell apoptosis via both intrinsic and extrinsic apoptotic pathways. Mechanistically, the CPS induced extrinsic cell apoptosis by upregulating FAS/FASL signaling proteins and cleaved-caspase-8 and promoted a ROS-dependent intrinsic cell apoptosis by activating a JNK and p38 signaling but not ERK1/2 signaling of mitogen-activated protein kinases (MAPK) pathways. These findings provide the first evidence that CPS of M. ovipneumoniae induces a caspase-dependent apoptosis via both intrinsic and extrinsic apoptotic pathways in sheep bronchial epithelial cells, which may be mainly attributed by a ROS-dependent JNK and p38 MAPK signaling pathways. PMID:28367270

  1. Comparative characterization of the virulence gene clusters (lipooligosaccharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species.

    PubMed

    Richards, Vincent P; Lefébure, Tristan; Pavinski Bitar, Paulina D; Stanhope, Michael J

    2013-03-01

    Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosaccharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain-Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking.

  2. Structure determination of Streptococcus suis serotype 9 capsular polysaccharide and assignment of functions of the cps locus genes involved in its biosynthesis.

    PubMed

    Vinogradov, Evgueny; Goyette-Desjardins, Guillaume; Okura, Masatoshi; Takamatsu, Daisuke; Gottschalk, Marcelo; Segura, Mariela

    2016-10-04

    Streptococcus suis serotype 9 is the most prevalent S. suis serotype in several European countries. In spite of its pathogenicity for pigs and increasing zoonotic potential, limited information is available on this serotype. Here we determined for the first time the chemical composition and structure of serotype 9 capsular polysaccharide (CPS), a major bacterial virulence factor and the antigen at the origin of S. suis classification into serotypes. Chemical and spectroscopic data gave the repeating unit sequence: [3)Glcol-6-P-3-[D-Gal(α1-2)]D-Gal(β1-3)D-Sug(β1-3)L-Rha(α1-)]n. Compared to previously characterized S. suis CPSs (serotypes 1, 1/2, 2 and 14), serotype 9 CPS does not contain sialic acid but contains a labile 4-keto sugar (2-acetamido-2,6-dideoxy-β-D-xylo-hexopyranos-4-ulose), one particular feature of this serotype. A correlation between S. suis serotype 9 CPS sequence and genes of this serotype cps locus encoding putative glycosyltransferases and polymerase responsible for the biosynthesis of the repeating unit was tentatively established. Knowledge of CPS structure and composition will contribute to better dissect the role of this bacterial component in the pathogenesis of S. suis serotype 9.

  3. Expression of type 8 capsular polysaccharide and production of toxic shock syndrome toxin 1 are associated among vaginal isolates of Staphylococcus aureus.

    PubMed Central

    Lee, J C; Liu, M J; Parsonnet, J; Arbeit, R D

    1990-01-01

    A colony immunoblot method was developed for serotyping the capsular polysaccharides expressed by Staphylococcus aureus isolates. The method was rapid and specific and was performed with either polyclonal or monoclonal antibodies specific for each of the capsule types. S. aureus isolates were obtained from patients with toxic shock syndrome (TSS) or other staphylococcal infections and from asymptomatic women with vaginal colonization. Among the vaginal isolates of S. aureus, expression of the type 8 capsule was significantly (P less than 0.001) more frequent among strains that produced TSS toxin 1 (TSST-1) than it was among TSST-1-negative strains. In contrast, the frequency of type 8 capsule expression was similar among both TSST-1-positive and -negative strains of S. aureus from patients with nonvaginal TSS. When all vaginal and nonvaginal isolates were compared, TSST-1-negative S. aureus strains were equally distributed among the type 5 and 8 and nontypeable capsule groups, whereas TSST-1-positive strains were predominantly capsule type 8. Images PMID:2279990

  4. CpsR, a GntR family regulator, transcriptionally regulates capsular polysaccharide biosynthesis and governs bacterial virulence in Streptococcus pneumoniae

    PubMed Central

    Wu, Kaifeng; Xu, Hongmei; Zheng, Yuqiang; Wang, Libin; Zhang, Xuemei; Yin, Yibing

    2016-01-01

    Transcriptional regulation of capsule expression is critical for pneumococcal transition from carriage to infection, yet the underlying mechanism remains incompletely understood. Here, we describe the regulation of capsular polysaccharide, one of the most important pneumococcal virulence factor by a GntR family regulator, CpsR. Electrophoretic mobility-shift assays have shown the direct interaction between CpsR and the cps promoter (cpsp), and their interaction could be competitively interfered by glucose. DNase I footprinting assays localized the binding site to a region −146 to −114 base pairs relative to the transcriptional start site of the cps locus in S. pneumoniae D39. We found that CpsR negatively controlled the transcription of the cps locus and hence CPS production, which was confirmed by fine-tuning expression of CpsR in a ΔcpsR complemented strain. Increased expression of CpsR in complemented strain led to a decreased resistance to the whole-blood-mediated killing, suggesting a protective role for CpsR-cpsp interaction in the establishment of invasive infection. Finally, animal experiments showed that CpsR-cpsp interaction was necessary for both pneumococcal colonization and invasive infection. Taken together, our results provide a thorough insight into the regulation of capsule production mediated by CpsR and its important roles in pneumococcal pathogenesis. PMID:27386955

  5. ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae.

    PubMed

    Zheng, Yuqiang; Zhang, Xuemei; Wang, Xiaofang; Wang, Libin; Zhang, Jinghui; Yin, Yibing

    2017-01-01

    The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA). Our results showed that phosphorylated mimetic of ComE (ComE(D58E)) bound specifically to the cps locus prompter in vitro, and phosphorylated ComE negatively impacted both cps locus transcription and CPS production attenuating the pneumococcal virulence in vivo. Compared with D39-WT strain, D39ΔcomE mutant exhibited much thicker capsule, attenuated nasopharyngeal colonization and enhanced virulence in both pneumonia and bacteremia models of Balb/c mice. Furthermore, it was demonstrated that CSP-ComD/E competence system involved in regulating negatively the CPS production during the progress of transformation in D39. Our CSP1 induction experiment results showed that the expression of ComE in D39-WT strain increased powerfully by 120% after 10 min of CSP1 induction, but the CPS production in D39-WT strain decreased sharply by 67.1% after 15 min of CSP1 induction. However, the CPS production in D39ΔcomE mutant was almost constant during the whole stage of induction. Additionally, we found that extracellular glucose concentration could affect both the expression of ComE and CPS production of D39 in vitro. Taken together, for the first time, we report that ComE, as a transcriptional regulator of cps locus, plays an important role in transcriptional regulation of cps locus and capsular production level.

  6. ComE, an Essential Response Regulator, Negatively Regulates the Expression of the Capsular Polysaccharide Locus and Attenuates the Bacterial Virulence in Streptococcus pneumoniae

    PubMed Central

    Zheng, Yuqiang; Zhang, Xuemei; Wang, Xiaofang; Wang, Libin; Zhang, Jinghui; Yin, Yibing

    2017-01-01

    The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA). Our results showed that phosphorylated mimetic of ComE (ComED58E) bound specifically to the cps locus prompter in vitro, and phosphorylated ComE negatively impacted both cps locus transcription and CPS production attenuating the pneumococcal virulence in vivo. Compared with D39-WT strain, D39ΔcomE mutant exhibited much thicker capsule, attenuated nasopharyngeal colonization and enhanced virulence in both pneumonia and bacteremia models of Balb/c mice. Furthermore, it was demonstrated that CSP-ComD/E competence system involved in regulating negatively the CPS production during the progress of transformation in D39. Our CSP1 induction experiment results showed that the expression of ComE in D39-WT strain increased powerfully by 120% after 10 min of CSP1 induction, but the CPS production in D39-WT strain decreased sharply by 67.1% after 15 min of CSP1 induction. However, the CPS production in D39ΔcomE mutant was almost constant during the whole stage of induction. Additionally, we found that extracellular glucose concentration could affect both the expression of ComE and CPS production of D39 in vitro. Taken together, for the first time, we report that ComE, as a transcriptional regulator of cps locus, plays an important role in transcriptional regulation of cps locus and capsular production level. PMID

  7. Rapid Detection of Contagious Caprine Pleuropneumonia Using a Mycoplasma capricolum subsp. capripneumoniae Capsular Polysaccharide-Specific Antigen Detection Latex Agglutination Test

    PubMed Central

    March, J. B.; Gammack, C.; Nicholas, R.

    2000-01-01

    Latex microspheres (diameter, 8 μm) were coated with anti-Mycoplasma capricolum subsp. capripneumoniae polyclonal immunoglobulin G (IgG) antiserum (anti-F38 biotype). The coated microspheres, when used in a latex agglutination test (LAT), detected M. capricolum subsp. capripneumoniae antigen in the serum of goats with contagious caprine pleuropneumoniae (CCPP). Beads also agglutinated strongly in the presence of purified M. capricolum subsp. capripneumoniae capsular polysaccharide (CPS). Preabsorption of CPS-specific antibodies prior to coating of the beads removed agglutinating activity in the presence of M. capricolum subsp. capripneumoniae, strongly suggesting that CPS is the likely soluble antigen recognized by the test. In addition, the specificity of the LAT exactly mirrored that of an M. capricolum subsp. capripneumoniae CPS-specific monoclonal antibody (WM25): of the 8 other mycoplasma species tested, agglutination was observed only with bovine serogroup 7. The LAT detected all 11 strains of M. capricolum subsp. capripneumoniae examined in this study, with a sensitivity level of 2 ng of CPS, or the equivalent of 1.7 × 104 CFU, in a reaction volume of 0.03 ml of serum. With field sera from goats with CCPP, the results of the LAT exhibited a 67% correlation with the results of the currently used complement fixation test (CFT), with the main discrepancy in diagnosis resulting from the increased sensitivity of the LAT compared to that of CFT. This antigen-detection LAT should prove particularly useful in identifying animals in the earliest stages of CCPP and combines sensitivity and low cost with ease of application in the field, without the need for any specialist training or equipment. PMID:11060083

  8. Design and optimization of a chromatographic purification process for Streptococcus pneumoniae serotype 23F capsular polysaccharide by a Design of Experiments approach.

    PubMed

    Ji, Yu; Tian, Yang; Ahnfelt, Mattias; Sui, Lili

    2014-06-27

    Multivalent pneumococcal vaccines were used worldwide to protect human beings from pneumococcal diseases. In order to eliminate the toxic organic solutions used in the traditional vaccine purification process, an alternative chromatographic process for Streptococcus pneumoniae serotype 23F capsular polysaccharide (CPS) was proposed in this study. The strategy of Design of Experiments (DoE) was introduced into the process development to solve the complicated design procedure. An initial process analysis was given to review the whole flowchart, identify the critical factors of chromatography through FMEA and chose the flowthrough mode due to the property of the feed. A resin screening study was then followed to select candidate resins. DoE was utilized to generate a resolution IV fractional factorial design to further compare candidates and narrow down the design space. After Capto Adhere was selected, the Box-Behnken DoE was executed to model the process and characterize all effects of factors on the responses. Finally, Monte Carlo simulation was used to optimize the process, test the chosen optimal conditions and define the control limit. The results of three scale-up runs at set points verified the DoE and simulation predictions. The final results were well in accordance with the EU pharmacopeia requirements: Protein/CPS (w/w) 1.08%; DNA/CPS (w/w) 0.61%; the phosphorus content 3.1%; the nitrogen 0.315% and the Methyl-pentose percentage 47.9%. Other tests of final pure CPS also met the pharmacopeia specifications. This alternative chromatographic purification process for pneumococcal vaccine without toxic organic solvents was successfully developed by the DoE approach and proved scalability, robustness and suitability for large scale manufacturing.

  9. Diversity of Capsular Polysaccharide Gene Clusters in Kpc-Producing Klebsiella pneumoniae Clinical Isolates of Sequence Type 258 Involved in the Italian Epidemic

    PubMed Central

    D’Andrea, Marco Maria; Amisano, Francesco; Giani, Tommaso; Conte, Viola; Ciacci, Nagaia; Ambretti, Simone; Santoriello, Luisa; Rossolini, Gian Maria

    2014-01-01

    Strains of Klebsiella pneumoniae producing KPC-type beta-lactamases (KPC-Kp) are broadly disseminating worldwide and constitute a major healthcare threat given their extensively drug resistant phenotypes and ability to rapidly disseminate in healthcare settings. In this work we report on the characterization of two different capsular polysaccharide (CPS) gene clusters, named cpsBO-4 and cps207-2, from two KPC-Kp clinical strains from Italy belonging in sequence type (ST) 258, which is one of the most successful ST of KPC-Kp spreading worldwide. While cpsBO-4 was different from known 78 K-types according to the recently proposed typing schemes based on the wzi or wzc gene sequences, cps207-2 was classified as K41 by one of these methods. Bioinformatic analysis revealed that they were represented in the genomic sequences of KPC-Kp from strains of ST258 from different countries, and cpsBO-4 was also detected in a KPC-Kp strain of ST442 from Brazil. Investigation of a collection of 46 ST258 and ST512 (a single locus variant of ST258) clinical strains representative of the recent Italian epidemic of KPC-Kp by means of a multiplex PCR typing approach revealed that cpsBO-4 was the most prevalent type, being detected both in ST258 and ST512 strains with a countrywide distribution, while cps207-2 was only detected in ST258 strains with a more restricted distribution. PMID:24823690

  10. USA300 and USA500 Clonal Lineages of Staphylococcus aureus Do Not Produce a Capsular Polysaccharide Due to Conserved Mutations in the cap5 Locus

    PubMed Central

    Li, Xue; Alam, Md Tauqeer; Read, Timothy D.; Sieth, Julia; Cywes-Bentley, Colette; Dobbins, Ginette; David, Michael Z.; Kumar, Neha; Eells, Samantha J.; Miller, Loren G.; Boxrud, David J.; Chambers, Henry F.; Lynfield, Ruth; Lee, Jean C.; Daum, Robert S.

    2015-01-01

    ABSTRACT The surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed against Staphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistant S. aureus (MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptible S. aureus (MSSA) isolates were CP negative (CP−). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP−. Whole-genome sequence analysis of 146 CP− USA300 MRSA isolates revealed they all carry a cap5 locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in the cap5 promoter, cap5D nucleotide 994, and cap5E nucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same four cap5 mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of the cap loci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specific cap5 mutations arose sequentially in S. aureus in a common ancestor of USA300 and USA500 isolates. PMID:25852165

  11. Diversity of capsular polysaccharide gene clusters in Kpc-producing Klebsiella pneumoniae clinical isolates of sequence type 258 involved in the Italian epidemic.

    PubMed

    D'Andrea, Marco Maria; Amisano, Francesco; Giani, Tommaso; Conte, Viola; Ciacci, Nagaia; Ambretti, Simone; Santoriello, Luisa; Rossolini, Gian Maria

    2014-01-01

    Strains of Klebsiella pneumoniae producing KPC-type beta-lactamases (KPC-Kp) are broadly disseminating worldwide and constitute a major healthcare threat given their extensively drug resistant phenotypes and ability to rapidly disseminate in healthcare settings. In this work we report on the characterization of two different capsular polysaccharide (CPS) gene clusters, named cpsBO-4 and cps207-2, from two KPC-Kp clinical strains from Italy belonging in sequence type (ST) 258, which is one of the most successful ST of KPC-Kp spreading worldwide. While cpsBO-4 was different from known 78 K-types according to the recently proposed typing schemes based on the wzi or wzc gene sequences, cps207-2 was classified as K41 by one of these methods. Bioinformatic analysis revealed that they were represented in the genomic sequences of KPC-Kp from strains of ST258 from different countries, and cpsBO-4 was also detected in a KPC-Kp strain of ST442 from Brazil. Investigation of a collection of 46 ST258 and ST512 (a single locus variant of ST258) clinical strains representative of the recent Italian epidemic of KPC-Kp by means of a multiplex PCR typing approach revealed that cpsBO-4 was the most prevalent type, being detected both in ST258 and ST512 strains with a countrywide distribution, while cps207-2 was only detected in ST258 strains with a more restricted distribution.

  12. Preparation and characterization of a Staphylococcus aureus capsular polysaccharide-protein conjugate prepared by a low cost technique: a proof-of-concept study.

    PubMed

    Pujato, Nazarena; Díaz, Germán; Barbagelata, María Sol; Vicco, Miguel Hernán; Calvinho, Luis Fernando; Marcipar, Iván Sergio

    2015-01-01

    Staphylococcus aureus is a worldwide distributed pathogen that produces several diseases in many species and is the major cause of mastitis in dairy cows. S. aureus capsular polysaccharide 5 (CP5) has been widely proposed as a vaccine candidate since it is expressed in a high proportion of isolates from intramammary infections and is able to induce opsonophagocytic antibodies. However, to reach immunological properties, polysaccharides need to be coupled to carrier proteins. The aim of this study was to evaluate a conjugation method employing p-benzoquinone (PBQ), which was not previously reported for the development of vaccine components. Purified S. aureus CP5 was coupled to human serum albumin (HSA) with high efficiency, reaching a rate PS/protein of 0.5. Mice groups were immunized at days 0, 14, 28, and 42, with the conjugate (CP5-HSAPBQ), free CP5, or PBS, formulated with incomplete Freund adjuvant, and after 3 months, they were challenged with free CP5 to evaluate the memory response. IgG and IgM isotypes were measured on serum samples all along the experiment, and IgG subclasses were determined to analyze the humoral profile. In contrast to the response obtained with free CP5, CP5-HSAPBQ induced IgG titers of 1/238,900 after three doses and a memory response was observed after the challenge. Results indicate that immunization with CP5-HSAPBQ effectively induce a T-dependent immune response against CP5. Moreover, besides IgG2a was the main subtype obtained, the joint production of specific IgG1, IgG2b, and IgG3 types indicated a balanced humoral response. As p-benzoquinone conjugation of CPs to proteins is far less expensive and straightforward than other methods commonly used in vaccine preparations, the robust humoral response obtained using this method points out that this can be an interesting alternative to prepare S. aureus CP5 conjugate vaccines.

  13. STEPWISE INTRATYPE TRANSFORMATION OF PNEUMOCOCCUS FROM R TO S BY WAY OF A VARIANT INTERMEDIATE IN CAPSULAR POLYSACCHARIDE PRODUCTION

    PubMed Central

    MacLeod, Colin M.; Krauss, Marjorie R.

    1947-01-01

    1. A variant intermediate between the classical R and S forms has been isolated by selective procedures from a rough strain of pneumococcus originally derived from Type II S. 2. The intermediate variant D39/Int53 is avirulent for mice, forms rough colonies, and does not possess a demonstrable capsule. However, it synthesizes SSSII which is immunologically indistinguishable from that produced by fully encapsulated pneumococcus Type II, though in much smaller amount. The polysaccharide is present as a surface component and as it exists in the cell is highly antigenic for rabbits. 3. An extract of the intermediate variant causes the transformation in vitro of an R strain into a variant resembling the intermediate in SSSII production but without any apparent alteration in the colonial characteristics of the R variant. 4. The intermediate variant is convertible in vivo, into a fully encapsulated strain of pneumococcus Type II. Transformation of the intermediate to a heterologous type of pneumococcus (Type III) was unsuccessful. 5. A method is described for the preparation of transforming extracts of pneumococci utilizing the massive growth of the organisms obtained in the presence of a large concentration of glucose. PMID:19871689

  14. The K1 Capsular Polysaccharide of Acinetobacter baumannii Strain 307-0294 Is a Major Virulence Factor ▿

    PubMed Central

    Russo, Thomas A.; Luke, Nicole R.; Beanan, Janet M.; Olson, Ruth; Sauberan, Shauna L.; MacDonald, Ulrike; Schultz, L. Wayne; Umland, Timothy C.; Campagnari, Anthony A.

    2010-01-01

    Acinetobacter baumannii is a pathogen of increasing medical importance with a propensity to be multidrug resistant, thereby making treatment challenging. Little is known of virulence traits in A. baumannii. To identify virulence factors and potential drug targets, random transposon (Tn) mutants derived from the A. baumannii strain AB307-0294 were screened to identify genes essential for growth in human ascites fluid in vitro, an inflammatory exudative fluid. These studies led to the identification of two genes that were predicted to be required for capsule polymerization and assembly. The first, ptk, encodes a putative protein tyrosine kinase (PTK), and the second, epsA, encodes a putative polysaccharide export outer membrane protein (EpsA). Monoclonal antibodies used in flow cytometric and Western analyses confirmed that these genes are required for a capsule-positive phenotype. A capsule-positive phenotype significantly optimized growth in human ascites fluid, survival in human serum, and survival in a rat soft tissue infection model. Importantly, the clearance of the capsule-minus mutants AB307.30 (ptk mutant, capsule minus) and AB307.45 (epsA mutant, capsule minus) was complete and durable. These data demonstrated that the K1 capsule from AB307-0294 was an important protectin. Further, these data suggested that conserved proteins, which contribute to the capsule-positive phenotype, are potential antivirulence drug targets. Therefore, the results from this study have important biologic and translational implications and, to the best of our knowledge, are the first to address the role of capsule in the pathogenesis of A. baumannii infection. PMID:20643860

  15. Constitutive Expression of the Vi Polysaccharide Capsular Antigen in Attenuated Salmonella enterica Serovar Typhi Oral Vaccine Strain CVD 909

    PubMed Central

    Wang, Jin Yuan; Noriega, Fernando R.; Galen, James E.; Barry, Eileen; Levine, Myron M.

    2000-01-01

    Live oral Ty21a and parenteral Vi polysaccharide vaccines provide significant protection against typhoid fever, albeit by distinct immune mechanisms. Vi stimulates serum immunoglobulin G Vi antibodies, whereas Ty21a, which does not express Vi, elicits humoral and cell-mediated immune responses other than Vi antibodies. Protection may be enhanced if serum Vi antibody as well as cell-mediated and humoral responses can be stimulated. Disappointingly, several new attenuated Salmonella enterica serovar Typhi oral vaccines (e.g., CVD 908-htrA and Ty800) that elicit serum O and H antibody and cell-mediated responses following a single dose do not stimulate serum Vi antibody. Vi expression is regulated in response to environmental signals such as osmolarity by controlling the transcription of tviA in the viaB locus. To investigate if Vi antibodies can be stimulated if Vi expression is rendered constitutive, we replaced PtviA in serovar Typhi vaccine CVD 908-htrA with the constitutive promoter Ptac, resulting in CVD 909. CVD 909 expresses Vi even under high-osmolarity conditions and is less invasive for Henle 407 cells. In mice immunized with a single intranasal dose, CVD 909 was more immunogenic than CVD 908-htrA in eliciting serum Vi antibodies (geometric mean titer of 160 versus 49, P = 0.0007), whereas O antibody responses were virtually identical (geometric mean titer of 87 versus 80). In mice challenged intraperitoneally with wild-type serovar Typhi 4 weeks after a single intranasal immunization, the mortality of those immunized with CVD 909 (3 of 8) was significantly lower than that of control mice (10 of 10, P = 0.043) or mice given CVD 908-htrA (9 of 10, P = 0.0065). PMID:10899868

  16. The vacuolar-sorting protein Snf7 is required for export of virulence determinants in members of the Cryptococcus neoformans complex.

    PubMed Central

    da C. Godinho, Rodrigo M.; Crestani, Juliana; Kmetzsch, Lívia; de S. Araujo, Glauber; Frases, Susana; Staats, Charley C.; Schrank, Augusto; Vainstein, Marilene H.; Rodrigues, Marcio L.

    2014-01-01

    Fungal pathogenesis requires a number of extracellularly released virulence factors. Recent studies demonstrating that most fungal extracellular molecules lack secretory tags suggest that unconventional secretion mechanisms and fungal virulence are strictly connected. Proteins of the endosomal sorting complex required for transport (ESCRT) have been recently associated with polysaccharide export in the yeast-like human pathogen Cryptococcus neoformans. Snf7 is a key ESCRT operator required for unconventional secretion in Eukaryotes. In this study we generated snf7Δ mutant strains of C. neoformans and its sibling species C. gattii. Lack of Snf7 resulted in important alterations in polysaccharide secretion, capsular formation and pigmentation. This phenotype culminated with loss of virulence in an intranasal model of murine infection in both species. Our data support the notion that Snf7 expression regulates virulence in C. neoformans and C. gattii by ablating polysaccharide and melanin traffic. These results are in agreement with the observation that unconventional secretion is essential for cryptococcal pathogenesis and strongly suggest the occurrence of still obscure mechanisms of exportation of non-protein molecules in Eukaryotes. PMID:25178636

  17. Molecular characterization of the early B cell response to pulmonary Cryptococcus neoformans infection.

    PubMed

    Rohatgi, Soma; Pirofski, Liise-anne

    2012-12-15

    The role of B cells in host defense against fungi has been difficult to establish. We quantified and determined the molecular derivation of B-1a, B-1b, and B-2 B cell populations in C57BL/6 mice after pulmonary infection with Cryptococcus neoformans. Total B-1 and B-2 cell numbers increased in lungs and peritoneal cavity as early as day 1 postinfection, but lacked signs of clonal expansion. Labeled capsular (24067) and acapsular (Cap67) C. neoformans strains were used to identify C. neoformans-binding B cell subsets by flow cytometry. Peritoneal cavity B-1a B cells exhibited the most acapsular and capsular C. neoformans binding in C. neoformans-infected mice, and C. neoformans-selected B-1 B cells secreted laminarin- and C. neoformans-binding IgM. Single-cell PCR-based sequence analysis of B-1a, B-1b, and B-2 cell IgH V region H chain (V(H)) genes revealed increased usage of V(H)11 and V(H)12, respectively, in acapsular and capsular C. neoformans-selected B-1a cells. Germline V(H) segments were used, with capsular C. neoformans-selected cells having less junctional diversity than acapsular C. neoformans-selected cells. Further studies in B-1 B cell-depleted mice showed that these mice had higher brain and lung fungal burdens and less alveolar macrophage phagocytosis of C. neoformans than did control and B-1a B cell-reconstituted mice. Taken together, these results establish a mechanistic role for B-1 B cells in the innate B cell response to pulmonary infection with C. neoformans and reveal that IgM-producing B-1a cells, which express germline V(H) genes, bind C. neoformans and contribute to early fungal clearance. Thus, B-1a B cells provide a first line of defense during pulmonary C. neoformans infection in mice.

  18. Type-specific polysaccharides of Cryptococcus neoformans. n.m.r.-spectral study of a glucuronomannan chemically derived from a Tremella mesenterica exopolysaccharide.

    PubMed

    Cherniak, R; Jones, R G; Slodki, M E

    1988-11-01

    A glucuronomannan (GM) was derived by removal, through Smith degradation, of xylose from the native (3-O-acetylglucurono)xylomannan exopolysaccharide isolated from Tremella mesenterica. 13C-N.m.r. chemical shifts measured at various pD values were compared for p-nitrophenyl beta-D-glucopyranosiduronic acid (1) and two GMs (2 and 3) differing in GlcA content (Man:GlcA; 2, 10:1; and 3, 5:1). Also measured and compared were pKa values for 1 and 2. One-dimensional and two-dimensional (COSY and HETCOR) n.m.r. data allowed unambiguous assignments of pD-sensitive chemical shifts due to 2-O-beta-D-GlcpA substituents attached to a (1----3)-linked alpha-D-Manp backbone. The pKa and n.m.r. data indicated that the CO2H groups in either GM are independent of each other, and are similar in behavior to those of p-nitrophenyl beta-D-glucopyranosiduronic acid molecules. The n.m.r. data confirmed the previous, chemically deduced, structural role of GlcpA in the native polysaccharide from T. mesenterica, and indicated that significant pD-induced changes occur in the stabilities of the glycosidic orientations in the GM. Previous 13C-n.m.r. assignments for 2-O-beta-D-GlcpA in polysaccharides derived from Cryptococcus neoformans serotype A-variant were confirmed, except for the signal due to the anomeric carbon atom. This signal is now known to be pD-sensitive. In acidic solutions, it is coincident with the signal (104.5 p.p.m.) due to the anomeric carbon atoms of the unsubstituted alpha-D-Manp backbone residues. In basic solutions, the 2-O-beta-D-GlcpA anomeric carbon resonance is shifted upfield by approximately 0.2 p.p.m., and is observed as a separate signal.

  19. What makes Cryptococcus neoformans a pathogen?

    PubMed Central

    Buchanan, K. L.; Murphy, J. W.

    1998-01-01

    Life-threatening infections caused by the encapsulated fungal pathogen Cryptococcus neoformans have been increasing steadily over the past 10 years because of the onset of AIDS and the expanded use of immunosuppressive drugs. Intricate host-organism interactions make the full understanding of pathogenicity and virulence of C. neoformans difficult. We discuss the current knowledge of the characteristics C. neoformans must possess to enter the host and establish progressive disease: basic growth requirements and virulence factors, such as the polysaccharide capsule; shed products of the organism; melanin production; mannitol secretion; superoxide dismutase; proteases; and phospholipases. PMID:9452400

  20. Deletion of Cryptococcus neoformans AIF ortholog promotes chromosome aneuploidy and fluconazole-resistance in a metacaspase-independent manner.

    PubMed

    Semighini, Camile P; Averette, Anna F; Perfect, John R; Heitman, Joseph

    2011-11-01

    Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLC(R)) in vitro and in vivo. FLC(R) isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLC(R) subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug

  1. Mannoprotein MP84 mediates the adhesion of Cryptococcus neoformans to epithelial lung cells

    PubMed Central

    Teixeira, Pedro A. C.; Penha, Luciana L.; Mendonça-Previato, Lucia; Previato, Jose O.

    2014-01-01

    The capsule is the most important virulence factor of the fungal pathogen Cryptococcus neoformans. This structure consists of highly hydrated polysaccharides, including glucuronoxylomannan (GXM), and galactoxylomannan (GalXM). It is also composed of mannoproteins (MPs) which corresponds to less than 1% of the capsular weight. Despite MPs being the minority and least studied components, four of these molecules with molecular masses of 115, 98, 88, and 84 kDa were identified and characterized as C. neoformans immunoreactive antigens involved in the pathogenesis, and are potential cryptococcosis vaccine candidates. With the aim to describe the adhesive property of MPs, we cloned and expressed the MP84, a mannoprotein with molecular weight of 84 kDa, on Pichia pastoris yeast, and performed interaction assays of C. neoformans with epithelial lung cells, in the presence or absence of capsule components. Two fungal strains, the wild type, NE-241, and a mutant, CAP67, deficient in GXM production, were used throughout this study. The adhesion assays were completed using epithelial lung cells, A549, and human prostate cancer cells, PC3, as a control. We observed that capsulated wild type (NE-241), and acapsular (CAP67) strains adhered significantly to A549 cells, compared with PC3 cells (p < 0.05). GXM inhibits the NE-241 adhesion, but not the CAP67. In contrast, CAP67 adhesion was only inhibited in the presence of MP84. These results demonstrate the involvement of MP in the adhesion of C. neoformans to epithelial lung cells. We conclude that this interaction possibly involves an adhesion-like interaction between MP on the fungal surface and the complementary receptor molecules on the epithelial cells. PMID:25191644

  2. Contribution of the mannan backbone of cryptococcal glucuronoxylomannan and a glycolytic enzyme of Staphylococcus aureus to contact-mediated killing of Cryptococcus neoformans.

    PubMed

    Ikeda, Reiko; Saito, Fumito; Matsuo, Miki; Kurokawa, Kenji; Sekimizu, Kazuhisa; Yamaguchi, Masashi; Kawamoto, Susumu

    2007-07-01

    The fungal pathogen Cryptococcus neoformans is killed by the bacterium Staphylococcus aureus, and the killing is inhibited by soluble capsular polysaccharides. To investigate the mechanism of killing, cells in coculture were examined by scanning and transmission electron microscopy. S. aureus attached to the capsule of C. neoformans, and the ultrastructure of the attached C. neoformans cells was characteristic of dead cells. To identify the molecules that contributed to the fungal-bacterial interaction, we treated each with NaIO(4) or protease. Treatment of C. neoformans with NaIO(4) promoted adherence. It was inferred that cleavage of xylose and glucuronic acid side chains of glucuronoxylomannan (GXM) allowed S. aureus to recognize mannose residues in the backbone, which resisted periodate oxidation. On the other hand, treatment of S. aureus with protease decreased adherence, suggesting that protein contributed to attachment in S. aureus. In confirmation, side chain-cleaved polysaccharide or defined alpha-(1-->3)-mannan inhibited the killing at lower concentrations than native GXM did. Also, these polysaccharides reduced the adherence of the two species and induced clumping of pure S. aureus cells. alpha-(1-->3)-Mannooligosaccharides with a degree of polymerization (DP) of >/=3 induced cluster formation of S. aureus in a dose-dependent manner. Surface plasmon resonance analyses showed interaction of GXM and surface protein from S. aureus; the interaction was inhibited by oligosaccharides with a DP of > or =3. Conformations of alpha-(1-->3) oligosaccharides were predicted. The three-dimensional structures of mannooligosaccharides larger than triose appeared curved and could be imagined to be recognized by a hypothetical staphylococcal lectin. Native polyacrylamide gel electrophoresis of staphylococcal protein followed by electroblotting, enzyme-linked immunolectin assay, protein staining, and N-terminal amino acid sequencing suggested that the candidate protein was

  3. Cryptococcus neoformans requires the ESCRT protein Vps23 for iron acquisition from heme, for capsule formation, and for virulence.

    PubMed

    Hu, Guanggan; Caza, Mélissa; Cadieux, Brigitte; Chan, Vivienne; Liu, Victor; Kronstad, James

    2013-01-01

    Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenized C. neoformans by Agrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomal sorting complex required for transport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably, vps23 mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression of Vps23 resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in the vps23 mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment in C. neoformans.

  4. Differential Regulation of IgG Anti-Capsular Polysaccharide and Antiprotein Responses to Intact Streptococcus pneumoniae in the Presence of Cognate CD4+ T Cell Help

    DTIC Science & Technology

    2004-01-01

    polysaccharide (PPS14), the phosphorylcholine determinant of the cell wall C-polysaccharide, and the cell wall protein, pneumococcal surface protein A...a more rapid delivery of CD4 T cell help. In contrast, the IgG anti- phosphorylcholine response, although also dependent on CD4 T cells, is TCR...and/or IgG isotypes specific for the phosphorylcholine (PC) determinant of the cell wall C-polysaccharide (teichoic acid) and for the cell wall

  5. Synthetic trimer and tetramer of 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate conjugated to protein induce antibody responses to Haemophilus influenzae type b capsular polysaccharide in mice and monkeys.

    PubMed Central

    Peeters, C C; Evenberg, D; Hoogerhout, P; Käyhty, H; Saarinen, L; van Boeckel, C A; van der Marel, G A; van Boom, J H; Poolman, J T

    1992-01-01

    Synthetic oligosaccharides derived from the capsular polysaccharide (PRP) of Haemophilus influenzae type b were conjugated to carrier proteins via a thioether linkage. Conjugates were made of trimeric and tetrameric ribose-ribitol-phosphate and tetanus toxoid or diphtheria toxin. All conjugates elicited anti-PRP antibody responses with an increasing immunoglobulin G/immunoglobulin M ratio in adult mice and monkeys. Trimer conjugates elicited lower anti-PRP antibody responses compared with tetramer conjugates. Adult monkeys responded equally well to the tetrameric oligosaccharide-tetanus toxoid conjugate as to the oligosaccharide-CRM197 conjugate (HbOC), which elicits protective levels of serum antibodies in human infants after two or three injections. PMID:1563770

  6. Modulation of Macrophage Inflammatory Nuclear Factor κB (NF-κB) Signaling by Intracellular Cryptococcus neoformans.

    PubMed

    Hayes, James B; Sircy, Linda M; Heusinkveld, Lauren E; Ding, Wandi; Leander, Rachel N; McClelland, Erin E; Nelson, David E

    2016-07-22

    Cryptococcus neoformans (Cn) is a common facultative intracellular pathogen that can cause life-threatening fungal meningitis in immunocompromised individuals. Shortly after infection, Cn is detectable as both extra- and intracellular yeast particles, with Cn being capable of establishing long-lasting latent infections within host macrophages. Although recent studies have shown that shed capsular polysaccharides and intact extracellular Cn can compromise macrophage function through modulation of NF-κB signaling, it is currently unclear whether intracellular Cn also affects NF-κB signaling. Utilizing live cell imaging and computational modeling, we find that extra- and intracellular Cn support distinct modes of NF-κB signaling in cultured murine macrophages. Specifically, in RAW 264.7 murine macrophages treated with extracellular glucuronoxylomannan (GXM), the major Cn capsular polysaccharide, LPS-induced nuclear translocation of p65 is inhibited, whereas in cells with intracellular Cn, LPS-induced nuclear translocation of p65 is both amplified and sustained. Mathematical simulations and quantification of nascent protein expression indicate that this is a possible consequence of Cn-induced "translational interference," impeding IκBα resynthesis. We also show that long term Cn infection induces stable nuclear localization of p65 and IκBα proteins in the absence of additional pro-inflammatory stimuli. In this case, nuclear localization of p65 is not accompanied by TNFα or inducible NOS (iNOS) expression. These results demonstrate that capsular polysaccharides and intact intracellular yeast manipulate NF-κB via multiple distinct mechanisms and provide new insights into how Cn might modulate cellular signaling at different stages of an infection.

  7. Cryptococcosis in the era of AIDS--100 years after the discovery of Cryptococcus neoformans.

    PubMed Central

    Mitchell, T G; Perfect, J R

    1995-01-01

    Although Cryptococcus neoformans and cryptococcosis have existed for several millennia, a century has passed since the discovery of this encapsulated yeast and its devastating disease. With the advent of the AIDS pandemic, cryptococcal meningitis has emerged as a leading cause of infectious morbidity and mortality and a frequently life-threatening opportunistic mycosis among patients with AIDS. Both basic and clinical research have accelerated in the 1990s, and this review attempts to highlight some of these advances. The discussion covers recent findings, current concepts, controversies, and unresolved issues related to the ecology and genetics of C. neoformans; the surface structure of the yeast; and the mechanisms of host defense. Regarding cell-mediated immunity, CD4+ T cells are crucial for successful resistance, but CD8+ T cells may also participate significantly in the cytokine-mediated activation of anticryptococcal effector cells. In addition to cell-mediated immunity, monoclonal antibodies to the major capsular polysaccharide, the glucuronoxylomannan, offer some protection in murine models of cryptococcosis. Clinical concepts are presented that relate to the distinctive features of cryptococcosis in patients with AIDS and the diagnosis, treatment, and prevention of cryptococcosis in AIDS patients. PMID:8665468

  8. Polysaccharide-Based Vaccines

    NASA Astrophysics Data System (ADS)

    Santana, Violeta Fernández; Balbin, Yury Valdés; Calderón, Janoi Chang; Icart, Luis Peña; Verez-Bencomo, Vicente

    Capsular polysaccharides (CPS) and lipopolysaccharides from bacteria are employed for the production of vaccines against human diseases. Initial development of CPS as a vaccine was followed by the development and introduction of conjugate polysaccharide-protein vaccines. The principles leading to both developments are reviewed.

  9. Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism.

    PubMed Central

    Hannesson, H H; Hagner-McWhirter, A; Tiedemann, K; Lindahl, U; Malmström, A

    1996-01-01

    The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin ([GlcA(beta 1-->3)GalNAc(beta 1-->4)]n) backbone, to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues. Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis. Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units. A Km of 67 x 10(-6) M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80 x 10(-6) M) obtained for dermatan (desulphated dermatan sulphate). Vmax was about 4 times higher with dermatan than with the K4 substrate. A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-[5-3H]glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme. Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA. A corresponding incubation of dermatan yielded approx. 22% GlcA, which contained virtually all the 3H label. These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base. Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed. PMID:8573097

  10. Nasal immunization of mice with AFCo1 or AFPL1 plus capsular polysaccharide Vi from Salmonella typhi induces cellular response and memory B and T cell responses.

    PubMed

    Romeu, Belkis; Lastre, Miriam; Reyes, Laura; González, Elizabeth; Borrero, Yusnaby; Lescaille, Diandra; Pérez, Rocmira; Nuñez, Darzy; Pérez, Oliver

    2014-12-05

    The response to infection against Salmonella involves both B and T cell mediated immunity. An effective immunization can activate an adequate immune response capable to control the primary infection and protect against a secondary infection. Mucosal vaccination, by inducing local pathogen-specific immune responses, has the potential to counter mucosally transmitted pathogens at the portal of entry, thereby increasing the efficacy of vaccines. The aim of this work was to explore the efficacy of AFCo1 or AFPL1, as mucosal adjuvants to stimulate cell immunity and memory responses against Vi polysaccharide antigen of Salmonella typhi (PsVi). Mice immunized with 3 intranasal doses exhibited high levels of PsVi-specific IgG (p<0.05), IgG2a and IgG2c subclasses. Also, an amplified recall response after a booster immunization with a plain polysaccharide vaccine was induced. Avidities index were higher in mice immunized with adjuvanted formulations at different chaotropic concentrations. Furthermore, IL-12 and IFN-γ levels in nasally vaccinated mice with both adjuvants were induced. Moreover, priming with 3 doses followed by booster immunization with VaxTyVi(®) resulted in high levels of anti-Vi specific IgG, IgG subclasses and antibody avidity. Long lived plasma cells in bone marrow, memory B cells and long-term memory T cells after booster dose were induced. The combined formulation of Vi polysaccharide with mucosal adjuvants provides an improved immunogenicity, in particular with regard to cellular responses and long lasting cells responses.

  11. Antibacterial Activity of Cold Atmospheric Pressure Argon Plasma against 78 Genetically Different (mecA, luk-P, agr or Capsular Polysaccharide Type) Staphylococcus aureus Strains.

    PubMed

    Matthes, Rutger; Lührman, Anne; Holtfreter, Silva; Kolata, Julia; Radke, Dörte; Hübner, Nils-Olaf; Assadian, Ojan; Kramer, Axel

    2016-01-01

    Previous studies on the antimicrobial activity of cold atmospheric pressure argon plasma showed varying effects against mecA+ or mecA-Staphylococcus aureus strains. This observation may have important clinical and epidemiological implications. Here, the antibacterial activity of argon plasma was investigated against 78 genetically different S. aureus strains, stratified by mecA, luk-P, agr1-4, or the cell wall capsule polysaccharide types 5 and 8. kINPen09® served as the plasma source for all experiments. On agar plates, mecA+luk-P-S. aureus strains showed a decreased susceptibility against plasma compared to other S. aureus strains. This study underlines the high complexity of microbial defence against antimicrobial treatment and confirms a previously reported strain-dependent susceptibility of S. aureus to plasma treatment.

  12. Acinetobacter baumannii K27 and K44 capsular polysaccharides have the same K unit but different structures due to the presence of distinct wzy genes in otherwise closely related K gene clusters.

    PubMed

    Shashkov, Alexander S; Kenyon, Johanna J; Senchenkova, Sof'ya N; Shneider, Mikhail M; Popova, Anastasiya V; Arbatsky, Nikolay P; Miroshnikov, Konstantin A; Volozhantsev, Nikolay V; Hall, Ruth M; Knirel, Yuriy A

    2016-05-01

    Capsular polysaccharides (CPSs), from Acinetobacter baumannii isolates 1432, 4190 and NIPH 70, which have related gene content at the K locus, were examined, and the chemical structures established using 2D(1)H and(13)C NMR spectroscopy. The three isolates produce the same pentasaccharide repeat unit, which consists of 5-N-acetyl-7-N-[(S)-3-hydroxybutanoyl] (major) or 5,7-di-N-acetyl (minor) derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7R), D-galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. However, the linkage between repeat units in NIPH 70 was different to that in 1432 and 4190, and this significantly alters the CPS structure. The KL27 gene cluster in 4190 and KL44 gene cluster in NIPH 70 are organized identically and contain lga genes for Leg5Ac7R synthesis, genes for the synthesis of the common sugars, as well as anitrA2 initiating transferase and four glycosyltransferases genes. They share high-level nucleotide sequence identity for corresponding genes, but differ in the wzy gene encoding the Wzy polymerase. The Wzy proteins, which have different lengths and share no similarity, would form the unrelated linkages in the K27 and K44 structures. The linkages formed by the four shared glycosyltransferases were predicted by comparison with gene clusters that synthesize related structures. These findings unambiguously identify the linkages formed by WzyK27 and WzyK44, and show that the presence of different wzy genes in otherwise closely related K gene clusters changes the structure of the CPS. This may affect its capacity as a protective barrier for A. baumannii.

  13. Eucalyptus Tree: A Potential Source of Cryptococcus neoformans in Egyptian Environment

    PubMed Central

    Hamza, Dalia; Elhelw, Rehab; Refai, Mohamed

    2016-01-01

    In Egypt, the River Red Gum (Eucalyptus camaldulensis) is a well-known tree and is highly appreciated by the rural and urban dwellers. The role of Eucalyptus trees in the ecology of Cryptococcus neoformans is documented worldwide. The aim of this survey was to show the prevalence of C. neoformans during the flowering season of E. camaldulensis at the Delta region in Egypt. Three hundred and eleven samples out of two hundred Eucalyptus trees, including leaves, flowers, and woody trunks, were collected from four governorates in the Delta region. Thirteen isolates of C. neoformans were recovered from Eucalyptus tree samples (4.2%). Molecular identification of C. neoformans was done by capsular gene specific primer CAP64 and serotype identification was done depending on LAC1 gene. This study represents an update on the ecology of C. neoformans associated with Eucalyptus tree in Egyptian environment. PMID:26884765

  14. Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide.

    PubMed Central

    Pazzani, C; Rosenow, C; Boulnois, G J; Bronner, D; Jann, K; Roberts, I S

    1993-01-01

    The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters. Images PMID:8397187

  15. Efficacy of immune sera from human immunoglobulin transgenic mice immunized with a peptide mimotope of Cryptococcus neoformans glucuronoxylomannan.

    PubMed

    Maitta, Robert W; Datta, Kausik; Pirofski, Liise-Anne

    2004-09-28

    The efficacy of antibody mediated immunity against Cryptococcus neoformans has not been established experimentally for human antibodies. Our group has previously shown that immunization with a conjugate consisting of a peptide mimotope of the C. neoformans capsular polysaccharide glucuronoxylomannan (GXM), P13, and diphtheria toxoid (P13-DT) prolonged survival of transgenic mice with human immunoglobulin loci, XenoMouse mice, which were challenged with a lethal dose of C. neoformans. In the study reported herein, we determined the efficacy of human antibodies in the sera of immunized XenoMouse mice against C. neoformans in passive transfer experiments in naïve BALB/c mice. Survival studies were performed with sera from XenoMouse mice expressing human IgG2/kappa (G2/k mice) or IgG4/kappa (G4/k mice) that had been immunized with P13-tetanus toxoid (TT)/Alhydrogel with or without CpG, and G2/k mice that had been immunized with P13-DT/Alhydrogel/CpG or Alhydrogel/CpG, obtained on day 7 (early sera) and days 30 or 35-59 (late sera) after primary immunization. Compared to mice receiving sera from G2/k-PBS-treated mice, the survival of naïve mice was prolonged by both early and late sera from G2/k-P13-DT/Alhydrogel/CpG-immunized mice, but only late sera from G2/k-P13-TT/Alhydrogel/CpG-immunized mice. Late, but not early sera from G2/k-Alhydrogel/CpG-immunized mice also prolonged survival. For all sera, prolongation of survival was associated with GXM-specific serum IgM. Sera from G2/k mice that received P13-TT without CpG, and all groups of G4/k mice had low to undetectable levels of antibody to GXM and were not protective. Our findings suggest that GXM-specific human IgM may be a functional mediator of protection against C. neoformans.

  16. Cryptococcus neoformans meningitis in the rat.

    PubMed

    Goldman, D L; Casadevall, A; Cho, Y; Lee, S C

    1996-12-01

    The primary clinical manifestation of Cryptococcus neoformans infection in humans is meningoencephalitis. To study the defense mechanisms that participate in the host response against C. neoformans infection of the central nervous system (CNS), we have developed a new model of cryptococcal meningitis in rats. Intracisternal inoculation of C. neoformans produced a granulomatous meningitis with minimal brain parenchymal involvement, resembling cryptococcal meningitis in immunocompetent patients. The granulomas were composed of T cells (CD4+ and CD8+) and macrophages (CD11b/c+); a subpopulation of the macrophages expressed inducible nitric oxide synthase (NOS2). In this model, C. neoformans disseminated to systemic organs early in the course of infection and provoked granuloma formation and NOS2 expression. The temporal profile of inflammation indicated that the CNS inflammatory response is delayed relative to that in the lung and the spleen, which suggests that the effective inflammatory response within the CNS may follow activation of T cells in the periphery and their subsequent entry into the CNS. Inflammation in the meninges was associated with signs of subpial and subependymal glial activation, including enhanced expression of CD11b/c and CD4 in microglia and glial fibrillary acidic protein in astrocytes. Neither cells, however, expressed NOS2. Although C. neoformans invasion to the brain parenchyma was rare, soluble polysaccharide was commonly associated with reactive glial cells. Necrosis was not a feature of C. neoformans granulomas, but, instead, inflammatory cells underwent apoptosis in inflamed organs. The current rat intrathecal cryptococcosis model has several unique advantages for the study of human cryptococcal meningoencephalitis that include close resemblance of histopathologic changes to those in humans, easy accessibility to the cerebrospinal fluid compartment, and no requirement of immunosuppressive agents for establishment of infection.

  17. The Effect of a BSA Conjugate of a Synthetic Hexasaccharide Related to the Fragment of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 on the Activation of Innate and Adaptive Immune Responses

    PubMed Central

    Akhmatova, Nelli K.; Kurbatova, Ekaterina A.; Akhmatov, Elvin A.; Egorova, Nadezhda B.; Logunov, Denis Yu.; Gening, Marina L.; Sukhova, Elena V.; Yashunsky, Dmitry V.; Tsvetkov, Yury E.; Nifantiev, Nikolay E.

    2016-01-01

    We report the effect of a bovine serum albumin (BSA) conjugate of a synthetic hexasaccharide (HS) related to the fragment of the capsular polysaccharide (PS) of Streptococcus pneumoniae type 14 on the stimulation of innate immune system and the subsequent development of a PS-specific antibody response. Glycoconjugate (GC) in the presence (GC + AL) or absence of aluminum hydroxide was administered to mice twice. GC increased the number of TLR2-expressing cells and induced the maturation of dendritic cells (CD11c+, CD80+ and, MHCII+), which secreted IL-1β, IL-6, and TNFα into the culture medium. The level of IL-1β, IL-10, IFNγ, and TNFα in the blood increased within 24 h after the single GC administration to mice. On day 7, the numbers of splenic CD4+ and CD8+ T lymphocytes and B lymphocytes increased. After the second immunization, the levels of CD4+ and CD8+ T lymphocytes were lower than in the control, whereas the B cell, NK cell, and MHC class II-expressing cell numbers remained enhanced. However, of the presence of anti-PS, IgG antibodies were not detected. The addition of aluminum hydroxide to GC stimulated the production of GM-CSF, IL-1β, IL-5, IL-6, IL-10, IL-17, IFNγ, and TNFα. Anti-PS IgG1 antibody titers 7 days after the second immunization were high. During that period, normal levels of splenic CD4+ T lymphocytes were maintained, whereas reduced CD8+ T lymphocyte numbers and increased levels of B lymphocytes, NK cells, and MHC class II-expressing cell numbers were observed. Anti-PS IgG levels diminished until day 92. A booster immunization with GC + AL stimulated the production of anti-PS IgG memory antibodies, which were determined within 97 days. The elucidation of specific features of the effect of the synthetic HS conjugate on the stimulation of innate, cell-mediated immunity, and antibody response can favor the optimization of GC vaccine design. PMID:27446078

  18. Eosinophil-Cryptococcus neoformans interactions in vivo and in vitro.

    PubMed Central

    Feldmesser, M; Casadevall, A; Kress, Y; Spira, G; Orlofsky, A

    1997-01-01

    Eosinophils are components of inflammatory responses to a variety of pathogens. Although a variety of beneficial and harmful functions have been ascribed to these cells, their role in protection against infectious agents remains uncertain. Previous studies have reported eosinophilic pneumonia in mice infected intratracheally with Cryptococcus neoformans. We confirmed this observation and studied the inflammatory response in the lung at day 14 by light and electron microscopy. Immunostaining for glucuronoxylomannan showed isolated cryptococci inside the eosinophilic cuffs. Eosinophils were found to be in close association with C. neoformans in vivo. Cryptococci were associated with eosinophils within eosinophilic perivascular cuffs, within granulomas, and lining the alveolar space. To further investigate this phenomenon in vitro, we isolated rat peritoneal eosinophils and studied cryptococcus-eosinophil interactions in the presence and absence of anti-capsular immunoglobulin G1 (IgG1) and IgE monoclonal antibody (MAb). Eosinophils phagocytosed C. neoformans only in the presence of specific antibody. Phagocytosis was rapid, and dense rings that appeared to consist of granule contents were formed around the organisms. Mast cells were observed to occasionally phagocytose C. neoformans in vitro in the presence of IgE MAb. Our observations suggest that eosinophils may be effector cells against C. neoformans. PMID:9125578

  19. Sequence diversity within the capsular genes of Streptococcus pneumoniae serogroup 6 and 19.

    PubMed

    Elberse, Karin; Witteveen, Sandra; van der Heide, Han; van de Pol, Ingrid; Schot, Corrie; van der Ende, Arie; Berbers, Guy; Schouls, Leo

    2011-01-01

    The main virulence factor of Streptococcus pneumoniae is the capsule. The polysaccharides comprising this capsule are encoded by approximately 15 genes and differences in these genes result in different serotypes. The aim of this study was to investigate the sequence diversity of the capsular genes of serotypes 6A, 6B, 6C, 19A and 19F and to explore a possible effect of vaccination on variation and distribution of these serotypes in the Netherlands. The complete capsular gene locus was sequenced for 25 serogroup 6 and for 20 serogroup 19 isolates. If one or more genes varied in 10 or more base pairs from the reference sequence, it was designated as a capsular subtype. Allele-specific PCRs and specific gene sequencing of highly variable capsular genes were performed on 184 serogroup 6 and 195 serogroup 19 isolates to identify capsular subtypes. This revealed the presence of 6, 3 and a single capsular subtype within serotypes 6A, 6B and 6C, respectively. The serotype 19A and 19F isolates comprised 3 and 4 capsular subtypes, respectively. For serogroup 6, the genetic background, as determined by multi locus sequence typing (MLST) and multiple-locus variable number of tandem repeat analysis (MLVA), seemed to be closely related to the capsular subtypes, but this was less pronounced for serogroup 19 isolates. The data also suggest shifts in the occurrence of capsular subtypes within serotype 6A and 19A after introduction of the 7-valent pneumococcal vaccine. The shifts within these non-vaccine serotypes might indicate that these capsular subtypes are filling the niche of the vaccine serotypes. In conclusion, there is considerable DNA sequence variation of the capsular genes within pneumococcal serogroup 6 and 19. Such changes may result in altered polysaccharides or in strains that produce more capsular polysaccharides. Consequently, these altered capsules may be less sensitive for vaccine induced immunity.

  20. Effects of Pimenta pseudocaryophyllus (Gomes) L. R. Landrum, on melanized and non-melanized Cryptococcus neoformans.

    PubMed

    de Fátima Lisboa Fernandes, Orionalda; Costa, Carolina Rodrigues; de Souza Lino Junior, Ruy; Vinaud, Marina Clare; Hasimoto E Souza, Lúcia Kioko; de Paula, Joelma Abadia Marciano; do Rosário Rodrigues Silva, Maria

    2012-12-01

    In the present study, the in vitro susceptibility and capsular width from both melanized and non-melanized Cryptococcus neoformans cells in the presence of Pimenta pseudocaryophyllus crude extract were determined. The results were compared with those obtained for voriconazole and amphotericin B. Melanization was obtained in minimal medium broth with the addition of L-dopa, and the antifungal susceptibility tests were performed using the broth microdilution method. Capsular width of 30 cells of each one of the isolates in medium with crude extracts of P. pseudocaryophyllus or voriconazole or amphotericin B at a concentration corresponding to 0.5 times the minimal inhibitory concentration (MIC) was measured, and the mean was calculated. The MICs and minimal fungicidal concentrations (MFCs) for plant extract and voriconazole were identical for both melanized and non-melanized C. neoformans isolates, but for amphotericin, the MFCs for melanized cells were up to 8 times higher than for non-melanized cells. The capsular width of C. neoformans cells was smaller (p < 0.001) in the presence crude extract of P. pseudocaryophyllus and of voriconazole regardless melanization. The findings of capsule alterations of C. neoformans verified in this study provide fertile ways for future research into the effects of antifungal agents on the pathogenesis of cryptococcosis.

  1. Specificity of Cryptococcus neoformans factor sera determined by enzyme-linked immunosorbent assay and dot enzyme assay.

    PubMed Central

    Belay, T; Cherniak, R; Shinoda, T

    1993-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA) and a dot enzyme assay (DEA) were used to determine the specificities of Cryptococcus neoformans factor sera to serotype type-specific capsular polysaccharides, glucuronoxylomannans (GXMs). Pure and chemically characterized GXMs were obtained from representative isolates of C. neoformans serotypes A, B, C, and D. Distinctive specificity patterns and quantitative differences were observed for each factor serum when the selected GXMs were studied by ELISA. The specificity patterns for each factor serum determined by DEA almost completely paralleled the ELISA results. The serotype specificities demonstrated by ELISA and DEA were similar to previously reported results that were obtained by slide agglutination studies of whole cells. On the basis of the ELISA and DEA activity patterns, factor sera 5, 6, and 8 were specific for serotypes B, C, and D, respectively; factor serum 1 was strongly reactive to all serotypes; factor serum 2 was specific for serotypes A, B, and D; factor serum 3 was specific for serotypes A and D; and factor serum 4 was specific for serotypes B and C. The specificity of factor serum 7 for serotype A was demonstrated by DEA only. Structural variation was indicated among the serotype C isolates studied because a unique activity pattern versus factor serum 6 was observed for each isolate. The quantitative differences in the activity of the GXMs from five serotype C isolates suggest that mannopyranoside residues substituted O-2 and O-4 with xylose are essential elements of the determinant responsible for the observed activity of factor 6. No significant differences in activity patterns and specificities of factor serum 6 were observed when O-deacetylated GXMs were substituted for the native GXMs. Our results show that ELISA and DEA are valuable techniques for the serological analysis of cryptococcal factor sera and GXMs. Images PMID:7685739

  2. The capsular network of Klebsiella pneumoniae.

    PubMed

    Cassone, A; Garaci, E

    1977-06-01

    Attempts at improving chemical fixation for electron-microscopic observation of the capsule of Klebsiella pneumoniae were made. The capsule was preserved by using alcian blue - lanthanum and tris-(1-aziridinyl) phosphine oxide (TAPO) - aldehyde - osmium procedures. Despite the different retention of the overall capsular material and minor variations in morphological details, in both cases the interpretation of ultrastructural patterns suggested that the capsule be composed of a meshed network of thin polysaccharide fibrils radiating from the cell wall. This organization is in keeping with all recognized chemical properties of bacterial polysaccharide capsules or, at least, does not contradict them. Moreover, an effective preservation of bacterial structures other than capsule has been obtained, mostly in specimens fixed by the TAPO-aldehyde-osmium method, a fact which gives further reliability to the technical approach used for capsule visualization.

  3. Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis.

    PubMed

    Kwon-Chung, Kyung J; Fraser, James A; Doering, Tamara L; Wang, Zhou; Janbon, Guilhem; Idnurm, Alexander; Bahn, Yong-Sun

    2014-07-01

    Cryptococcus neoformans and Cryptococcus gattii are the two etiologic agents of cryptococcosis. They belong to the phylum Basidiomycota and can be readily distinguished from other pathogenic yeasts such as Candida by the presence of a polysaccharide capsule, formation of melanin, and urease activity, which all function as virulence determinants. Infection proceeds via inhalation and subsequent dissemination to the central nervous system to cause meningoencephalitis. The most common risk for cryptococcosis caused by C. neoformans is AIDS, whereas infections caused by C. gattii are more often reported in immunocompetent patients with undefined risk than in the immunocompromised. There have been many chapters, reviews, and books written on C. neoformans. The topics we focus on in this article include species description, pathogenesis, life cycle, capsule, and stress response, which serve to highlight the specializations in virulence that have occurred in this unique encapsulated melanin-forming yeast that causes global deaths estimated at more than 600,000 annually.

  4. Cryptococcus neoformans and Cryptococcus gattii, the Etiologic Agents of Cryptococcosis

    PubMed Central

    Kwon-Chung, Kyung J.; Fraser, James A.; Doering, Tamara L.; Wang, Zhou; Janbon, Guilhem; Idnurm, Alexander; Bahn, Yong-Sun

    2014-01-01

    Cryptococcus neoformans and Cryptococcus gattii are the two etiologic agents of cryptococcosis. They belong to the phylum Basidiomycota and can be readily distinguished from other pathogenic yeasts such as Candida by the presence of a polysaccharide capsule, formation of melanin, and urease activity, which all function as virulence determinants. Infection proceeds via inhalation and subsequent dissemination to the central nervous system to cause meningoencephalitis. The most common risk for cryptococcosis caused by C. neoformans is AIDS, whereas infections caused by C. gattii are more often reported in immunocompetent patients with undefined risk than in the immunocompromised. There have been many chapters, reviews, and books written on C. neoformans. The topics we focus on in this article include species description, pathogenesis, life cycle, capsule, and stress response, which serve to highlight the specializations in virulence that have occurred in this unique encapsulated melanin-forming yeast that causes global deaths estimated at more than 600,000 annually. PMID:24985132

  5. The Cryptococcus neoformans capsule: lessons from the use of optical tweezers and other biophysical tools

    PubMed Central

    Pontes, Bruno; Frases, Susana

    2015-01-01

    The fungal pathogen Cryptococcus neoformans causes life-threatening infections in immunocompromised individuals, representing one of the leading causes of morbidity and mortality in AIDS patients. The main virulence factor of C. neoformans is the polysaccharide capsule; however, many fundamental aspects of capsule structure and function remain poorly understood. Recently, important capsule properties were uncovered using optical tweezers and other biophysical techniques, including dynamic and static light scattering, zeta potential and viscosity analysis. This review provides an overview of the latest findings in this emerging field, explaining the impact of these findings on our understanding of C. neoformans biology and resistance to host immune defenses. PMID:26157436

  6. In vitro synergistic effects of chlorpromazine and sertraline in combination with amphotericin B against Cryptococcus neoformans var. grubii.

    PubMed

    Rossato, Luana; Loreto, Érico S; Zanette, Régis A; Chassot, Francieli; Santurio, Janio M; Alves, Sydney H

    2016-09-01

    Cryptococcus neoformans is encapsulated yeast that causes cryptococcosis. The cryptococcal meningitis may cause neuropsychiatric symptoms. Here, we evaluated the in vitro activity of amphotericin B (AMB), chlorpromazine (CLOR), and sertraline (SERT) alone or in combination against clinical isolates of C. neoformans considering the capsular induction in vitro. Susceptibility tests were carried out using the broth microdilution method in accordance with the CLSI document M27-A3. The combination [CLOR + AMB] exhibited synergism for 50 and 67 % of strains before capsular induction (group I) and after capsular induction (group II), respectively. The combination [SERT + AMB] showed 60 % of synergism against the both groups. Antagonism was not observed. Our results show the therapeutic potential of chlorpromazine and sertraline in combination with amphotericin B against neurocryptococcosis.

  7. Interleukin-6 production by human monocytes stimulated with Cryptococcus neoformans components.

    PubMed

    Delfino, D; Cianci, L; Lupis, E; Celeste, A; Petrelli, M L; Curró, F; Cusumano, V; Teti, G

    1997-06-01

    In order to ascertain if Cryptococcus neoformans components can induce interleukin-6 (IL-6) production, we stimulated human whole blood with purified capsular products. Their potencies in stimulating IL-6 release were mannoproteins > galactoxylomannan = glucuronoxylomannan > alpha(1-3)glucan. IL-6 production was tumor necrosis factor alpha independent and required the presence of monocytes and plasma. Since IL-6 can stimulate replication of the human immunodeficiency virus in monocytic cells, these findings may be clinically relevant.

  8. Interleukin-6 production by human monocytes stimulated with Cryptococcus neoformans components.

    PubMed Central

    Delfino, D; Cianci, L; Lupis, E; Celeste, A; Petrelli, M L; Curró, F; Cusumano, V; Teti, G

    1997-01-01

    In order to ascertain if Cryptococcus neoformans components can induce interleukin-6 (IL-6) production, we stimulated human whole blood with purified capsular products. Their potencies in stimulating IL-6 release were mannoproteins > galactoxylomannan = glucuronoxylomannan > alpha(1-3)glucan. IL-6 production was tumor necrosis factor alpha independent and required the presence of monocytes and plasma. Since IL-6 can stimulate replication of the human immunodeficiency virus in monocytic cells, these findings may be clinically relevant. PMID:9169790

  9. The Cryptococcus neoformans Capsule: a Sword and a Shield

    PubMed Central

    O'Meara, Teresa R.

    2012-01-01

    Summary: The human fungal pathogen Cryptococcus neoformans is characterized by its ability to induce a distinct polysaccharide capsule in response to a number of host-specific environmental stimuli. The induction of capsule is a complex biological process encompassing regulation at multiple steps, including the biosynthesis, transport, and maintenance of the polysaccharide at the cell surface. By precisely regulating the composition of its cell surface and secreted polysaccharides, C. neoformans has developed intricate ways to establish chronic infection and dormancy in the human host. The plasticity of the capsule structure in response to various host conditions also underscores the complex relationship between host and parasite. Much of this precise regulation of capsule is achieved through the transcriptional responses of multiple conserved signaling pathways that have been coopted to regulate this C. neoformans-specific virulence-associated phenotype. This review focuses on specific host stimuli that trigger the activation of the signal transduction cascades and on the downstream transcriptional responses that are required for robust encapsulation around the cell. PMID:22763631

  10. The Anti-helminthic Compound Mebendazole Has Multiple Antifungal Effects against Cryptococcus neoformans

    PubMed Central

    Joffe, Luna S.; Schneider, Rafael; Lopes, William; Azevedo, Renata; Staats, Charley C.; Kmetzsch, Lívia; Schrank, Augusto; Del Poeta, Maurizio; Vainstein, Marilene H.; Rodrigues, Marcio L.

    2017-01-01

    Cryptococcus neoformans is the most lethal pathogen of the central nervous system. The gold standard treatment of cryptococcosis, a combination of amphotericin B with 5-fluorocytosine, involves broad toxicity, high costs, low efficacy, and limited worldwide availability. Although the need for new antifungals is clear, drug research and development (R&D) is costly and time-consuming. Thus, drug repurposing is an alternative to R&D and to the currently available tools for treating fungal diseases. Here we screened a collection of compounds approved for use in humans seeking for those with anti-cryptococcal activity. We found that benzimidazoles consist of a broad class of chemicals inhibiting C. neoformans growth. Mebendazole and fenbendazole were the most efficient antifungals showing in vitro fungicidal activity. Since previous studies showed that mebendazole reaches the brain in biologically active concentrations, this compound was selected for further studies. Mebendazole showed antifungal activity against phagocytized C. neoformans, affected cryptococcal biofilms profoundly and caused marked morphological alterations in C. neoformans, including reduction of capsular dimensions. Amphotericin B and mebendazole had additive anti-cryptococcal effects. Mebendazole was also active against the C. neoformans sibling species, C. gattii. To further characterize the effects of the drug a random C. gattii mutant library was screened and indicated that the antifungal activity of mebendazole requires previously unknown cryptococcal targets. Our results indicate that mebendazole is as a promising prototype for the future development of anti-cryptococcal drugs.

  11. Evolution of the capsular operon of Streptococcus iniae in response to vaccination.

    PubMed

    Millard, Candice M; Baiano, Justice C F; Chan, Candy; Yuen, Benedict; Aviles, Fabian; Landos, Matt; Chong, Roger S M; Benedict, Suresh; Barnes, Andrew C

    2012-12-01

    Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins.

  12. Evolution of the Capsular Operon of Streptococcus iniae in Response to Vaccination

    PubMed Central

    Millard, Candice M.; Baiano, Justice C. F.; Chan, Candy; Yuen, Benedict; Aviles, Fabian; Landos, Matt; Chong, Roger S. M.; Benedict, Suresh

    2012-01-01

    Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins. PMID:23001668

  13. Iron and fungal pathogenesis: a case study with Cryptococcus neoformans.

    PubMed

    Jung, Won Hee; Kronstad, James W

    2008-02-01

    The acquisition of iron from mammalian hosts is an important aspect of infection because microbes must compete with the host for this nutrient and iron perception often regulates virulence factor expression. For example, iron levels are known to influence the elaboration of two major virulence factors, the polysaccharide capsule and melanin, in the pathogenic fungus Cryptococcus neoformans. This pathogen, which causes meningoencephalitis in immunocompromised people, acquires iron through the use of secreted reductants, cell surface reductases, a permease/ferroxidase uptake system and siderophore transporters. In addition, a master regulator, Cir1, integrates iron sensing with the expression of virulence factors, with growth at 37 degrees C and with signalling pathways that also influence virulence. The challenge ahead is to develop mechanistic views of the iron acquisition functions and regulatory schemes that operate when C. neoformans is in host tissue. Achieving these goals may contribute to an understanding of the notable predilection of the fungus for the mammalian central nervous system.

  14. Identification of a capsular variant and characterization of capsular acetylation in Klebsiella pneumoniae PLA-associated type K57

    PubMed Central

    Hsu, Chun-Ru; Liao, Chun-Hsing; Lin, Tzu-Lung; Yang, Han-Ru; Yang, Feng-Ling; Hsieh, Pei-Fang; Wu, Shih-Hsiung; Wang, Jin-Town

    2016-01-01

    Klebsiella pneumoniae can cause community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) is important for its virulence. Among 79 capsular (K) types discovered thus far, K57 is often associated with PLA. Here, we report the identification of a K57 variant. Cps gene locus sequencing revealed differences between the K57 reference strain 4425/51 (Ref-K57) and a variant, the PLA isolate A1142. While Ref-K57 cps contained orf13 encoding a putative acetyltransferase, the insertion of a putative transposase-encoding gene at this position was detected in A1142. This variation was detected in other K57 clinical strains. Biochemical analyses indicated that A1142 was deficient in CPS acetylation. Genetic replacement and complementation verified that orf13 was responsible for CPS acetylation. Acetylation increased CPS immunoreactivity to antiserum and enhanced K. pneumoniae induction of pro-inflammatory cytokines through JNK and MAPK signaling. While acetylation diminished the serum resistance of bacteria, it promoted adhesion to intestinal epithelial cells possibly via increasing production of type I fimbriae. In conclusion, acetylation-mediated capsular variation in K57 was observed. Capsular acetylation contributed to the variety and antigenic diversity of CPS, influenced its biological activities, and was involved in K. pneumoniae-host interactions. These findings have implications for vaccine design and pathogenicity of K. pneumoniae. PMID:27550826

  15. Cryptococcus neoformans is internalized by receptor-mediated or 'triggered' phagocytosis, dependent on actin recruitment.

    PubMed

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both 'zipper' (receptor-mediated) and 'trigger' (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells.

  16. Chitosan, the deacetylated form of chitin, is necessary for cell wall integrity in Cryptococcus neoformans.

    PubMed

    Baker, Lorina G; Specht, Charles A; Donlin, Maureen J; Lodge, Jennifer K

    2007-05-01

    Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. The fungal cell wall is an excellent target for antifungal therapies as it is an essential organelle that provides cell structure and integrity, it is needed for the localization or attachment of known virulence factors, including the polysaccharide capsule, melanin, and phospholipase, and it is critical for host-pathogen interactions. In C. neoformans, chitosan produced by the enzymatic removal of acetyl groups from nascent chitin polymers has been implicated as an important component of the vegetative cell wall. In this study, we identify four putative chitin/polysaccharide deacetylases in C. neoformans. We have demonstrated that three of these deacetylases, Cda1, Cda2, and Cda3, can account for all of the chitosan produced during vegetative growth in culture, but the function for one, Fpd1, remains undetermined. The data suggest a model for chitosan production in vegetatively growing C. neoformans where the three chitin deacetylases convert chitin generated by the chitin synthase Chs3 into chitosan. Utilizing a collection of chitin/polysaccharide deacetylase deletion strains, we determined that during vegetative growth, chitosan helps to maintain cell integrity and aids in bud separation. Additionally, chitosan is necessary for maintaining normal capsule width and the lack of chitosan results in a "leaky melanin" phenotype. Our analysis indicates that chitin deacetylases and the chitosan made by them may prove to be excellent antifungal targets.

  17. Immunomodulatory Effects of Serotype B Glucuronoxylomannan from Cryptococcus gattii Correlate with Polysaccharide Diameter▿

    PubMed Central

    Fonseca, Fernanda L.; Nohara, Lilian L.; Cordero, Radames J. B.; Frases, Susana; Casadevall, Arturo; Almeida, Igor C.; Nimrichter, Leonardo; Rodrigues, Marcio L.

    2010-01-01

    Glucuronoxylomannan (GXM), the major capsular component in the Cryptococcus complex, interacts with the immune system in multiple ways, which include the activation of Toll-like receptors (TLRs) and the modulation of nitric oxide (NO) production by phagocytes. In this study, we analyzed several structural parameters of GXM samples from Cryptococcus neoformans (serotypes A and D) and Cryptococcus gattii (serotypes B and C) and correlated them with the production of NO by phagocytes and the activation of TLRs. GXM fractions were differentially recognized by TLR2/TLR1 (TLR2/1) and TLR2/6 heterodimers expressed on TLR-transfected HEK293A cells. Higher NF-κB luciferase reporter activity induced by GXM was observed in cells expressing TLR2/1 than in cells transfected with TLR2/6 constructs. A serotype B GXM from C. gattii was the most effective polysaccharide fraction activating the TLR-mediated response. This serotype B polysaccharide, which was also highly efficient at eliciting the production of NO by macrophages, was similar to the other GXM samples in monosaccharide composition, zeta potential, and electrophoretic mobility. However, immunofluorescence with four different monoclonal antibodies and dynamic light-scattering analysis revealed that the serotype B GXM showed particularities in serological reactivity and had the smallest effective diameter among the GXM samples analyzed in this study. Fractionation of additional serotype B GXMs, followed by exposure of these fractions to macrophages, revealed a correlation between NO production and reduced effective diameters. Our results demonstrate a great functional diversity in GXM samples from different isolates and establish their abilities to differentially activate cellular responses. We propose that serological properties as well as physical chemical parameters, such as the diameter of polysaccharide molecules, may potentially influence the inflammatory response against Cryptococcus spp. and may contribute to the

  18. Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene.

    PubMed

    Chae, H S; Park, G N; Kim, S H; Jo, H J; Kim, J T; Jeoung, H Y; An, D J; Kim, N H; Shin, B W; Kang, Y I; Chang, K S

    2012-08-01

    Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) μg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.

  19. Campylobacter jejuni capsular genotypes are related to Guillain-Barré syndrome.

    PubMed

    Heikema, A P; Islam, Z; Horst-Kreft, D; Huizinga, R; Jacobs, B C; Wagenaar, J A; Poly, F; Guerry, P; van Belkum, A; Parker, C T; Endtz, H P

    2015-09-01

    In about one in a thousand cases, a Campylobacter jejuni infection results in the severe polyneuropathy Guillain-Barré syndrome (GBS). It is established that sialylated lipo-oligosaccharides (LOS) of C. jejuni are a crucial virulence factor in GBS development. Frequent detection of C. jejuni with sialylated LOS in stools derived from patients with uncomplicated enteritis implies that additional bacterial factors should be involved. To assess whether the polysaccharide capsule is a marker for GBS, the capsular genotypes of two geographically distinct GBS-associated C. jejuni strain collections and an uncomplicated enteritis control collection were determined. Capsular genotyping of C. jejuni strains from the Netherlands revealed that three capsular genotypes, HS1/44c, HS2 and HS4c, were dominant in GBS-associated strains and capsular types HS1/44c and HS4c were significantly associated with GBS (p 0.05 and p 0.01, respectively) when compared with uncomplicated enteritis. In a GBS-associated strain collection from Bangladesh, capsular types HS23/36c, HS19 and HS41 were most prevalent and the capsular types HS19 and HS41 were associated with GBS (p 0.008 and p 0.02, respectively). Next, specific combinations of the LOS class and capsular genotypes were identified that were related to the occurrence of GBS. Multilocus sequence typing revealed restricted genetic diversity for strain populations with the capsular types HS2, HS19 and HS41. We conclude that capsular types HS1/44c, HS2, HS4c, HS19, HS23/36c and HS41 are markers for GBS. Besides a crucial role for sialylated LOS of C. jejuni in GBS pathogenesis, the identified capsules may contribute to GBS susceptibility.

  20. The heat shock protein (Hsp) 70 of Cryptococcus neoformans is associated with the fungal cell surface and influences the interaction between yeast and host cells.

    PubMed

    Silveira, Carolina P; Piffer, Alicia C; Kmetzsch, Lívia; Fonseca, Fernanda L; Soares, Danielle A; Staats, Charley C; Rodrigues, Marcio L; Schrank, Augusto; Vainstein, Marilene H

    2013-11-01

    The pathogenic yeast Cryptococcus neoformans secretes numerous proteins, such as heat shock proteins, by unconventional mechanisms during its interaction with host cells. Hsp70 is a conserved chaperone that plays important roles in various cellular processes, including the interaction of fungi with host immune cells. Here, we report that sera from individuals with cryptococcosis infection recognize a recombinant C. neoformans Hsp70 (Cn_rHsp70). Moreover, immunofluorescence assays using antibodies against Cn_rHsp70 revealed the localization of this protein at the cell surface mainly in association with the capsular network. We found that the addition of Cn_rHsp70 positively modulated the interaction of C. neoformans with human alveolar epithelial cells and decreased fungal killing by mouse macrophages, without affecting phagocytosis rates. Immunofluorescence analysis showed that there was a competitive association among the receptor, GXM and Cn_rHsp70, indicating that the Hsp70-binding sites in host cells appear to be shared by glucuronoxylomannan (GXM), the major capsular antigen in C. neoformans. Our observations suggest additional mechanisms by which Hsp70 influences the interaction of C. neoformans with host cells.

  1. Role of Cln1 during melanization of Cryptococcus neoformans.

    PubMed

    García-Rodas, Rocío; Trevijano-Contador, Nuria; Román, Elvira; Janbon, Guilhem; Moyrand, Frédérique; Pla, Jesús; Casadevall, Arturo; Zaragoza, Oscar

    2015-01-01

    Cryptococcus neoformans is an opportunistic fungal pathogen that has several well-described virulence determinants. A polysaccharide capsule and the ability to produce melanin are among the most important. Melanization occurs both in vitro, in the presence of catecholamine and indole compounds, and in vivo during the infection. Despite the importance of melanin production for cryptococcal virulence, the component and mechanisms involved in its synthesis have not been fully elucidated. In this work, we describe the role of a G1/S cyclin (Cln1) in the melanization process. Cln1 has evolved specifically with proteins present only in other basidiomycetes. We found that Cln1 is required for the cell wall stability and production of melanin in C. neoformans. Absence of melanization correlated with a defect in the expression of the LAC1 gene. The relation between cell cycle elements and melanization was confirmed by the effect of drugs that cause cell cycle arrest at a specific phase, such as rapamycin. The cln1 mutant was consistently more susceptible to oxidative damage in a medium that induces melanization. Our results strongly suggest a novel and hitherto unrecognized role for C. neoformans Cln1 in the expression of virulence traits.

  2. PCR for capsular typing of Haemophilus influenzae.

    PubMed Central

    Falla, T J; Crook, D W; Brophy, L N; Maskell, D; Kroll, J S; Moxon, E R

    1994-01-01

    A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine. Images PMID:7814470

  3. Cryptococcus neoformans, Cryptococcus gattii: serotypes in Venezuela.

    PubMed

    Pérez, C; Dolande, M; Moya, M; Roselló, A; de Capriles, Claudia R Hartung; Landaeta, M E; Mata-Essayag, S

    2008-09-01

    Cryptococcus neoformans is one of the medically important yeast-like fungi. C. neoformans var. gatti has been made a species: C. gatti. In our country, there are few studies about these two species and their serotypes. The aim of this study was to determine the distribution of C. neoformans and C. gattii, and their serotypes in Venezuelan clinical isolates. One hundred and twenty C. neoformans and 12 C. gattii clinical isolates were identified by L-canavanine, glycine, and bromothymol blue agar media (CGB). These were investigated by agglutination and adsorption studies with anticryptococcal sera, which were produced by rabbit immunization. Of the 132 isolates 59.8% were typed serotype A (C. neoformans), followed by 25.8% serotype D (C. neoformans), 5.3% serotype AD (C. neoformans), and 5.3% were typed serotype C (var. gattii). Additionally 3.8% were serotype B (C. gattii).

  4. Connecting virulence pathways to cell-cycle progression in the fungal pathogen Cryptococcus neoformans.

    PubMed

    Kelliher, Christina M; Haase, Steven B

    2017-03-06

    Proliferation and host evasion are critical processes to understand at a basic biological level for improving infectious disease treatment options. The human fungal pathogen Cryptococcus neoformans causes fungal meningitis in immunocompromised individuals by proliferating in cerebrospinal fluid. Current antifungal drugs target "virulence factors" for disease, such as components of the cell wall and polysaccharide capsule in C. neoformans. However, mechanistic links between virulence pathways and the cell cycle are not as well studied. Recently, cell-cycle synchronized C. neoformans cells were profiled over time to identify gene expression dynamics (Kelliher et al., PLoS Genet 12(12):e1006453, 2016). Almost 20% of all genes in the C. neoformans genome were periodically activated during the cell cycle in rich media, including 40 genes that have previously been implicated in virulence pathways. Here, we review important findings about cell-cycle-regulated genes in C. neoformans and provide two examples of virulence pathways-chitin synthesis and G-protein coupled receptor signaling-with their putative connections to cell division. We propose that a "comparative functional genomics" approach, leveraging gene expression timing during the cell cycle, orthology to genes in other fungal species, and previous experimental findings, can lead to mechanistic hypotheses connecting the cell cycle to fungal virulence.

  5. Effects of microplusin, a copper-chelating antimicrobial peptide, against Cryptococcus neoformans.

    PubMed

    Silva, Fernanda D; Rossi, Diego C P; Martinez, Luis R; Frases, Susana; Fonseca, Fernanda L; Campos, Claudia Barbosa L; Rodrigues, Marcio L; Nosanchuk, Joshua D; Daffre, Sirlei

    2011-11-01

    Microplusin is an antimicrobial peptide isolated from the cattle tick Rhipicephalus (Boophilus) microplus. Its copper-chelating ability is putatively responsible for its bacteriostatic activity against Micrococcus luteus as microplusin inhibits respiration in this species, which is a copper-dependent process. Microplusin is also active against Cryptococcus neoformans (MIC(50) = 0.09 μM), the etiologic agent of cryptococcosis. Here, we show that microplusin is fungistatic to C. neoformans and this inhibitory effect is abrogated by copper supplementation. Notably, microplusin drastically altered the respiratory profile of C. neoformans. In addition, microplusin affects important virulence factors of this fungus. We observed that microplusin completely inhibited fungal melanization, and this effect correlates with the inhibition of the related enzyme laccase. Also, microplusin significantly inhibited the capsule size of C. neoformans. Our studies reveal, for the first time, a copper-chelating antimicrobial peptide that inhibits respiration and growth of C. neoformans and modifies two major virulence factors: melanization and formation of a polysaccharide capsule. These features suggest that microplusin, or other copper-chelation approaches, may be a promising therapeutic for cryptococcosis.

  6. Origin of Cryptococcus neoformans var. neoformans Diploid Strains

    PubMed Central

    Cogliati, Massimo; Esposto, Maria C.; Clarke, David L.; Wickes, Brian L.; Viviani, Maria A.

    2001-01-01

    The basidiomycetous yeast Cryptococcus neoformans is an important human fungal pathogen. Two varieties, C. neoformans var. neoformans and C. neoformans var. gattii, have been identified. Both are heterothallic with two mating types, MATa and MATα. Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene Università Milano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)4 primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (MFa and MFα) as probes identified 36 of the 49 diploid isolates as MATa/α. The results of pheromone gene sequencing showed that two allelic MFα genes exist and are distinct for serotypes A and D. In contrast, the MFa gene sequence was conserved in both serotype alleles. Amplification of serotype-specific STE20 alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the C. neoformans population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction. PMID:11682503

  7. Opsonization of encapsulated Cryptococcus neoformans by specific anticapsular antibody.

    PubMed Central

    Kozel, T R; Follette, J L

    1981-01-01

    Antisera prepared in rabbits against either whole encapsulated cells of Cryptococcus neoformans or purified cryptococcal polysaccharide were opsonic for the encapsulated yeast. The opsonic activity was removed by absorption with whole cryptococci and was inhibited by free polysaccharide. As little as 0.13 microgram of cryptococcal polysaccharide produced a 50% inhibition of opsonization. Various degrees of neutralization by polysaccharides from the four cryptococcal serotypes suggested that the opsonins were type specific. Fractionation of antiserum on Bio-Gel A-5m (Bio-Rad Laboratories) and diethylaminoethyl cellulose showed that the opsonins were antibodies of the immunoglobulin G class. These opsonizing antibodies did not require heat-labile serum components for optimal phagocytosis of the yeast. Inhibition studies using 2-deoxy-D-glucose demonstrated that ingestion of encapsulated cryptococci opsonized with anticapsular antibody was a 2-deoxy-D-glucose-inhibitable process. This result differed from similar studies with non-encapsulated cryptococci which showed that ingestion of non-encapsulated cryptococci opsonized with normal serum was not inhibited by 2-deoxy-D-glucose. PMID:7014468

  8. Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

    PubMed Central

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

  9. Capsular Suspension Technique for Hip Arthroscopy

    PubMed Central

    Federer, Andrew E.; Karas, Vasili; Nho, Shane; Coleman, Struan H.; Mather, Richard C.

    2015-01-01

    Hip arthroscopy has recently become a common procedure to treat central and peripheral hip pathology. Capsulotomies are necessary in these procedures, and negotiating adequate visualization, as well as capsular preservation, is a challenge. We describe a capsular suspension technique that allows for adequate visualization of the central and peripheral compartments while facilitating preservation of the native hip capsule. This technique eliminates the need for additional personnel for retraction, potentially decreases iatrogenic hip injury, eliminates the need for excessive capsular debridement, and allows for capsular closure under minimal tension. PMID:26759769

  10. Quantification and assessment of viability of Cryptococcus neoformans by LightCycler amplification of capsule gene mRNA.

    PubMed

    Amjad, Muhammad; Kfoury, Najla; Cha, Raymond; Mobarak, Reem

    2004-12-01

    Cryptococcus neoformans is an opportunistic fungal pathogen. It infects the central nervous system causing meningitis, which is fatal if untreated, especially in AIDS and immunosuppressed patients. In this study a method of quantification and assessment of viability of C. neoformans by LightCycler RT-PCR amplification of the capsule gene mRNA is established. The sequence of primers and probes were derived from C. neoformans capsular CAP10 gene mRNA (GenBank accession number AF144574), and were species specific. Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp in length. In order to develop an internal control a 223 bp exon fragment of capsule mRNA was cloned in the pCR2.1 plasmid vector and RNA was generated by in vitro transcription. To determine the sensitivity of the assay, serial dilutions of in vitro-transcribed RNA with known concentrations and copy numbers, and serially diluted cultures of viable and nonviable C. neoformans were used. Under optimal conditions as little as 0.472 fg of capsule mRNA could be detected, corresponding to 1-10 c.f.u. ml(-1) of the sample. No amplification was observed from up to 10(5) heat/UV radiation-killed yeast cells and RNA of other bacterial and fungal pathogens and human genomic DNA or RNA. The amplification of capsule mRNA represents a sensitive, specific and quantitative means of detection of viable C. neoformans in clinical specimens and can be useful in the evaluation of the therapeutic efficacy of antifungal drugs in the treatment of C. neoformans meningitis.

  11. Cryptococcus neoformans STE12α Regulates Virulence but Is Not Essential for Mating

    PubMed Central

    Chang, Y.C.; Wickes, B.L.; Miller, G.F.; Penoyer, L.A.; Kwon-Chung, K.J.

    2000-01-01

    The Cryptococcus neoformans STE12α gene, a homologue of Saccharomyces cerevisiae STE12, exists only in mating type (MAT)α cells. In S. cerevisiae, STE12 was required for mating and filament formation. In C. neoformans, haploid fruiting on filament agar required STE12α. The ability to form hyphae, however, was not affected by deletion of STE12α when convergently growing MATa strains were present. Furthermore, ste12α disruptants were fertile when mated with MATa strains, albeit with reduced mating frequency. Most importantly, the virulence of a ste12α disruptant of serotype D strain was significantly reduced in a mouse model. When the ste12α locus was reconstituted with the wild-type allele by cotransformation, virulence was restored. Histopathological analysis demonstrated a reduction in capsular size of yeast cells, less severe cystic lesions, and stronger immune responses in meninges of mice infected with ste12α cells than those of mice infected with STE12α cells. Using reporter gene constructs, we found that STE12α controls the expression of several phenotypes known to be involved in virulence, such as capsule and melanin production. These results demonstrate a clear molecular link between mating type and virulence in C. neoformans. PMID:10704467

  12. Binding of the wheat germ lectin to Cryptococcus neoformans chitooligomers affects multiple mechanisms required for fungal pathogenesis

    PubMed Central

    Fonseca, Fernanda L.; Guimarães, Allan J.; Kmetzsch, Lívia; Dutra, Fabianno F.; Silva, Fernanda D.; Taborda, Carlos P.; Araujo, Glauber de S.; Frases, Susana; Staats, Charley C.; Bozza, Marcelo T.; Schrank, Augusto; Vainstein, Marilene H.; Nimrichter, Leonardo; Casadevall, Arturo; Rodrigues, Marcio L.

    2015-01-01

    The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis. PMID:23608320

  13. Antibiotic modulation of capsular exopolysaccharide and virulence in Acinetobacter baumannii.

    PubMed

    Geisinger, Edward; Isberg, Ralph R

    2015-02-01

    Acinetobacter baumannii is an opportunistic pathogen of increasing importance due to its propensity for intractable multidrug-resistant infections in hospitals. All clinical isolates examined contain a conserved gene cluster, the K locus, which determines the production of complex polysaccharides, including an exopolysaccharide capsule known to protect against killing by host serum and to increase virulence in animal models of infection. Whether the polysaccharides determined by the K locus contribute to intrinsic defenses against antibiotics is unknown. We demonstrate here that mutants deficient in the exopolysaccharide capsule have lowered intrinsic resistance to peptide antibiotics, while a mutation affecting sugar precursors involved in both capsule and lipopolysaccharide synthesis sensitizes the bacterium to multiple antibiotic classes. We observed that, when grown in the presence of certain antibiotics below their MIC, including the translation inhibitors chloramphenicol and erythromycin, A. baumannii increases production of the K locus exopolysaccharide. Hyperproduction of capsular exopolysaccharide is reversible and non-mutational, and occurs concomitantly with increased resistance to the inducing antibiotic that is independent of the presence of the K locus. Strikingly, antibiotic-enhanced capsular exopolysaccharide production confers increased resistance to killing by host complement and increases virulence in a mouse model of systemic infection. Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS. These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low

  14. Antibiotic Modulation of Capsular Exopolysaccharide and Virulence in Acinetobacter baumannii

    PubMed Central

    Geisinger, Edward; Isberg, Ralph R.

    2015-01-01

    Acinetobacter baumannii is an opportunistic pathogen of increasing importance due to its propensity for intractable multidrug-resistant infections in hospitals. All clinical isolates examined contain a conserved gene cluster, the K locus, which determines the production of complex polysaccharides, including an exopolysaccharide capsule known to protect against killing by host serum and to increase virulence in animal models of infection. Whether the polysaccharides determined by the K locus contribute to intrinsic defenses against antibiotics is unknown. We demonstrate here that mutants deficient in the exopolysaccharide capsule have lowered intrinsic resistance to peptide antibiotics, while a mutation affecting sugar precursors involved in both capsule and lipopolysaccharide synthesis sensitizes the bacterium to multiple antibiotic classes. We observed that, when grown in the presence of certain antibiotics below their MIC, including the translation inhibitors chloramphenicol and erythromycin, A. baumannii increases production of the K locus exopolysaccharide. Hyperproduction of capsular exopolysaccharide is reversible and non-mutational, and occurs concomitantly with increased resistance to the inducing antibiotic that is independent of the presence of the K locus. Strikingly, antibiotic-enhanced capsular exopolysaccharide production confers increased resistance to killing by host complement and increases virulence in a mouse model of systemic infection. Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS. These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low

  15. Cryptococcus neoformans myositis without dissemination.

    PubMed

    Sharma, Mamta; Khatib, Riad; Jones, Bruce A; Fakih, Mohamad G

    2002-01-01

    We report a case of isolated cryptococcal myositis involving the paraspinal muscle without evidence of disseminated disease in a patient with a large B-cell lymphoma diagnosed at the time of presentation. Biopsy of the muscle involved grew a pure culture of Cryptococcus neoformans and periodic acid-Schiff staining showed numerous budding yeast consistent with Cryptococcus spp. The patient responded to systemic antifungal therapy with complete resolution of his infection. We also present a review of 5 previously published cases of cryptococcal myositis.

  16. Isolation and characterization of Cryptococcus neoformans varieties recovered from natural sources in Bogotá, Colombia, and study of ecological conditions in the area.

    PubMed

    Granados, D P; Castañeda, E

    2005-02-01

    Cryptococcus neoformans, the etiological agent of cryptococcosis, has been associated with avian droppings and certain trees in different countries, including Colombia. C neoformans environmental isolates were obtained in urban areas in Bogotá, Colombia, and the strains recovered were phenotypically characterized. Attempts to determine the ecological conditions (micro- and macroclimatic) possibly related to their habitat were also undertaken. Four hundred and eighty samples from bark, soil around trunk bases, and detritus inside hollows of 32 trees were collected in three urban areas during a 5-month period, as well as 89 avian droppings samples from different places. Of plant samples, 6.7% collected from nine tree species yielded C. neoformans var. gattii, serotype B strains in 99% of the cases, and C. neoformans var. grubii, serotype A in 1%. The yeast was more frequently recovered from bark than from soil or detritus inside hollows, and from trees with hollows or rotted wood rather than from trees in which birds nest. C. neoformans was present with higher frequency and density in the rainy season than in the dry season; we found that slightly higher temperature and humidity values of the microhabitat, as compared to those of the environment, favored fungal occurrence, but the phenological state of the tree did not. Of dropping samples, 7.9% yielded C. neoformans strains, all of them C. neoformans var. grubii, serotype A. The yeast was obtained more frequently from dry droppings than from moist ones, but neither the sunlight exposure nor the site of collection of samples was correlated with this occurrence. Population density was significantly higher in droppings than in tree samples. Under laboratory conditions, isolates of different serotype showed similar capsular sizes. Water content and pH ranges were wide and did not show any significant difference between positive and negative samples.

  17. A Rare Case of Primary Supraclavicular Lymphadenitis due to Cryptococcus Neoformans in an HIV Infected Patient

    PubMed Central

    Sood, Anuradha; Chandel, Lata R; Chauhan, Smriti; Thakur, Kamlesh; Jaryal, S.C

    2014-01-01

    Cryptococcosis caused by encapsulated yeast Cryptococcus neoformans most commonly presents as disease of the central nervous system. Cryptococcus is a non–mycelial budding yeast found in soil, pigeon droppings and their nesting places. The three ‘classic’ virulence factors of cryptococci are: polysaccharide capsule, melanin production and growth at 37°C. Here, we present a rare case of cryptococcosis affecting left supraclavicular lymph node in a Human Immunodeficiency virus (HIV) infected individual. Culture of fine needle aspirate of the lymph node yielded Cryptococcus neoformans which was identified by standard microbiological techniques. Meyer’s mucicarmine stain imparted a typical rose burgundy colour to the capsule. Unusual characteristics of the isolate included poorly developed capsule and the presence of yeast in chains resembling pseudo-hyphae. This case highlights the importance of microbiological techniques for diagnosis and prompt treatment of cryptococcosis. PMID:24701506

  18. Pflanzen als Naehrsubstrat fuer Cryptococcus Neoformans (Plants as a Substratum for Growth of Cryptococcus Neoformans),

    DTIC Science & Technology

    The suitability of plants as a possible substratum for growth (in vitro) of C . neoformans has been investigated. A sample of hay collected from...the second left unsterile. The samples were inoculated by flooding them with suspensions prepared from two known strains of C . neoformans . The...month and subsequently at room temperature. Discrete colonies of C . neoformans appeared after five days on samples of hay as well as of dandelion

  19. Pseudomonas aeruginosa inactivation mechanism is affected by capsular extracellular polymeric substances reactivity with chlorine and monochloramine.

    PubMed

    Xue, Zheng; Hessler, Christopher M; Panmanee, Warunya; Hassett, Daniel J; Seo, Youngwoo

    2013-01-01

    The reactivity of capsular extracellular polymeric substances (EPS) to chlorine and monochloramine was assessed and compared in this study. The impact of capsular EPS on Gram-negative bacteria Pseudomonas aeruginosa inactivation mechanisms was investigated both qualitatively and quantitatively using a combination of batch experiments, viability tests with LIVE/DEAD staining, and Fourier transform infrared spectroscopy (FTIR). Both wild-type and isogenic mutant strains with different alginate EPS production capabilities were used to evaluate their susceptibility to chlorine and monochloramine. The mucA22 mutant strain, which overproduces the EPS composed largely of acidic polysaccharide alginate, exhibited high resistance and prolonged inactivation time to both chlorine and monochloramine relative to PAO1 (wild-type) and algT(U) mutant strains (alginate EPS deficient). Multiple analyses were combined to better understand the mechanistic role of EPS against chlorine-based disinfectants. The extracted EPS exhibited high reactivity with chlorine and very low reactivity with monochloramine, suggesting different mechanism of protection against disinfectants. Moreover, capsular EPS on cell membrane appeared to reduce membrane permeabilization by disinfectants as suggested by deformation of key functional groups in EPS and cell membrane (the C-O-C stretching of carbohydrate and the C=O stretching of ester group). The combined results supported that capsular EPS, acting either as a disinfectant consumer (for chlorine inactivation) or limiting access to reactive sites on cell membrane (for monochloramine inactivation), provide a protective role for bacterial cells against regulatory residual disinfectants by reducing membrane permeabilization.

  20. Iron acquisition by Cryptococcus neoformans.

    PubMed

    Vartivarian, S E; Cowart, R E; Anaissie, E J; Tashiro, T; Sprigg, H A

    1995-01-01

    Iron is an essential element for the growth and metabolism of microbial cells. Most pathogenic microbes elaborate powerful iron chelating agents (siderophores) to mobilize iron from ferric ligands. The pathogenic yeast, Cryptococcus neoformans has not been found to produce siderophores and its mechanism of iron acquisition is unknown. This investigation explored an alternative pathway for iron acquisition by examining the interactions of iron with the cell surface. Iron uptake experiments were conducted utilizing radiolabelled ferrous iron and ferric iron chelates, with evidence for the presence of iron(II) receptors and the generation of ferrous iron by surface reduction. Hyperbolic kinetics were found when 59FeII was presented to the organism and uptake was blocked with bathophenanthroline sulphonate, an Fe2+ chelator. The yeast also acquired iron as [59Fe3+]-citrate and [59Fe3+]-pyrophosphate while bathophenanthroline sulphonate reduced the acquisition of these ferric ligands by 48% and 52% respectively. Pre-incubation with either ferric ligand also reduced iron acquisition by 50%. KCN inhibited uptake of iron(II) by 90% and uptake of [59Fe3+]-pyrophosphate and [59Fe3+]-citrate by 46% and 56% respectively; dinitrophenol had no effect on these processes. The data suggest that C. neoformans can (i) generate ferrous iron at the cell surface via a reduction of ferric chelates, with the subsequent acquisition of the ferrous iron, and (ii) acquire iron through the interaction of ferric chelates with a surface component.

  1. Serogroup quantitation of multivalent polysaccharide and polysaccharide-conjugate meningococcal vaccines from China.

    PubMed

    Cook, Matthew C; Gibeault, Sabrina; Filippenko, Vasilisa; Ye, Qiang; Wang, Junzhi; Kunkel, Jeremy P

    2013-07-01

    The active components of most meningococcal vaccines are four antigenic serogroup capsular polysaccharides (A, C, Y, W135). The vaccines, monovalent or multivalent mixtures of either free polysaccharides or polysaccharides conjugated to antigenic carrier proteins, may be in liquid or lyophilised formulations, with or without excipients. Acid hydrolysis and chromatographic methods for serogroup quantitation, which were previously optimised and qualified using polysaccharide-based standards and a narrow range of real vaccines, are here challenged with multiple lots of a broad assortment of additional multivalent polysaccharide-based meningococcal vaccine products. Centrifugal filtration successfully removed all interfering lactose excipient without loss of polysaccharides to allow for the determination of Y and W135 serogroups. Replicate operations by three different analysts indicated high method reproducibility. Results indicated some lot-to-lot and product-to-product variations. However, all vaccines were within general specifications for each serogroup polysaccharide, with the exception of all lots of one polysaccharide vaccine - which by these methods were found to be deficient in the serogroup A component only. These robust techniques are very useful for the evaluation of antigen content and consistency of manufacture. The deformulation, hydrolysis and chromatographic methods may be adaptable for the evaluation of other types of polysaccharide-based vaccines.

  2. Streptococcus iniae cpsG alters capsular carbohydrate composition and is a cause of serotype switching in vaccinated fish.

    PubMed

    Heath, Candice; Gillen, Christine M; Chrysanthopoulos, Panagiotis; Walker, Mark J; Barnes, Andrew C

    2016-09-25

    Streptococcus iniae causes septicaemia and meningitis in marine and freshwater fish wherever they are farmed in warm-temperate and tropical regions. Although serotype specific, vaccination with bacterins (killed bacterial cultures) is largely successful and vaccine failure occurs only occasionally through emergence of new capsular serotypes. Previously we showed that mutations in vaccine escapes are restricted to a limited repertoire of genes within the 20-gene capsular polysaccharide (cps) operon. cpsG, a putative UDP-galactose 4-epimerase, has three sequence types based on the insertion or deletion of the three amino acids leucine, serine and lysine in the substrate binding site of the protein. To elucidate the role of cpsG in capsular polysaccharide (CPS) biosynthesis and capsular composition, we first prepared isogenic knockout and complemented mutants of cpsG by allelic exchange mutagenesis. Deletion of cpsG resulted in changes to colony morphology and cell buoyant density, and also significantly decreased galactose content relative to glucose in the capsular polysaccharide as determined by GC-MS, consistent with epimerase activity of CpsG. There was also a metabolic penalty of cpsG knockout revealed by slower growth in complex media, and reduced proliferation in whole fish blood. Moreover, whilst antibodies raised in fish against the wild type cross-reacted in whole cell and cps ELISA, they did not cross-opsonise the mutant in a peripheral blood neutrophil opsonisation assay, consistent with reported vaccine escape. We have shown here that mutation in cpsG results in altered CPS composition and this in turn results in poor cross-opsonisation that explains some of the historic vaccination failure on fish farms in Australia.

  3. EXTRACELLULAR POLYSACCHARIDES OF AZOTOBACTER VINELANDII1

    PubMed Central

    Cohen, Gary H.; Johnstone, Donald B.

    1964-01-01

    Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone. Extracellular polysaccharides of Azotobacter vinelandii. J. Bacteriol. 88:329–338. 1964.—Extracellular polysaccharides synthetized by Azotobacter vinelandii strains 155, 102, and 3A were shown to be carboxylic acid heteropolysaccharides of apparent high molecular weight. Cells were grown in a nitrogen-free, mineral broth medium with 2% sucrose. Extracellular slime was recovered by centrifugation and purified by repeated alcohol precipitation and Sevag deproteinization. Capsular polysaccharide was recovered from washed cells by mild alkaline digestion. Methods of isolation and purification appeared to provide polysaccharide showing no evidence of heterogeneity when examined by chemical and physical methods. Infrared analysis of purified slime from the three strains suggested fundamental structural similarities. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed slime polysaccharide indicated that the polymers contained in common galacturonic acid, [α] d-glucose, and rhamnose at a ratio of approximately 43:2:1, as well as a hexuronic acid lactone, probably mannurono-lactone. However, as shown by chemical and infrared analysis, minor differences did exist; namely, slime from strain 155 and 102 contained o-acetyl groups, whereas slime from strain 3A contained none. A sialic acid-like component (1.5% of dry weight of the polysaccharide, calculated as N-acetyl neuraminic acid), was found only in the slime of strain 155. Capsular polysaccharide composition closely resembled that for slime. It is of interest that the major slime components were identical whether the energy source provided for the cells was sucrose, glucose, fructose, or ethanol. PMID:14203348

  4. Host immunity to Cryptococcus neoformans

    PubMed Central

    Rohatgi, Soma; Pirofski, Liise-anne

    2015-01-01

    Cryptococcosis is caused by the fungal genus Cryptococcus. Cryptococcosis, predominantly meningoencephalitis, emerged with the HIV pandemic, primarily afflicting HIV-infected patients with profound T-cell deficiency. Where in use, combination antiretroviral therapy has markedly reduced the incidence of and risk for disease, but cryptococcosis continues to afflict those without access to therapy, particularly in sub-Saharan Africa and Asia. However, cryptococcosis also occurs in solid organ transplant recipients and patients with other immunodeficiencies as well as those with no known immunodeficiency. This article reviews innate and adaptive immune responses to C. neoformans, with an emphasis on recent studies on the role of B cells, natural IgM and Fc gamma receptor polymorphisms in resistance to cryptococcosis. PMID:25865194

  5. Toward an Integrated Model of Capsule Regulation in Cryptococcus neoformans

    PubMed Central

    Haynes, Brian C.; Skowyra, Michael L.; Spencer, Sarah J.; Gish, Stacey R.; Williams, Matthew; Held, Elizabeth P.; Brent, Michael R.; Doering, Tamara L.

    2011-01-01

    Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule

  6. Fine structure of Cryptococcus neoformans grown in vivo as observed by freeze-etching.

    PubMed

    Takeo, K; Uesaka, I; Uehira, K; Nishiura, M

    1973-03-01

    Cryptococcus neoformans grown in the parasitic state was observed by the freeze-etching technique and was compared with that grown on culture media. Unlike other yeasts, this organism grown in vivo is very often devoid of the "ordinary" invaginations. The membrane of the cell grown in vivo was almost free from concavity and convexity except for many round depressions which represent the surface view of paramural bodies. Some of the paramural bodies were found to be multivesicular systems. Most were spherical invaginations containing a single vesicle or its ghost remaining after secretion of the vesicles. In clear contrast to the cell grown in vitro, the in vivo cell contained a great number of vesicles in the cytoplasm. These seemed to show high-secretion activity in C. neoformans grown in the parasitic state. On transfer from in vitro to in vivo, this organism enlarged the cell wall, capsule, and cell body. The appearance of a large vacuole, accumulation of storage organelles, and the existence of rodlike structures, seemingly lipid deposits, were also noted in the cytoplasm of the cell grown in vivo. the meaning of these results as well as the mode of capsular production are discussed.

  7. Cryptococcus neoformans Intracellular Proliferation and Capsule Size Determines Early Macrophage Control of Infection.

    PubMed

    Bojarczuk, Aleksandra; Miller, Katie A; Hotham, Richard; Lewis, Amy; Ogryzko, Nikolay V; Kamuyango, Alfred A; Frost, Helen; Gibson, Rory H; Stillman, Eleanor; May, Robin C; Renshaw, Stephen A; Johnston, Simon A

    2016-02-18

    Cryptococcus neoformans is a significant fungal pathogen of immunocompromised patients. Many questions remain regarding the function of macrophages in normal clearance of cryptococcal infection and the defects present in uncontrolled cryptococcosis. Two current limitations are: 1) The difficulties in interpreting studies using isolated macrophages in the context of the progression of infection, and 2) The use of high resolution imaging in understanding immune cell behavior during animal infection. Here we describe a high-content imaging method in a zebrafish model of cryptococcosis that permits the detailed analysis of macrophage interactions with C. neoformans during infection. Using this approach we demonstrate that, while macrophages are critical for control of C. neoformans, a failure of macrophage response is not the limiting defect in fatal infections. We find phagocytosis is restrained very early in infection and that increases in cryptococcal number are driven by intracellular proliferation. We show that macrophages preferentially phagocytose cryptococci with smaller polysaccharide capsules and that capsule size is greatly increased over twenty-four hours of infection, a change that is sufficient to severely limit further phagocytosis. Thus, high-content imaging of cryptococcal infection in vivo demonstrates how very early interactions between macrophages and cryptococci are critical in the outcome of cryptococcosis.

  8. Development of an opsonin inhibition assay for evaluation of complex polysaccharide protective epitopes.

    PubMed

    McNeely, Tessie; Luo, Shengyuan; Manger, Walt; Herber, Wayne; Schofield, Tim; Tan, Charles; Newman, Kathy; Sadoff, Jerald; Donnelly, John; Cross, Alan

    2006-03-10

    The induction of opsonic antibodies directed against capsular polysaccharides (Ps) is an important mechanism by which immunization protects against the development of invasive pneumococcal (Pn) infection. In preparing Pn vaccines, it is necessary to compare different manufacturing lots of capsular Ps, or to compare oligosaccharides used for conjugate vaccines with native capsular Ps, in order to insure that important epitopes of the Ps are maintained. We have developed an opsonic-antibody inhibition assay (OIA) to compare the functional epitopes of different capsular Ps preparations in vitro. Components of the OIA are primary neutrophils, rabbit complement (C'), and type-specific antibody (Ab). After conditions for optimal opsonic killing were determined for each Pn serotype, anti-Pn Ab was pre-incubated with different dilutions of purified capsular Ps, then added to the OIA mix. Plotting the % bacteria killed versus Ps concentration (log transformed) yielded a linear curve that was used to quantify the concentration of capsular Ps which inhibited the bacteria killing by 50% (IC50). The IC50 was determined for 8 Pn Ps types. These ranged between 6 ng/ml for type 6B and 1268 ng/ml for type 23F. Importantly OIA curves were statistically identical for two different manufacturing lots of capsular Ps for the 8 Pn Ps types. We conclude that differences among capsular Ps used for Pn vaccines could be detected with an OIA assay and these differences may predict the ability of Ps preparations to induce functionally active antibody when formulated into vaccines.

  9. Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population.

    PubMed

    Cogliati, Massimo; Zani, Alberto; Rickerts, Volker; McCormick, Ilka; Desnos-Ollivier, Marie; Velegraki, Aristea; Escandon, Patricia; Ichikawa, Tomoe; Ikeda, Reiko; Bienvenu, Anne-Lise; Tintelnot, Kathrin; Tore, Okan; Akcaglar, Sevim; Lockhart, Shawn; Tortorano, Anna Maria; Varma, Ashok

    2016-02-01

    Cryptococcus neoformans var. neoformans (serotype D) represents about 30% of the clinical isolates in Europe and is present less frequently in the other continents. It is the prevalent etiological agent in primary cutaneous cryptococcosis as well as in cryptococcal skin lesions of disseminated cryptococcosis. Very little is known about the genotypic diversity of this Cryptococcus subtype. The aim of this study was to investigate the genotypic diversity among a set of clinical and environmental C. neoformans var. neoformans isolates and to evaluate the relationship between genotypes, geographical origin and clinical manifestations. A total of 83 globally collected C. neoformans var. neoformans isolates from Italy, Germany, France, Belgium, Denmark, Greece, Turkey, Thailand, Japan, Colombia, and the USA, recovered from different sources (primary and secondary cutaneous cryptococcosis, disseminated cryptococcosis, the environment, and animals), were included in the study. All isolates were confirmed to belong to genotype VNIV by molecular typing and they were further investigated by MLST analysis. Maximum likelihood phylogenetic as well as network analysis strongly suggested the existence of a recombinant rather than a clonal population structure. Geographical origin and source of isolation were not correlated with a specific MLST genotype. The comparison with a set of outgroup C. neoformans var. grubii isolates provided clear evidence that the two varieties have different population structures.

  10. Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population

    PubMed Central

    Cogliati, Massimo; Zani, Alberto; Rickerts, Volker; McCormick, Ilka; Desnos-Ollivier, Marie; Velegraki, Aristea; Escandon, Patricia; Ichikawa, Tomoe; Ikeda, Reiko; Bienvenue, Anne-Lise; Tintelnot, Kathrin; Tore, Okan; Akcaglar, Sevim; Lockhart, Shawn; Tortorano, Anna Maria; Varma, Ashok

    2016-01-01

    Cryptococcus neoformans var. neoformans (serotype D) represents about 30% of the clinical isolates in Europe and is present less frequently in the other continents. It is the prevalent etiological agent in primary cutaneous cryptococcosis as well as in cryptococcal skin lesions of disseminated cryptococcosis. Very little is known about the genotypic diversity of this Cryptococcus subtype. The aim of this study was to investigate the genotypic diversity among a set of clinical and environmental C. neoformans var. neoformans isolates and to evaluate the relationship between genotypes, geographical origin and clinical manifestations. A total of 83 globally collected C. neoformans var. neoformans isolates from Italy, Germany, France, Belgium, Denmark, Greece, Turkey, Thailand, Japan, Colombia, and the USA, recovered from different sources (primary and secondary cutaneous cryptococcosis, disseminated cryptococcosis, the environment, and animals), were included in the study. All isolates were confirmed to belong to genotype VNIV by molecular typing and they were further investigated by MLST analysis. Maximum likelihood phylogenetic as well as network analysis strongly suggested the existence of a recombinant rather than a clonal population structure. Geographical origin and source of isolation were not correlated with a specific MLST genotype. The comparison with a set of outgroup C. neoformans var. grubii isolates provided clear evidence that the two varieties have different population structures. PMID:26768709

  11. Hip Capsular Reconstruction Using Dermal Allograft.

    PubMed

    Chahla, Jorge; Dean, Chase S; Soares, Eduardo; Mook, William R; Philippon, Marc J

    2016-04-01

    Because hip arthroscopic procedures are increasing in number, complications related to the operation itself are starting to emerge. Whereas the capsule has been recognized as an important static stabilizer for the hip, it has not been until recently that surgeons have realized the importance of its preservation and restoration. Disruption of the capsule during arthroscopic procedures is a potential contributor to postoperative iatrogenic hip instability. In cases of a symptomatic deficient capsule, a capsular reconstruction is mandatory because instability may lead to detrimental chondral and labral changes. The purpose of this report was to describe our technique for arthroscopic hip capsular reconstruction using dermal allograft.

  12. Networks of fibers and factors: regulation of capsule formation in Cryptococcus neoformans

    PubMed Central

    de S. Araújo, Glauber R.; Frases, Susana; Kronstad, James W.

    2016-01-01

    The ability of the pathogenic fungus Cryptococcus neoformans to cause life-threatening meningoencephalitis in immunocompromised individuals is due in large part to elaboration of a capsule consisting of polysaccharide fibers. The size of the cell-associated capsule is remarkably responsive to a variety of environmental and host conditions, but the mechanistic details of the regulation, synthesis, trafficking, and attachment of the polysaccharides are poorly understood. Recent studies reveal a complex network of transcription factors that influence capsule elaboration in response to several different signals of relevance to disease (e.g., iron deprivation). The emerging complexity of the network is consistent with the diversity of conditions that influence the capsule and illustrates the responsiveness of the fungus to both the environment and mammalian hosts. PMID:27516877

  13. Role of capsular modified heptose in the virulence of Campylobacter jejuni.

    PubMed

    Wong, Anthony; Lange, Dirk; Houle, Sebastien; Arbatsky, Nikolay P; Valvano, Miguel A; Knirel, Yuriy A; Dozois, Charles M; Creuzenet, Carole

    2015-06-01

    The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.

  14. The Intracellular Life of Cryptococcus neoformans

    PubMed Central

    Coelho, Carolina; Bocca, Anamelia L.; Casadevall, Arturo

    2016-01-01

    Cryptococcus neoformans is a fungal pathogen with worldwide distribution. Serological studies of human populations show a high prevalence of human infection, which rarely progresses to disease in immunocompetent hosts. However, decreased host immunity places individuals at high risk for cryptococcal disease. The disease can result from acute infection or reactivation of latent infection, in which yeasts within granulomas and host macrophages emerge to cause disease. In this review, we summarize what is known about the cellular recognition, ingestion, and killing of C. neoformans and discuss the unique and remarkable features of its intracellular life, including the proposed mechanisms for fungal persistence and killing in phagocytic cells. PMID:24050625

  15. Antibiofilm polysaccharides

    PubMed Central

    Rendueles, Olaya; Kaplan, Jeffrey B.; Ghigo, Jean-Marc

    2012-01-01

    Summary Bacterial extracellular polysaccharides have been shown to mediate many of the cell-to cell and cell-to-surface interactions that are required for the formation, cohesion and stabilization of bacterial biofilms. However, recent studies have identified several bacterial polysaccharides that inhibit biofilm formation by a wide-spectrum of bacteria and fungi both in vitro and in vivo. This review discusses the composition, modes of action, and potential biological roles of antibiofilm polysaccharides recently identified in bacteria and eukaria. Some of these molecules may have technological applications as antibiofilm agents in industry and medicine. PMID:22730907

  16. Simvastatin Reduces Capsular Fibrosis around Silicone Implants.

    PubMed

    Chung, Kyu Jin; Park, Ki Rin; Lee, Jun Ho; Kim, Tae Gon; Kim, Yong-Ha

    2016-08-01

    Capsular fibrosis and contracture occurs in most breast reconstruction patients who undergo radiotherapy, and there is no definitive solution for its prevention. Simvastatin was effective at reducing fibrosis in various models. Peri-implant capsular formation is the result of tissue fibrosis development in irradiated breasts. The purpose of this study was to examine the effect of simvastatin on peri-implant fibrosis in rats. Eighteen male Sprague-Dawley rats were allocated to an experimental group (9 rats, 18 implants) or a control group (9 rats, 18 implants). Two hemispherical silicone implants, 10 mm in diameter, were inserted in subpanniculus pockets in each rat. The next day, 10-Gy of radiation from a clinical accelerator was targeted at the implants. Simvastatin (15 mg/kg/day) was administered by oral gavage in the experimental group, while animals in the control group received water. At 12 weeks post-implantation, peri-implant capsules were harvested and examined histologically and by real-time polymerase chain reaction. The average capsular thickness was 371.2 μm in the simvastatin group and 491.2 μm in the control group. The fibrosis ratio was significantly different, with 32.33% in the simvastatin group and 58.44% in the control group (P < 0.001). Connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β1 gene expression decreased significantly in the simvastatin group compared to the control group (P < 0.001). This study shows that simvastatin reduces radiation-induced capsular fibrosis around silicone implants in rats. This finding offers an alternative therapeutic strategy for reducing capsular fibrosis and contracture after implant-based breast reconstruction.

  17. Simvastatin Reduces Capsular Fibrosis around Silicone Implants

    PubMed Central

    2016-01-01

    Capsular fibrosis and contracture occurs in most breast reconstruction patients who undergo radiotherapy, and there is no definitive solution for its prevention. Simvastatin was effective at reducing fibrosis in various models. Peri-implant capsular formation is the result of tissue fibrosis development in irradiated breasts. The purpose of this study was to examine the effect of simvastatin on peri-implant fibrosis in rats. Eighteen male Sprague-Dawley rats were allocated to an experimental group (9 rats, 18 implants) or a control group (9 rats, 18 implants). Two hemispherical silicone implants, 10 mm in diameter, were inserted in subpanniculus pockets in each rat. The next day, 10-Gy of radiation from a clinical accelerator was targeted at the implants. Simvastatin (15 mg/kg/day) was administered by oral gavage in the experimental group, while animals in the control group received water. At 12 weeks post-implantation, peri-implant capsules were harvested and examined histologically and by real-time polymerase chain reaction. The average capsular thickness was 371.2 μm in the simvastatin group and 491.2 μm in the control group. The fibrosis ratio was significantly different, with 32.33% in the simvastatin group and 58.44% in the control group (P < 0.001). Connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β1 gene expression decreased significantly in the simvastatin group compared to the control group (P < 0.001). This study shows that simvastatin reduces radiation-induced capsular fibrosis around silicone implants in rats. This finding offers an alternative therapeutic strategy for reducing capsular fibrosis and contracture after implant-based breast reconstruction. PMID:27478339

  18. Cryptococcus neoformans: Tripping on Acid in the Phagolysosome

    PubMed Central

    DeLeon-Rodriguez, Carlos M.; Casadevall, Arturo

    2016-01-01

    Cryptococcus neoformans (Cn) is a basidiomycetous pathogenic yeast that is a frequent cause of meningoencephalitis in immunocompromised individuals. Cn is a facultative intracellular pathogen in mammals, insects and amoeba. Cn infection occurs after inhalation of spores or desiccated cells from the environment. After inhalation Cn localizes to the lungs where it can be phagocytosed by alveolar macrophages. Cn is surrounded by a polysaccharide capsule that helps the fungus survive in vivo by interfering with phagocytosis, quenching free radical bursts and shedding polysaccharides that negatively modulates the immune system. After phagocytosis, Cn resides within the phagosome that matures to become a phagolysosome, a process that results in the acidification of the phagolysosomal lumen. Cn replicates at a higher rate inside macrophages than in the extracellular environment, possibly as a result that the phagosomal pH is near that optimal for growth. Cn increases the phagolysosomal pH and modulates the dynamics of Rab GTPases interaction with the phagolysosome. Chemical manipulation of the phagolysosomal pH with drugs can result in direct and indirect killing of Cn and reduced non-lytic exocytosis. Phagolysosomal membrane damage after Cn infection occurs both in vivo and in vitro, and is required for Cn growth and survival. Macrophage treatment with IFN-γ reduces the phagolysosomal damage and increases intracellular killing of Cn. Studies on mice and humans show that treatment with IFN-γ can improve host control of the disease. However, the mechanism by which Cn mediates phagolysosomal membrane damage remains unknown but likely candidates are phospholipases and mechanical damage from an enlarging capsule. Here we review Cn intracellular interaction with a particular emphasis on phagosomal interactions and develop the notion that the extent of damage of the phagosomal membrane is a key determinant of the outcome of the Cn-macrophage interaction. PMID:26925039

  19. Dectin-2 Deficiency Promotes Th2 Response and Mucin Production in the Lungs after Pulmonary Infection with Cryptococcus neoformans

    PubMed Central

    Nakamura, Yuri; Sato, Ko; Yamamoto, Hideki; Matsumura, Kana; Matsumoto, Ikumi; Nomura, Toshiki; Miyasaka, Tomomitsu; Ishii, Keiko; Kanno, Emi; Tachi, Masahiro; Yamasaki, Sho; Saijo, Shinobu; Iwakura, Yoichiro

    2014-01-01

    Dectin-2 is a C-type lectin receptor that recognizes high mannose polysaccharides. Cryptococcus neoformans, a yeast-form fungal pathogen, is rich in polysaccharides in its cell wall and capsule. In the present study, we analyzed the role of Dectin-2 in the host defense against C. neoformans infection. In Dectin-2 gene-disrupted (knockout) (Dectin-2KO) mice, the clearance of this fungus and the inflammatory response, as shown by histological analysis and accumulation of leukocytes in infected lungs, were comparable to those in wild-type (WT) mice. The production of type 2 helper T (Th2) cytokines in lungs was higher in Dectin-2KO mice than in WT mice after infection, whereas there was no difference in the levels of production of Th1, Th17, and proinflammatory cytokines between these mice. Mucin production was significantly increased in Dectin-2KO mice, and this increase was reversed by administration of anti-interleukin 4 (IL-4) monoclonal antibody (MAb). The levels of expression of β1-defensin, cathelicidin, surfactant protein A (Sp-A), and Sp-D in infected lungs were comparable between these mice. In in vitro experiments, IL-12p40 and tumor necrosis factor alpha (TNF-α) production and expression of CD86 and major histocompatibility complex (MHC) class II by bone marrow-derived dendritic cells and alveolar macrophages were completely abrogated in Dectin-2KO mice. Finally, the disrupted lysates of C. neoformans, but not of whole yeast cells, activated Dectin-2-triggered signaling in an assay with nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Dectin-2 may oppose the Th2 response and IL-4-dependent mucin production in the lungs after infection with C. neoformans, and it may not be required for the production of Th1, Th17, and proinflammatory cytokines or for clearance of this fungal pathogen. PMID:25422263

  20. Transcriptomic and virulence factors analyses of Cryptococcus neoformans hypoxia response.

    PubMed

    Kong, Qingtao; Yang, Rui; Wang, Zhen; Zhou, Wenquan; Du, Xue; Huang, Suyang; Jiang, Yuan; Liu, Weida; Sang, Hong

    2017-03-01

    Cryptococcus neoformans is an environmental pathogen requiring atmospheric levels of oxygen for optimal growth. Upon inhalation, C. neoformans disseminates to the brain and causes meningoencephalitis. However, the mechanisms by which the pathogen adapts to the low-oxygen environment in the brain have not been investigated. We isolated a C. neoformans strain with a small capsule from a host tissue, although this strain produces large capsules in normoxic conditions. We hypothesize that this difference in capsule size is attributed to hypoxia caused by chronic inflammatory response. This study investigated the effect of hypoxia on virulence factors (including capsule, melanin, urease, and phospholipase) of C. neoformans and conducted transcriptomic analyses of the virulence-associated genes. We found that C. neoformans grew under hypoxic condition, albeit slowly, and that hypoxia may have inhibited the capsule size, melanin production, and phospholipase and urease activities in C. neoformans.

  1. Application of glyco-blotting for identification of structures of polysaccharides causing membrane fouling in a pilot-scale membrane bioreactor treating municipal wastewater.

    PubMed

    Kimura, Katsuki; Nishimura, Shin-Ichiro; Miyoshi, Risho; Hoque, Asiful; Miyoshi, Taro; Watanabe, Yoshimasa

    2015-03-01

    A new approach for the analysis of polysaccharides in membrane bioreactor (MBR) is proposed in this study. Enrichment of polysaccharides by glyco-blotting, in which polysaccharides are specifically collected via interactions between the aldehydes in the polysaccharides and aminooxy groups on glycoblotting beads, enabled MALDI-TOF/MS analysis at a high resolution. Structures of polysaccharides extracted from fouled membranes used in a pilot-scale MBR treating municipal wastewater and those in the supernatant of the mixed liquor suspension in the MBR were investigated. It was found that the overlap between polysaccharides found in the supernatants and those extracted from the fouled membrane was rather limited, suggesting that polysaccharides that dominate in supernatants may not be important in membrane fouling in MBRs. Analysis using a bacterial carbohydrate database suggested that capsular polysaccharides (CPS) and/or lipo-polysaccharides (LPS) produced by gram-negative bacteria are key players in the evolution of membrane fouling in MBRs.

  2. [Etiopathogenesis and treatment of breast capsular contracture].

    PubMed

    Pereira Leite, Luis; Correia Sá, Inês; Marques, Marisa

    2013-01-01

    Introdução: A contractura capsular é a complicação crónica mais frequente da mamoplastia de aumento com próteses mamárias e a principal causa de insatisfação da doente e do cirurgião plástico. A cápsula mamária consiste num tecido fibroso que circunda a prótese e que pode contrair, alterando a forma e a consistência da mama. No estádio mais avançado é acompanhada de deformidade acentuada, rigidez e dor, tendo indicação para tratamento cirúrgico.Material e Métodos: Foram revistos todos os artigos indexados na PubMed através da pesquisa ‘capsular contracture’ (2000 - Janeiro 2012), dos quais foram inseridos os artigos de maior interesse em termos de etiologia, profilaxia e tratamento. Artigos referenciados em publicações relevantes foram também analisados.Resultados: Tudo indica que a sua etiologia é multifactorial; a etiopatogenia da contractura capsular mamária continua a ser alvo de múltipla investigação pré-clínica. Vários são os estudos realizados de forma a prevenir a ocorrência de contractura capsular e, embora os resultados sejam promissores, pouco está definido em termos da sua aplicação na prática clínica. Relativamente ao tratamento a capsulectomia/capsulotomia continua a ser o gold-standard, no entanto o futuro poderá passar por técnicas não invasivas, pelo menos em estádios mais leves da doença.Conclusão: Apesar das técnicas cirúrgicas e a qualidade das próteses mamárias terem vindo a melhorar drasticamente nos últimos anos, a contractura capsular mamária mantém-se uma complicação real, com incidência elevada e que continua a afectar milhares de mulheres no mundo.

  3. Virulence-Associated Enzymes of Cryptococcus neoformans

    PubMed Central

    Almeida, Fausto; Wolf, Julie M.

    2015-01-01

    Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infection process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better understanding pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, transcriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This review focuses on recent developments in the virulence-associated enzymes from Cryptococcus neoformans. The enzymatic suite of C. neoformans has evolved for environmental survival, but several of these enzymes play a dual role in colonizing the mammalian host. We also discuss new therapeutic and diagnostic strategies that could be based on the underlying enzymology. PMID:26453651

  4. Isolation of Cryptococcus neoformans var. neoformans from bird droppings, fruits and vegetables in Mexico City.

    PubMed

    López-Martínez, R; Castañón-Olivares, L R

    1995-01-01

    The presence of Cryptococcus neoformans in various natural sources, such as bird droppings, fruits and vegetables, was investigated. A total of 711 samples were analyzed; C. neoformans var. neoformans was isolated from seven out of 74 bird droppings (9.5%), with parrots as one of the most significant sources. Fruits were positive in 9.5% of the 169 samples studied, specially citrus fruits, particularly grapefruit, in which the highest frequency was found. From the 468 vegetable samples, only 20 were positive (4.2%). It is emphasized that five of the positive vegetables species are autochthonous to Mexico: avocado (Nectandra salicifolia), beet (Beta vulgaris var. quinopodiace), chayote (Sechium edule), stringbean (Cassia sp), and nopal (Opuntia ficus-indica).

  5. Fungal polysaccharides.

    PubMed

    San-Blas, G; Suzuki, S; Hearn, V; Pinel, C; Kobayashi, H; Mendez, C; Niño, G; Nishikawa, A; San-Blas, F; Shibata, N

    1994-01-01

    Fungal polysaccharides are cell wall components which may act as antigens or as structural substrates. As antigens, the role of mannans in Saccharomyces cerevisiae and Candida albicans, and of glycoproteins in Aspergillus fumigatus are discussed. Analyses on beta-glucan synthetase in Paracoccidioides brasiliensis and the inhibitory effect of Hansenula mrakii killer toxin on beta-glucan biosynthesis are also considered.

  6. Traumatic Extra-capsular and Intra-capsular Floating Fat: Fat-fluid Levels of the Knee Revisited

    PubMed Central

    Davis, Derik L; Vachhani, Prasann

    2015-01-01

    Floating fat is a sign of acute bone injury at the knee following trauma. The goal of this article is to review the etiology, patterns, and mimickers of extra-capsular and intra-capsular floating fat, with the major emphasis on knee trauma in the acute setting. We will discuss the spectrum of multimodal imaging findings for rare presentations of extra-capsular floating fat, and contrast these with common and atypical forms of intra-capsular lipohemarthrosis, as an aid to the assessment of acute bone trauma at the knee. PMID:26713176

  7. Rapid identification of Cryptococcus neoformans var. grubii, C. neoformans var. neoformans, and C. gattii by use of rapid biochemical tests, differential media, and DNA sequencing.

    PubMed

    McTaggart, Lisa; Richardson, Susan E; Seah, Christine; Hoang, Linda; Fothergill, Annette; Zhang, Sean X

    2011-07-01

    Rapid identification of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii is imperative for facilitation of prompt treatment of cryptococcosis and for understanding the epidemiology of the disease. Our purpose was to evaluate a test algorithm incorporating commercial rapid biochemical tests, differential media, and DNA sequence analysis that will allow us to differentiate these taxa rapidly and accurately. We assessed 147 type, reference, and clinical isolates, including 6 other Cryptococcus spp. (10 isolates) and 14 other yeast species (24 isolates), using a 4-hour urea broth test (Remel), a 24-hour urea broth test (Becton Dickinson), a 4-hour caffeic acid disk test (Hardy Diagnostics and Remel), 40- to 44-hour growth assessment on l-canavanine glycine bromothymol blue (CGB) agar, and intergenic spacer (IGS) sequence analysis. All 123 Cryptococcus isolates hydrolyzed urea, along with 7 isolates of Rhodotorula and Trichosporon. Eighty-five of 86 C. neoformans (99%) and 26 of 27 C. gattii (96%) isolates had positive caffeic acid results, unlike the other cryptococci (0/10) and yeast species (0/24). Together, these two tests positively identified virtually all C. neoformans/C. gattii isolates (98%) within 4 h. CGB agar or IGS sequencing further differentiated these isolates within 48 h. On CGB, 25 of 27 (93%) C. gattii strains induced a blue color change, in contrast to 0 of 86 C. neoformans isolates. Neighbor-joining cluster analysis of IGS sequences differentiated C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii. Based on these results, we describe a rapid identification algorithm for use in a microbiology laboratory to distinguish clinically relevant Cryptococcus spp.

  8. The water channel protein aquaporin 1 regulates cellular metabolism and competitive fitness in a global fungal pathogen Cryptococcus neoformans.

    PubMed

    Meyers, Gena Lee; Jung, Kwang-Woo; Bang, Soohyun; Kim, Jungyeon; Kim, Sooah; Hong, Joohyeon; Cheong, Eunji; Kim, Kyoung Heon; Bahn, Yong-Sun

    2017-03-02

    In this study, an aquaporin protein, Aqp1, in Cryptococcus neoformans, which can lead either saprobic or parasitic lifestyles and causes life-threatening fungal meningitis was identified and characterized. AQP1 expression was rapidly induced (via the HOG pathway) by osmotic or oxidative stress. In spite of such transcriptional regulation, Aqp1 was found to be largely unnecessary for adaptation to diverse environmental stressors, regardless of the presence of the polysaccharide capsule. The latter is shown here to be a key environmental-stress protectant for C. neoformans. Furthermore, Aqp1 was not required for the development and virulence of C. neoformans. Deletion of AQP1 increased hydrophobicity of the cell surface. The comparative metabolic profiling analysis of the aqp1Δ mutant and AQP1-overexpressing strains revealed that deletion of AQP1 significantly increased cellular accumulation of primary and secondary metabolites, whereas overexpression of AQP1 depleted such metabolites, suggesting that this water channel protein performs a critical function in metabolic homeostasis. In line with this result, it was found that the aqp1Δ mutant (which is enriched with diverse metabolites) survived better than the wild type and a complemented strain, indicating that Aqp1 is likely to be involved in competitive fitness of this fungal pathogen.

  9. Multiplex PCR for Identification of Two Capsular Types in Epidemic KPC-Producing Klebsiella pneumoniae Sequence Type 258 Strains

    PubMed Central

    Chen, Liang; Chavda, Kalyan D.; Findlay, Jacqueline; Peirano, Gisele; Hopkins, Katie; Pitout, Johann D. D.; Bonomo, Robert A.; Woodford, Neil; DeLeo, Frank R.

    2014-01-01

    We developed a multiplex PCR assay capable of identifying two capsular polysaccharide synthesis sequence types (sequence type 258 [ST258] cps-1 and cps-2) in epidemic Klebsiella pneumoniae ST258 strains. The assay performed with excellent sensitivity (100%) and specificity (100%) for identifying cps types in 60 ST258 K. pneumoniae sequenced isolates. The screening of 419 ST258 clonal isolates revealed a significant association between cps type and K. pneumoniae carbapenemase (KPC) variant: cps-1 is largely associated with KPC-2, while cps-2 is primarily associated with KPC-3. PMID:24733470

  10. Macrophage mitochondrial and stress response to ingestion of Cryptococcus neoformans.

    PubMed

    Coelho, Carolina; Souza, Ana Camila Oliveira; Derengowski, Lorena da Silveira; de Leon-Rodriguez, Carlos; Wang, Bo; Leon-Rivera, Rosiris; Bocca, Anamelia Lorenzetti; Gonçalves, Teresa; Casadevall, Arturo

    2015-03-01

    Human infection with Cryptococcus neoformans, a common fungal pathogen, follows deposition of yeast spores in the lung alveoli. The subsequent host-pathogen interaction can result in eradication, latency, or extrapulmonary dissemination. Successful control of C. neoformans infection is dependent on host macrophages, but macrophages display little ability to kill C. neoformans in vitro. Recently, we reported that ingestion of C. neoformans by mouse macrophages induces early cell cycle progression followed by mitotic arrest, an event that almost certainly reflects host cell damage. The goal of the present work was to understand macrophage pathways affected by C. neoformans toxicity. Infection of macrophages by C. neoformans was associated with alterations in protein translation rate and activation of several stress pathways, such as hypoxia-inducing factor-1-α, receptor-interacting protein 1, and apoptosis-inducing factor. Concomitantly we observed mitochondrial depolarization in infected macrophages, an observation that was replicated in vivo. We also observed differences in the stress pathways activated, depending on macrophage cell type, consistent with the nonspecific nature of C. neoformans virulence known to infect phylogenetically distant hosts. Our results indicate that C. neoformans infection impairs multiple host cellular functions and undermines the health of these critical phagocytic cells, which can potentially interfere with their ability to clear this fungal pathogen.

  11. Arthroscopic Capsular Release of the Ankle Joint.

    PubMed

    Lui, Tun Hing

    2016-12-01

    Adhesive capsulitis of the ankle is also known as frozen ankle and results in marked fibrosis and contracture of the ankle capsule. Arthroscopic capsular release is indicated for symptomatic frozen ankle that is resistant to conservative treatment. It is contraindicated for ankle stiffness due to degenerative joint disease, intra-articular malunion, or adhesion of the extensors of the ankle. The procedure consists of endoscopic posterior ankle capsulectomy and arthroscopic anterior ankle capsulotomy. It has the advantages of being minimally invasive surgery and allowing early postoperative vigorous mobilization of the ankle joint.

  12. Iron regulation of the major virulence factors in the AIDS-associated pathogen Cryptococcus neoformans.

    PubMed

    Jung, Won Hee; Sham, Anita; White, Rick; Kronstad, James W

    2006-11-01

    Iron overload is known to exacerbate many infectious diseases, and conversely, iron withholding is an important defense strategy for mammalian hosts. Iron is a critical cue for Cryptococcus neoformans because the fungus senses iron to regulate elaboration of the polysaccharide capsule that is the major virulence factor during infection. Excess iron exacerbates experimental cryptococcosis and the prevalence of this disease in Sub-Saharan Africa has been associated with nutritional and genetic aspects of iron loading in the background of the HIV/AIDS epidemic. We demonstrate that the iron-responsive transcription factor Cir1 in Cr. neoformans controls the regulon of genes for iron acquisition such that cir1 mutants are "blind" to changes in external iron levels. Cir1 also controls the known major virulence factors of the pathogen including the capsule, the formation of the anti-oxidant melanin in the cell wall, and the ability to grow at host body temperature. Thus, the fungus is remarkably tuned to perceive iron as part of the disease process, as confirmed by the avirulence of the cir1 mutant; this characteristic of the pathogen may provide opportunities for antifungal treatment.

  13. Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates

    PubMed Central

    1980-01-01

    A method is presented for covalently bonding Haemophilus influenzae type b capsular polysaccharide (HIB Ps) to several proteins. The method is efficient and relies upon the use of adipic dihydrazide as a spacer between the capsular polysaccharide and the carrier protein. In contrast to the poor immunogenicity of the purified HIB Ps in mice and rabbits, the HIB Ps-protein conjugates induced serum anti-type b antibodies having bactericidal activity at levels shown to be protective in humans when low doses were injected subcutaneously in a saline solution. The antibody response in mice was related to the dose of the conjugates, increased with the number of injections, and could be primed by the previous injection of the carrier protein. The HIB Ps- protein conjugates were immunogenic in three different mouse strains. The importance of the carrier molecule for the enhanced immunogenicity of the HIB Ps-protein conjugates was shown by the failure of HIB Ps hybrids prepared with either the homologous polysaccharide or pneumococcus type 3 polysaccharide to induce antibodie in mice. Rabbits injected with the HIB Ps-protein conjugates emulsified in Freund's adjuvant produced high levels of serum anti-type b antibodies which induced a bactericidal effect upon H. influenzae type b organisms. It is proposed that the HIB Ps component of the polysaccharide protein conjugates has been converted to a thymic-dependent immunogen. This method may be used to prepare protein-polysaccharide conjugates with HIB Ps and other polysaccharides to be considered for human use. PMID:6967514

  14. Polysaccharide Degradation

    NASA Astrophysics Data System (ADS)

    Stone, Bruce A.; Svensson, Birte; Collins, Michelle E.; Rastall, Robert A.

    An overview of current and potential enzymes used to degrade polysaccharides is presented. Such depolymerases are comprised of glycoside hydrolases, glycosyl transferases, phosphorylases and lyases, and their classification, active sites and action patterns are discussed. Additionally, the mechanisms that these enzymes use to cleave glycosidic linkages is reviewed as are inhibitors of depolymerase activity; reagents which react with amino acid residues, glycoside derivatives, transition state inhibitors and proteinaceous inhibitors. The characterization of various enzymes of microbial, animal or plant origin has led to their widespread use in the production of important oligosaccharides which can be incorporated into food stuffs. Sources of polysaccharides of particular interest in this chapter are those from plants and include inulin, dextran, xylan and pectin, as their hydrolysis products are purported to be functional foods in the context of gastrointestinal health. An alternative use of degraded polysaccharides is in the treatment of disease. The possibility exists to treat bacterial exopolysaccharide with lyases from bacteriophage to produce oligosaccharides exhibiting bioactive sequences. Although this area is currently in its infancy the knowledge is available to investigate further.

  15. Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans

    PubMed Central

    Liu, Oliver W.; Chun, Cheryl D.; Chow, Eric D.; Chen, Changbin; Madhani, Hiten D.; Noble, Suzanne M.

    2008-01-01

    SUMMARY The fungus Cryptococcus neoformans is a leading cause of mortality and morbidity among HIV-infected individuals. We utilized the completed genome sequence and optimized methods for homologous DNA replacement using high-velocity particle bombardment to engineer 1,201 gene knockout mutants. We screened this resource in vivo for proliferation in murine lung tissue and in vitro for three well-recognized virulence attributes — polysaccharide capsule formation, melanization, and growth at body temperature. We identified dozens of previously uncharacterized genes that affect these known attributes as well as 40 infectivity mutants without obvious defects in these traits. The latter mutants affect predicted regulatory factors, secreted proteins, and immune-related factors, and represent powerful tools for elucidating novel virulence mechanisms. In particular, we describe a GATA family transcription factor that inhibits phagocytosis by murine macrophages independently of the capsule, indicating a previously unknown mechanism of innate immune modulation. PMID:18854164

  16. Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans.

    PubMed

    Liu, Oliver W; Chun, Cheryl D; Chow, Eric D; Chen, Changbin; Madhani, Hiten D; Noble, Suzanne M

    2008-10-03

    The fungus Cryptococcus neoformans is a leading cause of mortality and morbidity among HIV-infected individuals. We utilized the completed genome sequence and optimized methods for homologous DNA replacement using high-velocity particle bombardment to engineer 1201 gene knockout mutants. We screened this resource in vivo for proliferation in murine lung tissue and in vitro for three well-recognized virulence attributes-polysaccharide capsule formation, melanization, and growth at body temperature. We identified dozens of previously uncharacterized genes that affect these known attributes as well as 40 infectivity mutants without obvious defects in these traits. The latter mutants affect predicted regulatory factors, secreted proteins, and immune-related factors, and represent powerful tools for elucidating novel virulence mechanisms. In particular, we describe a GATA family transcription factor that inhibits phagocytosis by murine macrophages independently of the capsule, indicating a previously unknown mechanism of innate immune modulation.

  17. Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall*

    PubMed Central

    Chatterjee, Subhasish; Prados-Rosales, Rafael; Itin, Boris; Casadevall, Arturo; Stark, Ruth E.

    2015-01-01

    Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment “ghosts” and applied 2D 13C-13C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics. PMID:25825492

  18. Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall.

    PubMed

    Chatterjee, Subhasish; Prados-Rosales, Rafael; Itin, Boris; Casadevall, Arturo; Stark, Ruth E

    2015-05-29

    Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment "ghosts" and applied 2D (13)C-(13)C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics.

  19. Primary Frozen Shoulder Syndrome: Arthroscopic Capsular Release

    PubMed Central

    Arce, Guillermo

    2015-01-01

    Idiopathic adhesive capsulitis, or primary frozen shoulder syndrome, is a fairly common orthopaedic problem characterized by shoulder pain and loss of motion. In most cases, conservative treatment (6-month physical therapy program and intra-articular steroid injections) improves symptoms and restores shoulder motion. In refractory cases, arthroscopic capsular release is indicated. This surgical procedure carries several advantages over other treatment modalities. First, it provides precise and controlled release of the capsule and ligaments, reducing the risk of traumatic complications observed after forceful shoulder manipulation. Second, release of the capsule and the involved structures with a radiofrequency device delays healing, which prevents adhesion formation. Third, the technique is straightforward, and an oral postoperative steroid program decreases pain and allows for a pleasant early rehabilitation program. Fourth, the procedure is performed with the patient fully awake under an interscalene block, which boosts the patient's confidence and adherence to the physical therapy protocol. In patients with refractory primary frozen shoulder syndrome, arthroscopic capsular release emerges as a suitable option that leads to a faster and long-lasting recovery. PMID:26870652

  20. EPS-I Polysaccharide Protects Mycoplasma pulmonis from Phagocytosis

    PubMed Central

    Shaw, Brandon M.; Daubenspeck, James M.; Simmons, Warren L.; Dybvig, Kevin

    2012-01-01

    Few mycoplasmal polysaccharides have been described and little is known about their role in pathogenesis. The infection of mice with Mycoplasma pulmonis has been utilized in many in vivo and in vitro studies to gain a better understanding of host-pathogen interactions during chronic respiratory infection. Although alveolar macrophages have a primary role in host defense, M. pulmonis is killed inefficiently in vitro. One antiphagocytic factor produced by the mycoplasma is the family of phase- and size-variable Vsa lipoproteins. However, bacteria generally employ multiple strategies for combating host defenses, with capsular polysaccharide often having a key role. We show here that mutants lacking the EPS-I polysaccharide of M. pulmonis exhibit increased susceptibility to binding and subsequent killing by alveolar macrophages. These results give further insight into how mycoplasmas are able to avoid the host immune system and sustain a chronic infection. PMID:23190331

  1. Rapid presumptive identification of Cryptococcus neoformans.

    PubMed Central

    Muchmore, H G; Felton, F G; Scott, E N

    1978-01-01

    Carbohydrate-containing extracts were prepared from mature yeast colonies grown on Sabouraud dextrose agar by mixing a 0.001-ml loopful of yeast cells for 30 s in phenolized saline and removing the cells by centrifugation. Extracts were prepared from 54 Cryptococcus neoformans isolates, 29 isolates of other Cryptococcus species, 16 isolates of Candida species, 2 Rhodotorula, 2 Torulopsis, and 1 Saccharomyces species. Initially the carbohydrate content of each extract was estimated (Molisch method) and adjusted to 1, 5, and 10 microgram/ml. Twofold dilutions of each extract were tested for reactivity with the cryptococcal latex agglutination reagent of Bloomfield et al. (N. Bloomfield, M.A. Gordon, and D.F. Elmendorf, Jr., Proc. Soc. Exp. Biol. Med. 114:64-67, 1963). All 54 C. neoformans extracts gave strong agglutinations (3+ to 4+) in dilutions of 1:4 or greater. None of the other yeasts produced any agglutination, except for 1 of 15 C. laurentii isolates, which showed a 1+ reaction that disappeared at a dilution of 1:4 and above. Subsequent testing established that a single extract made from 0.001 ml of yeast cells in 6 ml of phenolized saline contained less than 5 microgram of carbohydrate per ml, was suitable for a single rapid screening dilution, and eliminated any cross-reaction from the C. laurentii isolates. In our hands this method has provided a reliable differentiation of C. neoformans from other unknown yeast colonies in less than 20 min exclusive of a Molisch determination. PMID:359587

  2. Cryptococcus neoformans isolation from swallow (Hirundo rustica) excreta in Iran.

    PubMed

    Hedayati, Mohammad T; Mayahi, Sabah; Fakhar, Mahdi; Shokohi, Tahereh; Majidi, Mohammad

    2011-01-01

    Cryptococcus neoformans is an encapsulated yeast that can cause cryptococcosis, a life-threatening infection that mainly occurs in immunocompromised patients. The major environmental sources of C. neoformans have been shown to be soil contaminated with avian droppings. In the present study, we evaluated the isolation of C. neoformans from swallow (Hirundo rustica) excreta in two northern cities of Iran. Ninety-seven swallow droppings were evaluated and 498 yeast-like colonies were isolated and identified as Rhodotorula spp. (62.8%), Candida spp. (28.5%)and C. neoformans (8.7%). Cryptococcus neoformans was isolated from 5/97 (5.2%) of collected samples. Min-Max colony forming units (CFU) per one gram for the positive samples were 3-10 C. neoformans colonies. The total mean CFU per one gram for the positive samples was 4.8. The results of this study demonstrate that excreta of swallow may harbor different species of potentially pathogenic yeasts, mainly C. neoformans, and may be capable of disseminating these fungi in the environment.

  3. Enzymatic characterisation of clinical isolates of Cryptococcus neoformans, Cryptococcus gattii and other environmental Cryptococcus spp.

    PubMed

    Chan, M Y; Tay, S T

    2010-01-01

    This study compared the enzymatic activity of clinical isolates of Cryptococcus neoformans, Cryptococcus gattii, environmental isolates of C. neoformans and non-neoformans Cryptococcus. Most of the cryptococcal isolates investigated in this study exhibited proteinase and phospholipase activities. Laccase activity was detected from all the C. neoformans and C. gattii isolates, but not from the non-neoformans Cryptococcus isolates. There was no significant difference in the proteinase, phospholipase and laccase activities of C. neoformans and C. gattii. However, significant difference in the enzymatic activities of beta-glucuronidase, alpha-glucosidase, beta-glucosidase and N-acetyl-beta-glucosaminidase between C. neoformans and C. gattii isolates was observed in this study. Environmental isolates of C. neoformans exhibited similar enzymatic profiles as the clinical isolates of C. neoformans, except for lower proteinase and laccase activities.

  4. Is the Capsular Bag Perimeter Round or Elliptical?

    PubMed Central

    Amigó, Alfredo; Bonaque-González, Sergio

    2016-01-01

    Purpose: To report findings that could suggest an elliptical shape of the capsular bag. Methods: Five eyes of three patients with axial length greater than 24 mm underwent phacoemulsification cataract surgery with plate-haptic multifocal toric intraocular lens (IOL) implantation oriented in the vertical meridian. Results: In all cases, correct orientation of the IOLs was verified 30 minutes after surgery. After 24 hours, all eyes demonstrated unwanted rotation of the IOLs ranging from 15 to 45 degrees. The IOLs remained stable in the new position in all cases until adhesion of the capsular bag took place. Conclusion: These observations could suggest that the perimeter of the capsular bag has an elliptical shape. Therefore, the IOL tends to become fixated in a meridian of the capsular bag that best fits the diagonal diameter of the IOL. PMID:27413495

  5. Capsular Plication for Treatment of Iatrogenic Hip Instability

    PubMed Central

    Levy, David M.; Grzybowski, Jeffrey; Salata, Michael J.; Mather, Richard C.; Aoki, Stephen K.; Nho, Shane J.

    2015-01-01

    The most commonly reported reasons for persistent hip pain after hip arthroscopy are residual femoroacetabular impingement, dysplasia and dysplasia variants, or extra-articular impingement. There are some cases in which the underlying osseous pathomorphology has been appropriately treated, and the cause of persistent hip pain can be soft-tissue injuries such as chondrolabral tears or capsular abnormalities. Capsular defects after hip arthroscopy may suggest an alteration of the biomechanical properties of the iliofemoral ligament and lead to iatrogenically induced hip instability. There are a growing number of biomechanical and clinical studies showing the importance of capsular management during hip arthroscopy. We describe the workup, examination under anesthesia, diagnostic arthroscopy, and technique of capsular plication for iatrogenic instability of the hip. PMID:26870636

  6. Cryptococcus neoformans shows a remarkable genotypic diversity in Brazil.

    PubMed

    Barreto de Oliveira, M T; Boekhout, T; Theelen, B; Hagen, F; Baroni, F A; Lazera, M S; Lengeler, K B; Heitman, J; Rivera, I N G; Paula, C R

    2004-03-01

    The genotypic diversity of Brazilian Cryptococcus neoformans strains was analyzed. The majority of the samples were alphaA (65%), followed by alphaB (17.5%), alphaD (9%), alphaAaD hybrids (5%), and alphaC (3.5%). A considerable genotypic diversity occurred within C. neoformans var. grubii, and a new amplified fragment length polymorphism genotype, 1B, was recognized.

  7. In vitro interactions of immune lymphocytes and Cryptococcus neoformans.

    PubMed Central

    Fung, P Y; Murphy, J W

    1982-01-01

    CBA/J mice immunized subcutaneously with emulsions of heat-killed Cryptococcus neoformans in complete Freund adjuvant displayed delayed-type hypersensitivity to cryptococcal culture filtrate antigen and developed sensitized splenic lymphoid cells which inhibited the growth of C. neoformans in vitro. The in vitro assay of growth inhibition served to investigate further the kinetics of the effect of sensitized lymphoid cells on the pathogen. There was a close correlation between the delayed-type hypersensitivity response in mice and inhibition of growth of C. neoformans by lymphoid cells. Sensitized splenic lymphocytes capable of inhibiting the growth of the cryptococci were detected at day 6 after immunization and reached maximum levels by days 8 through 16. Inhibition of growth was highest with effector-to-target cell ratios of 300:1 or greater. Inhibition of growth of C. neoformans by sensitized lymphoid cells was detectable as early as 4 h after effector and target cells were mixed and increased gradually, reaching a maximum at 24 h, but dropped significantly by 48 h. By supplementing the reaction mixtures with fresh medium or additional sensitized effector cells during incubation, the inhibition of growth of C. neoformans could be maintained through 48 h. C. neoformans-sensitized effector lymphoid populations not only inhibited the growth of the pathogen in vitro but also restricted C. neoformans proliferation in various vital organs upon transfer to naive recipient animals, indicating that the in vitro growth inhibition assay may be a means of assessing the resistance of animals to C. neoformans. The effector cells from sensitized animals were nylon wool-nonadherent Thy-1+ and Ia+ lymphocytes. PMID:7047393

  8. Targeted Genome Editing via CRISPR in the Pathogen Cryptococcus neoformans.

    PubMed

    Arras, Samantha D M; Chua, Sheena M H; Wizrah, Maha S I; Faint, Joshua A; Yap, Amy S; Fraser, James A

    2016-01-01

    Low rates of homologous integration have hindered molecular genetic studies in Cryptococcus neoformans over the past 20 years, and new tools that facilitate genome manipulation in this important pathogen are greatly needed. To this end, we have investigated the use of a Class 2 CRISPR system in C. neoformans (formerly C. neoformans var. grubii). We first expressed a derivative of the Streptococcus pyogenes Cas9 nuclease in C. neoformans, and showed that it has no effect on growth, production of virulence factors in vitro, or virulence in a murine inhalation model. In proof of principle experiments, we tested the CAS9 construct in combination with multiple self-cleaving guide RNAs targeting the well-characterized phosphoribosylaminoamidazole carboxylase-encoding ADE2 gene. Utilizing combinations of transient and stable expression of our constructs, we revealed that functionality of our CRISPR constructs in C. neoformans is dependent upon the CAS9 construct being stably integrated into the genome, whilst transient expression of the guide RNA is sufficient to enhance rates of homologous recombination in the CAS9 genetic background. Given that the presence of the CRISPR nuclease does not influence virulence in a murine inhalation model, we have successfully demonstrated that this system is compatible with studies of C. neoformans pathogenesis and represents a powerful tool that can be exploited by researchers in the field.

  9. Acapsular Cryptococcus neoformans activates the NLRP3 inflammasome.

    PubMed

    Guo, Caiqin; Chen, Mingkuan; Fa, Zhenzong; Lu, Ailing; Fang, Wei; Sun, Bing; Chen, Changbin; Liao, Wanqing; Meng, Guangxun

    2014-10-01

    Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen that mainly infects immunocompromised individuals such as AIDS patients. Although cell surface receptors for recognition of C. neoformans have been studies intensively, cytoplasmic recognition of this pathogen remains unclear. As an important detector of pathogen infection, inflammasome can sense and get activated by infection of various pathogens, including pathogenic fungi such as Candida albicans and Aspergillus fumigatus. Our present study showed that acapsular C. neoformans (cap59Δ) activated the NLRP3-, but not AIM2-nor NLRC4- inflammasome. During this process, viability of the fungus was required. Moreover, our in vivo results showed that during the pulmonary infection of cap59Δ, immune cell infiltration into the lung and effective clearance of the fungus were both dependent on the presence of NLRP3 inflammasome. In summary, our data suggest that the capsule of C. neoformans prevents recognition of the fungus by host NLRP3 inflammasome and indicate that manipulation of inflammasome activity maybe a novel approach to control C. neoformans infection.

  10. Targeted Genome Editing via CRISPR in the Pathogen Cryptococcus neoformans

    PubMed Central

    Arras, Samantha D. M.; Chua, Sheena M. H.; Wizrah, Maha S. I.; Faint, Joshua A.; Yap, Amy S.; Fraser, James A.

    2016-01-01

    Low rates of homologous integration have hindered molecular genetic studies in Cryptococcus neoformans over the past 20 years, and new tools that facilitate genome manipulation in this important pathogen are greatly needed. To this end, we have investigated the use of a Class 2 CRISPR system in C. neoformans (formerly C. neoformans var. grubii). We first expressed a derivative of the Streptococcus pyogenes Cas9 nuclease in C. neoformans, and showed that it has no effect on growth, production of virulence factors in vitro, or virulence in a murine inhalation model. In proof of principle experiments, we tested the CAS9 construct in combination with multiple self-cleaving guide RNAs targeting the well-characterized phosphoribosylaminoamidazole carboxylase-encoding ADE2 gene. Utilizing combinations of transient and stable expression of our constructs, we revealed that functionality of our CRISPR constructs in C. neoformans is dependent upon the CAS9 construct being stably integrated into the genome, whilst transient expression of the guide RNA is sufficient to enhance rates of homologous recombination in the CAS9 genetic background. Given that the presence of the CRISPR nuclease does not influence virulence in a murine inhalation model, we have successfully demonstrated that this system is compatible with studies of C. neoformans pathogenesis and represents a powerful tool that can be exploited by researchers in the field. PMID:27711143

  11. High-resolution melting analysis for identification of the Cryptococcus neoformans-Cryptococcus gattii complex.

    PubMed

    Gago, Sara; Zaragoza, Óscar; Cuesta, Isabel; Rodríguez-Tudela, Juan L; Cuenca-Estrella, Manuel; Buitrago, María J

    2011-10-01

    We have developed a two-step method based on high-resolution melting (HRM) that reliably identifies species from the Cryptococcus species complex (Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii). Our results indicate that HRM can provide a fast protocol to identify and distinguish among the main Cryptococcus species.

  12. [Characterization of Cryptococcus neoformans strains isolated from patients with acquired immunodeficiency syndrome (AIDS)].

    PubMed

    Garza-Garza, D; Buendía-Uribe, J L; Martínez-Cruz, E; Argüero-Licea, B

    1995-01-01

    In Mexico cryptococosis ranks third in frequency among the mycoses ocurring as complications in AIDS patients. Neither the prevalence of the two varieties of C. neoformans in these patients nor the morphological and physiological changes suffered by these strains in AIDS patients are known. A total of 60 isolates were obtained from patients with AIDS from the Hospital de Infectología, Centro Médico "La Raza" IMSS. The identity of each isolate was established by: growth at 37 degrees C, colony and microscopic characteristics, urease and phenoloxidase activity, carbon sources assimilation. The canavanine glycine-bromothymol blue agar was used to distinguish C. neoformans var. neoformans and C. neoformans var. gattii. Pathogenicity in mice was also tested. Fifty one isolates of C. neoformans var. neoformans and nine of C. neoformans var. gattii were identified. All strains grew well at 37 degrees C, urease and phenoloxidase were positive, the morphology and the auxanographic profile were variable. C. neoformans var. neoformans was more virulent in mouse than C. neoformans var. gattii. This study has confirmed the presence of the two varieties of C. neoformans in Mexico with 85% prevalence of var. neoformans and 15% of var. gattii in AIDS patients. This frequency was higher than in reports from other countries.

  13. The formation of titan cells in Cryptococcus neoformans depends on the mouse strain and correlates with induction of Th2-type responses.

    PubMed

    García-Barbazán, Irene; Trevijano-Contador, Nuria; Rueda, Cristina; de Andrés, Belén; Pérez-Tavárez, Raquel; Herrero-Fernández, Inés; Gaspar, María Luisa; Zaragoza, Oscar

    2016-01-01

    Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN-γ, TNF-α and IL17, while C57BL/BL mice had an increase in the anti-inflammatory cytokine IL-4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2-type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment.

  14. ALL2, a Homologue of ALL1, Has a Distinct Role in Regulating pH Homeostasis in the Pathogen Cryptococcus neoformans

    PubMed Central

    Jain, Neena; Bouklas, Tejas; Gupta, Anjali; Varshney, Avanish K.; Orner, Erika P.

    2015-01-01

    Cryptococcus neoformans is a facultative intracellular fungal pathogen that has a polysaccharide capsule and causes life-threatening meningoencephalitis. Its capsule, as well as its ability to survive in the acidic environment of the phagolysosome, contributes to the pathogen's resilience in the host environment. Previously, we reported that downregulation of allergen 1 (ALL1) results in the secretion of a shorter, more viscous exopolysaccharide with less branching and structural complexity, as well as altered iron homeostasis. Now, we report on a homologous coregulated gene, allergen 2 (ALL2). ALL2's function was characterized by generating null mutants in C. neoformans. In contrast to ALL1, loss of ALL2 attenuated virulence in the pulmonary infection model. The all2Δ mutant shed a less viscous exopolysaccharide and exhibited higher sensitivity to hydrogen peroxide than the wild type, and as a result, the all2Δ mutant was more resistant to macrophage-mediated killing. Transcriptome analysis further supported the distinct function of these two genes. Unlike ALL1's involvement in iron homeostasis, we now present data on ALL2's unique function in maintaining intracellular pH in low-pH conditions. Thus, our data highlight that C. neoformans, a human-pathogenic basidiomycete, has evolved a unique set of virulence-associated genes that contributes to its resilience in the human niche. PMID:26597983

  15. Capsular Weakness around Breast Implant: A Non-Recognized Complication

    PubMed Central

    Arquero, Pedro Salinero; Zanata, Fabiana Cristina; Ferreira, Lydia Masako; Nahas, Fabio Xerfan

    2015-01-01

    Capsular contraction is a frequent complication following breast augmentation. On the other hand, capsular weakness, a not widely recognized complication, may occur around the implant. A weak capsule allows the migration of the prosthesis to the lateral region of the thoracic region or inferiorly, towards the abdomen, due to gravitational forces. The cause of capsular weakness remains unresolved. Implant malposition, with lateral or downward displacement, breast asymmetry, improper contour, with implants moving in the pocket that compromise the aesthetic outcome of breast augmentation and require surgical correction may be different symptoms from the same clinical problem. Capsular weakness is a short or mid-term complication of breast augmentation. Most techniques aim to correct the malposition by making sutures to increase the resistance to the displacement of the implant, rearrange the structures using the capsule as flaps to remodel the envelope of the new pocket, obtaining a more stable and reliable result. In this article, four cases of displacement of breast prosthesis with capsular weakness are described and the surgical treatment that included a capsulotomy and capsulorraphy is described. PMID:26284187

  16. Enzymatic Modifications of Polysaccharides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polysaccharides are often modified chemically in order to improve its properties or to impart specific characteristics. Indeed quite a few commercial products are based on modified polysaccharides. In this talk, I shall describe a new set of modified polysaccharides based on enzymatic reactions. ...

  17. Intron retention-dependent gene regulation in Cryptococcus neoformans

    PubMed Central

    Gonzalez-Hilarion, Sara; Paulet, Damien; Lee, Kyung-Tae; Hon, Chung-Chau; Lechat, Pierre; Mogensen, Estelle; Moyrand, Frédérique; Proux, Caroline; Barboux, Rony; Bussotti, Giovanni; Hwang, Jungwook; Coppée, Jean-Yves; Bahn, Yong-Sun; Janbon, Guilhem

    2016-01-01

    The biological impact of alternative splicing is poorly understood in fungi, although recent studies have shown that these microorganisms are usually intron-rich. In this study, we re-annotated the genome of C. neoformans var. neoformans using RNA-Seq data. Comparison with C. neoformans var. grubii revealed that more than 99% of ORF-introns are in the same exact position in the two varieties whereas UTR-introns are much less evolutionary conserved. We also confirmed that alternative splicing is very common in C. neoformans, affecting nearly all expressed genes. We also observed specific regulation of alternative splicing by environmental cues in this yeast. However, alternative splicing does not appear to be an efficient method to diversify the C. neoformans proteome. Instead, our data suggest the existence of an intron retention-dependent mechanism of gene expression regulation that is not dependent on NMD. This regulatory process represents an additional layer of gene expression regulation in fungi and provides a mechanism to tune gene expression levels in response to any environmental modification. PMID:27577684

  18. DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans

    PubMed Central

    Heung, Lena J.

    2016-01-01

    Cryptococcus neoformans is an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in immunocompromised patients. Currently, the molecular mechanisms that regulate the mammalian immune response to respiratory cryptococcal challenge remain poorly defined. DAP12, a signaling adapter for multiple pattern recognition receptors in myeloid and natural killer (NK) cells, has been shown to play both activating and inhibitory roles during lung infections by different bacteria and fungi. In this study, we demonstrate that DAP12 plays an important inhibitory role in the immune response to C. neoformans. Infectious outcomes in DAP12−/− mice, including survival and lung fungal burden, are significantly improved compared to those in C57BL/6 wild-type (WT) mice. We find that eosinophils and macrophages are decreased while NK cells are increased in the lungs of infected DAP12−/− mice. In contrast to WT NK cells, DAP12−/− NK cells are able to repress C. neoformans growth in vitro. Additionally, DAP12−/− macrophages are more highly activated than WT macrophages, with increased production of tumor necrosis factor (TNF) and CCL5/RANTES and more efficient uptake and killing of C. neoformans. These findings suggest that DAP12 acts as a brake on the pulmonary immune response to C. neoformans by promoting pulmonary eosinophilia and by inhibiting the activation and antifungal activities of effector cells, including NK cells and macrophages. PMID:27068093

  19. Isolation and characterization of the Cryptococcus neoformans MATa pheromone gene.

    PubMed Central

    McClelland, Carol M; Fu, Jianmin; Woodlee, Gay L; Seymour, Tara S; Wickes, Brian L

    2002-01-01

    Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATalpha. The mating pathway of this fungus has a number of conserved genes, including a MATalpha-specific pheromone (MFalpha1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATalpha cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfalpha1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFalpha1 gene, which is found in MATalpha strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans. PMID:11901112

  20. Using solid-state NMR to monitor the molecular consequences of Cryptococcus neoformans melanization with different catecholamine precursors.

    PubMed

    Chatterjee, Subhasish; Prados-Rosales, Rafael; Frases, Susana; Itin, Boris; Casadevall, Arturo; Stark, Ruth E

    2012-08-07

    Melanins are a class of natural pigments associated with a wide range of biological functions, including microbial virulence, energy transduction, and protection against solar radiation. Because of their insolubility and structural heterogeneity, solid-state nuclear magnetic resonance (NMR) spectroscopy provides an unprecedented means to define the molecular architecture of these enigmatic pigments. The requirement of obligatory catecholamines for melanization of the pathogenic fungus Cryptococcus neoformans also offers unique opportunities for investigating melanin development. In the current study, pigments produced with L-dopa, methyl-L-dopa, epinephrine, and norepinephrine precursors are compared structurally using (13)C and (1)H magic-angle spinning (MAS) NMR. Striking structural differences were observed for both aromatic and aliphatic molecular constituents of the mature fungal pigment assemblies, thus making it possible to redefine the molecular prerequisites for formation of the aromatic domains of insoluble indole-based biopolymers, to rationalize their distinctive physical characteristics, and to delineate the role of cellular constituents in assembly of the melanized macromolecules with polysaccharides and fatty acyl chain-containing moieties. By achieving an augmented understanding of the mechanisms of C. neoformans melanin biosynthesis and cellular assembly, such studies can guide future drug discovery efforts related to melanin-associated virulence, resistance to tumor therapy, and production of melanin mimetics under cell-free conditions.

  1. Quality Control of Fungus-specific Glucosylceramide in Cryptococcus neoformans by Endoglycoceramidase-related Protein 1 (EGCrP1)*

    PubMed Central

    Ishibashi, Yohei; Ikeda, Kazutaka; Sakaguchi, Keishi; Okino, Nozomu; Taguchi, Ryo; Ito, Makoto

    2012-01-01

    A fungus-specific glucosylceramide (GlcCer), which contains a unique sphingoid base possessing two double bonds and a methyl substitution, is essential for pathogenicity in fungi. Although the biosynthetic pathway of the GlcCer has been well elucidated, little is known about GlcCer catabolism because a GlcCer-degrading enzyme (glucocerebrosidase) has yet to be identified in fungi. We found a homologue of endoglycoceramidase tentatively designated endoglycoceramidase-related protein 1 (EGCrP1) in several fungal genomic databases. The recombinant EGCrP1 hydrolyzed GlcCer but not other glycosphingolipids, whereas endoglycoceramidase hydrolyzed oligosaccharide-linked glycosphingolipids but not GlcCer. Disruption of egcrp1 in Cryptococcus neoformans, a typical pathogenic fungus causing cryptococcosis, resulted in the accumulation of fungus-specific GlcCer and immature GlcCer that possess sphingoid bases without a methyl substitution concomitant with a dysfunction of polysaccharide capsule formation. These results indicated that EGCrP1 participates in the catabolism of GlcCer and especially functions to eliminate immature GlcCer in vivo that are generated as by-products due to the broad specificity of GlcCer synthase. We conclude that EGCrP1, a glucocerebrosidase identified for the first time in fungi, controls the quality of GlcCer by eliminating immature GlcCer incorrectly generated in C. neoformans, leading to accurate processing of fungus-specific GlcCer. PMID:22072709

  2. Serotyping of 467 Cryptococcus neoformans Isolates from Clinical and Environmental Sources in Brazil: Analysis of Host and Regional Patterns

    PubMed Central

    Nishikawa, Marília M.; Lazera, Márcia S.; Barbosa, Glaucia G.; Trilles, Luciana; Balassiano, Beatriz R.; Macedo, Regina C. L.; Bezerra, Cláudia C. F.; Pérez, Maurício A.; Cardarelli, Paola; Wanke, Bodo

    2003-01-01

    Cryptococcus neoformans is an important zoopathogen, and it is one of the most prevalent lethal mycotic agents. Its polysaccharide capsule, synthesized in vivo and in vitro, is a virulence factor, contains predominantly glucuronoxylomannan, and is responsible for the antigenic differentiation of serotypes A, B, C, D, and AD. A total of 467 isolates of C. neoformans obtained from clinical and environmental sources from Brazilian regions were studied serologically by using the Crypto Check Iatron RM 304-K kit. Serotyping of the clinical isolates showed the following prevalences of the serotypes: A (77.95%), followed by B (18.2%), AD (1.3%), D (0.4%), C (0.2%), and untypeable (1.93%). The epidemiology of serotype A in the Brazilian southern and southeastern regions reproduces the picture observed worldwide. In contrast, serotype B was the most frequent agent of cryptococcosis in the northeastern region, occurring nearly equally in male and female healthy hosts. Among the isolates from environmental sources, serotypes A and B were found to occur in the hollows of tropical trees of the genera Cassia, Ficus, and Moquillea. The few isolates from Eucalyptus camaldulensis debris were serotypes A and B and untypeable. Overall, no association with a specific host tree was identified for these serotypes, denoting a distinct ecoepidemiological regional pattern. The one serotype C isolate was recovered from a human immunodeficiency virus-negative host. Serotype AD predominated over serotype D among both clinical and environmental isolates. PMID:12517828

  3. Threonine biosynthetic genes are essential in Cryptococcus neoformans

    PubMed Central

    Kingsbury, Joanne M.; McCusker, John H.

    2009-01-01

    Summary We identified and attempted to disrupt the Cryptococcus neoformans homoserine and/or threonine biosynthetic genes encoding aspartate kinase (HOM3), homoserine kinase (THR1), and threonine synthase (THR4), however, each gene proved recalcitrant to disruption. By replacing the endogenous promoters of HOM3 and THR1 with the copper-repressible CTR4-1 promoter, we showed that HOM3 and THR1 were essential for the growth of C. neoformans in rich media, when ammonium was the nitrogen source, or when threonine was supplied as an amino acid instead of a dipeptide. Moreover, the severity of the growth defect associated with HOM3- or THR1-repression increased with increasing incubation temperature. This study comprises the first demonstration of threonine biosynthetic genes being essential in a fungus. The necessity of these genes for C. neoformans growth, particularly at physiologically relevant temperatures, makes threonine biosynthetic genes ideal anti-cryptococcal drug targets. PMID:18757810

  4. Type-specific capsular antigen is associated with virulence in late-onset group B Streptococcal type III disease.

    PubMed Central

    Klegerman, M E; Boyer, K M; Papierniak, C K; Levine, L; Gotoff, S P

    1984-01-01

    Strain differences have been postulated to explain the observation that group B Streptococcus type III (GBS III) late-onset disease occurs in only a fraction of colonized infants. To determine the distribution of type-specific polysaccharide antigen (Ag) in GBS III, Ag was measured by rocket immunoelectrophoresis in both supernatant fluids and EDTA extracts and by radial immunodiffusion in multiple HCl extracts of the pellet from cultures of 10 strains of GBS III. Capsular Ag was defined as the sum of Ag in EDTA extracts + Ag in multiple HCl extracts. Both Ag in EDTA extracts and Ag in supernatant fluids correlated with capsular Ag (r = 0.94). GBS III strains were obtained from the blood of 19 infants with late-onset sepsis, from the cerebrospinal fluid or blood of 22 infants with late-onset meningitis, and from mucosal surfaces of both 18 infants and 12 mothers of infants with low levels of type-specific antibody and asymptomatic colonization. Mean values of Ag in supernatant fluids in strains from infants with late-onset sepsis (1.50 +/- 0.08 micrograms/ml) and late-onset meningitis (1.67 +/- 0.09 micrograms/ml) were significantly greater than those in asymptomatic colonization strains (1.14 +/- 0.05 micrograms/ml; P less than 0.001). The number of organisms required for a 50% lethal dose in the chick embryo, determined in 29 strains, was inversely related to Ag in supernatant fluids (r = -0.60). The demonstration that the quantity of capsular Ag produced by GBS III strains is related to their virulence in chick embryos and to their invasiveness in susceptible infants supports the hypothesis that Ag is a virulence factor in humans. Images PMID:6423540

  5. Experimental murine cryptococcal infection results in contamination of bedding with Cryptococcus neoformans.

    PubMed

    Nosanchuk, Joshua D; Mednick, Aron; Shi, Li; Casadevall, Arturo

    2003-07-01

    Cryptococcus neoformans is a fungal pathogen that survives in diverse environments. To determine whether cages of mice infected with C. neoformans posed an infection risk to animal caregivers, we investigated whether the fungus could be isolated from the bedding or stool of mice infected by intratracheal (i.t.), intravenous (i.v.), or intraperitoneal (i.p.) routes. The bedding of mice infected i.t. was contaminated with C. neoformans. In contrast, no contamination of bedding with C. neoformans was detected in cages of mice infected i.v. or i.p. C. neoformans was not isolated from murine feces. The C. neoformans strain recovered from bedding material was indistinguishable from the infecting strain by biochemical and molecular techniques. This result suggests that precautions may be warranted when disposing bedding from cages that housed mice with pulmonary C. neoformans infection.

  6. Ferric iron reduction by Cryptococcus neoformans.

    PubMed Central

    Nyhus, K J; Wilborn, A T; Jacobson, E S

    1997-01-01

    The pathogenic yeast Cryptococcus neoformans must reduce Fe(III) to Fe(II) prior to uptake. We investigated mechanisms of reduction using the chromogenic ferrous chelator bathophenanthroline disulfonate. Iron-depleted cells reduced 57 nmol of Fe(III) per 10(6) cells per h, while iron-replete cells reduced only 8 nmol of Fe(III). Exponential-phase cells reduced the most and stationary-phase cells reduced the least Fe(III), independent of iron status. Supernatants from iron-depleted cells reduced up to 2 nmol of Fe(III) per 10(6) cells per h, while supernatants from iron-replete cells reduced 0.5 nmol of Fe(III), implying regulation of the secreted reductant(s). One such reductant is 3-hydroxyanthranilic acid (3HAA), which was found at concentrations up to 29 microM in iron-depleted cultures but <2 microM in cultures supplemented with iron. Moreover, when washed and resuspended in low iron medium, iron-depleted cells secreted 20.4 microM 3HAA, while iron-replete cells secreted only 4.5 microM 3HAA. Each mole of 3HAA reduced 3 mol of Fe(III), and increasing 3HAA concentrations correlated with increasing reducing activity of supernatants; however, 3HAA accounted for only half of the supernatant's reducing activity, indicating the presence of additional reductants. Finally, we found that melanized stationary-phase cells reduced 2 nmol of Fe(III) per 10(6) cells per h--16 times the rate of nonmelanized cells--suggesting that this redox polymer participates in reduction of Fe(III). PMID:9009293

  7. Ferrous iron uptake in Cryptococcus neoformans.

    PubMed

    Jacobson, E S; Goodner, A P; Nyhus, K J

    1998-09-01

    Previous studies have implicated ferric reduction in the iron uptake pathway of the opportunistic pathogen Cryptococcus neoformans. Here we studied iron uptake directly, using 55Fe in the presence of reductants. Uptake was linear with respect to time and number of yeast cells. The plot of uptake versus concentration exhibited a steep rise up to about 1 microM, a plateau between 1 and 25 microM, and a second steep rise above 25 microM, consistent with high- and low-affinity uptake systems. A Km for high-affinity uptake was estimated to be 0.6 microM Fe(II); 1 microM was used for standardized uptake assays. At this concentration, the uptake rate was 110 +/- 3 pmol/10(6) cells/h. Iron repletion (15 microM) and copper starvation drastically decreased high-affinity iron uptake. Incubation at 0 degreesC or in the presence of 2 mM KCN abolished high-affinity iron uptake, suggesting that uptake requires metabolic energy. When exogenous reducing agents were not supplied and the culture was washed free of secreted reductants, uptake was reduced by 46%; the remaining uptake activity presumably was dependent upon the cell membrane ferric reductase. Further decreases in free Fe(II) levels achieved by trapping with bathophenanthroline disulfonate or reoxidizing with potassium nitrosodisulfonate reduced iron uptake very drastically, suggesting that it is the Fe(II) species which is transported by the high-affinity transporter. The uptake of Fe was stimulated two- to threefold by deferoxamine, but this increment could be abolished by copper starvation or inhibition of the ferric reductase by Pt, indicating that Fe solubilized by this molecule also entered the reductive iron uptake pathway.

  8. Chemical Modification of Polysaccharides

    PubMed Central

    Cumpstey, Ian

    2013-01-01

    This review covers methods for modifying the structures of polysaccharides. The introduction of hydrophobic, acidic, basic, or other functionality into polysaccharide structures can alter the properties of materials based on these substances. The development of chemical methods to achieve this aim is an ongoing area of research that is expected to become more important as the emphasis on using renewable starting materials and sustainable processes increases in the future. The methods covered in this review include ester and ether formation using saccharide oxygen nucleophiles, including enzymatic reactions and aspects of regioselectivity; the introduction of heteroatomic nucleophiles into polysaccharide chains; the oxidation of polysaccharides, including oxidative glycol cleavage, chemical oxidation of primary alcohols to carboxylic acids, and enzymatic oxidation of primary alcohols to aldehydes; reactions of uronic-acid-based polysaccharides; nucleophilic reactions of the amines of chitosan; and the formation of unsaturated polysaccharide derivatives. PMID:24151557

  9. Characterization of the Kingella kingae Polysaccharide Capsule and Exopolysaccharide

    PubMed Central

    Starr, Kimberly F.; Porsch, Eric A.; Heiss, Christian; Black, Ian; Azadi, Parastoo; St. Geme, Joseph W.

    2013-01-01

    Recent evidence indicates that Kingella kingae produces a polysaccharide capsule. In an effort to determine the composition and structure of this polysaccharide capsule, in the current study we purified capsular material from the surface of K. kingae strain 269–492 variant KK01 using acidic conditions to release the capsule and a series of steps to remove DNA, RNA, and protein. Analysis of the resulting material by gas chromatography and mass spectrometry revealed N-acetyl galactosamine (GalNAc), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), and galactose (Gal). Further analysis by NMR demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→. Disruption of the ctrA gene required for surface localization of the K. kingae polysaccharide capsule resulted in elimination of GalNAc and Kdo but had no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. Disruption of ctrA and deletion of pamABCDE resulted in a loss of all carbohydrates in surface extracts. These results establish that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→. The polysaccharide capsule and the exopolysaccharide require distinct genetic loci for surface localization. PMID:24098695

  10. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cryptococcus neoformans serological reagents. 866.3165 Section 866.3165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN.... Cryptococcosis infections are found most often as chronic meningitis (inflammation of brain membranes) and,...

  11. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Cryptococcus neoformans serological reagents. 866.3165 Section 866.3165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN.... Cryptococcosis infections are found most often as chronic meningitis (inflammation of brain membranes) and,...

  12. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Cryptococcus neoformans serological reagents. 866.3165 Section 866.3165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN.... Cryptococcosis infections are found most often as chronic meningitis (inflammation of brain membranes) and,...

  13. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cryptococcus neoformans serological reagents. 866.3165 Section 866.3165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN.... Cryptococcosis infections are found most often as chronic meningitis (inflammation of brain membranes) and,...

  14. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Cryptococcus neoformans serological reagents. 866.3165 Section 866.3165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN.... Cryptococcosis infections are found most often as chronic meningitis (inflammation of brain membranes) and,...

  15. Genotypic diversity of environmental Cryptococcus neoformans isolates from Northern Portugal.

    PubMed

    Ferreira, Ana Sofia; Sampaio, Ana; Maduro, Ana Paula; Silva, Inês; Teles, Fernando; Martins, Maria da Luz; Inácio, João

    2014-02-01

    The Cryptococcus neoformans/C. gattii species complex members are the main agents of systemic cryptococcosis. This disease is believed to be acquired from the environment via fungal cell inhalation. Often, isolates recovered from environmental and clinical sources have proven to be genotypically similar. We assessed the occurrence of C. neoformans and C. gattii in environmental substrates collected in a Portuguese region. Twenty-eight isolates were identified as C. neoformans - five from decaying Eucalyptus leaves and 23 from domestic pigeon droppings. The isolates were genotyped using a URA5-RFLP approach. The C. neoformans VNIV (53.6%, n = 15) and VNI (32.1%, n = 9) genotypes were abundantly present among environmental isolates. The hybrid VNIII (14.3%, n = 4) genotype was underrepresented and the VNII was not found. Cryptococcus gattii was also not found although some isolates yielded a positive canavanine-glycine-bromothymol blue test.

  16. Production of diagnostic pigment by phenoloxidase activity of cryptococcus neoformans.

    PubMed

    Shaw, C E; Kapica, L

    1972-11-01

    Cryptococcus neoformans produces brown pigmented colonies when grown on agar media made from an extract of potatoes and carrots, broad beans (Vicia faba), or Guizotia abyssinica seeds. Since other yeasts do not produce the pigment, these media are useful as differential isolation media for C. neoformans. Similar specific pigment was produced by C. neoformans on chemically defined agar media which contained six different substrates of phenoloxidase (o-diphenol: oxygen oxidoreductase EC 1.10.3.1) an enzyme which catalyses the oxidation of o-diphenols to melanin. Substrates were incorporated singly into the media and included L-3, 4-dihydroxyphenylalanine (L-DOPA), chlorogenic acid, protocatechuic acid, catechol, norepinephrine, and 3-hydroxytyramine hydrochloride (dopamine). No pigment was produced on media without substrate. Phenoloxidase activity in (NH(4))(2)SO(4) precipitates of C. neoformans cell-free extract was assayed by measuring increases in absorbance at 480 nm produced in solutions of L-DOPA. This reaction showed oxygen uptake and was effectively inhibited by copper chelators, but not by catalase. The enzyme also oxidized the five other substrates which induced pigment formation. Electron micrographs of cells incubated in L-DOPA showed deposition of the pigment in the cell wall.

  17. Temporal Kinetics and Quantitative Analysis of Cryptococcus neoformans Nonlytic Exocytosis

    PubMed Central

    Stukes, Sabriya A.; Cohen, Hillel W.

    2014-01-01

    Cryptococcus neoformans is a facultative intracellular pathogen and the causative agent of cryptococcosis, a disease that is often fatal to those with compromised immune systems. C. neoformans has the capacity to escape phagocytic cells through a process known as nonlytic exocytosis whereby the cryptococcal cell is released from the macrophage into the extracellular environment, leaving both the host and pathogen alive. Little is known about the mechanism behind nonlytic exocytosis, but there is evidence that both the fungal and host cells contribute to the process. In this study, we used time-lapse movies of C. neoformans-infected macrophages to delineate the kinetics and quantitative aspects of nonlytic exocytosis. We analyzed approximately 800 macrophages containing intracellular C. neoformans and identified 163 nonlytic exocytosis events that were further characterized into three subcategories: type I (complete emptying of macrophage), type II (partial emptying of macrophage), and type III (cell-to-cell transfer). The majority of type I and II events occurred after several hours of intracellular residence, whereas type III events occurred significantly (P < 0.001) earlier in the course of macrophage infection. Our results show that nonlytic exocytosis is a morphologically and temporally diverse process that occurs relatively rapidly in the course of macrophage infection. PMID:24595144

  18. Cryptococcus neoformans dual GDP-mannose transporters and their role in biology and virulence.

    PubMed

    Wang, Zhuo A; Griffith, Cara L; Skowyra, Michael L; Salinas, Nichole; Williams, Matthew; Maier, Ezekiel J; Gish, Stacey R; Liu, Hong; Brent, Michael R; Doering, Tamara L

    2014-06-01

    Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro. We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis.

  19. Cyclic AMP-Dependent Protein Kinase Controls Virulence of the Fungal Pathogen Cryptococcus neoformans

    PubMed Central

    D'Souza, Cletus A.; Alspaugh, J. Andrew; Yue, Changli; Harashima, Toshiaki; Cox, Gary M.; Perfect, John R.; Heitman, Joseph

    2001-01-01

    Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Gα protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Gα protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen. PMID:11287622

  20. Molecular typing of clinical Cryptococcus neoformans isolates collected in Germany from 2004 to 2010.

    PubMed

    Sanchini, Andrea; Smith, Ilka McCormick; Sedlacek, Ludwig; Schwarz, Roman; Tintelnot, Kathrin; Rickerts, Volker

    2014-10-01

    Cryptococcosis is a fungal infection mostly caused by Cryptococcus neoformans. We identified agents of cryptococcosis diagnosed in Germany from 2004 to 2010. We used multi-locus sequence typing (MLST) to understand the molecular epidemiology of cryptococcosis. Sero- and mating types of individual patient isolates were determined by PCR. MLST was performed using the seven-locus scheme. Allele and nucleotide diversity was calculated for each locus of C. neoformans var. grubii and C. neoformans var. neoformans. Phylogenetic relations were assessed by dendrograms. Clinical data were compared between infections caused by the two variants. We studied 101 isolates. Eight were identified as hybrids (8%). All non-hybrids were of the α mating type. Among 78 C. neoformans var. grubii (77%), 16 sequence types (STs) were identified including three novel STs. They clustered in four groups, previously isolated in Asia, Europe or worldwide. Among 15 C. neoformans var. neoformans (15%), 10 STs were identified, without clustering. These isolates showed higher allele, and nucleotide diversity compared with C. neoformans var. grubii. C. neoformans var. neoformans was more likely to cause soft-tissue infections (3/9, 33 vs. 1/63, 2%, p = 0.005) and to affect non-AIDS patients (7/14, 50 vs. 15/76, 20%, p = 0.036). C. neoformans var. grubii is the predominant agent of cryptococcosis in Germany. MLST suggests that a part of these cases are acquired abroad by immigrants or tourists. C. neoformans var. neoformans isolates represent a greater genetic diversity and are associated with more variable clinical presentations.

  1. A Novel ICOS-Independent, but CD28- and SAP-Dependent, Pathway of T Cell-Dependent, Polysaccharide-Specific Humoral Immunity in Response to Intact Streptococcus pneumoniae versus Pneumococcal Conjugate Vaccine

    DTIC Science & Technology

    2008-01-01

    PPS14) and the phosphorylcholine determinant (PC) of the cell wall C-polysaccharide (C-PS, teichoic acid). The protein- and PS-specific IgG responses...hydroxy-3-nitrophenyl)acetyl; PC, phosphorylcholine determi- nant of teichoic or lipoteichoic acid; Pn, intact Streptococcus pneumoniae; PPS14, capsular

  2. Characterizing the role of RNA silencing components in Cryptococcus neoformans

    PubMed Central

    Janbon, Guilhem; Maeng, Shinae; Yang, Dong-Hoon; Ko, Young-Joon; Jung, Kwang-Woo; Moyrand, Frédérique; Floyd, Anna; Heitman, Joseph; Bahn, Yong-Sun

    2010-01-01

    Summary The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RNA-dependent RNA polymerase (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. Comparative transcriptome analysis of the serotype A and D RNAi mutants revealed that only modest changes occur in the genome-wide transcriptome profiles when the RNAi process was perturbed. Notably, the serotype D rdp1Δ mutants showed an increase in transcript abundance of active retrotransposons and transposons, such as T2 and T3, the latter of which is a novel serotype D-specific transposon of C. neoformans. In a wild type background both T2 and T3 were found to be weakly active mobile elements, although we found no evidence of Cnl1 retrotransposon mobility. In contrast, all three transposable elements exhibited enhanced mobility in the rdp1Δ mutant strain. In conclusion, the RNAi pathway plays an important role in controlling transposon activity and genome integrity of C. neoformans. PMID:21067947

  3. Improved conjugation and purification strategies for the preparation of protein-polysaccharide conjugates.

    PubMed

    Suárez, N; Massaldi, H; Franco Fraguas, L; Ferreira, F

    2008-12-12

    A glycoconjugate constituted by the Streptococcus pneumoniae serotype 14 capsular polysaccharide (CPS14) and bovine serum albumin (BSA) was prepared, and the unique properties of Sephadex LH-20 were used to separate the conjugate from the unconjugated material. The strength of this approach consists in its capacity to produce pure polysaccharide-protein conjugate in good yield and free from unconjugated material, a common residual contaminant of this type of immunobiologicals. The CPS14-BSA conjugate prepared via an improved 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)-activation technique was characterized chemically and its immunogenicity was evaluated in mice. The purified conjugate, unlike the corresponding polysaccharide, produced a T-cell-dependent response in this species.

  4. Cryptococcus neoformans Typing by PCR Fingerprinting Using (GACA)4 Primers Based on C. neoformans Genome Project Data▿

    PubMed Central

    Cogliati, Massimo; Esposto, Maria Carmela; Liberi, Giordano; Tortorano, Anna Maria; Viviani, Maria Anna

    2007-01-01

    Four (GACA)4 PCR fingerprinting sequences, used as markers to identify serotypes A and D and AD hybrids, were retrieved in four Cryptococcus neoformans genome databases. Their locations, both in serotype A and D genomes, were confirmed by chromosomal hybridization with specific probes. Two sequences were recognized to code for hypothetical functional proteins. PMID:17670921

  5. Gene arrangement and sequence of the 5S rRNA in Filobasidiella neoformans (Cryptococcus neoformans) as a phylogenetic indicator.

    PubMed

    Kwon-Chung, K J; Chang, Y C

    1994-04-01

    We cloned the 5S rRNA gene and determined its organization in the four genes encoding rRNAs in a ribosomal DNA repeat unit of Filobasidiella neoformans, the teleomorph of Cryptococcus neoformans. The 5S rRNA gene contained 118 nucleotides and was located 1 kb upstream from the 18S rRNA gene within the 8.6-kb fragment of the ribosomal DNA repeat unit. The sequence of the 5S rRNA gene from F. neoformans was more similar to the sequence of the 5S rRNA gene from Tremella mesenterica than to the sequences of the 5S rRNA genes from Filobasidium species. The arrangement of the rRNA genes in F. neoformans closely resembles the arrangement of the rRNA genes in mushrooms such as Schizophyllum commune, Agaricus bisporus, and Coprinus cinereus in that the 5S rRNA-coding region not only is located within the repeat unit that encodes the other rRNAs but also is transcribed in the same direction as the other rRNA genes. This is the first description of the arrangement of rRNA genes in a species belonging to the Heterobasidiomycetes.

  6. Enhanced virulence of Histoplasma capsulatum through transfer and surface incorporation of glycans from Cryptococcus neoformans during co-infection

    PubMed Central

    Cordero, Radames J. B.; Liedke, Susie Coutinho; de S. Araújo, Glauber R.; Martinez, Luis R.; Nimrichter, Leonardo; Frases, Susana; Peralta, Jose Mauro; Casadevall, Arturo; Rodrigues, Marcio L.; Nosanchuk, Joshua D.; Guimaraes, Allan J.

    2016-01-01

    Cryptococcus neoformans (Cn) and Histoplasma capsulatum (Hc) co-exist in the environment and occasionally co-infect individuals, which can lead to severe disease/lethal outcomes. We investigated specific interactions between Cn-Hc to determine the impact of synchronous infection in virulence and disease. Co-infected mice had significantly higher mortality than infection with either species or acapsular Cn-Hc. Coating of Hc with cryptococcal glycans (Cn-gly) resulted in higher pulmonary fungal burden in co-infected animals relative to control. Co-cultivation or addition of Cn-gly resulted in enhanced pellicle formation with a hybrid polysaccharide matrix with higher reactivity to GXM mAbs. Transfer and incorporation of Cn polysaccharide onto Hc surface was time and temperature dependent. Cn-gly transfer altered the zeta potential of Hc and was associated with increased resistance to phagocytosis and killing by macrophages. Mice infected with Hc and subsequently injected with purified Cn-gly died significantly more rapidly than Hc alone infected, establishing the precedent that virulence factors from one fungus can enhance the virulence of unrelated species. These findings suggest a new mechanism of microbial interaction involving the transfer of virulence traits that translates into enhanced lethality during mixed fungal infections and highlights the importance of studying heterogeneous microbial populations in the setting of infection. PMID:26908077

  7. Enhanced virulence of Histoplasma capsulatum through transfer and surface incorporation of glycans from Cryptococcus neoformans during co-infection.

    PubMed

    Cordero, Radames J B; Liedke, Susie Coutinho; de S Araújo, Glauber R; Martinez, Luis R; Nimrichter, Leonardo; Frases, Susana; Peralta, Jose Mauro; Casadevall, Arturo; Rodrigues, Marcio L; Nosanchuk, Joshua D; Guimaraes, Allan J

    2016-02-24

    Cryptococcus neoformans (Cn) and Histoplasma capsulatum (Hc) co-exist in the environment and occasionally co-infect individuals, which can lead to severe disease/lethal outcomes. We investigated specific interactions between Cn-Hc to determine the impact of synchronous infection in virulence and disease. Co-infected mice had significantly higher mortality than infection with either species or acapsular Cn-Hc. Coating of Hc with cryptococcal glycans (Cn-gly) resulted in higher pulmonary fungal burden in co-infected animals relative to control. Co-cultivation or addition of Cn-gly resulted in enhanced pellicle formation with a hybrid polysaccharide matrix with higher reactivity to GXM mAbs. Transfer and incorporation of Cn polysaccharide onto Hc surface was time and temperature dependent. Cn-gly transfer altered the zeta potential of Hc and was associated with increased resistance to phagocytosis and killing by macrophages. Mice infected with Hc and subsequently injected with purified Cn-gly died significantly more rapidly than Hc alone infected, establishing the precedent that virulence factors from one fungus can enhance the virulence of unrelated species. These findings suggest a new mechanism of microbial interaction involving the transfer of virulence traits that translates into enhanced lethality during mixed fungal infections and highlights the importance of studying heterogeneous microbial populations in the setting of infection.

  8. Pigment production on L-tryptophan medium by Cryptococcus gattii and Cryptococcus neoformans.

    PubMed

    Chaskes, Stuart; Cammer, Michael; Nieves, Edward; Casadevall, Arturo

    2014-01-01

    In recent years strains previously grouped within Cryptococcus neoformans have been divided into two species C. neoformans and C. gattii, with Cryptococcus neoformans comprising serotypes A, D, and AD and C. gattii comprising serotypes B and C. Cryptococcus neoformans have also been subdivided into two varieties C. neoformans var. grubii, serotype A, and C. neoformans var. neoformans, serotype D. We analyzed the growth and pigment production characteristics of 139 strains of Cryptococcus spp. in L-tryptophan containing media. Nearly all strains of Cryptococcus, including each variety and serotype tested produced a pink water-soluble pigment (molecular weight of 535.2 Da) from L-tryptophan. Consequently, the partial separation of the species was based on whether the pink pigment was secreted into the medium (extracellular) or retained as an intracellular pigment. On L-tryptophan medium C. neoformans var. grubii and serotype AD produced a pink extracellular pigment. In contrast, for C. gattii, the pink pigment was localized intracellularly and masked by heavy production of brown pigments. Pigment production by C. neoformans var. neoformans was variable with some strains producing the pink extracellular pigment and others retained the pink pigment intracellularly. The pink intracellular pigment produced by strains of C. neoformans var. neoformans was masked by production of brown pigments. Cryptococcus laccase mutants failed to produce pigments from L-tryptophan. This is the first report that the enzyme laccase is involved in tryptophan metabolism. Prior to this report Cryptococcus laccase produced melanin or melanin like-pigments from heterocyclic compounds that contained ortho or para diphenols, diaminobenzenes and aminophenol compounds. The pigments produced from L-tryptophan were not melanin.

  9. Modulation of Replicative Lifespan in Cryptococcus neoformans: Implications for Virulence.

    PubMed

    Bouklas, Tejas; Jain, Neena; Fries, Bettina C

    2017-01-01

    The fungal pathogen, Cryptococcus neoformans, has been shown to undergo replicative aging. Old cells are characterized by advanced generational age and phenotypic changes that appear to mediate enhanced resistance to host and antifungal-based killing. As a consequence of this age-associated resilience, old cells accumulate during chronic infection. Based on these findings, we hypothesized that shifting the generational age of a pathogenic yeast population would alter its vulnerability to the host and affect its virulence. SIR2 is a well-conserved histone deacetylase, and a pivotal target for the development of anti-aging drugs. We tested its effect on C. neoformans' replicative lifespan (RLS). First, a mutant C. neoformans strain (sir2Δ) was generated, and confirmed a predicted shortened RLS in sir2Δ cells consistent with its known role in aging. Next, RLS analysis showed that treatment of C. neoformans with Sir2p-agonists resulted in a significantly prolonged RLS, whereas treatment with a Sir2p-antagonist shortened RLS. RLS modulating effects were dependent on SIR2 and not observed in sir2Δ cells. Because SIR2 loss resulted in a slightly impaired fitness, effects of genetic RLS modulation on virulence could not be compared with wild type cells. Instead we chose to chemically modulate RLS, and investigated the effect of Sir2p modulating drugs on C. neoformans cells in a Galleria mellonella infection model. Consistent with our hypothesis that shifts in the generational age of the infecting yeast population alters its vulnerability to host cells, we observed decreased virulence of C. neoformans in the Galleria host when RLS was prolonged by treatment with Sir2p agonists. In contrast, treatment with a Sir2p antagonist, which shortens RLS enhanced virulence in Galleria. In addition, combination of Sir2p agonists with antifungal therapy enhanced the antifungal's effect. Importantly, no difference in virulence was observed with drug treatment when sir2Δ cells were

  10. Amino Acid Permeases and Virulence in Cryptococcus neoformans

    PubMed Central

    Takahashi, Juliana Possato Fernandes; Guerra, Juliana Mariotti; Santos, Dayane Cristina da Silva; Purisco, Sônia Ueda; Melhem, Márcia de Souza Carvalho; Fazioli, Raquel dos Anjos; Phanord, Clerlune; Sartorelli, Patrícia; Vallim, Marcelo A.

    2016-01-01

    Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i

  11. Amino Acid Permeases and Virulence in Cryptococcus neoformans.

    PubMed

    Martho, Kevin Felipe Cruz; de Melo, Amanda Teixeira; Takahashi, Juliana Possato Fernandes; Guerra, Juliana Mariotti; Santos, Dayane Cristina da Silva; Purisco, Sônia Ueda; Melhem, Márcia de Souza Carvalho; Fazioli, Raquel Dos Anjos; Phanord, Clerlune; Sartorelli, Patrícia; Vallim, Marcelo A; Pascon, Renata C

    2016-01-01

    Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i

  12. Capsules from Pathogenic and Non-Pathogenic Cryptococcus spp. Manifest Significant Differences in Structure and Ability to Protect against Phagocytic Cells

    PubMed Central

    Araujo, Glauber de S.; Fonseca, Fernanda L.; Pontes, Bruno; Torres, Andre; Cordero, Radames J. B.; Zancopé-Oliveira, Rosely M.; Casadevall, Arturo; Viana, Nathan B.; Nimrichter, Leonardo; Rodrigues, Marcio L.; Garcia, Eloi S.; de Souza, Wanderley; Frases, Susana

    2012-01-01

    Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular

  13. A Simple Technique for Capsular Repair After Hip Arthroscopy

    PubMed Central

    Camp, Christopher L.; Reardon, Patrick J.; Levy, Bruce A.; Krych, Aaron J.

    2015-01-01

    Capsulotomy is typically performed during arthroscopic treatment for femoroacetabular impingement. As the frequency of hip arthroscopy continues to expand rapidly, increased attention is being paid to the implications of interportal capsulotomy and the need for repair. To minimize the risk of postoperative instability, capsular closure has been recommended to restore the anatomy and biomechanical function of the capsule. We present a reliable, efficient, and effective method for arthroscopic closure of the interportal capsulotomy after hip arthroscopy. PMID:26870655

  14. The isolation, characterization and antifungal susceptibilities of Cryptococcus neoformans from bird excreta in Klang Valley, Malaysia.

    PubMed

    Tay, S T; Chai, H C; Na, S L; Hamimah, H; Rohani, M Y; Soo-Hoo, T S

    2005-06-01

    The occurrence of Cryptococcus neoformans in bird excreta in Klang valley, Malaysia was determined in this study. Of 544 samples of bird excreta collected from a local zoo, pet shops and public areas, 20 strains of C. neoformans were isolated. All C. neoformans strains were serotype A and thus identified as C. neoformans variety grubii. All did not produce color changes on canavanine-glycine-bromothymol blue agar. All were of alpha-mating types, as determined by a pheromone-specific PCR assay. The antifungal susceptibility testing using agar diffusion method Neo-sensitabs showed that all were susceptible to amphotericin B, fluconazole and itraconazole.

  15. Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence.

    PubMed

    Davis, Michael J; Eastman, Alison J; Qiu, Yafeng; Gregorka, Brian; Kozel, Thomas R; Osterholzer, John J; Curtis, Jeffrey L; Swanson, Joel A; Olszewski, Michal A

    2015-03-01

    Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans-induced lysosome damage was observed in infected murine bone marrow-derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans-containing lung cells compared with C. neoformans-free cells. Among C. neoformans-containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-γ abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections.

  16. Differentiation of Cryptococcus neoformans serotypes A and D using creatinine dextrose bromothymol blue thymine medium.

    PubMed

    Irokanulo, E A; Akueshi, C O; Makinde, A A

    1994-06-01

    A serotype differentiation of the encapsulated yeast Cryptococcus neoformans is described using creatinine dextrose bromothymol blue thymine (CDBT) medium. On CDBT medium C. neoformans serotype D grew as bright red colonies, turning the medium a bright orange after five days incubation at 28 degrees C. C. neoformans serotype A grew as pale colonies with no apparent colour effect on the medium. Serotypes B and C caused a slight greening of the medium. The reaction of the four serotypes of C. neoformans on CDBT medium is considered useful in the differentiation of the closely related serotype A and D.

  17. Reduced virulence of melanized Cryptococcus neoformans in Galleria mellonella.

    PubMed

    Eisenman, Helene C; Duong, Raymond; Chan, Hsi; Tsue, Ryan; McClelland, Erin E

    2014-07-01

    Fungal melanins are important in the virulence of many pathogenic fungi. In this study, we examined the role of melanin in the interaction between Cryptococcus neoformans and the invertebrate host, Galleria mellonella. C. neoformans was able to melanize in the presence of G. mellonella homogenate, indicating the presence of melanin substrates. Melanization was confirmed by the recovery of acid-resistant particles that were recognized by anti-melanin antibodies. In addition, we tested the effect of fungal melanization on virulence. Surprisingly, G. mellonella larvae infected with melanized fungal cells lived longer than those infected with non-melanized fungi. When the cellular immune response of G. mellonella to melanized and non-melanized cells was compared, inflammatory nodules were observed in both groups. However the response was stronger in larvae infected with melanized cells. These results suggest that fungal melanin activates the immune response of G. mellonella, thereby resulting in the decreased virulence observed with melanized cells.

  18. Titan cells in Cryptococcus neoformans: cells with a giant impact.

    PubMed

    Zaragoza, Oscar; Nielsen, Kirsten

    2013-08-01

    Cryptococcus neoformans is a pathogenic yeast that commonly infects immunocompromised individuals, yet has developed multiple adaptation mechanisms to the host. Several virulence factors (capsule and melanin) have been known for many years. However, this yeast also possesses a morphogenetic program that is still not well characterized. C. neoformans has the ability to dramatically enlarge its size during infection to form 'titan cells' that can reach up to 100μm in cell body diameter, in contrast to typical size cells of 5-7μm. These titan cells pose a problem for the host because they contribute to fungal survival, dissemination to the central nervous system, and possibly even latency. In this review, we will provide an overview of these cells, covering current knowledge about their phenotypic features, mechanism of formation, and their significance during infection.

  19. Molecular epidemiology of Italian clinical Cryptococcus neoformans var. grubii isolates.

    PubMed

    Cogliati, Massimo; Zamfirova, Ralika R; Tortorano, Anna Maria; Viviani, Maria Anna

    2013-07-01

    Cryptococcus neoformans variety grubii is the major etiological agent of cryptococcal meningitis in both immunocompromised and immunocompetent patients. The current PCR-based molecular methods are not sufficient to discriminate among the different populations of this yeast. Therefore, the aim of the present study was to investigate the genotypes of the Italian clinical C. neoformans var. grubii isolates by multilocus sequence typing (MLST). A total of 53 isolates, each representative of a single case, were studied. Genotyping was performed using the ISHAM Cryptococcus MLST consensus scheme and the results were compared to the publically available global C. neoformans var. grubii MLST dataset. A total of 16 genotypes were identified; 14 were new genotypes, one was identical to sequence type (ST) ST81, which had been previously reported from Thailand, and one to ST23 already identified in Uganda, the USA and Korea. Sequence type ST61 was the most numerous, including 16 isolates. Network phylogenetic analysis showed that the Italian isolates could be divided into at least three clusters with similarities with those recovered in Africa, Asia and Americas. Distribution of the STs among the isolates could not be correlated to the hospital in which they were recovered or to the HIV status of the patients. The majority of the isolates belonged to the molecular type VNI; three belonged to the rare molecular type VNII and one to the VNB group, which until now had not been described in Europe. The results reveal that the Italian C. neoformans var. grubii population presents a distinct variability, displaying a high number of new genotypes, and probably recombines sexually.

  20. Effects of murine natural killer cells on Cryptococcus neoformans

    SciTech Connect

    Nabavi Nouri, N.

    1985-01-01

    Previous data generated by Murphy and McDaniel indicate that normal murine nylon wool nonadherent splenic cells, with the characteristics of natural killer (NK) cells, effectively inhibit the in vitro growth of Cryptococcus neoformans, a yeast-like pathogen. Nylon wood nonadherent cells from spleens of 7-8 week old mice were further fractionated on discontinuous Percoll gradients. The enrichment of NK cells in Percoll fractions 1 and 2 was confirmed by morphological examination, immunofluorescent staining, and by assessing the cytolytic activity of each Percoll cell fraction against YAC-1 targets in the 4 h /sup 51/Cr release assay. Cells isolated from each Percoll fraction were tested for growth inhibitory activity against C neoformans, using an in vitro 18 h growth inhibition assay. The results showed that NK cell enrichment was concomitant with the enrichment of anti-cryptococcal activity the Percoll fractions 1 and 2. An immunolabeling method combined with scanning electron microscopy was used to demonstrate that the effector cells attached to C. neoformans were asialo GM/sub 1/ positive and, therefore, had NK cell characteristics. NK cells have Fc receptors on their surfaces , and are capable of antibody-dependent cell-mediated cytotoxicity (ADCC) against IgG-coated target cells. The author examined the effects of the IgG fraction of rabbit anti-cryptococcal antibody on the NK cell-mediated growth inhibition of C. neoformans. The data indicated that the effector cells involved in antibody-dependent growth inhibition of cryptococci are either NK cells or copurify and coexist in the same population with NK cells.

  1. Histone deacetylases inhibitors effects on Cryptococcus neoformans major virulence phenotypes.

    PubMed

    Brandão, Fabiana As; Derengowski, Lorena S; Albuquerque, Patrícia; Nicola, André M; Silva-Pereira, Ildinete; Poças-Fonseca, Marcio J

    2015-01-01

    Cryptococcus neoformans undergoes phenotypical changes during host infection in order to promote persistence and survival. Studies have demonstrated that such adaptations require alterations in gene transcription networks by distinct mechanisms. Drugs such as the histone deacetylases inhibitors (HDACi) Sodium Butyrate (NaBut) and Trichostatin A (TSA) can alter the chromatin conformation and have been used to modulate epigenetic states in the treatment of diseases such as cancer. In this work, we have studied the effect of NaBut and TSA on the expression of C. neoformans major virulence phenotypes and on the survival rate of an animal model infected with drugs-treated yeasts. Both drugs affected fungal growth at 37°C more intensely than at 30°C; nonetheless, drugs did not affect cell viability at the concentrations we studied. HDACi also provoked the reduction of the fungal capsule expansion. Phospholipases enzyme activity decreased; mating process and melanin synthesis were also affected by both inhibitors. NaBut led to an increase in the population of cells in G2/M. Treated yeast cells, which were washed in order to remove the drugs from the culture medium prior to the inoculation in the Galleria mellonela infection model, did not cause significant difference at the host survival curve when compared to non-treated cells. Overall, NaBut effects on the impairment of C. neoformans main virulence factors were more intense and stable than the TSA effects.

  2. Systematic functional profiling of transcription factor networks in Cryptococcus neoformans

    PubMed Central

    Jung, Kwang-Woo; Yang, Dong-Hoon; Maeng, Shinae; Lee, Kyung-Tae; So, Yee-Seul; Hong, Joohyeon; Choi, Jaeyoung; Byun, Hyo-Jeong; Kim, Hyelim; Bang, Soohyun; Song, Min-Hee; Lee, Jang-Won; Kim, Min Su; Kim, Seo-Young; Ji, Je-Hyun; Park, Goun; Kwon, Hyojeong; Cha, Suyeon; Meyers, Gena Lee; Wang, Li Li; Jang, Jooyoung; Janbon, Guilhem; Adedoyin, Gloria; Kim, Taeyup; Averette, Anna K.; Heitman, Joseph; Cheong, Eunji; Lee, Yong-Hwan; Lee, Yin-Won; Bahn, Yong-Sun

    2015-01-01

    Cryptococcus neoformans causes life-threatening meningoencephalitis in humans, but its overall biological and pathogenic regulatory circuits remain elusive, particularly due to the presence of an evolutionarily divergent set of transcription factors (TFs). Here, we report the construction of a high-quality library of 322 signature-tagged gene-deletion strains for 155 putative TF genes previously predicted using the DNA-binding domain TF database, and examine their in vitro and in vivo phenotypic traits under 32 distinct growth conditions. At least one phenotypic trait is exhibited by 145 out of 155 TF mutants (93%) and ∼85% of them (132/155) are functionally characterized for the first time in this study. The genotypic and phenotypic data for each TF are available in the C. neoformans TF phenome database (http://tf.cryptococcus.org). In conclusion, our phenome-based functional analysis of the C. neoformans TF mutant library provides key insights into transcriptional networks of basidiomycetous fungi and human fungal pathogens. PMID:25849373

  3. Extracellular proteins of Cryptococcus neoformans and host antibody response.

    PubMed Central

    Chen, L C; Pirofski, L A; Casadevall, A

    1997-01-01

    Proteins secreted by the fungal pathogen Cryptococcus neoformans may be involved in invasion and could be useful in vaccine design. Despite the medical importance of this fungus, little is known about its extracellular proteins or the immune response to these antigens. To study C. neoformans extracellular proteins, 12 strains were metabolically radiolabeled and protein supernatants were analyzed. Both strain- and growth condition-dependent differences were observed. Enzymatic assays of filtered culture supernatants revealed butyrate esterase and caprylate esterase lipase activity for 11 of 12 strains, as well as acid phosphatase, naphthol-AS-BI-phosphohydrolase, and beta-glucosidase activities in some strains. Serum from infected rodents immunoprecipitated several secreted proteins, consistent with in vivo expression and development of an antibody response. For strain 24067, two immunodominant species, of approximately 75 and 30 kDa, were recognized. The relative intensity of the autoradiographic bands depended on the route of infection for both rats and mice. In summary, our results indicate that (i) there are multiple proteins in C. neoformans culture supernatants, (ii) there are strain differences in supernatant protein profiles, (iii) there are differences in supernatant protein profile depending on the growth conditions, (iv) there are several new extracellular and/or cell-associated enzymatic activities, and (v) antibodies to several supernatant proteins are made in the course of infection. PMID:9199426

  4. The Neurospora crassa CPS-1 polysaccharide synthase functions in cell wall biosynthesis.

    PubMed

    Fu, Ci; Sokolow, Eleanor; Rupert, Christopher B; Free, Stephen J

    2014-08-01

    The Neurospora crassa cps-1 gene encodes a polysaccharide synthase with homology to the Cryptococcus neoformans hyaluronic acid synthase Cps1p. Homologs of the cps-1 gene are found in the genomes of many fungi. Loss of CPS-1 results in a cell wall defect that affects all stages of the N. crassa life cycle, including vegetative growth, protoperithecia (female mating structure) development, and conidia (asexual spore) development. The cell wall of cps-1 deletion mutants is sensitive to cell wall perturbation reagents. Our results demonstrate that CPS-1 is required for the incorporation of cell wall proteins into the cell wall and plays a critical role in cell wall biogenesis. We found that the N. crassa cell wall is devoid of hyaluronic acid, and conclude that the polysaccharide produced by the CPS-1 is not hyaluronic acid.

  5. The discovery of creatinine assimilation in Cryptococcus neoformans, and subsequent work on the characterization of the two varieties of C. neoformans.

    PubMed

    Kwon-Chung, K J

    1991-08-01

    The discovery of creatinine assimilation in C. neoformans by Staib served as the foundation for our biochemical, genetical, ecological, epidemiological and taxonomic studies on the two varieties of C. neoformans for the past 15 years. The two varietal concept is now widely accepted and the arrival of AIDS epidemic has promoted the recognition of the differences between the two varieties, especially in their epidemiology and ecology. Since the agent of cryptococcosis in AIDS patients almost always belongs to the var. neoformans even in geographical areas prevalent for var. gattii, it is important to study the possible differences in the pathogenesis of the two varieties.

  6. Pivotal Roles of the Outer Membrane Polysaccharide Export and Polysaccharide Copolymerase Protein Families in Export of Extracellular Polysaccharides in Gram-Negative Bacteria

    PubMed Central

    Cuthbertson, Leslie; Mainprize, Iain L.; Naismith, James H.; Whitfield, Chris

    2009-01-01

    Summary: Many bacteria export extracellular polysaccharides (EPS) and capsular polysaccharides (CPS). These polymers exhibit remarkably diverse structures and play important roles in the biology of free-living, commensal, and pathogenic bacteria. EPS and CPS production represents a major challenge because these high-molecular-weight hydrophilic polymers must be assembled and exported in a process spanning the envelope, without compromising the essential barrier properties of the envelope. Emerging evidence points to the existence of molecular scaffolds that perform these critical polymer-trafficking functions. Two major pathways with different polymer biosynthesis strategies are involved in the assembly of most EPS/CPS: the Wzy-dependent and ATP-binding cassette (ABC) transporter-dependent pathways. They converge in an outer membrane export step mediated by a member of the outer membrane auxiliary (OMA) protein family. OMA proteins form outer membrane efflux channels for the polymers, and here we propose the revised name outer membrane polysaccharide export (OPX) proteins. Proteins in the polysaccharide copolymerase (PCP) family have been implicated in several aspects of polymer biogenesis, but there is unequivocal evidence for some systems that PCP and OPX proteins interact to form a trans-envelope scaffold for polymer export. Understanding of the precise functions of the OPX and PCP proteins has been advanced by recent findings from biochemistry and structural biology approaches and by parallel studies of other macromolecular trafficking events. Phylogenetic analyses reported here also contribute important new insight into the distribution, structural relationships, and function of the OPX and PCP proteins. This review is intended as an update on progress in this important area of microbial cell biology. PMID:19258536

  7. In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil.

    PubMed

    de F L Fernandes, Orionalda; Passos, Xisto S; Souza, Lúcia K H; Miranda, André T B; Cerqueira, Carlos Henrique P V; Silva, Maria do Rosário R

    2003-09-01

    Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiânia, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.

  8. A rapid method for detecting extracellular proteinase activity in Cryptococcus neoformans and a survey of 63 isolates.

    PubMed

    Ruma-Haynes, P; Brownlee, A G; Sorrell, T C

    2000-08-01

    A rapid method to detect extracellular proteolytic activity around colonies of Cryptococcus neoformans was developed with tannic acid used to complex with residual protein in a solid medium. A survey was conducted with 32 isolates of C. neoformans var. gattii and 31 isolates of C. neoformans var. neoformans which were cultured on medium containing gelatin as the sole nitrogen source. The annulus of clearing around fungal colonies was > 1.2 mm in 24 (77%) isolates of C. neoformans var. neoformans compared with only 7 (22%) isolates of C. neoformans var. gattii. There was no difference in proteolytic activity between environmental and human clinical isolates of C. neoformans. However, there was a difference between the size of the annulus around animal isolates of C. neoformans var. neoformans and isolates of the same variety from other sources. The annuli around the 14 animal isolates were all >1.2 mm, while 7 (70%) of 10 human clinical isolates and only 3 (43%) of 7 environmental isolates were scored in the high proteinase range. A difference between the genetic types (as characterised by RAPD typing) of C. neoformans var. gattii was also evident with 17 (77%) of 22 VG-I isolates having a small annulus compared with only 1 (17%) of 6 VG-II and VG-III isolates with annuli of similar size. Relatively low proteinase production by C. neoformans var. gattii may reduce local and systemic spread of infection in mammalian hosts.

  9. Modulation of Replicative Lifespan in Cryptococcus neoformans: Implications for Virulence

    PubMed Central

    Bouklas, Tejas; Jain, Neena; Fries, Bettina C.

    2017-01-01

    The fungal pathogen, Cryptococcus neoformans, has been shown to undergo replicative aging. Old cells are characterized by advanced generational age and phenotypic changes that appear to mediate enhanced resistance to host and antifungal-based killing. As a consequence of this age-associated resilience, old cells accumulate during chronic infection. Based on these findings, we hypothesized that shifting the generational age of a pathogenic yeast population would alter its vulnerability to the host and affect its virulence. SIR2 is a well-conserved histone deacetylase, and a pivotal target for the development of anti-aging drugs. We tested its effect on C. neoformans’ replicative lifespan (RLS). First, a mutant C. neoformans strain (sir2Δ) was generated, and confirmed a predicted shortened RLS in sir2Δ cells consistent with its known role in aging. Next, RLS analysis showed that treatment of C. neoformans with Sir2p-agonists resulted in a significantly prolonged RLS, whereas treatment with a Sir2p-antagonist shortened RLS. RLS modulating effects were dependent on SIR2 and not observed in sir2Δ cells. Because SIR2 loss resulted in a slightly impaired fitness, effects of genetic RLS modulation on virulence could not be compared with wild type cells. Instead we chose to chemically modulate RLS, and investigated the effect of Sir2p modulating drugs on C. neoformans cells in a Galleria mellonella infection model. Consistent with our hypothesis that shifts in the generational age of the infecting yeast population alters its vulnerability to host cells, we observed decreased virulence of C. neoformans in the Galleria host when RLS was prolonged by treatment with Sir2p agonists. In contrast, treatment with a Sir2p antagonist, which shortens RLS enhanced virulence in Galleria. In addition, combination of Sir2p agonists with antifungal therapy enhanced the antifungal’s effect. Importantly, no difference in virulence was observed with drug treatment when sir2Δ cells

  10. Uronic polysaccharide degrading enzymes.

    PubMed

    Garron, Marie-Line; Cygler, Miroslaw

    2014-10-01

    In the past several years progress has been made in the field of structure and function of polysaccharide lyases (PLs). The number of classified polysaccharide lyase families has increased to 23 and more detailed analysis has allowed the identification of more closely related subfamilies, leading to stronger correlation between each subfamily and a unique substrate. The number of as yet unclassified polysaccharide lyases has also increased and we expect that sequencing projects will allow many of these unclassified sequences to emerge as new families. The progress in structural analysis of PLs has led to having at least one representative structure for each of the families and for two unclassified enzymes. The newly determined structures have folds observed previously in other PL families and their catalytic mechanisms follow either metal-assisted or Tyr/His mechanisms characteristic for other PL enzymes. Comparison of PLs with glycoside hydrolases (GHs) shows several folds common to both classes but only for the β-helix fold is there strong indication of divergent evolution from a common ancestor. Analysis of bacterial genomes identified gene clusters containing multiple polysaccharide cleaving enzymes, the Polysaccharides Utilization Loci (PULs), and their gene complement suggests that they are organized to process completely a specific polysaccharide.

  11. Antipneumococcal effects of C-reactive protein and monoclonal antibodies to pneumococcal cell wall and capsular antigens.

    PubMed Central

    Briles, D E; Forman, C; Horowitz, J C; Volanakis, J E; Benjamin, W H; McDaniel, L S; Eldridge, J; Brooks, J

    1989-01-01

    Antibodies to pneumococcal capsular polysaccharides are well known for their ability to protect against pneumococcal infection. Recent studies indicate that antibodies to cell wall antigens, including pneumococcal surface protein A and the phosphocholine (PC) determinant of teichoic acids as well as human C-reactive protein (which also binds to PC), can protect mice against pneumococcal infection. In the present study we compared the protective effects of these agents as measured by mouse protection, the blood bactericidal assay, and clearance of pneumococci from the blood and peritoneal cavity. Our findings extend previous results indicating that human C-reactive protein and antibodies to noncapsular antigens are generally less protective than anticapsular antibodies. The new results obtained indicate the following: (i) mouse protection studies with intraperitoneal and intravenous infections provide very similar results; (ii) monoclonal immunoglobulin G2a (IgG2a) antibodies to PC, like IgG1, IgG2b, and IgG3 antibodies to PC, are highly protective against pneumococcal infection in mice; (iii) human antibody to PC is able to protect against pneumococcal infection in mice; (iv) antibodies to PspA are effective at mediating blood and peritoneal clearance of pneumococci; (v) complement is required for the in vivo protective effects of both IgG and IgM antibodies to PC; (vi) IgG1, IgG2b, and IgG3 anti-PC antibodies all mediate complement-dependent lysis of PC-conjugated erythrocytes; and (vii) antibodies and human C-reactive proteins that are reactive with capsular antigens but not cell wall antigens are able to mediate significant antibacterial activity in the blood bactericidal assay. PMID:2707854

  12. Neisseria lactamica and Neisseria meningitidis share lipooligosaccharide epitopes but lack common capsular and class 1, 2, and 3 protein epitopes.

    PubMed

    Kim, J J; Mandrell, R E; Griffiss, J M

    1989-02-01

    Neisseria lactamica, a common human pharyngeal commensal, contributes to acquired immunity to Neisseria meningitidis. To define the surface antigens shared between these two species, we used monoclonal antibodies (MAbs) to study 35 N. lactamica strains isolated in various parts of the world for cross-reactivity with meningococcal capsules, outer membrane proteins, and lipooligosaccharides (LOS). No N. lactamica strain reacted significantly with MAbs specific for capsular group A, B, C, Y, or W, and we were unable to extract capsular polysaccharide from them. Only 2 of 33 strains reacted weakly with MAbs against class 2 serotype proteins P2b and P2c. None reacted with MAbs specific for meningococcal class 1 protein P1.2 or P1.16 or class 2/3 serotype protein P2a or P15. Most N. lactamica strains (30 of 35) bound one or more of seven LOS-specific MAbs. Two LOS epitopes, defined by MAbs O6B4 and 3F11, that are commonly found on pathogenic Neisseria species were found on 25 of 35 N. lactamica. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the LOS of N. lactamica are composed of multiple components that are physically and antigenically similar to the LOS of pathogenic Neisseria species. Among four other commensal neisserial species, only Neisseria cinerea shared LOS epitopes defined by MAbs O6B4 and 3F11. Previous studies have shown that pharyngeal colonization with N. lactamica induces bactericidal antibodies against the meningococcus. We postulate that shared N. lactamica and meningococcal LOS epitopes may play an important role in the development of natural immunity to the meningococcus.

  13. Identification of QTLs Associated with Virulence Related Traits and Drug Resistance in Cryptococcus neoformans.

    PubMed

    Vogan, Aaron A; Khankhet, Jordan; Samarasinghe, Himeshi; Xu, Jianping

    2016-09-08

    Cryptococcus neoformans is a basidiomycete fungus capable of causing deadly meningoenchephilitis, primarily in immunocompromised individuals. Formerly, C. neoformans was composed of two divergent lineages, but these have recently been elevated to species status, now C. neoformans (formerly C. neoformans var. grubii) and C. deneoformans (formerly C. neoformans var. neoformans). While both species can cause deadly infections in humans, C. neoformans is much more prevalent in clinical settings than C. deneoformans However, the genetic factors contributing to their significant differences in virulence remain largely unknown. Quantitative trait locus (QTL) mapping is a powerful tool that can be used to identify genomic regions associated with phenotypic differences between strains. Here, we analyzed a hybrid cross between these two species and identified a total of 23 QTL, including five for melanin production, six for cell size, one for cell wall thickness, five for the frequency of capsule production, three for minimal inhibitory concentration (MIC) of fluconazole in broth, and three for MIC on solid medium. For the fluconazole resistance-associated QTL, three showed environment and/or concentration-specific effects. Our results provide a large number of candidate gene regions from which to explore the molecular bases for phenotypic differences between C. neoformans and C. deneoformans.

  14. Identification of QTLs Associated with Virulence Related Traits and Drug Resistance in Cryptococcus neoformans

    PubMed Central

    Vogan, Aaron A.; Khankhet, Jordan; Samarasinghe, Himeshi; Xu, Jianping

    2016-01-01

    Cryptococcus neoformans is a basidiomycete fungus capable of causing deadly meningoenchephilitis, primarily in immunocompromised individuals. Formerly, C. neoformans was composed of two divergent lineages, but these have recently been elevated to species status, now C. neoformans (formerly C. neoformans var. grubii) and C. deneoformans (formerly C. neoformans var. neoformans). While both species can cause deadly infections in humans, C. neoformans is much more prevalent in clinical settings than C. deneoformans. However, the genetic factors contributing to their significant differences in virulence remain largely unknown. Quantitative trait locus (QTL) mapping is a powerful tool that can be used to identify genomic regions associated with phenotypic differences between strains. Here, we analyzed a hybrid cross between these two species and identified a total of 23 QTL, including five for melanin production, six for cell size, one for cell wall thickness, five for the frequency of capsule production, three for minimal inhibitory concentration (MIC) of fluconazole in broth, and three for MIC on solid medium. For the fluconazole resistance-associated QTL, three showed environment and/or concentration-specific effects. Our results provide a large number of candidate gene regions from which to explore the molecular bases for phenotypic differences between C. neoformans and C. deneoformans. PMID:27371951

  15. Diagnosis and successful treatment of Cryptococcus neoformans variety grubii in a domestic ferret

    PubMed Central

    Hanley, Christopher S.; MacWilliams, Peter; Giles, Steve; Paré, Jean

    2006-01-01

    A domestic ferret was presented for episodic regurgitation. Cytologic examination and culture of an enlarged submandibular lymph node revealed Cryptococcus neoformans variety grubii (serotype A). The ferret was successfully treated with itraconazole. This is the first documented case of Cryptococcus neoformans variety grubii in a ferret in the United States. PMID:17078253

  16. Analysis of the Protein Kinase A-Regulated Proteome of Cryptococcus neoformans Identifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation

    PubMed Central

    Geddes, J. M. H.; Caza, M.; Croll, D.; Stoynov, N.; Foster, L. J.

    2016-01-01

    ABSTRACT The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningitis in immunocompromised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA) signal transduction pathway. In this study, we investigated the influence of PKA on the composition of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Through quantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA regulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-related functions. PKA regulation of the ubiquitin-proteasome pathway in C. neoformans showed a striking parallel with connections between PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigation of proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibility for strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stress with capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalytic subunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elaboration of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathway on the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis. PMID:26758180

  17. KRE genes are required for beta-1,6-glucan synthesis, maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans.

    PubMed

    Gilbert, Nicole M; Donlin, Maureen J; Gerik, Kimberly J; Specht, Charles A; Djordjevic, Julianne T; Wilson, Christabel F; Sorrell, Tania C; Lodge, Jennifer K

    2010-04-01

    The polysaccharide beta-1,6-glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in beta-1,6-glucan synthesis. The H99 deletion mutants kre5Delta and kre6Deltaskn1Delta contained less cell wall beta-1,6-glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI-anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Delta and kre6Deltaskn1Delta. Our results indicate that KRE5, KRE6 and SKN1 are involved in beta-1,6-glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.

  18. Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex.

    PubMed

    Hagen, Ferry; Khayhan, Kantarawee; Theelen, Bart; Kolecka, Anna; Polacheck, Itzhack; Sionov, Edward; Falk, Rama; Parnmen, Sittiporn; Lumbsch, H Thorsten; Boekhout, Teun

    2015-05-01

    Phylogenetic analysis of 11 genetic loci and results from many genotyping studies revealed significant genetic diversity with the pathogenic Cryptococcus gattii/Cryptococcus neoformans species complex. Genealogical concordance, coalescence-based, and species tree approaches supported the presence of distinct and concordant lineages within the complex. Consequently, we propose to recognize the current C. neoformans var. grubii and C. neoformans var. neoformans as separate species, and five species within C. gattii. The type strain of C. neoformans CBS132 represents a serotype AD hybrid and is replaced. The newly delimited species differ in aspects of pathogenicity, prevalence for patient groups, as well as biochemical and physiological aspects, such as susceptibility to antifungals. MALDI-TOF mass spectrometry readily distinguishes the newly recognized species.

  19. Human astrocytes inhibit Cryptococcus neoformans growth by a nitric oxide-mediated mechanism

    PubMed Central

    1994-01-01

    Cryptococcus neoformans is an opportunistic fungus that causes life- threatening meningoencephalitis in 5-10% of patients with acquired immune deficiency syndrome. Cryptococcal meningoencephalitis is characterized by a lymphohistiocytic infiltrate, accumulation of encapsulated forms of C. neoformans, and varying degrees of glial reaction. Little is known about the contribution of endogenous central nervous system cells to the pathogenesis of cryptococcal infections. In this study, we investigated the role of astrocytes as potential effector cells against C. neoformans. Primary cultures of human fetal astrocytes, activated with interleukin 1 beta plus interferon gamma inhibited the growth of C. neoformans. The inhibition of C. neoformans growth was paralleled by production of nitrite, and reversed by the inhibitors of nitric oxide (NO.) synthase, NG-methyl-mono-arginine and NG-nitro-arginine methyl ester. The results suggest a novel function for human astrocytes in host defence and provide a precedent for the use of NO. as an antimicrobial effector molecule by human cells. PMID:8006595

  20. Staphylococcus aureus capsular types and antibody response to lung infection in patients with cystic fibrosis.

    PubMed Central

    Albus, A; Fournier, J M; Wolz, C; Boutonnier, A; Ranke, M; Høiby, N; Hochkeppel, H; Döring, G

    1988-01-01

    Chronic respiratory tract infections caused by Staphylococcus aureus are common in patients with cystic fibrosis (CF). Recently, it was shown in a few CF patients that S. aureus isolates produce capsular polysaccharides (CPs). However, it is not known whether this is a common feature and whether an immune response to CPs in CF is detectable. Therefore, we examined 170 S. aureus isolates from CF patients and healthy individuals for production of CP types 5 and 8 by using monoclonal antibodies. We found that CP-producing staphylococcal isolates were randomly distributed among CF patients and healthy carriers. Eighty-five percent of all isolates produced CPs, 77% of which were type 8. Examination of one sputum sample by an immunofluorescence technique suggested that production of CPs is not an in vitro phenomenon. S. aureus isolates from various sites of a single person often yielded more than one CP type. A random distribution of S. aureus strains with CP type 5 or 8 from the skin and respiratory tracts of patients and from the skin of healthy individuals was found. Antibody response to CP types 5 and 8, measured by enzyme-linked immunosorbent assay, was not elevated in CF patients with chronic S. aureus lung infection in comparison with healthy carriers. On the contrary, in CF patients the ratios of antibodies to CP 8 were significantly lower (P less than 0.005; alpha = 0.025). The ratios of antibodies to CP types did not change when monitored longitudinally over several months. This study suggests that the production of CPs is a universal property of S. aureus and that infected CF patients do not have elevated ratios of antibodies to these antigens. Images PMID:3230130

  1. Interleukin-1-like activity in capsular material from Haemophilus actinomycetemcomitans.

    PubMed Central

    Harvey, W; Kamin, S; Meghji, S; Wilson, M

    1987-01-01

    This paper describes the activity of a bacterial surface component (capsular material, CM) in biological assays for interleukin-1 (IL-1). CM from the periodontal pathogen Haemophilus actinomycetemcomitans was tested in the following in vitro assays: mouse thymocyte proliferation (LAF assay), stimulation of collagenase and prostaglandin (PG) E2 synthesis by articular chondrocytes, and stimulation of PGE2 synthesis by fibroblasts. In all these assays, CM gave a response similar to an IL-1 preparation. This ability to mimic IL-1 suggests an important role for CM in both cell-mediated immunity and connective tissue destruction in localized juvenile periodontitis (LJP). PMID:3032779

  2. Interferon-γ promotes phagocytosis of Cryptococcus neoformans but not Cryptococcus gattii by murine macrophages.

    PubMed

    Ikeda-Dantsuji, Yurika; Ohno, Hideaki; Tanabe, Koichi; Umeyama, Takashi; Ueno, Keigo; Nagi, Minoru; Yamagoe, Satoshi; Kinjo, Yuki; Miyazaki, Yoshitsugu

    2015-12-01

    Among invasive fungal infections, cryptococcosis caused by inhalation of Cryptococcus neoformans or Cryptococcus gattii is particularly dangerous because it can disseminate to the central nervous system and cause life-threatening meningitis or meningoencephalitis. Previous reports described significant differences in the histopathological features of C. neoformans and C. gattii infection, such as greater pathogen proliferation and a limited macrophage response in mouse lung infected by C. gattii. To elucidate the difference in pathogenicity of these two Cryptococcus species, we investigated the interaction of C. neoformans and C. gattii with murine macrophages, the first line of host defense, by confocal laser microscopy. Only thin-capsulated, and not thick-capsulated C. neoformans and C. gattii were phagocytosed by macrophages. Preactivation with interferon-γ increased the phagocytic rate of thin-capsulated C. neoformans up to two-fold, but did not promote phagocytosis of thin-capsulated C. gattii. Lipopolysaccharide preactivation or Aspergillus fumigatus conidia co-incubation had no effect on internalization of thin-capsulated C. neoformans or C. gattii by macrophages. Phagocytosis of live thin-capsulated C. neoformans, but not that of live thin-capsulated C. gattii, induced interleukin-12 release from macrophages. However, phagocytosis of heat-killed or paraformaldehyde-fixed thin-capsulated C. neoformans did not increase IL-12 release, showing that the internalization of live yeast is important for initiating the immune response during C. neoformans-macrophage interactions. Our data suggest that macrophage response to C. gattii is limited compared with that to C. neoformans and that these results may partially explain the limited immune response and the greater pathogenicity of C. gattii.

  3. An OMV Vaccine Derived from a Capsular Group B Meningococcus with Constitutive FetA Expression: Preclinical Evaluation of Immunogenicity and Toxicity

    PubMed Central

    Norheim, Gunnstein; Sanders, Holly; Mellesdal, Jardar W.; Sundfør, Idunn; Chan, Hannah; Brehony, Carina; Vipond, Caroline; Dold, Chris; Care, Rory; Saleem, Muhammad; Maiden, Martin C. J.; Derrick, Jeremy P.; Feavers, Ian; Pollard, Andrew J.

    2015-01-01

    Following the introduction of effective protein-polysaccharide conjugate vaccines against capsular group C meningococcal disease in Europe, meningococci of capsular group B remain a major cause of death and can result in debilitating sequelae. The outer membrane proteins PorA and FetA have previously been shown to induce bactericidal antibodies in humans. Despite considerable antigenic variation among PorA and FetA OMPs in meningococci, systematic molecular epidemiological studies revealed this variation is highly structured so that a limited repertoire of antigenic types is congruent with the hyperinvasive meningococcal lineages that have caused most of the meningococcal disease in Europe in recent decades. Here we describe the development of a prototype vaccine against capsular group B meningococcal infection based on a N. meningitidis isolate genetically engineered to have constitutive expression of the outer membrane protein FetA. Deoxycholate outer membrane vesicles (dOMVs) extracted from cells cultivated in modified Frantz medium contained 21.8% PorA protein, 7.7% FetA protein and 0.03 μg LPS per μg protein (3%). The antibody response to the vaccine was tested in three mouse strains and the toxicological profile of the vaccine was tested in New Zealand white rabbits. Administration of the vaccine, MenPF-1, when given by intramuscular injection on 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 μg/dose, with no evidence of systemic toxicity. These data indicated that the MenPF-1 vaccine had a toxicological profile suitable for testing in a phase I clinical trial. PMID:26390123

  4. Prevalence, capsular type and antimicrobial susceptibility of Streptococcus suis isolated from slaughter pigs in Korea.

    PubMed Central

    Han, D U; Choi, C; Ham, H J; Jung, J H; Cho, W S; Kim, J; Higgins, R; Chae, C

    2001-01-01

    This study was undertaken to determine the prevalence, capsular serotype, and antimicrobial susceptibility of Streptococcus suis isolated from slaughter pigs. Capsular serotype and antimicrobial susceptibility were determined by coagglutination test and agar dilution minimum inhibitory concentration, respectively. Streptococcus suis was isolated from 55 of the 406 palatine tonsillar samples tested (13.8%) and 14 of the 29 sampled herds (48.3%). Of the 55 isolates recovered from slaughter pigs, 26 (47.3%) were untypeable. Of the remaining 29 isolates, capsular serotypes 9 (9 isolates) and 16 (4 isolates) were the most common, followed by capsular serotypes 4 (3 isolates) and 7 (3 isolates). Every capsulated isolate was typeable and no palatine tonsillar sample yielded more than one serotype. Most of isolates were susceptible to low concentrations (MIC90) of amoxicillin (2 microg/mL), ceftiofur (1 microg/mL), and penicillin (1 microg/mL). No correlation was found between antimicrobial susceptibility and capsular serotype. PMID:11480519

  5. Tumor necrosis factor-inducing activities of Cryptococcus neoformans components.

    PubMed Central

    Delfino, D; Cianci, L; Migliardo, M; Mancuso, G; Cusumano, V; Corradini, C; Teti, G

    1996-01-01

    Cryptococcus neoformans-induced tumor necrosis factor alpha (TNF-alpha) production may lead to increased human immunodeficiency virus replication in patients with AIDS. In order to identify cryptococcal components that are predominantly responsible for stimulating TNF production, various concentrations of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), mannoproteins (MP), and alpha(1-3) [corrected] glucan were added to whole-blood cultures. All of the cryptococcal components tested, as well as whole heat-killed cryptococci, were capable of inducing TNF-alpha release in a dose-dependent manner. MP were significantly more potent than any of the other cryptococcal components tested or heat-killed cryptococci in stimulating TNF-alpha production (P < 0.05). GXM, in contrast, was significantly less potent in this activity than either GalXM or MP (P < 0.05). As little as 0.5 microg of MP per ml was sufficient to produce moderate but significant elevations of TNF-alpha release. Maximal MP-induced TNF-alpha levels were similar to those induced by Salmonella enteritidis lipopolysaccharide, our positive control. Further experiments using isolated leukocytes suggested that monocytes were the cell population mainly responsible for TNF-alpha production, although the participation of other cell types could not be excluded. The presence of complement-sufficient plasma was a necessary requirement for TNF-alpha induction by GXM, GalXM, and low doses of MP. High MP concentrations (100 microg/ml) were also capable of stimulating TNF-alpha production in the absence of plasma. These data indicate that soluble products released by C. neoformans are capable of inducing TNF-alpha secretion in human leukocytes. This may be clinically relevant, since high concentrations of such products are frequently found in the body fluids of AIDS patients infected with C. neoformans. PMID:8945566

  6. Endophytic fungal compounds active against Cryptococcus neoformans and C. gattii.

    PubMed

    Pereira, Cristiane B; de Oliveira, Djalma M; Hughes, Alice Fs; Kohlhoff, Markus; LA Vieira, Mariana; Martins Vaz, Aline B; Ferreira, Mariana C; Carvalho, Camila R; Rosa, Luiz H; Rosa, Carlos A; Alves, Tânia Ma; Zani, Carlos L; Johann, Susana; Cota, Betania B

    2015-07-01

    Infections with Cryptococcus are invasive mycoses associated with significant morbidity and mortality, mainly in immunosuppressed patients. Several drugs have been introduced to combat these opportunistic infections. However, resistance of this organism to antifungal drugs has increased, causing difficulties in the treatment. The goal of this work was to evaluate the antifungal activity of ethanol extracts from endophytic fungi isolated from plants collected from different Brazilian ecosystems and to perform the fractionation of the most promising extract. Four-hundred fungal extracts were investigated by microdilution broth assays against Cryptococcus neoformans and Cryptococcus gattii at a concentration of 500 μg ml(-1). Among them, the extract of Mycosphaerella sp. UFMGCB 2032, an endophytic fungus isolated from the plant Eugenia bimarginata DC. (Myrtaceae) exhibited outstanding antifungal activity against C. neoformans and C. gattii, with MIC values of 31.2 μg ml(-1) and 7.8 μg ml(-1), respectively. The fractionation of this extract using liquid-liquid partitioning and semi-preparative HPLC afforded two eicosanoic acids with antifungal activity, compound 1, (2S,3R,4R)-(E)-2-amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxoeicos-6,12-dienoic acid with MIC values ranging from 1.3-2.50 μg ml(-1), and compound 2, known as myriocin, with MIC values of 0.5 μg ml(-1) against C. neoformans and C. gattii. These compounds are reported for the first time in the Mycosphaerella genus.

  7. Human Neutrophil-Mediated Nonoxidative Antifungal Activity against Cryptococcus neoformans

    PubMed Central

    Mambula, Salamatu S.; Simons, Elizabeth R.; Hastey, Ryan; Selsted, Michael E.; Levitz, Stuart M.

    2000-01-01

    It has long been appreciated that polymorphonuclear leukocytes (PMN) kill Cryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing of C. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation of C. neoformans with these fractions for 18 h resulted in percents inhibition of growth of 67.4 ± 3.4, 84.6 ± 4.4, and 29.2 ± 10.5 (mean ± standard error, n = 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C4 column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules. PMID:11035733

  8. Natural cellular resistance of beige mice against Cryptococcus neoformans

    SciTech Connect

    Hidore, M.R.; Murphy, J.W.

    1986-12-01

    Previous reports have demonstrated that natural killer (NK) cells are capable of inhibiting the growth of Cryptococcus neoformans in vitro, and recent studies indicate that adoptively transferred NK cell-enriched spleen cell populations enhance clearance of cryptococci from the tissues of cyclophosphamide-pretreated recipients. The primary objective of these studies was to confirm that NK cells participate in early clearance of C. neoformans in vivo. Secondarily, the anti-cryptococcal activities of polymorphonuclear leukocytes and macrophages were examined. Seven-week-old C57BL/6 bg/+ mice, which have normal levels of NK cell activity, were compared with their bg/bg littermates, which have impaired NK cell function. One and 3 days after injecting both groups of mice i.v. with 2 x 10/sup 4/ cryptococci, the authors assessed the NK cell activities in spleens, lungs, and livers and clearance of the organism from corresponding tissues as determined by the mean log/sub 1//sup 0/ numbers of cryptococcal colony-forming units (CFU) per organ. Although the data indicated a correlation between early clearance of cryptococci from tissues and levels of NK cell activities in the corresponding tissues, it was also possible that differences in phagocytic cell function between the bg/+ and bg/bg animals could account for the observed differences in clearance of cryptococci from the tissues. These data indicate that NK cells were the effector cells responsible for enhanced early clearance of cryptococci from the tissues of bg/+ animals when compared with clearance from the tissues of the bg/bg littermates. Furthermore, they confirm the hypothesis that NK cells can affect C. neoformans under in vivo conditions.

  9. Effect of nonablative laser energy on joint capsular properties

    NASA Astrophysics Data System (ADS)

    Hayashi, Kei; Markel, Mark D.; Thabit, George, III; Bogdanske, John J.; Thielke, Robert J.

    1995-05-01

    Recent scientific studies evaluating laser energy for tissue welding and thermokeratoplasty have demonstrated that the application of laser energy at non-ablative levels can alter collagen's structural and biochemical properties. The application of non-ablative laser to the human shoulder joint capsule in patients with glenohumeral instability has been found to enhance stability of the joint. Based on the collective findings of these studies, we hypothesized that thermal modification of dense collagenous tissues such as joint capsule, ligament, and tendon can be achieved by applying non-ablative laser energy. The purpose of this study was to evaluate the effect of laser energy at non-ablative levels on joint capsular mechanical properties in an in vitro rabbit model. Twelve mature New Zealand white rabbits, ranging from 3.73 to 5.33 kg (4.49 +/- 0.44; mean +/- SD), were used for this experiment. Animals were euthanized and two 5 mm X 20 mm specimens were collected from the medial and lateral portion of the femoropatellar joint of each rabbit under a dissecting microscope; therefore four specimens were collected from each rabbit (right medial, right lateral, left medial, left lateral). Specimens were divided into four groups using a randomized block design; a control group and 3 laser power settings (5 watts (5 W), 10 watts (10 W), 15 watts (15 W)). Laser energy was applied using the Ho:YAG laser in four transverse passes across the tissue at a velocity of 2 mm/sec and distance from the tip of the handpiece to the synovial surface of the specimen set at 1.5 mm in a 37 degree(s)C tissue bath of lactated Ringer's solution. Forty-eight specimens (n equals 12) were mechanically tested to determine single cycle structural properties (stiffness) and viscoelastic properties (% relaxation) before and after laser treatment. Shrinkage of the tissue and the loads required to return specimens to their original length were recorded after laser treatment. The application of laser

  10. An encapsulation of iron homeostasis and virulence in Cryptococcus neoformans.

    PubMed

    Kronstad, James W; Hu, Guanggan; Jung, Won Hee

    2013-09-01

    Vertebrate hosts actively sequester iron, and fungal and other pathogens must therefore adapt to a severe limitation in iron availability to cause disease. Recent studies reveal that the pathogenic fungus Cryptococcus neoformans overcomes iron limitation by multiple mechanisms that target transferrin and heme. The regulation of iron uptake is mediated by an interconnected set of transcription factors that include the master iron regulator Cir1 and the pH-responsive factor Rim101. These factors integrate iron homeostasis with a myriad of other functions including pH sensing, nutrient and stress signaling pathways, virulence factor elaboration, and cell wall biogenesis.

  11. SUPPRESSION OF BLOOD GROUP AGGLUTINABILITY OF HUMAN ERYTHROCYTES BY CERTAIN BACTERIAL POLYSACCHARIDES

    PubMed Central

    Ceppellini, Ruggero; Landy, Maurice

    1963-01-01

    Erythrocytes coated with bacterial capsular polysaccharides, notably the Vi antigen, were no longer agglutinated by antibodies directed against the various antigens native to the red cell surface. These effects could not be attributed to prevention of antibody uptake even though in some systems the uptake of antibody was diminished. In fact, agglutination by Rh-incomplete antibody was brought back to the original titer only after the sensitized Vi-coated cells had been subjected to ten alternating exposures to globulin and antiglobulin. Hemagglutination by Newcastle, mumps, and influenza viruses was also suppressed. Erythrocytes coated with Vi polysaccharide assumed the distinctive physicochemical attributes of this acidic polymer which results in a stabilization of the erythrocyte suspension as manifested by increased electrophoretic mobility and a striking decrease in the rate of sedimentation. Among the possible models for explaining the nature of the Vi effect on immune agglutination, the data favor interference with lattice formation. PMID:14019651

  12. STAT1 signaling within macrophages is required for antifungal activity against Cryptococcus neoformans.

    PubMed

    Leopold Wager, Chrissy M; Hole, Camaron R; Wozniak, Karen L; Olszewski, Michal A; Mueller, Mathias; Wormley, Floyd L

    2015-12-01

    Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an opportunistic fungal pathogen that primarily affects AIDS patients and patients undergoing immunosuppressive therapy. In immunocompromised individuals, C. neoformans can lead to life-threatening meningoencephalitis. Studies using a virulent strain of C. neoformans engineered to produce gamma interferon (IFN-γ), denoted H99γ, demonstrated that protection against pulmonary C. neoformans infection is associated with the generation of a T helper 1 (Th1)-type immune response and signal transducer and activator of transcription 1 (STAT1)-mediated classical (M1) macrophage activation. However, the critical mechanism by which M1 macrophages mediate their anti-C. neoformans activity remains unknown. The current studies demonstrate that infection with C. neoformans strain H99γ in mice with macrophage-specific STAT1 ablation resulted in severely increased inflammation of the pulmonary tissue, a dysregulated Th1/Th2-type immune response, increased fungal burden, deficient M1 macrophage activation, and loss of protection. STAT1-deficient macrophages produced significantly less nitric oxide (NO) than STAT1-sufficient macrophages, correlating with an inability to control intracellular cryptococcal proliferation, even in the presence of reactive oxygen species (ROS). Furthermore, macrophages from inducible nitric oxide synthase knockout mice, which had intact ROS production, were deficient in anticryptococcal activity. These data indicate that STAT1 activation within macrophages is required for M1 macrophage activation and anti-C. neoformans activity via the production of NO.

  13. Inhibition of heat-shock protein 90 enhances the susceptibility to antifungals and reduces the virulence of Cryptococcus neoformans/Cryptococcus gattii species complex.

    PubMed

    Cordeiro, Rossana de Aguiar; Evangelista, Antonio José de Jesus; Serpa, Rosana; Marques, Francisca Jakelyne de Farias; de Melo, Charlline Vládia Silva; de Oliveira, Jonathas Sales; Franco, Jônatas da Silva; de Alencar, Lucas Pereira; Bandeira, Tereza de Jesus Pinheiro Gomes; Brilhante, Raimunda Sâmia Nogueira; Sidrim, José Júlio Costa; Rocha, Marcos Fébio Gadelha

    2016-02-01

    Heat-shock proteins (Hsps) are chaperones required for the maintenance of cellular homeostasis in different fungal pathogens, playing an important role in the infectious process. This study investigated the effect of pharmacological inhibition of Hsp90 by radicicol on the Cryptococcus neoformans/Cryptococcus gattii species complex--agents of the most common life-threatening fungal infection amongst immunocompromised patients. The influence of Hsp90 inhibition was investigated regarding in vitro susceptibility to antifungal agents of planktonic and sessile cells, ergosterol concentration, cell membrane integrity, growth at 37 °C, production of virulence factors in vitro, and experimental infection in Caenorhabditis elegans. Hsp90 inhibition inhibited the in vitro growth of planktonic cells of Cryptococcus spp. at concentrations ranging from 0.5 to 2 μg ml(-1) and increased the in vitro inhibitory effect of azoles, especially fluconazole (FLC) (P < 0.05). Inhibition of Hsp90 also increased the antifungal activity of azoles against biofilm formation and mature biofilms of Cryptococcus spp., notably for Cryptococcus gattii. Furthermore, Hsp90 inhibition compromised the permeability of the cell membrane, and reduced planktonic growth at 37 °C and the capsular size of Cryptococcus spp. In addition, Hsp90 inhibition enhanced the antifungal activity of FLC during experimental infection using Caenorhabditis elegans. Therefore, our results indicate that Hsp90 inhibition can be an important strategy in the development of new antifungal drugs.

  14. Putatively novel serotypes and the potential for reduced vaccine effectiveness: capsular locus diversity revealed among 5405 pneumococcal genomes

    PubMed Central

    van Tonder, Andries J.; Bray, James E.; Quirk, Sigríður J.; Haraldsson, Gunnsteinn; Jolley, Keith A.; Maiden, Martin C. J.; Hoffmann, Steen; Bentley, Stephen D.; Haraldsson, Ásgeir; Erlendsdóttir, Helga; Kristinsson, Karl G.; Brueggemann, Angela B.

    2017-01-01

    The pneumococcus is a leading global pathogen and a key virulence factor possessed by the majority of pneumococci is an antigenic polysaccharide capsule (‘serotype’), which is encoded by the capsular (cps) locus. Approximately 100 different serotypes are known, but the extent of sequence diversity within the cps loci of individual serotypes is not well understood. Investigating serotype-specific sequence variation is crucial to the design of sequence-based serotyping methodology, understanding pneumococcal conjugate vaccine (PCV) effectiveness and the design of future PCVs. The availability of large genome datasets makes it possible to assess population-level variation among pneumococcal serotypes and in this study 5405 pneumococcal genomes were used to investigate cps locus diversity among 49 different serotypes. Pneumococci had been recovered between 1916 and 2014 from people of all ages living in 51 countries. Serotypes were deduced bioinformatically, cps locus sequences were extracted and variation was assessed within the cps locus, in the context of pneumococcal genetic lineages. Overall, cps locus sequence diversity varied markedly: low to moderate diversity was revealed among serogroups/types 1, 3, 7, 9, 11 and 22; whereas serogroups/types 6, 19, 23, 14, 15, 18, 33 and 35 displayed high diversity. Putative novel and/or hybrid cps loci were identified among all serogroups/types apart from 1, 3 and 9. This study demonstrated that cps locus sequence diversity varied widely between serogroups/types. Investigation of the biochemical structure of the polysaccharide capsule of major variants, particularly PCV-related serotypes and those that appear to be novel or hybrids, is warranted. PMID:28133541

  15. Inositol phosphosphingolipid phospholipase C1 regulates plasma membrane ATPase (Pma1) stability in Cryptococcus neoformans.

    PubMed

    Farnoud, Amir M; Mor, Visesato; Singh, Ashutosh; Del Poeta, Maurizio

    2014-11-03

    Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.

  16. Molecular Typing of IberoAmerican Cryptococcus neoformans Isolates

    PubMed Central

    Castañeda, Alexandra; Jackson, Stuart; Huynh, Matthew; Castañeda, Elizabeth

    2003-01-01

    A network was established to acquire basic knowledge of Cryptococcus neoformans in IberoAmerican countries. To this effect, 340 clinical, veterinary, and environmental isolates from Argentina, Brazil, Chile, Colombia, Mexico, Peru, Venezuela, Guatemala, and Spain were typed by using M13 polymerase chain reaction-fingerprinting and orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphsm analysis with HhaI and Sau96I in a double digest. Both techniques grouped all isolates into eight previously established molecular types. The majority of the isolates, 68.2% (n=232), were VNI (var. grubii, serotype A), which accords with the fact that this variety causes most human cryptococcal infections worldwide. A smaller proportion, 5.6% (n=19), were VNII (var. grubii, serotype A); 4.1% (n=14), VNIII (AD hybrid), with 9 isolates having a polymorphism in the URA5 gene; 1.8% (n=6), VNIV (var. neoformans, serotype D); 3.5% (n=12), VGI; 6.2% (n=21), VGII; 9.1% (n=31), VGIII, and 1.5% (n=5) VGIV, with all four VG types containing var. gattii serotypes B and C isolates. PMID:12603989

  17. Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology.

    PubMed

    Guerra, Caroline Rezende; Ishida, Kelly; Nucci, Marcio; Rozental, Sonia

    2012-08-01

    Cryptococcus neoformans is an encapsulated fungus that causes cryptococcosis. Central nervous system infection is the most common clinical presentation followed by pulmonary, skin and eye manifestations. Cryptococcosis is primarily treated with amphotericin B (AMB), fluconazole (FLC) and itraconazole (ITC). In the present work, we evaluated the in vitro effect of terbinafine (TRB), an antifungal not commonly used to treat cryptococcosis. We specifically examined the effects of TRB, either alone or in conjunction with AMB, FLC and ITC, on clinical C. neoformans isolates, including some isolates resistant to AMB and ITC. Broth microdilution assays showed that TRB was the most effective drug in vitro. Antifungal combinations demonstrated synergism of TRB with AMB, FLC and ITC. The drug concentrations used for the combination formulations were as much as 32 and 16-fold lower than the minimum inhibitory concentration (MIC) values of FLC and AMB alone, respectively. In addition, calcofluor white staining revealed the presence of true septa in hyphae structures that were generated after drug treatment. Ultrastructural analyses demonstrated several alterations in response to drug treatment, such as cell wall alterations, plasma membrane detachment, presence of several cytoplasmic vacuoles and mitochondrial swelling. Therefore, we believe that the use of TRB alone or in combination with AMB and azoles should be explored as an alternative treatment for cryptococcosis patients who do not respond to standard therapies.

  18. Mannoproteins from Cryptococcus neoformans promote dendritic cell maturation and activation.

    PubMed

    Pietrella, Donatella; Corbucci, Cristina; Perito, Stefano; Bistoni, Giovanni; Vecchiarelli, Anna

    2005-02-01

    Our previous data show that mannoproteins (MPs) from Cryptococcus neoformans are able to induce protective responses against both C. neoformans and Candida albicans. Here we provide evidence that MPs foster maturation and activation of human dendritic cells (DCs). Maturation was evaluated by the ability of MPs to facilitate expression of costimulatory molecules such as CD40, CD86, CD83, and major histocompatibility complex classes I and II and to inhibit receptors such as CD14, CD16, and CD32. Activation of DCs was measured by the capacity of MPs to promote interleukin-12 and tumor necrosis factor alpha secretion. DC-induced maturation and interleukin-12 induction are largely mediated by engagement of mannose receptors and presume MP internalization and degradation. DC activation leads to IkappaBalpha phosphorylation, which is necessary for nuclear factor kappaB transmigration into the nucleus. MP-loaded DCs are efficient stimulators of T cells and show a remarkable capacity to promote CD4 and CD8 proliferation. In conclusion, we have evidenced a novel regulatory role of MPs that promotes their candidacy as a vaccine against fungi.

  19. Mannoproteins from Cryptococcus neoformans Promote Dendritic Cell Maturation and Activation

    PubMed Central

    Pietrella, Donatella; Corbucci, Cristina; Perito, Stefano; Bistoni, Giovanni; Vecchiarelli, Anna

    2005-01-01

    Our previous data show that mannoproteins (MPs) from Cryptococcus neoformans are able to induce protective responses against both C. neoformans and Candida albicans. Here we provide evidence that MPs foster maturation and activation of human dendritic cells (DCs). Maturation was evaluated by the ability of MPs to facilitate expression of costimulatory molecules such as CD40, CD86, CD83, and major histocompatibility complex classes I and II and to inhibit receptors such as CD14, CD16, and CD32. Activation of DCs was measured by the capacity of MPs to promote interleukin-12 and tumor necrosis factor alpha secretion. DC-induced maturation and interleukin-12 induction are largely mediated by engagement of mannose receptors and presume MP internalization and degradation. DC activation leads to IκBα phosphorylation, which is necessary for nuclear factor κB transmigration into the nucleus. MP-loaded DCs are efficient stimulators of T cells and show a remarkable capacity to promote CD4 and CD8 proliferation. In conclusion, we have evidenced a novel regulatory role of MPs that promotes their candidacy as a vaccine against fungi. PMID:15664921

  20. Incorporation of bacterial extracellular polysaccharide by black fly larvae (Simuliidae)

    USGS Publications Warehouse

    Couch, C.A.; Meyer, J.L.; Hall, R.O.

    1996-01-01

    Black fly larvae (Simulium) assimilated, with high efficiency (80-90%), bacterial extracellular polysaccharide (EPS) extracted from laboratory cultures of a pseudomonad isolated from the Ogeechee River. Incorporation was traced using 13C-labelled EPS offered to larvae as a coating on a mixture of 1-??m latex beads and kaolin particles. These EPS-coated particles were used to simulate natural particles, both living and dead. Solubility, protein, and nitrogen content of the EPS suggested it was a slime rather than a capsular polysaccharide. Glycosyl composition of the EPS was glucose and galactose in ?? and ?? linkages, with pyruvate, succinate, and possibly malonate constituent groups. To evaluate the incorporation of C derived from protein associated with the EPS matrix, feeding experiments were conducted using EPS with and without proteins extracted. Black fly larvae incorporated 7.2 ??g EPS C larva-1 d-1 from EPS that did not have proteins extracted, and 19.5 ??g EPS C larva-1 d-1 from EPS with proteins extracted. Carbon in protein that is typically associated with EPS was not solely or selectively incorporated. EPS incorporation rates are similar to rates of cellular bacterial carbon incorporation previously estimated for Ogeechee River black fly larvae. If EPS is generally available as a food resource, the importance of bacteria in detrital food webs may be underestimated by studies that examine only the consumption of bacterial cells.

  1. The Relationship of Bacterial Biofilms and Capsular Contracture in Breast Implants

    PubMed Central

    Ajdic, Dragana; Zoghbi, Yasmina; Gerth, David; Panthaki, Zubin J.; Thaller, Seth

    2016-01-01

    Capsular contracture is a common sequelae of implant-based breast augmentation. Despite its prevalence, the etiology of capsular contracture remains controversial. Numerous studies have identified microbial biofilms on various implantable materials, including breast implants. Furthermore, biofilms have been implicated in subclinical infections associated with other surgical implants. In this review, we discuss microbial biofilms as a potential etiology of capsular contracture. The review also outlines the key diagnostic modalities available to identify the possible infectious agents found in biofilm, as well as available preventative and treatment measures. PMID:26843099

  2. A novel microactuator for microbiopsy in capsular endoscopes

    NASA Astrophysics Data System (ADS)

    Park, Sunkil; Koo, Kyo-in; Bang, Seoung Min; Youp Park, Jeong; Song, Si Young; 'Dan'Cho, Dongil

    2008-02-01

    This paper presents a LiGA (a German acronym for lithographie, galvanoformung, abformung) process based microactuator to be used for microbiopsy in capsular endoscopes. This microactuator is designed to be integrated into a capsular endoscope and to extract tissue samples inside the small gastrointestine which a conventional endoscope cannot reach. The proposed microactuator was fabricated as a cylindrical shape of diameter 10 mm and length 1.8 mm. This actuator consists of three parts: a microbiopsy part with a microspike, an actuating part with a torsion spring and a triggering part with a shape memory alloy (SMA) heating wire and polymer string. In order to extract sample tissue, a microspike in the developed actuator moves forward and backward using the slider-crank mechanism. For low power consumption triggering, a polymer-melting scheme was applied. The SMA heating wire consumed approximately 1.5 V × 160 mA × 1 second (66.67 µWH) for triggering. The precise components of the microactuator were fabricated using the LiGA process in order to overcome the limitations in accuracy of conventional precision machining. The developed microactuator was evaluated by extracting tissue samples from the small intestine of a pig ex vivo, and examining the tissue with hematoxylin and eosin (H&E) staining protocol. The experimental tests demonstrated that the developed microactuator with microspike successfully extracted tissue samples from the pig's small intestines. This paper is an extended version of an oral paper presented at Transducers 2007: 14th International Conference on Solid State Sensors and Actuators, Lyon, France, 10-14 June, 2007.

  3. Kinetics of lymphocyte transformation in mice immunized with viable avirulent forms of Cryptococcus neoformans.

    PubMed Central

    Fromtling, R A; Blackstock, R; Hall, N K; Bulmer, G S

    1979-01-01

    A murine model was developed to study the cell-mediated immune response of mice immunized with one of two live, avirulent forms of Cryptococcus neoformans: a nonencapsulated mutant and a thinly encapsulated pseudohyphal variant. A lymphocyte transformation assay was used to evaluate the cellular response of control and sensitized spleen cells after in vitro incubation with three merthiolate-killed whole-cell antigens of C. neoformans. An antigen-to-spleen cell ratio of 10:1 and 5 days of incubation of antigen-spleen cell mixtures were established as optimal conditions for maximum lymphocyte transformation. Maximum responses occurred from 2 to 3 weeks after the last of eight weekly intraperitoneal inoculations of C. neoformans. This assay provided an accurate, reproducible method of studying cell-mediated immunity to C. neoformans, and applications to the study of cryptococcal pathogenesis are proposed. PMID:378854

  4. Aging: an emergent phenotypic trait that contributes to the virulence of Cryptococcus neoformans.

    PubMed

    Bouklas, Tejas; Fries, Bettina C

    2015-01-01

    The pathogenic fungus, Cryptococcus neoformans, is known to undergo phenotypic variation, which affects its virulence in the host. Recent investigations on C. neoformans cells in humans have validated the concept that phenotypic variation is present and relevant for the outcome of chronic cryptococcosis. The C. neoformans capsule is not the only trait that varies among strains. An emerging variant is the "old cell phenotype" generated when C. neoformans undergoes replicative aging. This phenotype, which other than larger size also exhibits a thickened cell wall, inhibits phagocytosis and killing by antifungals in vitro. In concert with the finding that old cells accumulate in vivo, this emergent trait could have significant impact on cryptococcal virulence and infection, and contribute to treatment failure.

  5. Aging as an emergent factor that contributes to phenotypic variation in Cryptococcus neoformans.

    PubMed

    Bouklas, Tejas; Fries, Bettina C

    2015-05-01

    Cryptococcus neoformans, similar to other eukaryotes, undergoes replicative aging. Replicative life spans have been determined for clinical C. neoformans strains, and although they are a reproducible trait, life spans vary considerably among strains. C. neoformans has been proposed as an ideal model organism to investigate the contribution of replicative aging in a fungal pathogen population to emerging phenotypic variation during chronic cryptococcal infections. C. neoformans cells of advanced generational age manifest a distinct phenotype; specifically, a larger cell size, a thicker cell wall, drug resistance, as well as resistance to hydrogen peroxide-mediated killing. Consequently, old cells are selected in the host environment during chronic infection and aging could be an unanticipated mechanism of pathogen adaptation that contributes to persistent disease. Aging as a natural process of phenotypic variation should be further studied as it likely is also relevant for other eukaryotic pathogen populations that undergo asymmetric replicative aging.

  6. Genotypes of Clinical and Environmental Isolates of Cryptococcus neoformans and Cryptococcus gattii in Korea

    PubMed Central

    Park, So Hae; Choi, Seok Cheol; Lee, Kyung Won; Kim, Mi-Na

    2015-01-01

    Multilocus sequence typing analysis was applied to determine the genotypes of 147 (137 clinical and 10 environmental) Cryptococcus neoformans and three clinical Cryptococcus gattii isolates from 1993 to 2014 in Korea. Among the 137 clinical isolates of C. neoformans, the most prevalent genotype was ST5 (n = 131), followed by ST31 (n = 5) and ST127 (n = 1). Three C. gattii strains were identified as ST57, ST7, and ST113. All environmental isolates were identified as C. neoformans with two genotypes, ST5 (n = 7) and ST31 (n = 3). Our results show that C. neoformans isolates in Korea are genetically homogeneous, and represent a close genetic relationship between clinical and environmental isolates. PMID:26539057

  7. The Role of Bacterial Protein Tyrosine Phosphatases in the Regulation of the Biosynthesis of Secreted Polysaccharides

    PubMed Central

    Morona, Renato

    2014-01-01

    Abstract Significance: Tyrosine phosphorylation and associated protein tyrosine phosphatases are gaining prominence as critical mechanisms in the regulation of fundamental processes in a wide variety of bacteria. In particular, these phosphatases have been associated with the control of the biosynthesis of capsular polysaccharides and extracellular polysaccharides, critically important virulence factors for bacteria. Recent Advances: Deletion and overexpression of the phosphatases result in altered polysaccharide biosynthesis in a range of bacteria. The recent structures of associated auto-phosphorylating tyrosine kinases have suggested that the phosphatases may be critical for the cycling of the kinases between monomers and higher order oligomers. Critical Issues: Additional substrates of the phosphatases apart from cognate kinases are currently being identified. These are likely to be critical to our understanding of the mechanism by which polysaccharide biosynthesis is regulated. Future Directions: Ultimately, these protein tyrosine phosphatases are an attractive target for the development of novel antimicrobials. This is particularly the case for the polymerase and histidinol phosphatase family, which is predominantly found in bacteria. Furthermore, the determination of bacterial tyrosine phosphoproteomes will likely help to uncover the fundamental roles, mechanism, and critical importance of these phosphatases in a wide range of bacteria. Antioxid. Redox Signal. 20, 2274–2289. PMID:24295407

  8. Magnesium Ion Acts as a Signal for Capsule Induction in Cryptococcus neoformans.

    PubMed

    Rathore, Sudarshan S; Raman, Thiagarajan; Ramakrishnan, Jayapradha

    2016-01-01

    Cryptococcal meningitis caused by Cryptococcus neoformans, is a common opportunistic neural infection in immunocompromised individuals. Cryptococcus meningitis is associated with fungal burden with larger capsule size in cerebrospinal fluid (CSF). To understand the role of CSF constituents in capsule enlargement, we have evaluated the effect of artificial CSF on capsule induction in comparison with various other capsule inducing media. Two different strains of C. neoformans, an environmental and a clinical isolates were used in the present study. While comparing the various capsule inducing media for the two different strains of C. neoformans, it was observed that the capsule growth was significantly increased when grown in artificial CSF at pH 5.5, temperature 34°C for ATCC C. neoformans and 37°C for Clinical C. neoformans and with an incubation period of 72 h. In addition, artificial CSF supports biofilm formation in C. neoformans. While investigating the individual components of artificial CSF, we found that Mg(2+) ions influence the capsule growth in both environmental and clinical strains of C. neoformans. To confirm our results we studied the expression of four major CAP genes namely, CAP10, CAP59, CAP60, and CAP64 in various capsule inducing media and in different concentrations of Mg(2+) and Ca(2+). Our results on gene expression suggest that, Mg(2+) does have an effect on CAP gene expression, which are important for capsule biosynthesis and virulence. Our findings on the role of Mg(2+) ion as a signal for capsule induction will promote a way to elucidate the control mechanisms for capsule biosynthesis in C. neoformans.

  9. The Cell Wall and Membrane of Cryptococcus neoformans Possess a Mitogen for Human T Lymphocytes

    PubMed Central

    Mody, Christopher H.; Wood, Cynthia J.; Syme, Rachel M.; Spurrell, Jason C. L.

    1999-01-01

    The mechanism of human T-lymphocyte activation by the pathogenic yeast Cryptococcus neoformans has not been established. Previous investigations have suggested that C. neoformans contains a mitogen for T lymphocytes, while other investigators have attributed lymphocyte proliferation in vitro to a recall antigen. Because of the potential importance of the mechanism of T-cell activation for our understanding of the immune response to C. neoformans, the present studies were performed to determine whether C. neoformans contains a mitogen for T lymphocytes. C. neoformans stimulates fetal blood lymphocytes to proliferate and stimulates proliferation of CD45RA+ cells from adults, indicating that it stimulates naive T cells. The T-cell response to C. neoformans was dependent upon the presence of accessory cells. However, allogeneic cells were sufficient for accessory cell function, indicating that the response was not major histocompatibility complex restricted. The percentage of T cells in the cell cycle was higher than that with the recall antigen tetanus toxoid but lower than that with the mitogenic lectin phytohemagglutinin A or the superantigen Staphylococcus enterotoxin B. Precursor frequency analysis established that 1 in 7,750 ± 2,270 T cells proliferated in response to the cryptococcal cell wall and membrane. Compared to the case for most mitogens or superantigens, the proliferative response is late and the number of T cells that enter the cell cycle and the precursor frequency are low, indicating that the mitogenic effect is modest. However, the mitogenic effect of C. neoformans should be considered when interpreting the immune response to C. neoformans, since even weak mitogens can have profound effects on host defense. PMID:9916111

  10. Magnesium Ion Acts as a Signal for Capsule Induction in Cryptococcus neoformans

    PubMed Central

    Rathore, Sudarshan S.; Raman, Thiagarajan; Ramakrishnan, Jayapradha

    2016-01-01

    Cryptococcal meningitis caused by Cryptococcus neoformans, is a common opportunistic neural infection in immunocompromised individuals. Cryptococcus meningitis is associated with fungal burden with larger capsule size in cerebrospinal fluid (CSF). To understand the role of CSF constituents in capsule enlargement, we have evaluated the effect of artificial CSF on capsule induction in comparison with various other capsule inducing media. Two different strains of C. neoformans, an environmental and a clinical isolates were used in the present study. While comparing the various capsule inducing media for the two different strains of C. neoformans, it was observed that the capsule growth was significantly increased when grown in artificial CSF at pH 5.5, temperature 34°C for ATCC C. neoformans and 37°C for Clinical C. neoformans and with an incubation period of 72 h. In addition, artificial CSF supports biofilm formation in C. neoformans. While investigating the individual components of artificial CSF, we found that Mg2+ ions influence the capsule growth in both environmental and clinical strains of C. neoformans. To confirm our results we studied the expression of four major CAP genes namely, CAP10, CAP59, CAP60, and CAP64 in various capsule inducing media and in different concentrations of Mg2+ and Ca2+. Our results on gene expression suggest that, Mg2+ does have an effect on CAP gene expression, which are important for capsule biosynthesis and virulence. Our findings on the role of Mg2+ ion as a signal for capsule induction will promote a way to elucidate the control mechanisms for capsule biosynthesis in C. neoformans. PMID:27014245

  11. Capsular Block Syndrome Following Combined Cataract and Vitrectomy Surgery in a Patient With Intraocular Gas.

    PubMed

    Goodwin, Diamond M; Casey, Richard; Tsui, Irena

    2015-10-01

    The aim of this report is to describe the diagnosis, treatment, and prevention of the rare complication of capsular block syndrome following combined cataract and vitrectomy surgery in a patient with intraocular gas.

  12. Occurrence and susceptibilities to disinfectants of Cryptococcus neoformans in fecal droppings from pigeons in Bangkok, Thailand.

    PubMed

    Krangvichain, Prathomporn; Niyomtham, Waree; Prapasarakul, Nuvee

    2016-03-01

    Cryptococcus neoformans is an opportunistic pathogenic yeast that causes meningoencephalitis and deep skin dermatitis in humans and animals. A hygienic strategy using disinfectants on environmental samples can reduce the risk to the public. The objectives were to survey the distribution of C. neoformans in pigeon fecal droppings collected in 11 districts in Bangkok during 2011-2012 and to evaluate the efficacy of three commercial disinfectant products (based on potassium monopersulfate, sodium hypochlorite and quaternary ammonium compounds, respectively). These were evaluated against pure C. neoformans and yeasts resuspended in sterile pigeon feces using the dilution-neutralization method [Europäische NORM (EN) 1656]. In total, 18 of 164 (11%) samples were positive for C. neoformans. These came from only three of the 11 districts, with a prevalence of between 13-56%. Using multiplex PCR, serotype A was the sole group found. For all disinfectants, C. neoformans mixed in feces was tolerated at a higher dose and time exposure than pure isolates. The most effective disinfectant in this study was a 0.12% quaternary ammonium compound that could rapidly eradicate the yeasts mixed in feces. This finding highlights the occurrence and distribution of C. neoformans in the capital city of Thailand and the need to prolong the duration of exposure to disinfectants with pigeon feces.

  13. Occurrence and susceptibilities to disinfectants of Cryptococcus neoformans in fecal droppings from pigeons in Bangkok, Thailand

    PubMed Central

    KRANGVICHAIN, Prathomporn; NIYOMTHAM, Waree; PRAPASARAKUL, Nuvee

    2015-01-01

    Cryptococcus neoformans is an opportunistic pathogenic yeast that causes meningoencephalitis and deep skin dermatitis in humans and animals. A hygienic strategy using disinfectants on environmental samples can reduce the risk to the public. The objectives were to survey the distribution of C. neoformans in pigeon fecal droppings collected in 11 districts in Bangkok during 2011–2012 and to evaluate the efficacy of three commercial disinfectant products (based on potassium monopersulfate, sodium hypochlorite and quaternary ammonium compounds, respectively). These were evaluated against pure C. neoformans and yeasts resuspended in sterile pigeon feces using the dilution-neutralization method [Europäische NORM (EN) 1656]. In total, 18 of 164 (11%) samples were positive for C. neoformans. These came from only three of the 11 districts, with a prevalence of between 13–56%. Using multiplex PCR, serotype A was the sole group found. For all disinfectants, C. neoformans mixed in feces was tolerated at a higher dose and time exposure than pure isolates. The most effective disinfectant in this study was a 0.12% quaternary ammonium compound that could rapidly eradicate the yeasts mixed in feces. This finding highlights the occurrence and distribution of C. neoformans in the capital city of Thailand and the need to prolong the duration of exposure to disinfectants with pigeon feces. PMID:26596636

  14. Hydroxyurea enhances post-fusion hyphal extension during sexual development in C. neoformans var. grubii.

    PubMed

    Zulkifli, M Naim; Kaur, Jan Naseer; Panepinto, John C

    2012-03-01

    Mating and sexual development in C. neoformans var. grubii strains of the H99 background is often less robust than that laboratory generated isogenic C. neoformans var. neoformans strains in the JEC21 background. In Candida albicans and Saccharomyces serevisiae, slowing of DNA synthesis and engagement of the replication stress response, such as that caused by treatment with hydroxyurea (HU), induces filamentation and pseudohyphal growth, respectively. In this study, we investigated the effect of HU treatment on C. neoformans var. grubii morphogenesis. Treatment with HU did not induce filamentation of yeast cells either in liquid culture or on solid YPD or V8 agar. In the presence of the opposite mating partner, we observed early emergence of hyphae in the presence of HU. Semi-quantitative analysis of fusion using marked strains demonstrated that no significant enhancement of fusion in the presence of HU. Transfer of fusion colonies from crosses performed in the absence of HU to V8 + HU revealed enhanced hyphal growth in the presence of HU. Analysis of expression of the target of HU, ribonucleotide reductase, revealed that a phylogenetically divergent catalytic subunit is replication stress responsive in C. neoformans. These results suggest that induction of replication stress promotes post-fusion hyphal growth of C. neoformans var. grubii strains in the H99 background.

  15. CSN1201, a subunit of the COP9 signalosome, regulates the virulence in Cryptococcus neoformans infection.

    PubMed

    Fan, Yibin; Tao, Xiaohua; Pan, Weili; Fang, Wei; Huang, Youming; Jia, Mingyan

    2016-11-30

    The COP9 signalosome (CSN) is a multisubunit protein complex, and it now has been found to participate in diverse cellular and developmental processes in various eukaryotic organisms. Cryptococcus neoformans (C. neoformans) is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immune compromised population. Here, we generated CSN deletion mutants to investigate the role in Cryptococcus infection. Compared to other CSN mutants, we identified a CSN1201 mutant exhibited severely attenuated virulence. Deletion of CSN1201 made cryptococcal cells more susceptible to nearly all in vitro stresses. Furthermore, deletion of CSN1201 obviously impaired survival of C. neoformans. At the same time, in vivo virulence assay of mouse infection models demonstrated that CSN1201 significantly enhanced the virulence of C. neoformans compared with the other CSN subunit strains, while ELISA analysis of C. neoformans infection in innate or adaptive immune response showed that deletion of CSN1201 significantly impaired cytokines and interferon expression. In vitro model of the blood-brain barrier (BBB) analysis indicated that deletion of CSN1201 reduced the invasion efficacy of Cryptococcusto cross BBB. Taken together, our findings reveal a novel mechanism of CSN1201, which plays a critical role for the virulence composite of C. neoformans, and also provides an additional yeast survival and propagation advantage in the host.

  16. Induction by Klebsiella aerogenes of a melanin-like pigment in Cryptococcus neoformans.

    PubMed

    Frases, Susana; Chaskes, Stuart; Dadachova, Ekaterina; Casadevall, Arturo

    2006-02-01

    While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37 degrees C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.

  17. Infective capacity of Cryptococcus neoformans and Cryptococcus gattii in a human astrocytoma cell line.

    PubMed

    Olave, M C; Vargas-Zambrano, J C; Celis, A M; Castañeda, E; González, J M

    2017-03-24

    Pathogenesis of cryptococcosis in the central nervous system (CNS) is a topic of ongoing research, including the mechanisms by which this fungus invades and infects the brain. Astrocytes, the most common CNS cells, play a fundamental role in the local immune response. Astrocytes might participate in cryptococcosis either as a host or by responding to fungal antigens. To determine the infectivity of Cryptococcus neoformans var. grubii and Cryptococcus gattii in a human astrocytoma cell line and the induction of major histocompatibility complex (MHC) molecules. A glioblastoma cell line was infected with C. neoformans var. grubii and C. gattii blastoconidia labelled with FUN-1 fluorescent stain. The percentage of infection and expression of HLA class I and II molecules were determined by flow cytometry. The interactions between the fungi and cells were observed by fluorescence microscopy. There was no difference between C. neoformans var. grubii and C. gattii in the percentage infection, but C. neoformans var. grubii induced higher expression of HLA class II than C. gattii. More blastoconidia were recovered from C. neoformans-infected cells than from C. gattii infected cells. Cryptococcus neoformans var. grubii may have different virulence mechanisms that allow its survival in human glia-derived cells.

  18. Microcomputer system for automatic identification of the Cryptococcus neoformans and its clinical application.

    PubMed

    Zheng, Y C; Xie, J M; Wei, B; Zhu, Z R; Wu, Y Q; Ni, S H; Tan, Z J; Luo, C M; Liu, X; Zhou, Y

    1995-01-01

    In this study, microcomputer image processing and pattern recognition technology, and the knowledge of morphology and optical characteristics of Cryptococcus neoformans were used for identification of Cryptococcus neoformans. Four groups of mice were lethally infected with standard strain, Wuhan strain, American B-2643 strain and Var. Shanghainesis of the Cryptococcus neoformans. The samples collected included mice brain, lung, kidney, liver, small intestine tissue and were observed under a light microscope. More than 600 images of the fungus were input into a microcomputer. A system of computer for automatic identification of the Cryptococcus neoformans was developed. The technique involved image preprocessing, image segmenting, coding of line-length on the edge, curve fitting, extracting of image feature, building of image library and feature data bank etc.. And then, 768 images of the clinical samples and other fungus samples whose morphological features tend to be confused with Cryptococcus neoformans were input into microcomputer and subjected to automatic identification. The Cryptococcus neoformans was accurately identified within 15 min, and the consistency rate with results of routine culture was 98%.

  19. Cryptococcus neoformans as a Model for Radioimmunotherapy of Infections

    PubMed Central

    Dadachova, Ekaterina; Casadevall, Arturo

    2011-01-01

    There is an obvious and urgent need for novel approaches to treat infectious diseases. The use of monoclonal antibodies in therapy of infectious diseases is now experiencing renewed interest. During the last 5 years radioimmunotherapy (RIT), a modality previously developed only for cancer treatment, has been successfully adapted for the treatment of experimental fungal, bacterial, and viral infections. As our model organism for studying the efficacy, mechanisms, potential toxicity, and radioresistance to RIT, as well as for comparison of RIT with the existing antimicrobial therapies we have chosen the encapsulated yeast Cryptococcus neoformans (CN). The success of RIT approach in laboratory studies provides encouragement for feasibility of therapeutically targeting microbes with labeled antibodies. In addition, the creation of “panantibodies” for RIT which would recognize antigens shared by the whole class of pathogens such as fungi, for example, would facilitate the introduction of RIT into the clinic. PMID:21747848

  20. Immunity to Cryptococcus neoformans and C. gattii during cryptococcosis.

    PubMed

    Gibson, Josie F; Johnston, Simon A

    2015-05-01

    The vast majority of infection with cryptococcal species occurs with Cryptococcus neoformans in the severely immunocompromised. A significant exception to this is the infections of those with apparently normal immune systems by Cryptococcus gattii. Susceptibility to cryptococcosis can be broadly categorised as a defect in adaptive immune responses, especially in T cell immunity. However, innate immune cells such as macrophages play a key role and are likely the primary effector cell in the killing and ultimate clearance of cryptococcal infection. In this review we discuss the current state of our understanding of how the immune system responds to cryptococcal infection in health and disease, with reference to the work communicated at the 9th International Conference on Cryptococcus and Cryptococcosis (ICCC9). We have focussed on cell mediated responses, particularly early in infection, but with the aim of presenting a broad overview of our understanding of immunity to cryptococcal infection, highlighting some recent advances and offering some perspectives on future directions.

  1. Interactions between Triazoles and Amphotericin B against Cryptococcus neoformans

    PubMed Central

    Barchiesi, Francesco; Schimizzi, Anna M.; Caselli, Francesca; Novelli, Andrea; Fallani, Stefania; Giannini, Daniele; Arzeni, Daniela; Di Cesare, Simona; Di Francesco, Luigi Falconi; Fortuna, Moira; Giacometti, Andrea; Carle, Flavia; Mazzei, Teresita; Scalise, Giorgio

    2000-01-01

    The interaction of amphotericin B (AmB) and azole antifungal agents in the treatment of fungal infections is still a controversial issue. A checkerboard titration broth microdilution-based method that adhered to the recommendations of the National Committee for Clinical Laboratory Standards was applied to study the in vitro interactions of AmB with fluconazole (FLC), itraconazole (ITC), and the new investigational triazole SCH 56592 (SCH) against 15 clinical isolates of Cryptococcus neoformans. Synergy, defined as a fractional inhibitory concentration (FIC) index of ≤0.50, was observed for 7% of the isolates in studies of the interactions of both FLC-AmB and ITC-AmB and for 33% of the isolates in studies of the SCH-AmB interactions; additivism (FICs, >0.50 to 1.0) was observed for 67, 73, and 53% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively; indifference (FICs, >1.0 to ≤2.0) was observed for 26, 20, and 14% of the isolates in studies of the FLC-AmB, ITC-AmB, and SCH-AmB interactions, respectively. Antagonism (FIC >2.0) was not observed. When synergy was not achieved, there was still a decrease, although not as dramatic, in the MIC of one or both drugs when they were used in combination. To investigate the effects of FLC-AmB combination therapy in vivo, we established an experimental model of systemic cryptococcosis in BALB/c mice by intravenous injection of cells of C. neoformans 2337, a clinical isolate belonging to serotype D against which the combination of FLC and AmB yielded an additive interaction in vitro. Both survival and tissue burden studies showed that combination therapy was more effective than FLC alone and that combination therapy was at least as effective as AmB given as a single drug. On the other hand, when cells of C. neoformans 2337 were grown in FLC-containing medium, a pronounced increase in resistance to subsequent exposures to AmB was observed. In particular, killing experiments conducted

  2. Dancing cheek to cheek: Cryptococcus neoformans and phagocytes.

    PubMed

    Zhang, Mingshun; Sun, Donglei; Shi, Meiqing

    2015-01-01

    Meningoencephalitis caused by Cryptococcus neoformans (Cn) has become one of the leading causes of mortality in AIDS patients. Understanding the interactions between Cn and phagocytes is fundamental in exploring the pathogenicity of cryptococcal meningoencephalitis. Cn may be extracellular or contained in the monocytes, macrophages, neutrophils, dendritic cells and even endothelial cells. The internalized Cn may proliferate inside the host cells, or cause the lysis of host cells, or leave the host cells via non-lytic exocytosis, or even hijack the host cells (Trojan horse) for the brain dissemination, which are regulated by microbe factors and also immune molecules. Coexistence of protective and deleterious roles of phagocytes in the progression of cryptococcosis warrant further investigation.

  3. Anterior dislocation of an empty capsular bag in a pseudophakic eye: A rare case report

    PubMed Central

    Hwang, Hyung Bin; Yim, Hye Bin; Kim, Hyun Seung

    2015-01-01

    Spontaneous intraocular lens (IOL) dislocation is uncommon in the absence of any ocular areas with zonular weakness or trauma. There have been no reports of spontaneous capsular bag dislocation into the anterior chamber without an IOL. We report a rare, interesting case of spontaneous capsular bag anterior dislocation, without an IOL, into the anterior chamber with no history of genetic disease, ocular trauma, or pseudoexfoliation that might predispose to a zonular abnormality. PMID:25971181

  4. Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl

    PubMed Central

    Franklin, Michael J.; Nivens, David E.; Weadge, Joel T.; Howell, P. Lynne

    2011-01-01

    Pseudomonas aeruginosa thrives in many aqueous environments and is an opportunistic pathogen that can cause both acute and chronic infections. Environmental conditions and host defenses cause differing stresses on the bacteria, and to survive in vastly different environments, P. aeruginosa must be able to adapt to its surroundings. One strategy for bacterial adaptation is to self-encapsulate with matrix material, primarily composed of secreted extracellular polysaccharides. P. aeruginosa has the genetic capacity to produce at least three secreted polysaccharides; alginate, Psl, and Pel. These polysaccharides differ in chemical structure and in their biosynthetic mechanisms. Since alginate is often associated with chronic pulmonary infections, its biosynthetic pathway is the best characterized. However, alginate is only produced by a subset of P. aeruginosa strains. Most environmental and other clinical isolates secrete either Pel or Psl. Little information is available on the biosynthesis of these polysaccharides. Here, we review the literature on the alginate biosynthetic pathway, with emphasis on recent findings describing the structure of alginate biosynthetic proteins. This information combined with the characterization of the domain architecture of proteins encoded on the Psl and Pel operons allowed us to make predictive models for the biosynthesis of these two polysaccharides. The results indicate that alginate and Pel share certain features, including some biosynthetic proteins with structurally or functionally similar properties. In contrast, Psl biosynthesis resembles the EPS/CPS capsular biosynthesis pathway of Escherichia coli, where the Psl pentameric subunits are assembled in association with an isoprenoid lipid carrier. These models and the environmental cues that cause the cells to produce predominantly one polysaccharide over the others are subjects of current investigation. PMID:21991261

  5. Agrobacterium rubi(T) DSM 6772 produces a lipophilic polysaccharide capsule whose degree of acetylation is growth modulated.

    PubMed

    De Castro, Cristina; Gargiulo, Valentina; Lanzetta, Rosa; Parrilli, Michelangelo

    2007-03-01

    The structure of the capsular polysaccharide produced from the type strain of Agrobacterium rubi DSM 6772 is demonstrated by means of chemical and spectroscopical methodologies. It is constituted from the quite rare monosaccharide 6-deoxy-L-talose, involved in alternating alpha-(1 --> 2) and alpha-(1 --> 3) linkages. This simple backbone is further complicated from the occurrence of O-acetyl substituents located always at O-2 of the O-3 substituted 6-deoxy-talose. This decoration is not stoichiometric and it depends on the growth stadium of the bacterium, leading to an almost regular acetylation pattern only at the stationary phase, where all the potential positions are substituted.

  6. The role of host microfilaments and microtubules during opsonin-independent interactions of Cryptococcus neoformans with mammalian lung cells.

    PubMed

    Choo, K K; Chong, P P; Ho, A S H; Yong, P V C

    2015-12-01

    The purpose of this investigation was to characterise the interactions of Cryptococcus neoformans with mammalian host alveolar epithelial cells and alveolar macrophages, with emphasis on the roles of the cryptococcal capsule and the host cell cytoskeletons. The adherence and internalisation of C. neoformans into mammalian lung cells and the roles of host cell cytoskeletons in host-pathogen interactions were studied using in vitro models coupled with a differential fluorescence assay, fluorescence staining, immunofluorescence and drug inhibition of actin and microtubule polymerisation. Under conditions devoid of opsonin and macrophage activation, C. neoformans has a high affinity towards MH-S alveolar macrophages, yet associated poorly to A549 alveolar epithelial cells. Acapsular C. neoformans adhered to and internalised into the mammalian cells more effectively compared to encapsulated cryptococci. Acapsular C. neoformans induced prominent actin reorganisation at the host-pathogen interface in MH-S alveolar macrophages, but minimally affected actin reorganisation in A549 alveolar epithelial cells. Acapsular C. neoformans also induced localisation of microtubules to internalised cryptococci in MH-S cells. Drug inhibition of actin and microtubule polymerisation both reduced the association of acapsular C. neoformans to alveolar macrophages. The current study visualises and confirms the interactions of C. neoformans with mammalian alveolar cells during the establishment of infection in the lungs. The acapsular form of C. neoformans effectively adhered to and internalised into alveolar macrophages by inducing localised actin reorganisation, relying on the host's actin and microtubule activities.

  7. Prevalence, serotypes and mating patterns of Cryptococcus neoformans in the pellets of different avifauna in Madras, India.

    PubMed

    Gokulshankar, S; Ranganathan, S; Ranjith, M S; Ranjithsingh, A J A

    2004-08-01

    A total of 887 pellets of different avifauna were screened for the presence of Cryptococcus neoformans. One hundred and six of 887 samples (12%) yielded Cr. neoformans in culture. The report on the isolation of Cr. neoformans from the pellets of the crow appears to be new and of greater significance because of the ubiquitous prevalence of this bird in India. The prevalence of both MAT a and MAT alpha mating types were recorded. The serotype D was predominant over serotype A. The findings of the present study reveal the growing diverse ecological niche of Cr. neoformans in a the pellets of various avifauna in India.

  8. Genetic and pathological characteristics of Cryptococcus gattii and Cryptococcus neoformans var. neoformans from meningoencephalitis in autochthonous goats and mouflons, Sardinia, Italy.

    PubMed

    Maestrale, Caterina; Masia, Mariangela; Pintus, Davide; Lollai, Stefano; Kozel, Thomas R; Gates-Hollingsworth, Marcellene A; Cancedda, Maria Giovanna; Cabras, Pierangela; Pirino, Salvatore; D'Ascenzo, Vittoria; Ligios, Ciriaco

    2015-06-12

    In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouflons which were found affected by neurological signs. We found 6 goats and one mouflon with meningoencephalitis caused by Cryptococcus sp. There was no evidence of cryptococcal infections in any of the examined sheep. MLST genotyping on Cryptococcus sp. isolates identified Cryptococcus gatti genotype AFLP4/VGI and Cryptococcus neoformans var. neoformans genotype AFLP2/VNIV. Phylogenetically, all Cryptococcus gattii isolates fell within the autochthonous animal, human and environmental Mediterranean isolate cluster, forming a distinct branch along with environmental strains from Alicante, in the southern Mediterranean coast of Spain.

  9. Biomechanical Comparison of the Latarjet Procedure with and without Capsular Repair

    PubMed Central

    Kleiner, Matthew T.; Payne, William B.; McGarry, Michelle H.; Tibone, James E.

    2016-01-01

    Background The purpose of this study was to determine if capsular repair used in conjunction with the Latarjet procedure results in significant alterations in glenohumeral rotational range of motion and translation. Methods Glenohumeral rotational range of motion and translation were measured in eight cadaveric shoulders in 90° of abduction in both the scapular and coronal planes under the following four conditions: intact glenoid, 20% bony Bankart lesion, modified Latarjet without capsular repair, and modified Latarjet with capsular repair. Results Creation of a 20% bony Bankart lesion led to significant increases in anterior and inferior glenohumeral translation and rotational range of motion (p < 0.005). The Latarjet procedure restored anterior and inferior stability compared to the bony Bankart condition. It also led to significant increases in glenohumeral internal and external rotational range of motion relative to both the intact and bony Bankart conditions (p < 0.05). The capsular repair from the coracoacromial ligament stump to the native capsule did not significantly affect translations relative to the Latarjet condition; however it did cause a significant decrease in external rotation in both the scapular and coronal planes (p < 0.005). Conclusions The Latarjet procedure is effective in restoring anteroinferior glenohumeral stability. The addition of a capsular repair does not result in significant added stability; however, it does appear to have the effect of restricting glenohumeral external rotational range of motion relative to the Latarjet procedure performed without capsular repair. PMID:26929804

  10. The Timing of Implant Exchange in the Development of Capsular Contracture After Breast Reconstruction

    PubMed Central

    Weintraub, Jennifer L.; Kahn, David M.

    2008-01-01

    Objective: Capsular contracture is a common complication associated with reconstructive breast surgery. The optimal time interval between the completion of tissue expansion and placement of the permanent implant is arbitrary and incompletely studied in the literature. The aim of the study was to determine whether the time interval between completion of expansion and placement of the permanent implant would affect the incidence of capsular contracture. Methods: We conducted a retrospective study of 112 patients with breast cancer, including 140 breasts, who underwent postmastectomy tissue expander placement between 1997 and 2004. All patients underwent replacement of tissue expander with a permanent prosthesis. Data were collected retrospectively, including whether the patient smoked, underwent radiation therapy, had saline or silicone implant reconstruction, required reoperation after tissue expander placement or after permanent implant placement, Baker classification, and the interval between completion of expansion and placement of permanent implant. Results: We used a logistic regression model to incorporate the predictors of capsular contracture. Keeping all other predictors constant, we found that the time interval between implant exchange had no effect on capsular contracture. The only significant predictor of capsular contracture was whether the patient required a reoperation after the permanent implant was placed (P = .0001). Conclusions: Allowing the capsule around a tissue expander to mature does not significantly affect development of capsular contracture. However, a complication that necessitates disrupting the periprosthetic capsule of the permanent implant with an operation significantly increases odds of developing contracture. PMID:18587490

  11. Endocapsular hyperthermia probe to prevent posterior capsular opacification

    NASA Astrophysics Data System (ADS)

    Orozco, Marcia; Nose, Izuru; Lee, William; Acosta, Ana C.; Fragoso, Miryam; Fernandez, Viviana; Salas, Nelson; Parel, Jean M.

    2005-04-01

    Purpose: To evaluate the feasibility to induce lens epithelial cell death with intraoperative hyperthermia for prevention of secondary cataract. Methods: A prototype miniature resistive hyperthermia probe was designed. The probe contained a thermocouple for temperature feed-back. A timer allowed monitoring of the electrical driving of the hyperthermia probe and the temperature induced as a function of time. To model the heating response, a simple model of the lens capsule was constructed using a thin acrylic plastic shell embedded in a sponge immersed in a water bath at 37°C. The shell was filled with sodium hyaluronate. The probe was positioned at the center of the shell with the thermocouple next to the wall. An experimental protocol was developed to assess the feasibility of increasing the temperature of the human lens to hyperthermia levels in fresh cadaver eyes: An annular metal ring was bonded just below the limbus, the cornea and iris were sectioned, the lens material was removed through a central 5mm diameter capsulorhexis, the capsule was filled with SHA and the globe was set on a temperature-controlled cylindrical vial. Preliminary Results: At 3.3W (2.2V, 1.5A) the shell's content increases from 37°C to 51°C in 30s. At that temperature, LEC death is expected to occur within 1sec. Conclusion: This preliminary study demonstrates the feasibility of increasing the temperature of the capsular bag to kill LECs by hyperthermia.

  12. Improvements in interpretation of posterior capsular opacification (PCO) images

    NASA Astrophysics Data System (ADS)

    Paplinski, Andrew P.; Boyce, James F.; Barman, Sarah A.

    2000-06-01

    We present further improvements to the methods of interpretation of the Posterior Capsular Opacification (PCO) images. These retro-illumination images of the back surface of the implanted lens are used to monitor the state of patient's vision after cataract operation. A common post-surgical complication is opacification of the posterior eye capsule caused by the growth of epithelial cells across the back surface of the capsule. Interpretation of the PCO images is based on their segmentation into transparent image areas and opaque areas, which are affected by the growth of epithelial cells and can be characterized by the increase in the image local variance. This assumption is valid in majority of cases. However, for different materials used for the implanted lenses it sometimes happens that the epithelial cells grow in a way characterized by low variance. In such a case segmentation gives a relatively big error. We describe an application of an anisotropic diffusion equation in a non-linear pre-processing of PCO images. The algorithm preserves the high-variance areas of PCO images and performs a low-pass filtering of small low- variance features. The algorithm maintains a mean value of the variance and guarantees existence of a stable solution and improves segmentation of the PCO images.

  13. Internalized Cryptococcus neoformans Activates the Canonical Caspase-1 and the Noncanonical Caspase-8 Inflammasomes.

    PubMed

    Chen, Mingkuan; Xing, Yue; Lu, Ailing; Fang, Wei; Sun, Bing; Chen, Changbin; Liao, Wanqing; Meng, Guangxun

    2015-11-15

    Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcosis in immunocompromised patients as well as immunocompetent individuals. Host cell surface receptors that recognize C. neoformans have been widely studied. However, intracellular sensing of this pathogen is still poorly understood. Our previous studies have demonstrated that both biofilm and acapsular mutant of C. neoformans are able to activate the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome. In the current study, it was found that opsonization-mediated internalization of encapsulated C. neoformans also activated the canonical NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC)-caspase-1 inflammasome. In addition, the internalized C. neoformans activated the noncanonical NLRP3-ASC-caspase-8 inflammasome as well, which resulted in robust IL-1β secretion and cell death from caspase-1-deficient primary dendritic cells. Interestingly, we found that caspase-1 was inhibitory for the activation of caspase-8 in dendritic cells upon C. neorformans challenge. Further mechanistic studies showed that both phagolysosome membrane permeabilization and potassium efflux were responsible for C. neoformans-induced activation of either the canonical NLRP3-ASC-caspase-1 inflammasome or the noncanonical NLRP3-ASC-caspase-8 inflammasome. Moreover, challenge with zymosan also led to the activation of the noncanonical NLRP3-ASC-caspase-8 inflammasome in cells absent for caspase-1. Collectively, these findings uncover a number of novel signaling pathways for the innate immune response of host cells to C. neoformans infection and suggest that manipulating NLRP3 signaling may help to control fungal challenge.

  14. Cryptococcus neoformans phospholipase B1 activates host cell Rac1 for traversal across the blood-brain barrier.

    PubMed

    Maruvada, Ravi; Zhu, Longkun; Pearce, Donna; Zheng, Yi; Perfect, John; Kwon-Chung, Kyung J; Kim, Kwang Sik

    2012-10-01

    Cryptococcus neoformans penetration into the central nervous system (CNS) requires traversal of the blood-brain barrier that is composed of a single layer of human brain microvascular endothelial cells (HBMEC), but the underlying mechanisms of C. neoformans traversal remain incompletely understood. C. neoformans transcytosis of HBMEC monolayer involves rearrangements of the host cell actin cytoskeleton and small GTP-binding Rho family proteins such as Rac1 are shown to regulate host cell actin cytoskeleton. We, therefore, examined whether C. neoformans traversal of the blood-brain barrier involves host Rac1. While the levels of activated Rac1 (GTP-Rac1) in HBMEC increased significantly upon incubation with C. neoformans strains, pharmacological inhibition and down-modulation of Rac1 significantly decreased C. neoformans transcytosis of HBMEC monolayer. Also, Rac1 inhibition was efficient in preventing C. neoformans penetration into the brain. In addition, C. neoformans phospholipase B1 (Plb1) was shown to contribute to activating host cell Rac1, andSTAT3 was observed to associate with GTP-Rac1 in HBMEC that were incubated with C. neoformans strain but not with its Δplb1 mutant. These findings demonstrate for the first time that C. neoformans Plb1 aids fungal traversal across the blood-brain barrier by activating host cell Rac1 and its association with STAT3, and suggest that pharmacological intervention of host-microbial interaction contributing to traversal of the blood-brain barrier may prevent C. neoformans penetration into the brain.

  15. Polymorphism, duplication, and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA and capsular K antigens.

    PubMed

    Drummelsmith, J; Amor, P A; Whitfield, C

    1997-05-01

    Individual Escherichia coli strains produce several cell surface polysaccharides. In E. coli E69, the his region of the chromosome contains the rfb (serotype O9 lipopolysaccharide O-antigen biosynthesis) and cps (serotype K30 group IA capsular polysaccharide biosynthesis) loci. Polymorphisms in this region of the Escherichia coli chromosome reflect extensive antigenic diversity in the species. Previously, we reported a duplication of the manC-manB genes, encoding enzymes involved in GDP-mannose formation, upstream of rfb in strain E69 (P. Jayaratne et al., J. Bacteriol. 176:3126-3139, 1994). Here we show that one of the manC-manB copies is flanked by IS1 elements, providing a potential mechanism for the gene duplication. Adjacent to manB1 on the IS1-flanked segment is a further open reading frame (ugd), encoding uridine-5'-diphosphoglucose dehydrogenase. The Ugd enzyme is responsible for the production of UDP-glucuronic acid, a precursor required for K30 antigen synthesis. Construction of a chromosomal ugd::Gm(r) insertion mutation demonstrated the essential role for Ugd in the biosynthesis of the K30 antigen and confirmed that there is no additional functional ugd copy in strain E69. PCR amplification and Southern hybridization were used to examine the distribution of IS1 elements and ugd genes in the vicinity of rfb in other E. coli strains, producing different group IA K antigens. The relative order of genes and, where present, IS1 elements was established in these strains. The regions adjacent to rfb in these strains are highly variable in both size and gene order, but in all cases where a ugd homolog was present, it was found near rfb. The presence of IS1 elements in the rfb regions of several of these strains provides a potential mechanism for recombination and deletion events which could contribute to the antigenic diversity seen in surface polysaccharides.

  16. Genetic, Biochemical, and Serological Characterization of a New Pneumococcal Serotype, 6H, and Generation of a Pneumococcal Strain Producing Three Different Capsular Repeat Units

    PubMed Central

    Park, In Ho; Geno, K. Aaron; Yu, Jigui; Oliver, Melissa B.

    2015-01-01

    Streptococcus pneumoniae clinical isolates were recently described that produced capsular polysaccharide with properties of both serotypes 6A and 6B. Their hybrid serological property correlated with mutations affecting the glycosyltransferase WciP, which links rhamnose to ribitol by an α(1-3) linkage for serotypes 6A and 6C and an α(1-4) linkage for serotypes 6B and 6D. The isolates had mutations in the triad residues of WciP that have been correlated with enzyme specificity. The canonical triad residues of WciP are Ala192-Ser195-Arg254 for serotypes 6A and 6C and Ser192-Asn195-Gly254 for serotypes 6B and 6D. To prove that the mutations in the triad residues are responsible for the hybrid serotype, we introduced the previously described Ala192-Cys195-Arg254 triad into a 6A strain and found that the change made WciP bispecific, resulting in 6A and 6B repeat unit expression, although 6B repeat unit production was favored over production of 6A repeat units. Likewise, this triad permitted a 6C strain to express 6C and 6D repeat units. With reported bispecificity in WciN, which adds either glucose or galactose as the second sugar in the serogroup 6 repeat unit, the possibility exists for a strain to simultaneously produce all four serogroup 6 repeat units; however, when genes encoding both bispecific enzymes were introduced into a 6A strain, only 6A, 6B, and 6D repeat units were detected serologically. Nonetheless, this may be the first example of a bacterial polysaccharide with three different repeat units. This strategy of expressing multiple repeat units in a single polymer is a novel approach to broadening vaccine coverage by eliminating the need for multiple polysaccharide sources to cover multiple serogroup members. PMID:25589550

  17. Iodine-Catalyzed Polysaccharide Esterification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A review is provided of the recent reports to use iodine-catalyzed esterification reaction to produce esters from polysaccharides. The process entails reaction of the polysaccharide with an acid anhydride in the presence of a catalytic level of iodine, and in the absence of additional solvents. T...

  18. Polysaccharides of the red algae.

    PubMed

    Usov, Anatolii I

    2011-01-01

    Red algae (Rhodophyta) are known as the source of unique sulfated galactans, such as agar, agarose, and carrageenans. The wide practical uses of these polysaccharides are based on their ability to form strong gels in aqueous solutions. Gelling polysaccharides usually have molecules built up of repeating disaccharide units with a regular distribution of sulfate groups, but most of the red algal species contain more complex galactans devoid of gelling ability because of various deviations from the regular structure. Moreover, several red algae may contain sulfated mannans or neutral xylans instead of sulfated galactans as the main structural polysaccharides. This chapter is devoted to a description of the structural diversity of polysaccharides found in the red algae, with special emphasis on the methods of structural analysis of sulfated galactans. In addition to the structural information, some data on the possible use of red algal polysaccharides as biologically active polymers or as taxonomic markers are briefly discussed.

  19. Heteroimmunization to the capsular polysaccharide of Haemophilus influenzae type b induced by enteric cross-reacting bacteria.

    PubMed

    Handzel, Z T; Argaman, M; Parke, J C; Schneerson, R; Robbins, J B

    1975-05-01

    Cross-reacting Escherichia coli strains Easter and 89 and Bacillus pumilis fed to newborn rabbits and E. coli fed to adult rhesus monkeys did not exert untoward reactions. The E. coli regularly colonized the newborns' intestinal tract from 1 to 7 weeks. High doses of E. coli were necessary to colonize adult primates. Colonization occurred in fewer newborn rabbits and lasted only 1 to 3 weeks with B. pumilis. Colonized newborn rabbits and adult rhesus had an active Haemophilus influenzae type b (HITB) immune response. In the rabbit, colonization resulted in accelerated induction of immunoglobulin (Ig) M-. IgA-, and IgG-producing cells in the spleen, mesenteric lymph nodes, and Peyer's patches after HITB challenge. E. coli-fed and control newborn primates were naturally colonized with nasopharyngeal and enteric cross-reacting bacteria and both groups rapidly developed HITB antibodies in the absence of the homologous organisms. Human newborn stool cultures, taken at the time of discharge from the nursery, showed a 0.9% carriage rate for cross-reacting E. coli. These "carrier" infants acquired HITB antibodies more rapidly than their age-matched "noncarrier" controls.

  20. INFLUENCE OF METAL IONS, TEMPERATURE, AND POLYMER CONCENTRATION ON THE CONFORMATION OF ESCHERICHIA COLI K1 CAPSULAR POLYSACCHARIDE. (R825503)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  1. Why were polysaccharides necessary?

    PubMed

    Tolstoguzov, Vladimir

    2004-12-01

    The main idea of this paper is that the primordial soup may be modelled by food systems whose structure-property relationship is based on non-specific interactions between denatured biopolymers. According to the proposed hypothesis, polysaccharides were the first biopolymers that decreased concentration of salts in the primordial soup, 'compatibilised' and drove the joint evolution of proto-biopolymers. Synthesis of macromolecules within the polysaccharide-rich medium could have resulted in phase separation of the primordial soup and concentration of the polypeptides and nucleic acids in the dispersed phase particles. The concentration of proto-biopolymer mixtures favoured their cross-linking in hybrid supermacromolecules of conjugates. The cross-linking of proto-biopolymers could occur by hydrophobic, electrostatic interactions, H-bonds due to freezing aqueous mixed biopolymer dispersions and/or by covalent bonds due to the Maillard reaction. Cross-linking could have increased the local concentration of chemically different proto-biopolymers, fixed their relative positions and made their interactions reproducible. Attractive-repulsive interactions between cross-linked proto-biopolymer chains could develop pairing of the monomer units, improved chemical stability (against hydrolysis) and led to their mutual catalytic activity and coding. Conjugates could probably evolve to the first self-reproduced entities and then to specialized cellular organelles. Phase separation of the primordial soup with concentration of conjugates in the dispersed particles has probably resulted in proto-cells.

  2. Why Were Polysaccharides Necessary?

    NASA Astrophysics Data System (ADS)

    Tolstoguzov, Vladimir

    2004-12-01

    The main idea of this paper is that the primordial soup may be modelled by food systems whose structure-property relationship is based on non-specific interactions between denatured biopolymers. According to the proposed hypothesis, polysaccharides were the first biopolymers that decreased concentration of salts in the primordial soup, `compatibilised' and drove the joint evolution of proto-biopolymers. Synthesis of macromolecules within the polysaccharide-rich medium could have resulted in phase separation of the primordial soup and concentration of the polypeptides and nucleic acids in the dispersed phase particles. The concentration of proto-biopolymer mixtures favoured their cross-linking in hybrid supermacromolecules of conjugates. The cross-linking of proto-biopolymers could occur by hydrophobic, electrostatic interactions, H-bonds due to freezing aqueous mixed biopolymer dispersions and/or by covalent bonds due to the Maillard reaction. Cross-linking could have increased the local concentration of chemically different proto-biopolymers, fixed their relative positions and made their interactions reproducible. Attractive-repulsive interactions between cross-linked proto-biopolymer chains could develop pairing of the monomer units, improved chemical stability (against hydrolysis) and led to their mutual catalytic activity and coding. Conjugates could probably evolve to the first self-reproduced entities and then to specialized cellular organelles. Phase separation of the primordial soup with concentration of conjugates in the dispersed particles has probably resulted in proto-cells.

  3. Polysaccharides and bacterial plugging

    SciTech Connect

    Fogler, H.S.

    1991-11-01

    Before any successful application of Microbial Enhanced Oil Recovery process can be realized, an understanding of the cells' transport and retentive mechanisms in porous media is needed. Cell transport differs from particle transport in their ability to produce polysaccharides, which are used by cells to adhere to surfaces. Cell injection experiments have been conducted using Leuconostoc cells to illustrate the importance of cellular polysaccharide production as a transport mechanism that hinders cell movement and plugs porous media. Kinetic studies of the Leuconostoc cells, carried out to further understand the plugging rates of porous media, have shown that the cells' growth rates are approximately equal when provided with monosaccharide (glucose and fructose) or sucrose. The only difference in cell metabolism is the production of dextran when sucrose is supplied as a carbon source. Experimentally it has also been shown that the cells' growth rate is weakly dependent upon the sucrose concentration in the media, and strongly dependent upon the concentration of yeast extract. The synthesis of cellular dextran has been found to lag behind cell generation, thus indicating that the cells need to reach maturity before they are capable of expressing the detransucrase enzyme and synthesizing insoluble dextran. Dextran yields were found to be dependent upon the sucrose concentration in the media. 10 refs., 9 figs., 9 tabs.

  4. Polysaccharides from Extremophilic Microorganisms

    NASA Astrophysics Data System (ADS)

    Nicolaus, B.; Moriello, V. Schiano; Lama, L.; Poli, A.; Gambacorta, A.

    2004-02-01

    Several marine thermophilic strains were analyzed for exopolysaccharide production. The screening process revealed that a significant number of thermophilic microorganisms were able to produce biopolymers, and some of them also revealed interesting chemical compositions. We have identified four new polysaccharides from thermophilic marine bacteria, with complex primary structures and with different repetitive units: a galacto-mannane type from strain number 4004 and mannane type for the other strains. The thermophilic Bacillus thermantarcticus produces two exocellular polysaccharides (EPS 1, EPS 2) that give the colonies a typical mucous character. The exopolysaccharide fraction was produced with all substrates assayed, although a higher yield 400 mg liter-1 was obtained with mannose as carbon and energy source. NMR spectra confirmed that EPS 1 was a heteropolysaccharide of which the repeating unit was constituted by four different α-D-mannoses and three different β-D-glucoses. It seems to be close to some xantan polymers. EPS 2 was a mannan. Four different α-D-mannoses were found as the repeating unit. Production and chemical studies of biopolymers produced by halophilic archaea, Haloarcula species were also reported.

  5. Cryptococcus neoformans hyperfilamentous strain is hypervirulent in a murine model of cryptococcal meningoencephalitis.

    PubMed

    Feretzaki, Marianna; Hardison, Sarah E; Wormley, Floyd L; Heitman, Joseph

    2014-01-01

    Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated.

  6. Cryptococcus neoformans capsule protects cell from oxygen reactive species generated by antimicrobial photodynamic inactivation

    NASA Astrophysics Data System (ADS)

    Prates, Renato Araujo; Hamblin, Michael R.; Kato, Ilka T.; Fuchs, Beth; Mylonakis, Eleytherios; Simões Ribeiro, Martha; Tegos, George

    2011-03-01

    Antimicrobial photodynamic inactivation (APDI) is based on the utilization of substances that can photosensitize biological tissues and are capable of being activated in the presence of light. Cryptococcus neoformans is an yeast surrounded by a capsule composed primarily of glucoronoxylomannan that plays an important role in its virulence. This yeast causes infection on skin, lungs and brain that can be associated with neurological sequelae and neurosurgical interventions, and its conventional treatment requires prolonged antifungal therapy, which presents important adverse effects. The aim of this study was to evaluate the protective effect of Cryptococcus neoformans capsule against reactive oxygen species generated by APDI. Cryptococcus neoformans KN99α, which is a strain able to produce capsule, and CAP59 that does not present capsule production were submitted to APDI using methylene blue (MB), rose bengal (RB), and pL-ce6 as photosensitizers (PS). Then microbial inactivation was evaluated by counting colony form units following APDI and confocal laser scanning microscopy (CLSM) illustrated localization as well as the preferential accumulation of PS into the fungal cells. C. neoformans KN99α was more resistant to APDI than CAP59 for all PSs tested. CLSM showed incorporation of MB and RB into the cytoplasm and a preferential uptake in mitochondria. A nuclear accumulation of MB was also observed. Contrarily, pL-ce6 appears accumulated in cell wall and cell membrane and minimal florescence was observed inside the fungal cells. In conclusion, the ability of C. neoformans to form capsule enhances survival following APDI.

  7. Rewiring of Signaling Networks Modulating Thermotolerance in the Human Pathogen Cryptococcus neoformans.

    PubMed

    Yang, Dong-Hoon; Jung, Kwang-Woo; Bang, Soohyun; Lee, Jang-Won; Song, Min-Hee; Floyd-Averette, Anna; Festa, Richard A; Ianiri, Giuseppe; Idnurm, Alexander; Thiele, Dennis J; Heitman, Joseph; Bahn, Yong-Sun

    2017-01-01

    Thermotolerance is a crucial virulence attribute for human pathogens, including the fungus Cryptococcus neoformans that causes fatal meningitis in humans. Loss of the protein kinase Sch9 increases C. neoformans thermotolerance, but its regulatory mechanism has remained unknown. Here, we studied the Sch9-dependent and Sch9-independent signaling networks modulating C. neoformans thermotolerance by using genome-wide transcriptome analysis and reverse genetic approaches. During temperature upshift, genes encoding for molecular chaperones and heat shock proteins were upregulated, whereas those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process, Sch9 regulated basal expression levels or induced/repressed expression levels of some temperature-responsive genes, including heat shock transcription factor (HSF1) and heat shock proteins (HSP104 and SSA1). Notably, we found that the HSF1 transcript abundance decreased but the Hsf1 protein became transiently phosphorylated during temperature upshift. Nevertheless, Hsf1 is essential for growth and its overexpression promoted C. neoformans thermotolerance. Transcriptome analysis using an HSF1 overexpressing strain revealed a dual role of Hsf1 in the oxidative stress response and thermotolerance. Chromatin immunoprecipitation demonstrated that Hsf1 binds to the step-type like heat shock element (HSE) of its target genes more efficiently than to the perfect- or gap-type HSE. This study provides insight into the thermotolerance of C. neoformans by elucidating the regulatory mechanisms of Sch9 and Hsf1 through the genome-scale identification of temperature-dependent genes.

  8. Susceptibility profile and epidemiological cut-off values of Cryptococcus neoformans species complex from Argentina.

    PubMed

    Córdoba, Susana; Isla, Maria G; Szusz, Wanda; Vivot, Walter; Altamirano, Rodrigo; Davel, Graciela

    2016-06-01

    Epidemiological cut-off values (ECVs) based on minimal inhibitory concentration (MIC) distribution have been recently proposed for some antifungal drug/Cryptococcus neoformans combinations. However, these ECVs vary according to the species studied, being serotypes and the geographical origin of strains, variables to be considered. The aims were to define the wild-type (WT) population of the C. neoformans species complex (C. neoformans) isolated from patients living in Argentina, and to propose ECVs for six antifungal drugs. A total of 707 unique C. neoformans isolates obtained from HIV patients suffering cryptococcal meningitis were studied. The MIC of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole was determined according to the EDef 7.2 (EUCAST) reference document. The MIC distribution, MIC50 , MIC90 and ECV for each of these drugs were calculated. The highest ECV, which included ≥95% of the WT population modelled, was observed for flucytosine and fluconazole (32 μg ml(-1) each). For amphotericin B, itraconazole, voriconazole and posaconazole, the ECVs were: 0.5, 0.5, 0.5 and 0.06 μg ml(-1) respectively. The ECVs determined in this study may aid in identifying the C. neoformans strains circulating in Argentina with decreased susceptibility to the antifungal drugs tested.

  9. Neutrophil swarming toward Cryptococcus neoformans is mediated by complement and leukotriene B4.

    PubMed

    Sun, Donglei; Shi, Meiqing

    2016-09-02

    Swarming behavior of neutrophils has been noticed in both sterile injury and infection models and the mechanisms are being unveiled. So far, no in vitro model has been established to study neutrophil swarming to microbes. In the current study, using live-cell imaging, we observed in vitro neutrophil swarming toward Cryptococcus neoformans, a fungal pathogen causing human meningoencephalitis. Complement C3 and CD11b expression are essential for neutrophils to form cell swarms surrounding C. neoformans. Leukotriene B4 (LTB4) was quickly released by neutrophils during their interactions with C. neoformans. Blockade of LTB4 synthesis inhibited the swarming response to C. neoformans. Importantly, blockade of LTB4 synthesis also significantly reduced neutrophil recruitment in the lung vasculature of mice infected intravenously with C. neoformans, demonstrating a critical role of LTB4 in intravascular neutrophil swarming during infection. Together, this is the first report of neutrophil dynamics of swarming toward a microorganism in vitro, mediated by complement and LTB4.

  10. Structures of Cryptococcus neoformans protein farnesyltransferase reveal strategies for developing inhibitors that target fungal pathogens.

    PubMed

    Hast, Michael A; Nichols, Connie B; Armstrong, Stephanie M; Kelly, Shannon M; Hellinga, Homme W; Alspaugh, J Andrew; Beese, Lorena S

    2011-10-07

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

  11. Environmental prevalence of Cryptococcus neoformans and Cryptococcus gattii in India: an update.

    PubMed

    Chowdhary, Anuradha; Rhandhawa, Harbans S; Prakash, Anupam; Meis, Jacques F

    2012-02-01

    An overview of work done to-date in India on environmental prevalence, population structure, seasonal variations and antifungal susceptibility of Cryptococcus neoformans and Cryptococcus gattii is presented. The primary ecologic niche of both pathogens is decayed wood in trunk hollows of a wide spectrum of host trees, representing 18 species. Overall, C. neoformans showed a higher environmental prevalence than that of C. gattii which was not found in the avian habitats. Apart from their arboreal habitat, both species were demonstrated in soil and air in close vicinity of their tree hosts. In addition, C. neoformans showed a strong association with desiccated avian excreta. An overwhelming number of C. neoformans strains belonged to genotype AFLP1/VNI, var. grubii (serotype A), whereas C. gattii strains were genotype AFLP4/VGI, serotype B. All of the environmental strains of C. neoformans and C. gattii were mating type α (MATα). Contrary to the Australian experience, Eucalyptus trees were among the epidemiologically least important and, therefore, the hypothesis of global spread of C. gattii through Australian export of infected Eucalyptus seeds is rebutted. Reference is made to long-term colonization of an abandoned, old timber beam of sal wood (Shorea robusta) by a melanin positive (Mel(+)) variant of Cryptococcus laurentii that was pathogenic to laboratory mice.

  12. Molecular characterization of environmental Cryptococcus neoformans VNII isolates in Jos, Plateau State, Nigeria.

    PubMed

    Nnadi, N E; Enweani, I B; Cogliati, M; Ayanbimpe, G M; Okolo, M O; Kim, E; Sabitu, M Z; Criseo, G; Romeo, O; Scordino, F

    2016-12-01

    Cryptococcus neoformans and Cryptococcus gattii are encapsulated yeasts able to cause fatal neurological infections in both human and other mammals. Cryptococcosis is the most common fungal infection of the central nervous system and has a huge burden in sub-Saharan Africa and South East Asia. Bird excreta are considered an environmental reservoir for C. neoformans in urban areas, therefore a study aimed at isolating and characterizing this yeast is important in disease management. In this study, one hundred samples of pigeon droppings were collected in Jos, Plateau State, Nigeria. C. neoformans was isolated from three samples and initially identified using standard phenotypic and biochemical tests. Molecular analysis revealed that all three isolates belonged to C. neoformans genotype VNII, mating type α and were assigned to the sequence type ST43 by multilocus sequence typing analysis. This study reports, for the first time, the molecular characterization of C. neoformans in Nigeria, where little is still known about the environmental distribution of the genotypes, serotypes and mating types of this important human pathogen.

  13. Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

    SciTech Connect

    Hast, Michael A.; Nichols, Connie B.; Armstrong, Stephanie M.; Kelly, Shannon M.; Hellinga, Homme W.; Alspaugh, J. Andrew; Beese, Lorena S.

    2012-09-17

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

  14. Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin.

    PubMed

    Cogliati, Massimo; D'Amicis, Roberta; Zani, Alberto; Montagna, Maria Teresa; Caggiano, Giuseppina; De Giglio, Osvalda; Balbino, Stella; De Donno, Antonella; Serio, Francesca; Susever, Serdar; Ergin, Cagri; Velegraki, Aristea; Ellabib, Mohamed S; Nardoni, Simona; Macci, Cristina; Oliveri, Salvatore; Trovato, Laura; Dipineto, Ludovico; Rickerts, Volker; McCormick-Smith, Ilka; Akcaglar, Sevim; Tore, Okan; Mlinaric-Missoni, Emilija; Bertout, Sebastien; Mallié, Michele; Martins, Maria da Luz; Vencà, Ana C F; Vieira, Maria L; Sampaio, Ana C; Pereira, Cheila; Griseo, Giuseppe; Romeo, Orazio; Ranque, Stéphane; Al-Yasiri, Mohammed H Y; Kaya, Meltem; Cerikcioglu, Nilgun; Marchese, Anna; Vezzulli, Luigi; Ilkit, Macit; Desnos-Ollivier, Marie; Pasquale, Vincenzo; Korem, Maya; Polacheck, Itzhack; Scopa, Antonio; Meyer, Wieland; Ferreira-Paim, Kennio; Hagen, Ferry; Theelen, Bart; Boekhout, Teun; Lockhart, Shawn R; Tintelnot, Kathrin; Tortorano, Anna Maria; Dromer, Françoise; Varma, Ashok; Kwon-Chung, Kyung J; Inácio, Joäo; Alonso, Beatriz; Colom, Maria F

    2016-06-01

    In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

  15. Two Rac paralogs regulate polarized growth in the human fungal pathogen Cryptococcus neoformans

    PubMed Central

    Ballou, Elizabeth Ripley; Selvig, Kyla; Narloch, Jessica L.; Nichols, Connie B.; Alspaugh, J. Andrew

    2013-01-01

    A genome wide analysis of the human fungal pathogen Cryptococcus neoformans var. grubii has revealed a number of duplications of highly conserved genes involved in morphogenesis. Previously, we reported that duplicate Cdc42 paralogs provide C. neoformans with niche-specific responses to environmental stresses: Cdc42 is required for thermotolerance, while Cdc420 supports the formation of titan cells. The related Rho-GTPase Rac1 has been shown in C. neoformans var. neoformans to play a major role in filamentation and to share Cdc42/Cdc420 binding partners. Here we report the characterization of a second Rac paralog in C. neoformans, Rac2, and describe its overlapping function with the previously described CnRac, Rac1. Further, we demonstrate that the Rac paralogs play a primary role in polarized growth via the organization of reactive oxygen species and play only a minor role in the organization of actin. Finally, we provide preliminary evidence that pharmacological inhibitors of Rac activity and actin stability have synergistic activity. PMID:23748012

  16. Two CDC42 paralogs modulate C. neoformans thermotolerance and morphogenesis under host physiological conditions

    PubMed Central

    Ballou, Elizabeth R.; Nichols, Connie B.; Miglia, Kathleen J; Kozubowski, Lukasz; Alspaugh, J. Andrew

    2013-01-01

    The precise regulation of morphogenesis is a key mechanism by which cells respond to a variety of stresses, including those encountered by microbial pathogens in the host. The polarity protein Cdc42 regulates cellular morphogenesis throughout eukaryotes, and we explore the role of Cdc42 proteins in the host survival of the human fungal pathogen Cryptococcus neoformans. Uniquely, C. neoformans has two functional Cdc42 paralogs, Cdc42 and Cdc420. Here we investigate the contribution of each paralog to resistance to host stress. In contrast to non-pathogenic model organisms, C. neoformans Cdc42 proteins are not required for viability under non-stress conditions. In the presence of cell stress, strains deleted for either paralog show defects in thermotolerance and morphogenesis, likely as a result of their roles in the organization of actin and septin structures during bud growth and cytokinesis. These proteins act downstream of C. neoformans Ras1 to regulate its morphogenesis subpathway, but not its effects on mating. Cdc42, and not Cdc420, is required for virulence in a murine model of cryptococcosis. The C. neoformans Cdc42 proteins likely perform complementary functions with other Rho-like GTPases to control cell polarity, septin organization, and hyphal transitions that allow survival in the environment and in the host. PMID:20025659

  17. Proteogenomic analysis of pathogenic yeast Cryptococcus neoformans using high resolution mass spectrometry

    PubMed Central

    2014-01-01

    Background Cryptococcus neoformans, a basidiomycetous fungus of universal occurrence, is a significant opportunistic human pathogen causing meningitis. Owing to an increase in the number of immunosuppressed individuals along with emergence of drug-resistant strains, C. neoformans is gaining importance as a pathogen. Although, whole genome sequencing of three varieties of C. neoformans has been completed recently, no global proteomic studies have yet been reported. Results We performed a comprehensive proteomic analysis of C. neoformans var. grubii (Serotype A), which is the most virulent variety, in order to provide protein-level evidence for computationally predicted gene models and to refine the existing annotations. We confirmed the protein-coding potential of 3,674 genes from a total of 6,980 predicted protein-coding genes. We also identified 4 novel genes and corrected 104 predicted gene models. In addition, our studies led to the correction of translational start site, splice junctions and reading frame used for translation in a number of proteins. Finally, we validated a subset of our novel findings by RT-PCR and sequencing. Conclusions Proteogenomic investigation described here facilitated the validation and refinement of computationally derived gene models in the intron-rich genome of C. neoformans, an important fungal pathogen in humans. PMID:24484775

  18. The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases

    PubMed Central

    Frandsen, Kristian E. H.; Simmons, Thomas J.; Dupree, Paul; Poulsen, Jens-Christian N.; Hemsworth, Glyn R.; Ciano, Luisa; Johnston, Esther M.; Tovborg, Morten; Johansen, Katja S.; von Freiesleben, Pernille; Marmuse, Laurence; Fort, Sébastien; Cottaz, Sylvain; Driguez, Hugues; Henrissat, Bernard; Lenfant, Nicolas; Tuna, Floriana; Baldansuren, Amgalanbaatar; Davies, Gideon J.; Leggio, Leila Lo; Walton, Paul H.

    2016-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes which oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here the first structural determination of an LPMO–oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains. PMID:26928935

  19. Alleviation of capsular formations on silicone implants in rats using biomembrane-mimicking coatings.

    PubMed

    Park, Ji Ung; Ham, Jiyeon; Kim, Sukwha; Seo, Ji-Hun; Kim, Sang-Hyon; Lee, Seonju; Min, Hye Jeong; Choi, Sunghyun; Choi, Ra Mi; Kim, Heejin; Oh, Sohee; Hur, Ji An; Choi, Tae Hyun; Lee, Yan

    2014-10-01

    Despite their popular use in breast augmentation and reconstruction surgeries, the limited biocompatibility of silicone implants can induce severe side effects, including capsular contracture - an excessive foreign body reaction that forms a tight and hard fibrous capsule around the implant. This study examines the effects of using biomembrane-mimicking surface coatings to prevent capsular formations on silicone implants. The covalently attached biomembrane-mimicking polymer, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), prevented nonspecific protein adsorption and fibroblast adhesion on the silicone surface. More importantly, in vivo capsule formations around PMPC-grafted silicone implants in rats were significantly thinner and exhibited lower collagen densities and more regular collagen alignments than bare silicone implants. The observed decrease in α-smooth muscle actin also supported the alleviation of capsular formations by the biomembrane-mimicking coating. Decreases in inflammation-related cells, myeloperoxidase and transforming growth factor-β resulted in reduced inflammation in the capsular tissue. The biomembrane-mimicking coatings used on these silicone implants demonstrate great potential for preventing capsular contracture and developing biocompatible materials for various biomedical applications.

  20. Correlations between MRI findings and outcome of capsular distension in adhesive capsulitis of the shoulder

    PubMed Central

    Park, Yun Hee; Park, Young Sook; Chang, Hyun Jung; Kim, Yeongmi

    2016-01-01

    [Purpose] The aim of this study was to investigate the association between magnetic resonance imaging (MRI) findings in patients with adhesive shoulder capsulitis and the therapeutic effect of capsular distension. [Subjects and Methods] We retrospectively reviewed the medical records of 57 patients who underwent capsular distension therapy after a diagnosis of adhesive capsulitis with clinical and MRI scans. Axillary joint capsular thickness by MRI was graded as I (≤3.6 mm), II (3.7–4.2 mm), and III (≥4.3 mm). Subcoracoid fat obliteration of the rotator interval was graded subjectively as absent, partial, and complete. [Results] Capsular thickness and fat replacement were correlated with passive range of motion (PROM) and pain score on a visual analog scale (VAS) by analysis of variance with a Bonferroni correction before treatment and by analysis of covariance with a Bonferroni correction after treatment. Visual analog scale (VAS) for patients with all grades decreased significantly after treatment and passive range of motion (PROM) for patients with all grades improved. No difference was detected between grades. [Conclusion] Although MRI is useful to evaluate adhesive capsulitis, MRI findings of shoulder did not predict the prognosis after capsular distension treatment. PMID:27821938

  1. Experience with pneumococcal polysaccharide conjugate vaccine (conjugated to CRM197 carrier protein) in children and adults.

    PubMed

    Durando, P; Faust, S N; Fletcher, M; Krizova, P; Torres, A; Welte, T

    2013-10-01

    Streptococcus pneumoniae-related infections are a major cause of morbidity and mortality in people of all ages worldwide. Pneumococcal vaccine development started in 1911 with a whole cell vaccine and more recently multivalent plain polysaccharide and polysaccharide conjugate vaccines have been developed. The recent vaccines rely on capsular polysaccharide antigens to induce serotype-specific immune responses. We summarize here the presentations on pneumococcal polysaccharide conjugate vaccine (conjugated to CRM197 carrier protein) given during the integrated symposium organized and funded by Pfizer International Operations during the 22nd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) 31 March to 3 April 2012, London, UK. A dramatic reduction in the incidence of invasive pneumococcal diseases (IPD) due to vaccine serotypes (VST-IPD) has been reported since the introduction of a hepta-valent pneumococcal conjugate vaccine (PCV7). An indirect (herd) effect has been demonstrated to be associated with PCV7 infant vaccination programmes, with many studies reporting reductions in VST-IPD in populations that are not eligible for PCV7 vaccination. Since 2010, a 13-valent pneumococcal conjugate vaccine (PCV13) has been introduced into national immunization programmes and results from early surveillance suggest that this vaccine also has an impact on the serotypes unique to PCV13, as well as continuing to protect against the PCV7 serotypes. Data from a passive surveillance system in Europe in 2009, for instance, showed that the highest incidence of IPD remains in those aged >65 years and in children <5 years. PCV13 has now been licensed for vaccination of adults >50 years based on safety and immunogenicity data; an efficacy trial is being conducted. Regardless of previous pneumococcal vaccination status, if the use of 23-valent polysaccharide is considered appropriate, it is recommended to give PCV13 first. Novel immunization strategies remain

  2. Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1) in Cryptococcus neoformans reveals unique biochemical features.

    PubMed

    Henry, Jennifer; Guillotte, Aimee; Luberto, Chiara; Del Poeta, Maurizio

    2011-02-18

    In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.

  3. Requirement of the isocitrate lyase gene ICL1 for VPS41-mediated starvation response in Cryptococcus neoformans.

    PubMed

    Xu, Zhe; Zhi, Yafei; Dong, Jianzhang; Lin, Benfeng; Ye, Di; Liu, Xiaoguang

    2016-07-01

    Cryptococcus neoformans is a major cause of fungal meningitis in individuals with impaired immunity. Our previous studies have shown that the VPS41 gene plays a critical role in the survival of Cryptococcus neoformans under nitrogen starvation; however, the molecular mechanisms underlying VPS41-mediated starvation response remain to be elucidated. In the present study, we show that, under nitrogen starvation, VPS41 strongly enhanced ICL1 expression in C. neoformans and that overexpression of ICL1 in the vps41 mutant dramatically suppressed its defects in starvation response due to the loss of VPS41 function. Moreover, targeted deletion of ICL1 resulted in a dramatic decline in viability of C. neoformans cells under nitrogen deprivation. Taken together, our data suggest a model in which VPS41 up-regulates ICL1 expression, directly or indirectly, to promote survival of C. neoformans under nitrogen starvation.

  4. GWT1 encoding an inositol acyltransferase homolog is required for laccase repression and stress resistance in the basidiomycete Cryptococcus neoformans.

    PubMed

    Zhao, Qiang; Wei, Dongsheng; Li, Zhongming; Wang, Yu; Zhu, Xiangyang; Zhu, Xudong

    2015-12-01

    The transcriptional expression of laccase, which has been confirmed to contribute to the virulence of Cryptococcus neoformans, is often repressed by a high concentration of glucose in many fungi, including C. neoformans. The underlying mechanism of the repression remains largely unknown. In this study, we found that a GWT1 gene that encodes a glycosylphosphatidylinositol (GPI) anchor biosynthesis-related protein is required for laccase repression by glucose in the basidiomycete C. neoformans. Disruption of GWT1 with the Agrobacterium tumefaciens-mediated T-DNA random insertional mutagenesis (ATMT) method resulted in constitutive expression of the laccase gene LAC1 and constant melanin formation. The loss of GWT1 also dramatically affected the cell membrane integrity and stress resistance. Our results revealed a GPI-dependent glucose repression mechanism in C. neoformans, and it may be helpful for understanding the virulence of C. neoformans.

  5. Following fungal melanin biosynthesis with solid-state NMR: biopolymer molecular structures and possible connections to cell-wall polysaccharides.

    PubMed

    Zhong, Junyan; Frases, Susana; Wang, Hsin; Casadevall, Arturo; Stark, Ruth E

    2008-04-22

    Melanins serve a variety of protective functions in plants and animals, but in fungi such as Cryptococcus neoformans they are also associated with virulence. A recently developed solid-state nuclear magnetic resonance (NMR) strategy, based on the incorporation of site-specific (13)C-enriched precursors into melanin, followed by spectroscopy of both powdered and solvent-swelled melanin ghosts, was used to provide new molecular-level insights into fungal melanin biosynthesis. The side chain of an l-dopa precursor was shown to cyclize and form a proposed indole structure in C. neoformans melanin, and modification of the aromatic rings revealed possible patterns of polymer chain elongation and cross-linking within the biopolymer. Mannose supplied in the growth medium was retained as a beta-pyranose moiety in the melanin ghosts even after exhaustive degradative and dialysis treatments, suggesting the possibility of tight binding or covalent incorporation of the pigment into the polysaccharide fungal cell walls. In contrast, glucose was scrambled metabolically and incorporated into both polysaccharide cell walls and aliphatic chains present in the melanin ghosts, consistent with metabolic use as a cellular nutrient as well as covalent attachment to the pigment. The prominent aliphatic groups reported previously in several fungal melanins were identified as triglyceride structures that may have one or more sites of chain unsaturation. These results establish that fungal melanin contains chemical components derived from sources other than l-dopa polymerization and suggest that covalent linkages between l-dopa-derived products and polysaccharide components may serve to attach this pigment to cell wall structures.

  6. Pulmonary coinfection by Pneumocystis jiroveci and Cryptococcus neoformans.

    PubMed

    Javier, Bava; Susana, Lloveras; Santiago, Garro; Alcides, Troncoso

    2012-01-01

    We communicate the diagnosis by microscopy of a pulmonary coinfection produced by Cryptococcus neoformans and Pneumocystis jiroveci, from a respiratory secretion obtained by bronchoalveolar lavage of an AIDS patient. Our review of literature identified this coinfection as unusual presentation. Opportunistic infections associated with HIV infection are increasingly recognized. It may occur at an early stage of HIV-infection. Whereas concurrent opportunistic infections may occur, coexisting Pneumocystis jiroveci pneumonia (PCP) and disseminated cryptococcosis with cryptococcal pneumonia is uncommon. The lungs of individuals infected with HIV are often affected by opportunistic infections and tumours and over two-thirds of patients have at least one respiratory episode during the course of their disease. Pneumonia is the leading HIV-associated infection. We present the case of a man who presented dual Pneumocystis jiroveci and cryptococcal pneumonia in a patient with HIV. Definitive diagnosis of PCP and Cryptococcus requires demonstration of these organisms in pulmonary tissues or fluid. In patients with < 200/microliter CD4-lymphocytes, a bronchoalveolar lavage should be performed. This patient was successfully treated with amphotericin B and trimethoprim sulfamethoxazole. After 1 week the patient showed clinical and radiologic improvement and was discharged 3 weeks later.

  7. Macrophage mitochondrial and stress response to ingestion of Cryptococcus neoformans

    PubMed Central

    Coelho, Carolina; Souza, Ana Camila Oliveira; Derengowski, Lorena da Silveira; de Leon-Rodriguez, Carlos; Wang, Bo; Leon-Rivera, Rosiris; Bocca, Anamelia Lorenzetti; Gonçalves, Teresa; Casadevall, Arturo

    2015-01-01

    Human infection with Cryptococcus neoformans (Cn), a common fungal pathogen follows deposition of yeast spores in the lung alveoli. The subsequent host-pathogen interaction can result in either eradication, latency or extra-pulmonary dissemination. Successful control of Cn infection is dependent on host macrophages but macrophages display little ability to kill Cn in vitro. Recently, we reported that ingestion of Cn by mouse macrophages induces early cell cycle progression followed by mitotic arrest, an event that almost certainly reflects host cell damage. The goal of the present work was to understand macrophage pathways affected by Cn toxicity. Infection of macrophages by Cn was associated with alterations in protein translation rate and activation of several stress pathways such as Hypoxia Inducing Factor-1α (HIF-1α), Receptor-interacting Protein 1 (RIP1) and Apoptosis Inducing Factor (AIF). Concomitantly we observed mitochondrial depolarization in infected macrophages, an observation that was replicated in vivo. We also observed differences in the stress pathways activated depending on macrophage cell type, consistent with the non-specific nature of Cn virulence known to infect phylogenetically distant hosts. Our results indicate that Cn infection impairs multiple host cellular functions and undermines the health of these critical phagocytic cells, which can potentially interfere with their ability to clear this fungal pathogen. PMID:25646306

  8. Mechanisms of Uniparental Mitochondrial DNA Inheritance in Cryptococcus neoformans.

    PubMed

    Gyawali, Rachana; Lin, Xiaorong

    2011-12-01

    In contrast to the nuclear genome, the mitochondrial genome does not follow Mendelian laws of inheritance. The nuclear genome of meiotic progeny comes from the recombination of both parental genomes, whereas the meiotic progeny could inherit mitochondria from one, the other, or both parents. In fact, one fascinating phenomenon is that mitochondrial DNA in the majority of eukaryotes is inherited from only one particular parent. Typically, such unidirectional and uniparental inheritance of mitochondrial DNA can be explained by the size of the gametes involved in mating, with the larger gamete contributing towards mitochondrial DNA inheritance. However, in the human fungal pathogen Cryptococcus neoformans, bisexual mating involves the fusion of two isogamous cells of mating type (MAT) a and MATα, yet the mitochondrial DNA is inherited predominantly from the MATa parent. Although the exact mechanism underlying such uniparental mitochondrial inheritance in this fungus is still unclear, various hypotheses have been proposed. Elucidating the mechanism of mitochondrial inheritance in this clinically important and genetically amenable eukaryotic microbe will yield insights into general mechanisms that are likely conserved in higher eukaryotes. In this review, we highlight studies on Cryptococcus mitochondrial inheritance and point out some important questions that need to be addressed in the future.

  9. Isolation of Cryptococcus neoformans in dry droppings of captive birds in Santiago, Chile.

    PubMed

    González-Hein, Gisela; González-Hein, Jaime; Díaz Jarabrán, Maria C

    2010-09-01

    To investigate the prevalence of Cryptococcus in droppings from captive birds in Chile, dry droppings from 113 captive birds of various species were cultured for Cryptococcus neoformans. The yeast was recovered from 17 of the 113 samples (15% [95% confidence intervals, 8.4%-21.6%]). Other yeast organisms recovered from psittacine bird droppings were Cryptococcus albidus and Cryptococcus uniguttulatus. Secreted phospholipase has been proposed as a virulence determinant in C neoformans. Phospholipase production by the egg yolk plate method, and in vitro susceptibility to fluconazole by using the disk diffusion test were performed on 17 C neoformans isolates. Two of the 17 strains (11.7%) did not produce phospholipase. Two (11.7%) were resistant to fluconazole, and 5 of 17 (29.4%) were susceptible dose-dependent. The Cryptococcus species isolated from droppings from captive birds could be potential pathogens in humans.

  10. In vitro antifungal and antibiofilm activities of halogenated quinoline analogues against Candida albicans and Cryptococcus neoformans.

    PubMed

    Zuo, Ran; Garrison, Aaron T; Basak, Akash; Zhang, Peilan; Huigens, Robert W; Ding, Yousong

    2016-08-01

    With the increasing prevalence of fungal infections coupled with emerging drug resistance, there is an urgent need for new and effective antifungal agents. Here we report the antifungal activities of 19 diverse halogenated quinoline (HQ) small molecules against Candida albicans and Cryptococcus neoformans. Four HQ analogues inhibited C. albicans growth with a minimum inhibitory concentration (MIC) of 100 nM, whilst 16 analogues effectively inhibited C. neoformans at MICs of 50-780 nM. Remarkably, two HQ analogues eradicated mature C. albicans and C. neoformans biofilms [minimum biofilm eradication concentration (MBEC) = 6.25-62.5 µM]. Several active HQs were found to penetrate into fungal cells, whilst one inactive analogue was unable to, suggesting that HQs elicit their antifungal activities through an intracellular mode of action. HQs are a promising class of small molecules that may be useful in future antifungal treatments.

  11. Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

    PubMed Central

    Fox, Deborah

    2009-01-01

    Background The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS), resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface. Methodology The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen. Conclusions The results of this study provide

  12. STAT1 signaling is essential for protection against Cryptococcus neoformans infection in mice.

    PubMed

    Leopold Wager, Chrissy M; Hole, Camaron R; Wozniak, Karen L; Olszewski, Michal A; Wormley, Floyd L

    2014-10-15

    Nonprotective immune responses to highly virulent Cryptococcus neoformans strains, such as H99, are associated with Th2-type cytokine production, alternatively activated macrophages, and inability of the host to clear the fungus. In contrast, experimental studies show that protective immune responses against cryptococcosis are associated with Th1-type cytokine production and classical macrophage activation. The protective response induced during C. neoformans strain H99γ (C. neoformans strain H99 engineered to produce murine IFN-γ) infection correlates with enhanced phosphorylation of the transcription factor STAT1 in macrophages; however, the role of STAT1 in protective immunity to C. neoformans is unknown. The current studies examined the effect of STAT1 deletion in murine models of protective immunity to C. neoformans. Survival and fungal burden were evaluated in wild-type and STAT1 knockout (KO) mice infected with either strain H99γ or C. neoformans strain 52D (unmodified clinical isolate). Both strains H99γ and 52D were rapidly cleared from the lungs, did not disseminate to the CNS, or cause mortality in the wild-type mice. Conversely, STAT1 KO mice infected with H99γ or 52D had significantly increased pulmonary fungal burden, CNS dissemination, and 90-100% mortality. STAT1 deletion resulted in a shift from Th1 to Th2 cytokine bias, pronounced lung inflammation, and defective classical macrophage activation. Pulmonary macrophages from STAT1 KO mice exhibited defects in NO production correlating with inefficient inhibition of fungal proliferation. These studies demonstrate that STAT1 signaling is essential not only for regulation of immune polarization but also for the classical activation of macrophages that occurs during protective anticryptococcal immune responses.

  13. Cryptococcus neoformans Directly Stimulates Perforin Production and Rearms NK Cells for Enhanced Anticryptococcal Microbicidal Activity▿

    PubMed Central

    Marr, Kaleb J.; Jones, Gareth J.; Zheng, Chunfu; Huston, Shaunna M.; Timm-McCann, Martina; Islam, Anowara; Berenger, Byron M.; Ma, Ling Ling; Wiseman, Jeremy C. D.; Mody, Christopher H.

    2009-01-01

    NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, particularly whether the pathogen itself provides an activation or rearming signal, are largely unknown. The current studies were performed to determine whether exposure to this fungus alters subsequent NK cell anticryptococcal activity. NK cells lost perforin and mobilized lysosome-associated membrane protein 1 to the cell surface following incubation with the fungus, indicating that degranulation had occurred. Despite a reduced perforin content during killing, NK cells acquired an enhanced ability to kill C. neoformans, as demonstrated using auxotrophs that allowed independent assessment of the killing of two strains. De novo protein synthesis was required for optimal killing; however, there was no evidence that a soluble factor contributed to the enhanced anticryptococcal activity. Exposure of NK cells to C. neoformans caused the cells to rearm, as demonstrated by increased perforin mRNA levels and enhanced loss of perforin when transcription was blocked. Degranulation alone was insufficient to provide the activation signal as NK cells lost anticryptococcal activity following treatment with strontium chloride. However, NK cells regained the activity upon prolonged exposure to C. neoformans, which is consistent with activation by the microbe. The enhanced cytotoxicity did not extend to tumor killing since NK cells exposed to C. neoformans failed to kill NK-sensitive tumor targets (K562 cells). These studies demonstrate that there is contact-mediated microbe-specific rearming and activation of microbicidal activity that are necessary for optimal killing of C. neoformans. PMID:19307209

  14. Identification of novel hybrids between Cryptococcus neoformans var. grubii VNI and Cryptococcus gattii VGII.

    PubMed

    Aminnejad, Mojgan; Diaz, Mara; Arabatzis, Michael; Castañeda, Elizabeth; Lazera, Marcia; Velegraki, Aristea; Marriott, Deborah; Sorrell, Tania C; Meyer, Wieland

    2012-06-01

    Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts causing meningoencephalitis in immunocompromised and immunocompetent hosts. The fungus is typically haploid, and sexual reproduction occurs normally between individuals with opposite mating types, α and a. C. neoformans var. grubii (serotype A) is comprised of molecular types VNI, VNII, and VNB, and C. neoformans var. neoformans (serotype D) contains the molecular type VNIV. Additionally, diploid or aneuploid AD hybrids (VNIII) have been reported. C. gattii contains the molecular types VGI, VGII, VGIII, and VGIV, which encompass both serotypes B and C. To identify possible hybrid strains, URA5-RFLP analysis was performed on 350 globally obtained clinical, environmental, and veterinary isolates. Four clinical isolates from cerebrospinal fluid showed combination patterns of C. neoformans var. grubii and C. gattii: Brazil (n = 2), Colombia (n = 1), and India (n = 1). These strains were monokaryotic and diploid or aneuploid. M13 PCR fingerprinting showed that they contained fragments of both proposed parental groups. Luminex IGS genotyping identified these isolates as hybrids with two different molecular type combinations: three VNI/VGII and one VNI/VGI. Blue color development on CGB agar was delayed in three isolates and absent in one. C. gattii-specific PCR confirmed the presence of C. gattii in the hybrids. CAP59 allele-specific PCR revealed that all the hybrids contained both serotype A and B alleles. Determination of mating-type allelic patterns by PCR revealed that the isolates were αA aB. This is the first study discovering novel natural hybrids between C. neoformans molecular type VNI and C. gattii molecular type VGII.

  15. Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans

    PubMed Central

    Huang, Wei; Liao, Guojian; Baker, Gregory M.; Wang, Yina; Lau, Richard; Paderu, Padmaja; Perlin, David S.

    2016-01-01

    ABSTRACT Cryptococcus neoformans is a human fungal pathogen and a major cause of fungal meningitis in immunocompromised individuals. Treatment options for cryptococcosis are limited. Of the two major antifungal drug classes, azoles are active against C. neoformans but exert a fungistatic effect, necessitating long treatment regimens and leaving open an avenue for emergence of azole resistance. Drugs of the echinocandin class, which target the glucan synthase and are fungicidal against a number of other fungal pathogens, such as Candida species, are ineffective against C. neoformans. Despite the sensitivity of the target enzyme to the drug, the reasons for the innate resistance of C. neoformans to echinocandins remain unknown. To understand the mechanism of echinocandin resistance in C. neoformans, we screened gene disruption and gene deletion libraries for mutants sensitive to the echinocandin-class drug caspofungin and identified a mutation of CDC50, which encodes the β-subunit of membrane lipid flippase. We found that the Cdc50 protein localized to membranes and that its absence led to plasma membrane defects and enhanced caspofungin penetration into the cell, potentially explaining the increased caspofungin sensitivity. Loss of CDC50 also led to hypersensitivity to the azole-class drug fluconazole. Interestingly, in addition to functioning in drug resistance, CDC50 was also essential for fungal resistance to macrophage killing and for virulence in a murine model of cryptococcosis. Furthermore, the surface of cdc50Δ cells contained increased levels of phosphatidylserine, which has been proposed to act as a macrophage recognition signal. Together, these results reveal a previously unappreciated role of membrane lipid flippase in C. neoformans drug resistance and virulence. PMID:27165800

  16. Topoisomerase I is essential in Cryptococcus neoformans: role In pathobiology and as an antifungal target.

    PubMed Central

    Del Poeta, M; Toffaletti, D L; Rude, T H; Dykstra, C C; Heitman, J; Perfect, J R

    1999-01-01

    Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOP1 gene was fused to the GAL7 gene promoter. The resulting GAL7::TOP1 fusion gene was modestly regulated by carbon source in a serotype A strain of C. neoformans. Modest overexpression of topoisomerase I conferred sensitivity to heat shock, gamma-rays, and camptothecin. In contrast, alterations in topoisomerase I levels had no effect on the toxicity of a novel class of antifungal agents, the dicationic aromatic compounds (DACs), indicating that topoisomerase I is not the target of DACs. In an animal model of cryptococcal meningitis, topoisomerase I regulation was not critically important to established infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the human pathogen C. neoformans and represents a novel target for antifungal agents. PMID:10224251

  17. Open Capsulotomy: An Effective but Overlooked Treatment for Capsular Contracture after Breast Augmentation

    PubMed Central

    2016-01-01

    Background: The prevailing theory for capsular contracture after breast augmentation is a subclinical capsular infection. A capsulectomy, site change, and implant replacement are recommended. An open capsulotomy leaves the capsule in the patient. Theoretically, such a procedure would be ineffective because it does not remove the infected tissue. Recurrences occurred frequently in women treated in the 1970s when leaky silicone gel implants were in use. Open capsulotomy has not been studied in women implanted with third-generation devices. Methods: Seventy-five consecutive women with Baker III/IV capsular contractures after breast augmentation treated with open capsulotomies between 1996 and 2016 were retrospectively evaluated. The original implants were usually saline-filled (72.2%). Replacements were all smooth and round, and 92.6% were also saline-filled. Results: Seventeen women (22.7%) developed a recurrent capsular contracture. Two patients (2.7%) experienced a second recurrence. Patients with ruptured silicone gel implants (n = 13) had a significantly greater risk of recurrence (P = 0.01). There was no significant difference in recurrence rates comparing patients whose intact implants were reinserted (12.5%) with women whose intact implants were replaced (18.2%). Povidone–iodine irrigation did not affect the recurrence rate. Capsular contracture was corrected with 1 procedure in 77.3% of patients and 2 procedures in 97.3% of patients. Conclusions: Open capsulotomy is a safe and effective treatment that avoids the additional morbidity and cost of a capsulectomy. The findings challenge the infected biofilm theory of capsular contracture. Open capsulotomy deserves reconsideration by plastic surgeons. PMID:27826488

  18. In vitro susceptibilities of clinical and environmental isolates of Cryptococcus neoformans to five antifungal drugs.

    PubMed Central

    Franzot, S P; Hamdan, J S

    1996-01-01

    A total of 53 Cryptococcus neoformans strains, including clinical and environmental Brazilian isolates, were tested for their susceptibilities to amphotericin B, 5-flucytosine, ketoconazole, fluconazole, and itraconazole. The tests were performed according to the National Committee of Clinical Laboratory Standards recommendations (document M27-P). In general, there was a remarkable homogeneity of results for all strains, and comparable MICs were found for environmental and clinical isolates. This paper represents the first contribution in which susceptibility data for Brazilian C. neoformans isolates are provided. PMID:8851624

  19. The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans.

    PubMed

    Loftus, Brendan J; Fung, Eula; Roncaglia, Paola; Rowley, Don; Amedeo, Paolo; Bruno, Dan; Vamathevan, Jessica; Miranda, Molly; Anderson, Iain J; Fraser, James A; Allen, Jonathan E; Bosdet, Ian E; Brent, Michael R; Chiu, Readman; Doering, Tamara L; Donlin, Maureen J; D'Souza, Cletus A; Fox, Deborah S; Grinberg, Viktoriya; Fu, Jianmin; Fukushima, Marilyn; Haas, Brian J; Huang, James C; Janbon, Guilhem; Jones, Steven J M; Koo, Hean L; Krzywinski, Martin I; Kwon-Chung, June K; Lengeler, Klaus B; Maiti, Rama; Marra, Marco A; Marra, Robert E; Mathewson, Carrie A; Mitchell, Thomas G; Pertea, Mihaela; Riggs, Florenta R; Salzberg, Steven L; Schein, Jacqueline E; Shvartsbeyn, Alla; Shin, Heesun; Shumway, Martin; Specht, Charles A; Suh, Bernard B; Tenney, Aaron; Utterback, Terry R; Wickes, Brian L; Wortman, Jennifer R; Wye, Natasja H; Kronstad, James W; Lodge, Jennifer K; Heitman, Joseph; Davis, Ronald W; Fraser, Claire M; Hyman, Richard W

    2005-02-25

    Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its approximately 20-megabase genome, which contains approximately 6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.

  20. The Genome of the Basidiomycetous Yeast and Human Pathogen Cryptococcus neoformans

    PubMed Central

    Loftus, Brendan J.; Fung, Eula; Roncaglia, Paola; Rowley, Don; Amedeo, Paolo; Bruno, Dan; Vamathevan, Jessica; Miranda, Molly; Anderson, Iain J.; Fraser, James A.; Allen, Jonathan E.; Bosdet, Ian E.; Brent, Michael R.; Chiu, Readman; Doering, Tamara L.; Donlin, Maureen J.; D’Souza, Cletus A.; Fox, Deborah S.; Grinberg, Viktoriya; Fu, Jianmin; Fukushima, Marilyn; Haas, Brian J.; Huang, James C.; Janbon, Guilhem; Jones, Steven J. M.; Koo, Hean L.; Krzywinski, Martin I.; Kwon-Chung, June K.; Lengeler, Klaus B.; Maiti, Rama; Marra, Marco A.; Marra, Robert E.; Mathewson, Carrie A.; Mitchell, Thomas G.; Pertea, Mihaela; Riggs, Florenta R.; Salzberg, Steven L.; Schein, Jacqueline E.; Shvartsbeyn, Alla; Shin, Heesun; Shumway, Martin; Specht, Charles A.; Suh, Bernard B.; Tenney, Aaron; Utterback, Terry R.; Wickes, Brian L.; Wortman, Jennifer R.; Wye, Natasja H.; Kronstad, James W.; Lodge, Jennifer K.; Heitman, Joseph; Davis, Ronald W.; Fraser, Claire M.; Hyman, Richard W.

    2012-01-01

    Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its ~20-megabase genome, which contains ~6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes. PMID:15653466

  1. Cytoskeletal Drugs Prevent Posterior Capsular Opacification in Human Lens Capsule in vitro

    PubMed Central

    Sureshkumar, Jeyalakshmi; Haripriya, Aravind; Muthukkaruppan, Veerappan; Kaufman, Paul L.; Tian, Baohe

    2012-01-01

    Background To determine whether the cytoskeletal drugs H-7 and Latrunculin B (LAT-B) inhibit posterior capsular opacification (PCO) in the cultured human lens capsular bag. Methods Following extracapsular cataract (lens) extraction in human donor eyes, the capsular bag was prepared and cultured by standard techniques. Forty-eight capsular bags were studied, in which 13 were treated with H-7 (50, 100 or 300μM), 12 with 1% BSS (vehicle of H-7), 11 with LAT-B (2, 5 or 10μM), and 12 with 0.25% DMSO (vehicle of LAT-B). Forty out of the 48 capsular bags were from paired eyes of 20 donors with 1 bag being treated with H-7/LAT-B and the other with BSS/DMSO for each pair, including 20 for the H-7-BSS protocol and 20 for the LAT-B-DMSO protocol. The medium with the cytoskeletal drug/vehicle was replaced every 3–4 days for 4 weeks. PCO was assessed daily using inverted phase-contrast microscopy and scored on a 4-point scale. Results In all cultures with BSS or DMSO, residual lens epithelial cells (LECs) on the anterior capsule migrated to and proliferated on the posterior capsule by 3–7 days and apparent LEC growth on the posterior capsule with severe capsular wrinkling (PCO Grade 3) was seen by 2-3weeks. When treated continuously with H-7 or LAT-B, the migration and proliferation of LECs and the capsular wrinkling were inhibited in a dose-dependent manner, with the inhibition being complete (PCO Grade 0) in the 300μM H-7 (n=8, p<0.001) or 10μM LAT-B culture (n=3, p=0.002). Conclusion H-7 and LAT-B dose-dependently inhibited PCO formation in the cultured human lens capsular bags, suggesting that cytoskeletal drugs might prevent PCO formation after surgery in the human eye. PMID:22138731

  2. Capsular Management in Hip Arthroscopy: An Anatomic, Biomechanical, and Technical Review

    PubMed Central

    Kuhns, Benjamin D.; Weber, Alexander E.; Levy, David M.; Bedi, Asheesh; Mather, Richard C.; Salata, Michael J.; Nho, Shane J.

    2016-01-01

    Hip arthroscopy has become an increasingly utilized surgical technique for the treatment of the young, active patients with hip pain. The clinical outcomes of hip arthroscopy in this patient population have been largely successful; however, there is increasing interest in the contribution of hip capsule in postoperative clinical and functional outcomes. The structure and function of the normal hip capsule will be reviewed. Capsular contributions to hip stability will be discussed in the setting of hip arthroscopy with an emphasis on diagnosis-based considerations. Lastly, clinical outcomes following hip arthroscopy will be discussed as they relate to capsular management. PMID:26973840

  3. A threading receptor for polysaccharides

    NASA Astrophysics Data System (ADS)

    Mooibroek, Tiddo J.; Casas-Solvas, Juan M.; Harniman, Robert L.; Renney, Charles M.; Carter, Tom S.; Crump, Matthew P.; Davis, Anthony P.

    2016-01-01

    Cellulose, chitin and related polysaccharides are key renewable sources of organic molecules and materials. However, poor solubility tends to hamper their exploitation. Synthetic receptors could aid dissolution provided they are capable of cooperative action, for example by multiple threading on a single polysaccharide molecule. Here we report a synthetic receptor designed to form threaded complexes (polypseudorotaxanes) with these natural polymers. The receptor binds fragments of the polysaccharides in aqueous solution with high affinities (Ka up to 19,000 M-1), and is shown—by nuclear Overhauser effect spectroscopy—to adopt the threading geometry. Evidence from induced circular dichroism and atomic force microscopy implies that the receptor also forms polypseudorotaxanes with cellulose and its polycationic analogue chitosan. The results hold promise for polysaccharide solubilization under mild conditions, as well as for new approaches to the design of biologically active molecules.

  4. SYNTHESIS OF TYPE III PNEUMOCOCCAL POLYSACCHARIDE BY SUSPENSIONS OF RESTING CELLS

    PubMed Central

    Bernheimer, Alan W.

    1953-01-01

    The capsular polysaccharide (SIII) of type III pneumococci was removed enzymatically, and the cells thus deprived of preformed SIII were washed and examined for capacity to synthesize SIII anew. The washed, decapsulated cocci lost their capacity to be agglutinated in type-specific antiserum but again became agglutinable and formed readily measurable amounts of SIII, after suspension in a solution containing only glucose and salts. Maximal SIII synthesis required the presence of glucose, magnesium, potassium and phosphate ions, and oxygen. Other fermentable sugars could be substituted for glucose but then the yield of SIII was reduced. Synthesis of SIII occurred anaerobically but was increased four- to fivefold by oxygenation of the suspension. The effects of pH and of enzyme poisons on the capacity of the cocci to form SIII are described. PMID:13052821

  5. Isolation of Cryptococcus gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India.

    PubMed

    Gugnani, H C; Mitchell, T G; Litvintseva, A P; Lengeler, K B; Heitman, J; Kumar, A; Basu, S; Paliwal-Joshi, A

    2005-09-01

    The association of Cryptococcus gattii with Eucalyptus trees has been well established. Here we report the isolation of both C. gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India. We investigated a total of 233 samples of Eucalyptus trees: 120 flowers, 81 fragments of bark, and 32 leaves. C. gattii was isolated from two samples of flowers of Eucalyptus terreticornis. C. neoformans var. grubii was recovered twice from the bark of Eucalyptus camaldulensis, initially from one of three samples, and again 2 months later, from one of four samples collected beneath the canopy of the tree. The primary isolation medium was Nigerseed agar, and brown colonies were presumptively identified as C. gattii or C. neoformans. The species identification was confirmed by morphological and biochemical characteristics. Using the Crypto-Check kit (Iatron, Tokyo, Japan), the first two isolates were identified as serotype B (C. gattii) and the other two were serotype A (C. neoformans var. grubii). PCR analysis of the isolates of C. neoformans var. grubii revealed that they possessed the MATalpha mating type allele. Molecular typing by amplified fragment length polymorphism markers indicated that both isolates of C. neoformans var. grubii possessed the same genotype. This study demonstrates that C. neoformans var. grubii, as well as C. gattii, may be associated with Eucalyptus trees.

  6. Cryptococcus neoformans induces antimicrobial responses and behaves as a facultative intracellular pathogen in the non mammalian model Galleria mellonella.

    PubMed

    Trevijano-Contador, Nuria; Herrero-Fernández, Inés; García-Barbazán, Irene; Scorzoni, Liliana; Rueda, Cristina; Rossi, Suélen Andreia; García-Rodas, Rocío; Zaragoza, Oscar

    2015-01-01

    Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that is found in multiple niches in the environment and that can cause fatal meningoencephalitis in susceptible patients, mainly HIV+ individuals. Cryptococcus also infects environmental hosts such as nematodes, insects and plants. In particular, C. neoformans can kill the lepidopteran Galleria mellonella, which offers a useful tool to study microbial virulence and drug efficacy. Galleria mellonella immunity relies on innate responses based on melanization, accumulation of antimicrobial peptides, and cellular responses as phagocytosis or multicellular encapsulation. In this work we have investigated the immune response of G. mellonella during cryptococcal infection. We found that G. mellonella infected with C. neoformans had a high lytic activity in their hemolymph. This response was temperature- and capsule-dependent. During interaction with phagocytic cells, C. neoformans behaved as an intracellular pathogen since it could replicate within hemocytes. Non-lytic events were also observed. In contrast to Candida species, C. neoformans did not induce melanization of G. mellonella after infection. Finally, passage of C. neoformans through G. mellonella resulted in changes in capsule structure as it has been also reported during infection in mammals. Our results highlight that G. mellonella is an optimal model to investigate innate immune responses against C. neoformans.

  7. Cryptococcus neoformans induces antimicrobial responses and behaves as a facultative intracellular pathogen in the non mammalian model Galleria mellonella

    PubMed Central

    Trevijano-Contador, Nuria; Herrero-Fernández, Inés; García-Barbazán, Irene; Scorzoni, Liliana; Rueda, Cristina; Rossi, Suélen Andreia; García-Rodas, Rocío; Zaragoza, Oscar

    2015-01-01

    Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that is found in multiple niches in the environment and that can cause fatal meningoencephalitis in susceptible patients, mainly HIV+ individuals. Cryptococcus also infects environmental hosts such as nematodes, insects and plants. In particular, C. neoformans can kill the lepidopteran Galleria mellonella, which offers a useful tool to study microbial virulence and drug efficacy. Galleria mellonella immunity relies on innate responses based on melanization, accumulation of antimicrobial peptides, and cellular responses as phagocytosis or multicellular encapsulation. In this work we have investigated the immune response of G. mellonella during cryptococcal infection. We found that G. mellonella infected with C. neoformans had a high lytic activity in their hemolymph. This response was temperature- and capsule-dependent. During interaction with phagocytic cells, C. neoformans behaved as an intracellular pathogen since it could replicate within hemocytes. Non-lytic events were also observed. In contrast to Candida species, C. neoformans did not induce melanization of G. mellonella after infection. Finally, passage of C. neoformans through G. mellonella resulted in changes in capsule structure as it has been also reported during infection in mammals. Our results highlight that G. mellonella is an optimal model to investigate innate immune responses against C. neoformans. PMID:25531532

  8. The expanding host tree species spectrum of Cryptococcus gattii and Cryptococcus neoformans and their isolations from surrounding soil in India.

    PubMed

    Randhawa, H S; Kowshik, T; Chowdhary, Anuradha; Preeti Sinha, K; Khan, Z U; Sun, Sheng; Xu, Jianping

    2008-12-01

    This study reports the widespread prevalence of Cryptococcus neoformans and Cryptococcus gattii in decayed wood inside trunk hollows of 14 species representing 12 families of trees and from soil near the base of various host trees from Delhi and several places in the Indian states of Uttar Pradesh, Haryana, Tamil Nadu and Chandigarh Union Territory. Of the 311 trees from which samples were obtained, 64 (20.5%) were found to contain strains of the C. neoformans species complex. The number of trees positive for C. neoformans var grubii (serotypeA) was 51 (16.3%), for C. gattii (serotype B) 24 (7.7%) and for both C. neoformans and C. gattii 11 (3.5%). The overall prevalence of C. neoformans species complex in decayed wood samples was 19.9% (111/556). There was no obvious correlation between the prevalence of these two yeast species and the species of host trees. The data on prevalence of C. gattii (24%) and C. neoformans (26%) in soil around the base of some host trees indicated that soil is another important ecologic niche for these two Cryptococcus species in India. Among our sampled tree species, eight and six were recorded for the first time as hosts for C. neoformans var grubii and C. gattii, respectively. A longitudinal surveillance of 8 host tree species over 0.7 to 2.5 years indicated long term colonization of Polyalthia longifolia, Mimusops elengi and Manilkara hexandra trees by C. gattii and/or C. neoformans. The mating type was determined for 153 of the isolates, including 98 strains of serotype A and 55 of serotype B and all proved to be mating type alpha (MAT alpha). Our observations document the rapidly expanding spectrum of host tree species for C. gattii and C. neoformans and indicate that decayed woods of many tree species are potentially suitable ecological niches for both pathogens.

  9. Preparation of polysaccharides from wax gourd.

    PubMed

    Huang, Gangliang; Tan, Jiantao; Tan, Xianchun; Peng, Daquan

    2011-08-01

    Preparation of polysaccharides from the wax gourd was studied. The crude polysaccharides were extracted by ethanol precipitation, and deproteinized by the hydrochloric acid method. The deproteinized polysaccharides were separated by column chromatography to obtain the pure polysaccharides. The pure polysaccharides have a β-D-pyranosidic bond, and their molecular weight distribution is about 22,500. It was indicated that the final product had much more purity by IR spectrum analysis, UV absorption spectrum analysis, and phenol-sulfuric acid method, respectively. It was proved that wax gourd polysaccharides were composed of rhamnose, xylose, arabinose, mannose, glucose, and galactose by thin layer chromatography.

  10. Biochemical And Genetic Modification Of Polysaccharides

    NASA Technical Reports Server (NTRS)

    Kern, Roger G.; Petersen, Gene R.; Richards, Gil F.

    1993-01-01

    Bacteriophages producing endopolysaccharase-type enzymes used to produce, isolate, and purify high yields of modified polysaccharides from polysaccharides produced by, and incorporated into capsules of, certain bacteria. Bacteriophages used in conversion of native polysaccharide materials into polymers of nearly uniform high molecular weight or, alternatively, into highly pure oligosaccharides. Also used in genetic selection of families of polysaccharides structurally related to native polysaccharide materials, but having altered properties. Resulting new polysaccharides and oligosaccharides prove useful in variety of products, including pharmaceutical chemicals, coating materials, biologically active carbohydrates, and drag-reducing additives for fluids.

  11. Development and validation of benomyl birdseed agar for the isolation of Cryptococcus neoformans and Cryptococcus gattii from environmental samples.

    PubMed

    Pham, Cau D; Ahn, Stacey; Turner, Lance A; Wohrle, Ron; Lockhart, Shawn R

    2014-05-01

    One of the difficulties of isolating Cryptococcus neoformans and Cryptococcus gattii from environmental samples is the abundant overgrowth of other yeast and mold species that occurs on the plates. Here we report the application of benomyl to Guizotia abyssinica seed extract growth medium to improve the isolation of C. neoformans and C. gattii from environmental samples. We validated this medium by recovering C. neoformans and C. gattii from convenience soils and swabs from a region of the United States where these yeasts are endemic.

  12. Experimental infection of almond trees seedlings (Terminalia catappa) with an environmental isolate of Cryptococcus neoformans var. gattii, serotype C.

    PubMed

    Huérfano, S; Castañeda, A; Castañeda, E

    2001-09-01

    Recently, our laboratory reported the isolation of Cryptococcus neoformans var. gattii, serotype C for the first time from almond trees (Terminalia catappa) detritus. The aim of the present study was to establish the survival of C. neoformans in almond trees seedlings. Thirty seedlings were infected in the stems and samples were taken and processed at different times and by different techniques. No morphological alterations (macro or microscopic) were observed in the infected plants. However, C. neoformans was found to be viable for at least 100 days after infection. These data constitute our first approach towards the understanding of the yeast interactions with a host-plant.

  13. The Cryptococcus neoformans catalase gene family and its role in antioxidant defense.

    PubMed

    Giles, Steven S; Stajich, Jason E; Nichols, Connie; Gerrald, Quincy D; Alspaugh, J Andrew; Dietrich, Fred; Perfect, John R

    2006-09-01

    In the present study, we sought to elucidate the contribution of the Cryptococcus neoformans catalase gene family to antioxidant defense. We employed bioinformatics techniques to identify four members of the C. neoformans catalase gene family and created mutants lacking single or multiple catalase genes. Based on a phylogenetic analysis, CAT1 and CAT3 encode putative spore-specific catalases, CAT2 encodes a putative peroxisomal catalase, and CAT4 encodes a putative cytosolic catalase. Only Cat1 exhibited detectable biochemical activity in vitro, and Cat1 activity was constitutive in the yeast form of this organism. Although they were predicted to be important in spores, neither CAT1 nor CAT3 was essential for mating or spore viability. Consistent with previous studies of Saccharomyces cerevisiae, the single (cat1, cat2, cat3, and cat4) and quadruple (cat1 cat2 cat3 cat4) catalase mutant strains exhibited no oxidative-stress phenotypes under conditions in which either exogenous or endogenous levels of reactive oxygen species were elevated. In addition, there were no significant differences in the mean times to mortality between groups of mice infected with C. neoformans catalase mutant strains (the cat1 and cat1 cat2 cat3 cat4 mutants) and those infected with wild-type strain H99. We conclude from the results of this study that C. neoformans possesses a robust antioxidant system, composed of functionally overlapping and compensatory components that provide protection against endogenous and exogenous oxidative stresses.

  14. Role of IL-17A on resolution of pulmonary C. neoformans infection.

    PubMed

    Wozniak, Karen L; Hardison, Sarah E; Kolls, Jay K; Wormley, Floyd L

    2011-02-17

    The current studies evaluated the role of interleukin (IL)-17A in the induction of protective immunity against pulmonary cryptococcosis in mice. Protection against pulmonary infection with C. neoformans strain H99γ was associated with increased IL-17A production. Signaling through the IFN-γ receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed. Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response. Depletion of IL-17A in mice during pulmonary infection with C. neoformans strain H99γ resulted in an initial increase in pulmonary fungal burden, but had no effect on cryptococcal burden at later time points. Also, depletion of IL-17A did not affect the local production of other cytokines. IL-17RA⁻/⁻ mice infected with C. neoformans strain H99γ survived the primary infection as well as a secondary challenge with wild-type cryptococci. However, dissemination of the wild-type strain to the brain was noted in the surviving IL-17RA⁻/⁻ mice. Altogether, our results suggested that IL-17A may be important for optimal protective immune responsiveness during pulmonary C. neoformans infection, but protective Th1-type immune responses are sufficient for protection against cryptococcal infection.

  15. Molecular characterisation of clinical Cryptococcus neoformans and Cryptococcus gattii isolates from Sichuan province, China.

    PubMed

    Wu, Si-Ying; Lei, Yao; Kang, Mei; Xiao, Yu-Ling; Chen, Zhi-Xing

    2015-05-01

    Previous reports on the molecular characteristics of clinical isolates of Cryptococcus species in China have focused on isolates from southeast China. To obtain a more detailed molecular epidemiology, a total of 92 cryptococcal isolates were collected from Sichuan province. A total of 24 isolates from 12 other provinces were collected for comparative study. Genotypes and mating types of 116 Cryptococcus isolates were determined. Among the 116 isolates, 43 isolates (19 isolates from Sichuan and 24 isolates outside of Sichuan) were analysed by multi-locus sequence typing (MLST). All 116 clinical isolates were mating type α. Most isolates (114/116) were molecular type VNI and the remaining two isolates were VGI and VGII respectively. MLST results revealed five sequence types (STs) of C. neoformans including two novel STs, with most isolates identified as ST5. The two C. gattii isolates identified in our study were ST44 and ST159. Based on our report and previous studies, there are 15 C. neoformans STs in China which can be divided into three subgroups. The C. gattii isolate from Sichuan could be a scattered subtype of VGII (ST44). Our findings demonstrated that C. neoformans isolates in Sichuan are genetically homogeneous, and ST5 is the epidemic clone of C. neoformans in China.

  16. Cryptococcus neoformans Yap1 is required for normal fluconazole and oxidative stress resistance.

    PubMed

    Paul, Sanjoy; Doering, Tamara L; Moye-Rowley, W Scott

    2015-01-01

    Cryptococcus neoformans is a pathogen that is the most common cause of fungal meningitis. As with most fungal pathogens, the most prevalent clinical antifungal used to treat Cryptococcosis is orally administered fluconazole. Resistance to this antifungal is an increasing concern in treatment of fungal disease in general. Our knowledge of the specific determinants involved in fluconazole resistance in Cryptococcus is limited. Here we report the identification of an important genetic determinant of fluconazole resistance in C. neoformans that encodes a basic region-leucine zipper transcription factor homologous to Saccharomyces cerevisiae Yap1. Expression of a codon-optimized form of the Cn YAP1 cDNA in S. cerevisiae complemented defects caused by loss of the endogenous S. cerevisiae YAP1 gene and activated transcription from a reporter gene construct. Mutant strains of C. neoformans lacking YAP1 were hypersensitive to a range of oxidative stress agents but importantly also to fluconazole. Loss of Yap1 homologues from other fungal pathogens like Candida albicans or Aspergillus fumigatus was previously found to cause oxidant hypersensitivity but had no detectable effect on fluconazole resistance. Our data provide evidence for a unique biological role of Yap1 in wild-type fluconazole resistance in C. neoformans.

  17. A multi-host approach for the systematic analysis of virulence factors in Cryptococcus neoformans.

    PubMed

    Desalermos, Athanasios; Tan, Xiaojiang; Rajamuthiah, Rajmohan; Arvanitis, Marios; Wang, Yan; Li, Dedong; Kourkoumpetis, Themistoklis K; Fuchs, Beth Burgwyn; Mylonakis, Eleftherios

    2015-01-15

    A multi-host approach was followed to screen a library of 1201 signature-tagged deletion strains of Cryptococcus neoformans mutants to identify previously unknown virulence factors. The primary screen was performed using a Caenorhabditis elegans-C. neoformans infection assay. The hits among these strains were reconfirmed as less virulent than the wild type in the insect Galleria mellonella-C. neoformans infection assay. After this 2-stage screen, and to prioritize hits, we performed serial evaluations of the selected strains, using the C. elegans model. All hit strains identified through these studies were validated in a murine model of systemic cryptococcosis. Twelve strains were identified through a stepwise screening assay. Among them, 4 (CSN1201, SRE1, RDI1, and YLR243W) were previously discovered, providing proof of principle for this approach, while the role of the remaining 8 genes (CKS101, CNC5600, YOL003C, CND1850, MLH3, HAP502, MSL5, and CNA2580) were not previously described in cryptococcal virulence. The multi-host approach is an efficient method of studying the pathogenesis of C. neoformans. We used diverse model hosts, C. elegans, G. mellonella, and mice, with physiological differences and identified 12 genes associated with mammalian infection. Our approach may be suitable for large pathogenesis screens.

  18. Pleiotropic Roles of the Msi1-Like Protein Msl1 in Cryptococcus neoformans

    PubMed Central

    Yang, Dong-Hoon; Maeng, Shinae; Strain, Anna K.; Floyd, Anna; Nielsen, Kirsten; Heitman, Joseph

    2012-01-01

    Msi1-like (MSIL) proteins contain WD40 motifs and have a pleiotropic cellular function as negative regulators of the Ras/cyclic AMP (cAMP) pathway and components of chromatin assembly factor 1 (CAF-1), yet they have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans, which causes life-threatening meningoencephalitis in humans. Notably, Msl1 plays pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners largely independent of Ras. Msl1 negatively controls antioxidant melanin production and sexual differentiation, and this was repressed by the inhibition of the cAMP-signaling pathway. In contrast, Msl1 controls thermotolerance, diverse stress responses, and antifungal drug resistance in a Ras/cAMP-independent manner. Cac2, which is the second CAF-1 component, appears to play both redundant and distinct functions compared to the functions of Msl1. Msl1 is required for the full virulence of C. neoformans. Transcriptome analysis identified a group of Msl1-regulated genes, which include stress-related genes such as HSP12 and HSP78. In conclusion, this study demonstrates pleiotropic roles of Msl1 in the human fungal pathogen C. neoformans, providing insight into a potential novel antifungal therapeutic target. PMID:23042129

  19. [Cryptococcus Neoformans Var. Gattii meningoencephalitis with cryptococcoma in an immunocompetent patient successfully treated by surgical resection].

    PubMed

    Inada, Taku; Imamura, Hirotoshi; Kawamoto, Michi; Sekiya, Hiroaki; Imai, Yukihiro; Tani, Shoichi; Adachi, Hidemitsu; Ishikawa, Tatsuya; Mineharu, Yohei; Asai, Katsunori; Ikeda, Hiroyuki; Ogura, Takenori; Shibata, Teishiki; Beppu, Mikiya; Agawa, Yuji; Shimizu, Kanpei; Sakai, Nobuyuki; Kikuchi, Haruhiko

    2014-02-01

    Cryptococcosis is a fungal infection, which mainly invades the lungs and central nervous system. In Japan, most cases of cryptococcosis are caused by Cryptococcus neoformans(C. neoformans). Until now, only three cases which the infectious agent was Cryptococcus neoformans var. gattii(C. gattii)have been reported. As compared with cryptococcosis caused by C. neoformans, which is often observed in immunocompromised hosts, cryptococcosis caused by C. gattii occurs predominantly in immunocompetent hosts and is resistant to antifungal drugs. Here, we report a case of refractory cerebral cryptococcoma that was successfully treated by surgical resection of the lesions. A 33-year-old man with no medical history complained of headache, hearing disturbance, and irritability. Pulmonary CT showed a nodular lesion in the left lung. Cerebrospinal fluid examination with Indian ink indicated cryptococcal meningitis, and PCR confirmed infection with C. gattii. C. gattii is usually seen in the tropics and subtropics. Since this patient imported trees and soils from abroad to feed stag beetles, parasite or fungal infection was, as such, suspected. Although he received 2 years of intravenous and intraventricular antifungal treatment, brain cryptococcomas were formed and gradually increased. Because of the refractory clinical course, the patient underwent surgical resection of the cerebral lesions. With continuation of antifungal drugs for 6 months after the surgeries, Cryptococcus could not be cultured from cerebrospinal fluid, and no lesions were seen on MR images. If cerebral cryptococcosis responds poorly to antifungal agents, surgical treatment of the cerebral lesion should be considered.

  20. Cryptococcus neoformans Mediator Protein Ssn8 Negatively Regulates Diverse Physiological Processes and Is Required for Virulence

    PubMed Central

    Wang, Lin-Ing; Lin, Yu-Sheng; Liu, Kung-Hung; Jong, Ambrose Y.; Shen, Wei-Chiang

    2011-01-01

    Cryptococcus neoformans is a ubiquitously distributed human pathogen. It is also a model system for studying fungal virulence, physiology and differentiation. Light is known to inhibit sexual development via the evolutionarily conserved white collar proteins in C. neoformans. To dissect molecular mechanisms regulating this process, we have identified the SSN8 gene whose mutation suppresses the light-dependent CWC1 overexpression phenotype. Characterization of sex-related phenotypes revealed that Ssn8 functions as a negative regulator in both heterothallic a-α mating and same-sex mating processes. In addition, Ssn8 is involved in the suppression of other physiological processes including invasive growth, and production of capsule and melanin. Interestingly, Ssn8 is also required for the maintenance of cell wall integrity and virulence. Our gene expression studies confirmed that deletion of SSN8 results in de-repression of genes involved in sexual development and melanization. Epistatic and yeast two hybrid studies suggest that C. neoformans Ssn8 plays critical roles downstream of the Cpk1 MAPK cascade and Ste12 and possibly resides at one of the major branches downstream of the Cwc complex in the light-mediated sexual development pathway. Taken together, our studies demonstrate that the conserved Mediator protein Ssn8 functions as a global regulator which negatively regulates diverse physiological and developmental processes and is required for virulence in C. neoformans. PMID:21559476

  1. Defects in phosphate acquisition and storage influence virulence of Cryptococcus neoformans.

    PubMed

    Kretschmer, Matthias; Reiner, Ethan; Hu, Guanggan; Tam, Nicola; Oliveira, Debora L; Caza, Melissa; Yeon, Ju Hun; Kim, Jeongmi; Kastrup, Christian J; Jung, Won Hee; Kronstad, James W

    2014-07-01

    Nutrient acquisition and sensing are critical aspects of microbial pathogenesis. Previous transcriptional profiling indicated that the fungal pathogen Cryptococcus neoformans, which causes meningoencephalitis in immunocompromised individuals, encounters phosphate limitation during proliferation in phagocytic cells. We therefore tested the hypothesis that phosphate acquisition and polyphosphate metabolism are important for cryptococcal virulence. Deletion of the high-affinity uptake system interfered with growth on low-phosphate medium, perturbed the formation of virulence factors (capsule and melanin), reduced survival in macrophages, and attenuated virulence in a mouse model of cryptococcosis. Additionally, analysis of nutrient sensing functions for C. neoformans revealed regulatory connections between phosphate acquisition and storage and the iron regulator Cir1, cyclic AMP (cAMP)-dependent protein kinase A (PKA), and the calcium-calmodulin-activated protein phosphatase calcineurin. Deletion of the VTC4 gene encoding a polyphosphate polymerase blocked the ability of C. neoformans to produce polyphosphate. The vtc4 mutant behaved like the wild-type strain in interactions with macrophages and in the mouse infection model. However, the fungal load in the lungs was significantly increased in mice infected with vtc4 deletion mutants. In addition, the mutant was impaired in the ability to trigger blood coagulation in vitro, a trait associated with polyphosphate. Overall, this study reveals that phosphate uptake in C. neoformans is critical for virulence and that its regulation is integrated with key signaling pathways for nutrient sensing.

  2. Quantitation of cellular components in Cryptococcus neoformans for system biology analysis.

    PubMed

    Singh, Arpita; Qureshi, Asfia; Del Poeta, Maurizio

    2011-01-01

    Methods and procedures in molecular biology used to study fungal pathogenesis have significantly improved during the last decade. In this chapter, we provide step-by-step procedures for performing genetics and biochemical studies in the human pathogenic fungal microorganism Cryptococcus neoformans (Cn). These methods are employed for studying the pathobiology of Cn and for experimental validation of theoretical models of fungal pathogenicity.

  3. First Case of Human Cryptococcosis Due to Cryptococcus neoformans var. gattii in Spain

    PubMed Central

    Colom, M. Francisca; Frasés, Susana; Ferrer, Consuelo; Jover, Alejandro; Andreu, Mariano; Reus, Sergio; Sánchez, Manuel; Torres-Rodríguez, Josep M.

    2005-01-01

    We report the first case of human cryptococcosis due to Cryptococcus neoformans var. gattii described in our country, which was presented as brain cryptococcoma in an immunocompetent patient. An extensive sampling of the patient's environment was carried out to find the source of infection. PMID:16000503

  4. Efficient implementation of RNA interference in the pathogenic yeast Cryptococcus neoformans

    PubMed Central

    Bose, Indrani; Doering, Tamara L.

    2011-01-01

    An improved method has been developed for RNA interference in Cryptococcus neoformans, using opposing promoters to facilitate cloning and RNA interference targeting URA5 to allow selection of cells in which silencing is most effective. These advances significantly reduce the variability of silencing and the effort required for interference plasmid construction. PMID:21554906

  5. Microevolution During Serial Mouse Passage Demonstrates FRE3 as a Virulence Adaptation Gene in Cryptococcus neoformans

    PubMed Central

    Hu, Guowu; Chen, Shu Hui; Qiu, Jin; Bennett, John E.; Myers, Timothy G.; Williamson, Peter R.

    2014-01-01

    ABSTRACT Passage in mice of opportunistic pathogens such as Cryptococcus neoformans is known to increase virulence, but little is known about the molecular mechanisms involved in virulence adaptation. Serial mouse passage of nine environmental strains of serotype A C. neoformans identified two highly adapted virulent strains that showed a 4-fold reduction in time to death after four passages. Transcriptome sequencing expression studies demonstrated increased expression of a FRE3-encoded iron reductase in the two strains but not in a control strain that did not demonstrate increased virulence during mouse passage. FRE3 was shown to express an iron reductase activity and to play a role in iron-dependent growth of C. neoformans. Overexpression of FRE3 in the two original environmental strains increased growth in the macrophage cell line J774.16 and increased virulence. These data demonstrate a role for FRE3 in the virulence of C. neoformans and demonstrate how the increased expression of such a “virulence acquisition gene” during the environment-to-mammal transition, can optimize the virulence of environmental strains in mammalian hosts. PMID:24692633

  6. Cryptococcus neoformans meningitis with negative cryptococcal antigen: Evaluation of a new immunochromatographic detection assay.

    PubMed

    Opota, O; Desgraz, B; Kenfak, A; Jaton, K; Cavassini, M; Greub, G; Prod'hom, G; Giulieri, S

    2015-03-01

    Detection of cryptococcal antigen in serum or cerebrospinal fluid allows cryptococcal meningitis diagnosis within few hours with >90% sensitivity. In an HIV-positive patient with Cryptococcus neoformans meningitis, initial antigen detection by immunoagglutination was negative. We thus evaluated a new immunochromatographic detection assay that exhibited a higher sensitivity.

  7. Dectin-3 Is Not Required for Protection against Cryptococcus neoformans Infection

    PubMed Central

    Campuzano, Althea; Castro-Lopez, Natalia; Wozniak, Karen L.; Leopold Wager, Chrissy M.; Wormley, Floyd L.

    2017-01-01

    C-type lectin receptors (CLRs) are diverse, trans-membrane proteins that function as pattern recognition receptors (PRRs) which are necessary for orchestrating immune responses against pathogens. CLRs have been shown to play a major role in recognition and protection against fungal pathogens. Dectin-3 (also known as MCL, Clecsf8, or Clec4d) is a myeloid cell-specific CLR that recognizes mycobacterial trehalose 6,6’-dimycolate (TDM) as well as α-mannans present in the cell wall of fungal pathogens. To date, a potential role for Dectin-3 in the mediation of protective immune responses against C. neoformans has yet to be determined. Consequently, we evaluated the impact of Dectin-3 deficiency on the development of protective immune responses against C. neoformans using an experimental murine model of pulmonary cryptococcosis. Dectin-3 deficiency did not lead to increased susceptibility of mice to experimental pulmonary C. neoformans infection. Also, no significant differences in pulmonary leukocyte recruitment and cytokine production were observed in Dectin-3 deficient mice compared to wild type infected mice. In addition, we observed no differences in uptake and anti-cryptococcal activity of Dectin-3 deficient dendritic cells and macrophages. Altogether, our studies show that Dectin-3 is dispensable for mediating protective immune responses against pulmonary C. neoformans infection. PMID:28107361

  8. Occurrence and significance of Cryptococcus neoformans in the respiratory tract of patients with bronchopulmonary disorders.

    PubMed Central

    Randhawa, H S; Pal, M

    1977-01-01

    Cryptococcus neoformans was cultured from 13 (3%) of 469 clinical specimens examined from the respiratory tract of patients with bronchopulmonary diseases. These isolations came from 5 (2%) of 207 patients; 11 isolates were from sputum and 1 each were from bronchoscopic aspirate and empyema pus. The fungus was not cultured from the oropharyngeal washings of 101 apparently healthy volunteers. Of the 5 patients, 3 had pulmonary tuberculosis, including one with pyopneumothorax and 2 with allergic bronchopulmonary aspergillosis as the underlying disease. In the tuberculosis patient with pyopneumothorax and C. neoformans in empyema pus, the fungus was presumably a tissue invader, whereas its role could not be unequivocally ascertained in the remaining 4 patients from whom it was isolated from sputum or bronchial aspirate on at least two consecutive occasions. The question of C. neoformans being a transient resident, commensal, or incitant of benign minimal lesions in the tracheobronchial tree is discussed. A comprehensive laboratory and clinical follow-up is warranted in patients from whose sputum or bronchial aspirate C. neoformans may be cultured even though definitive signs of cryptococcosis may be lacking. PMID:319109

  9. Molecular identification, antifungal resistance and virulence of Cryptococcus neoformans and Cryptococcus deneoformans isolated in Seville, Spain.

    PubMed

    Gago, Sara; Serrano, Carmen; Alastruey-Izquierdo, Ana; Cuesta, Isabel; Martín-Mazuelos, Estrella; Aller, Ana Isabel; Gómez-López, Alicia; Mellado, Emilia

    2017-01-01

    Cryptococcal meningitis is one of the leading causes of death in HIV/AIDS patients. Our aim was to in order to characterise the epidemiology, antifungal susceptibility pattern and virulence of 28 Cyptococcus sp. strains recovered from 12 AIDS patients during two years in a Spanish single institution. Antifungal susceptibility testing was performed according to the CLSI protocols. Clinical strains were molecularly characterised by serotyping, mating type, PCR fingerprinting (M13 and GACA4 microsatellites) and analysis of two rDNA regions (IGS1 and ITS). Sequencing of the ERG11 gene was used to explore mechanisms of fluconazole resistance. Differences in virulence between species were studied in a Galleria mellonella infection model. Cryptococcus deneoformans and C. deneoformans x Cryptococcus neoformans hybrids were the most frequent variety (65%) followed by C. neoformans (35%). Strains were categorised according to 13 microsatellite genotypes and mixed infections could be detected in three patients. Twenty-nine per cent of the strains were fluconazole resistant. In one of the patients, the fluconazole resistance phenotype was associated with a point mutation in the ERG11 gene responsible for the amino acid substitution G470R. C. neoformans strains were able to kill G. mellonella larvae more efficiently than C. deneoformans and hybrids between both species. Precisely molecular characterisation of C. neoformans species is important for an accurate patient's management.

  10. Evidence that the Human Pathogenic Fungus Cryptococcus neoformans var. grubii May Have Evolved in Africa

    PubMed Central

    Litvintseva, Anastasia P.; Carbone, Ignazio; Rossouw, Jenny; Thakur, Rameshwari; Govender, Nelesh P.; Mitchell, Thomas G.

    2011-01-01

    Most of the species of fungi that cause disease in mammals, including Cryptococcus neoformans var. grubii (serotype A), are exogenous and non-contagious. Cryptococcus neoformans var. grubii is associated worldwide with avian and arboreal habitats. This airborne, opportunistic pathogen is profoundly neurotropic and the leading cause of fungal meningitis. Patients with HIV/AIDS have been ravaged by cryptococcosis – an estimated one million new cases occur each year, and mortality approaches 50%. Using phylogenetic and population genetic analyses, we present evidence that C. neoformans var. grubii may have evolved from a diverse population in southern Africa. Our ecological studies support the hypothesis that a few of these strains acquired a new environmental reservoir, the excreta of feral pigeons (Columba livia), and were globally dispersed by the migration of birds and humans. This investigation also discovered a novel arboreal reservoir for highly diverse strains of C. neoformans var. grubii that are restricted to southern Africa, the mopane tree (Colophospermum mopane). This finding may have significant public health implications because these primal strains have optimal potential for evolution and because mopane trees contribute to the local economy as a source of timber, folkloric remedies and the edible mopane worm. PMID:21589919

  11. ISOLATION OF Cryptococcus neoformans FROM ENVIRONMENTAL SAMPLES COLLECTED IN SOUTHEASTERN NIGERIA

    PubMed Central

    NWEZE, Emeka I.; KECHIA, Fred A.; DIBUA, Uju E.; EZE, Charles; ONOJA, Uwakwe S.

    2015-01-01

    SUMMARY Cryptococcosis caused by Cryptococcus neoformans is the second most common fungal opportunistic pathogen and a lifethreatening infection with serious clinical manifestations especially in HIV/AIDS and other immunocompromised patients. In Nigeria, HIV/AIDS infection has reached an alarming level. Despite this, information on the presence of this fungus in clinical and environmental samples is very scanty in Nigeria and many other parts of Africa. We set out to evaluate the presence of Cryptococcus neoformans or C. gattii in pigeon droppings obtained from Southeastern Nigeria. One hundred and seventy-seven samples of pigeon droppings from six sample types were collected. The area covered comprised of ten cities and other locations spanning across five States in Nigeria. Using established techniques, Cryptococcus neoformans was isolated from 39 of the 177 (22.0%) samples overall. No C. gattiiwas isolated. Most of the isolates (32.4%) were recovered from dovecotes (11 of 34) followed closely by samples taken from markets (31.8%; seven of 22) and least from the church (4.0%; one of 25). The highest isolation rate (38.9%) was found in samples from Enugu-Ezike(seven of 23) while the least came from Afikpoand the other locations each with 9.1% isolation rate. This is the first large-scale screening of Cryptococcus neoformans from pigeon droppings in Nigeria. The ecological and epidemiological significance of these findings are discussed. PMID:26422152

  12. Analyses of Pediatric Isolates of Cryptococcus neoformans from South Africa ▿ †

    PubMed Central

    Miglia, Kathleen J.; Govender, Nelesh P.; Rossouw, Jenny; Meiring, Susan; Mitchell, Thomas G.

    2011-01-01

    Compared to the incidence in adults, cryptococcosis is inexplicably rare among children, even in sub-Saharan Africa, which has the highest prevalence of coinfection with HIV and Cryptococcus neoformans. To explore any mycological basis for this age-related difference in the incidence of cryptococcosis, we investigated isolates of C. neoformans recovered from pediatric and adult patients during a 2-year period in South Africa. From reports to the Group for Enteric, Respiratory, and Meningeal Disease Surveillance in South Africa (GERMS-SA), we reviewed all cases of cryptococcosis in 2005 and 2006. We analyzed one isolate of C. neoformans from each of 82 pediatric patients (<15 years of age) and determined the multilocus sequence type (ST), mating type, ploidy, and allelic profile. This sample included isolates of all three molecular types of serotype A or C. neoformans var. grubii (molecular types VNI, VNII, and VNB) and one AD hybrid. Seventy-seven (94%) of the strains possessed the MATα mating type allele, and five were MATa. Seventy-five (91%) were haploid, and seven were diploid. A total of 24 different STs were identified. The ratios of each mating type and the proportion of haploids were comparable to those for the isolates that were obtained from 86 adult patients during the same period. Notably, the most prevalent pediatric ST was significantly associated with male patients. Overall, these pediatric isolates exhibited high genotypic diversity. They included a relatively large percentage of diploids and the rarely reported MATa mating type. PMID:20980574

  13. Inferior capsular shift operation for multidirectional instability of the shoulder in players of contact sports

    PubMed Central

    Choi, C; Ogilvie-Harris, D

    2002-01-01

    Objectives: To assess the results of inferior capsular shift for multidirectional instability of the shoulder in athletes. Methods: Multidirectional instability was surgically corrected in 53 shoulders in 47 athletes who engaged in contact sports. A history of major trauma was found in eight patients, the others having had minor episodes. Before surgery, all patients had complex combinations of instabilities. The surgical approach was selected according to the predominant direction of instability. Results: Anterior inferior capsular shift was carried out in 37 shoulders, and anterior dislocation recurred in three. In one of these, it was anterior alone, one was anterior and inferior, and one was unstable in all three directions. After posterior inferior capsular shift in 16 shoulders, one dislocation occurred anteriorly and one posteriorly. With the anterior approach, four athletes could not return to sport. Two patients treated with the posterior approach could not return to sport. Of these six failures, five patients had had bilateral repairs. Successful repair based on the criteria of the American Shoulder and Elbow Association was achieved in 92% of anterior repairs and 81% of posterior repairs. Successful return to sport was noted in 82% of patients with anterior repairs, 75% with posterior repairs, and 17% with bilateral repairs. Overall, there were five subsequent dislocations, three in the anterior repair group (8%), and two in the posterior repair group (12%). Conclusions: Inferior capsular shift can successfully correct multidirectional instability in most players of contact sports, but the results in bilateral cases are poor. PMID:12145120

  14. Enhanced bioavailability of EPA from emulsified fish oil preparations versus capsular triacylglycerol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pre-emulsified fish oil supplements, an alternative to capsular triacylglycerol, may enhance the uptake of LCn3 fatty acids it contains. A randomized, Latin-square crossover design was used to compare the effects of four fish oil supplement preparations on phospholipid (PLFA) and chylomicron fatty ...

  15. Epidemiology, Etiology, and Prevention of Late IOL-Capsular Bag Complex Dislocation: Review of the Literature

    PubMed Central

    Ascaso, Francisco J.; Huerva, Valentín; Grzybowski, Andrzej

    2015-01-01

    Posterior chamber intraocular lens (PC-IOL) subluxation is uncommon but represents one of the most serious complications following phacoemulsification. Late spontaneous IOL-capsular bag complex dislocation is defined as occurring three months or later following cataract surgery. Unlike early IOL dislocation, late spontaneous IOL dislocation is due to a progressive zonular dehiscence and contraction of the capsular bag many years what seemed to be uneventful surgery. In recent years, late in-the-bag IOL subluxation or dislocation has been reported with increasing frequency, having a cumulative risk of IOL dislocation following cataract extraction of 0.1% after 10 years and 1.7% after 25 years. A predisposition to zonular insufficiency and capsular contraction is identified in 90% of reviewed cases. Multiple conditions likely play a role in contributing to this zonular weakness and capsular contraction. Pseudoexfoliation is the most common risk factor, accounting for more than 50% of cases. Other associated conditions predisposing to zonular dehiscence are aging, high myopia, uveitis, trauma, previous vitreoretinal surgery, retinitis pigmentosa, diabetes mellitus, atopic dermatitis, previous acute angle-closure glaucoma attack, and connective tissue disorders. The recognition of these predisposing factors suggests a modified approach in cases at risk. We review certain measures to prevent IOL-bag complex luxation that have been proposed. PMID:26798506

  16. The significance of the shape of the lens and capsular energy changes in accommodation

    PubMed Central

    Fisher, R. F.

    1969-01-01

    1. A method for the estimation of the energy released by the anterior part of the lens capsule during accommodation is described. This includes (i) A determination of the pressure required to distend the capsule by a standard volume. (ii) The calculation from the photographed lens profiles of the degree of capsular contraction which occurs when the lens changes from the unaccommodated to the accommodated form. (iii) Capsular volume changes in vitro are then related to the surface area changes calculated for the lens in vivo. 2. A correlation exists between the stored capsular energy per unit area or surface tension and the accommodation power of different species. The human lens capsule releases 1170 ergs/cm2 while the more spherical lenses of the cat and rabbit release 520 and 485 ergs/cm2 respectively for a 10% change in lens diameter. The amount of energy which can be stored depends on the degree of flatness of the lens and the volume of the anterior segment. The flatter the lens and the smaller the volume of the anterior segment, the greater the capsular surface tension. 3. The anterior surface of the human lens remains ellipsoidal throughout life. The changes of accommodation which occur in presbyopia may therefore be related to the lens profiles at various ages. It is found that a coefficient obtained by dividing the anterior volume of the lens by the 5th power of the equatorial radius of the lens modifies the degree of accommodation for a given change of lens diameter. 4. The loss of accommodation is proportional to the effective capsular surface energy until about the age of 45. The effective capsular surface energy can be defined as the energy which gives the same change in lens dioptric power per erg regardless of the lenticular profile changes which occur with age. It is obtained by multiplying capsular surface tension at a given age by a ratio. This is obtained by dividing the profile coefficient mentioned in paragraph 3 of the given lens, by the

  17. Organization of genes required for gellan polysaccharide biosynthesis in Sphingomonas elodea ATCC 31461.

    PubMed

    Harding, Nancy E; Patel, Yamini N; Coleman, Russell J

    2004-02-01

    Sphingomonas elodea ATCC 31461 produces gellan, a capsular polysaccharide that is useful as a gelling agent for food and microbiological media. Complementation of nonmucoid S. elodea mutants with a gene library resulted in identification of genes essential for gellan biosynthesis. A cluster of 18 genes spanning 21 kb was isolated. These 18 genes are homologous to genes for synthesis of sphingan polysaccharide S-88 from Sphingomonas sp. ATCC 31554, with predicted amino acid identities varying from 61% to 98%. Both polysaccharides have the same tetrasaccharide repeat unit, comprised of [-->4)-alpha- l-rhamnose-(1-->3)-beta- d-glucose-(1-->4)-beta- d-glucuronic acid-(1-->4)-beta- d-glucose-(1-->]. Polysaccharide S-88, however, has mannose or rhamnose in the fourth position and has a rhamnosyl side chain, while gellan has no sugar side chain but is modified by glyceryl and acetyl substituents. Genes for synthesis of the precursor dTDP- l-rhamnose were highly conserved. The least conserved genes in this cluster encode putative glycosyl transferases III and IV and a gene of unknown function, gelF. Three genes ( gelI, gelM, and gelN) affected the amount and rheology of gellan produced. Four additional genes present in the S-88 sphingan biosynthetic gene cluster did not have homologs in the gene cluster for gellan biosynthesis. Three of these gene homologs, gelR, gelS, and gelG, were found in an operon unlinked to the main gellan biosynthetic gene cluster. In a third region, a gene possibly involved in positive regulation of gellan biosynthesis was identified.

  18. Value of the CT "capsular sign" as a potential indicator of acute adrenal ischemia.

    PubMed

    Moschetta, Marco; Telegrafo, Michele; Pignatelli, Armando; Stabile Ianora, Amato Antonio; Angelelli, Giuseppe

    2015-10-01

    Acute adrenal ischemia represents a rare cause of adrenal insufficiency which should be promptly diagnosed in order to preserve adrenal vitality and function. Our study aims to retrospectively evaluate the diagnostic accuracy of the CT capsular sign as an indicator of adrenal ischemia and its association with vascular involvement. Between January 2013 and January 2014, 69 consecutive patients (47 men, 22 women; mean age 46; range 22-67) with suspected adrenal insufficiency based on clinical and biochemical data underwent 320-row CT examination in our Emergency Department. Written informed consent was obtained for the CT examinations, and the institutional review board approval was obtained for our retrospective study. CT multi-planar images were retrospectively and independently analyzed by two radiologists searching for the patency of adrenal vessels, enlarged adrenals, the presence of the "capsular sign" represented by a peripheral subtle hyperdense line around a hypodense enlarged adrenal, and the presence of any periadrenal inflammatory changes. All CT findings were then compared with the surgical findings (n = 5), follow-up examinations (n = 20), or autopsy (n = 4). Sensitivity, specificity, diagnostic accuracy (DA), positive predictive value (PPV), and negative predictive value (NPV) were calculated for the "capsular sign" and were further evaluated by ROC analysis. Acute adrenal ischemia occurred in 29/69 patients (42 %), unilateral in 20, and bilateral in 9. Forty of sixty-nine patients (58 %) had no evidence of adrenal disease on CT. Thrombosis of the main adrenal vein was found in 20/29 (69 %) and non-venous ischemia in 9/29 (31 %). The capsular sign was found in 24/29 patients (83 %). Sensitivity, specificity, DA, PPV, and NPV values of 83, 100, 93, 100, and 89 %, respectively, were obtained. The capsular sign represents a CT indicator of acute adrenal ischemia, with a specificity of 100 % and leading to a prompt diagnosis in the early

  19. Characterization of IL-22 and antimicrobial peptide production in mice protected against pulmonary Cryptococcus neoformans infection.

    PubMed

    Wozniak, Karen L; Hole, Camaron R; Yano, Junko; Fidel, Paul L; Wormley, Floyd L

    2014-07-01

    Cryptococcus neoformans is a significant cause of fungal meningitis in patients with impaired T cell-mediated immunity (CMI). Experimental pulmonary infection with a C. neoformans strain engineered to produce IFN-γ, H99γ, results in the induction of Th1-type CMI, resolution of the acute infection, and protection against challenge with WT Cryptococcus. Given that individuals with suppressed CMI are highly susceptible to pulmonary C. neoformans infection, we sought to determine whether antimicrobial peptides were produced in mice inoculated with H99γ. Thus, we measured levels of antimicrobial peptides lipocalin-2, S100A8, S100A9, calprotectin (S100A8/A9 heterodimer), serum amyloid A-3 (SAA3), and their putative receptors Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) in mice during primary and recall responses against C. neoformans infection. Results showed increased levels of IL-17A and IL-22, cytokines known to modulate antimicrobial peptide production. We also observed increased levels of lipocalin-2, S100A8, S100A9 and SAA3 as well as TLR4(+) and RAGE(+) macrophages and dendritic cells in mice inoculated with H99γ compared with WT H99. Similar results were observed in the lungs of H99γ-immunized, compared with heat-killed C. neoformans-immunized, mi