Induction of T helper type 1 responses by a polysaccharide deacetylase from Cryptococcus neoformans.
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ABSTRACT:
SUBMITTER: Biondo C
PROVIDER: S-EPMC187367 | biostudies-other | 2003 Sep
REPOSITORIES: biostudies-other
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Project description:Cryptococcus neoformans is an important human, fungal pathogen that sheds polysaccharide (exo-PS) into host tissues. While shed exo-PS mediates numerous untoward effects (including promoting increased intracranial pressure), little is known about the regulation of this phenomenon. Since downregulation of the Allergen 1 (ALL1) gene is associated with high ICP, we investigated the relationship between ALL1 expression and exo-PS structure using a variety of biophysical techniques. The ?all1 mutants of two serotypes produced a shorter exo-PS with less branching and structural complexity than the parental strains. Consistent with lower branching, these exo-PSs manifested higher intrinsic viscosity than the parental strains. The ?all1 mutant strains manifested differences in epitope expression and significant resistance to phagocytosis. Exo-PS of ?all1 mutant exhibited anti-phagocytic properties. Comparative transcriptome analysis of mutant and parental strain under iron-deprived conditions indicated a role of ALL1 in iron homeostasis, characterized by differential regulation of genes that mediate iron reduction and transport. Together, our results demonstrate a role of ALL1 in regulating conformational aspects of PS structure and iron homeostasis. These findings provide a mechanism to explain how changes in ALL1 expression influence virulence of switch variants and suggest that structural changes and polymer length are epigenetically regulated.
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Project description:Cryptococcus neoformans capsular polysaccharide is composed of at least two components, glucuronoxylomannan (GXM) and galactoxylomannans (GalXM). Although GXM has been extensively studied, little is known about the location of GalXM in the C. neoformans capsule, in part because there are no serological reagents specific to this antigen. To circumvent the poor immunogenicity of GalXM, this antigen was conjugated to protective antigen from Bacillus anthracis as a protein carrier. The resulting conjugate elicited antibodies that reacted with GalXM in mice and yielded an immune serum that proved useful for studying GalXM in the polysaccharide capsule. In acapsular cells, immune serum localized GalXM to the cell wall. In capsulated cells, immune serum localized GalXM to discrete pockets near the capsule edge. GalXM was abundant on the nascent capsules of budding daughter cells. The constituent sugars of GalXM were found in vesicle fractions consistent with vesicular transport for this polysaccharide. In addition, we generated a single-chain fraction variable fragment antibody with specificity to oxidized carbohydrates that also produced punctate immunofluorescence on encapsulated cells that partially colocalized with GalXM. The results are interpreted to mean that GalXM is a transient component of the polysaccharide capsule of mature cells during the process of secretion. Hence, the function of GalXM appears to be more consistent with that of an exopolysaccharide than a structural component of the cryptococcal capsule.
| S-EPMC2620754 | biostudies-literature
Project description: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.
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Project description: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.
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Project description: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.
| S-EPMC3630864 | biostudies-literature
Project description:Chitin is an essential component of the cell wall of Cryptococcus neoformans conferring structural rigidity and integrity under diverse environmental conditions. Chitin deacetylase genes encode the enyzmes (chitin deacetylases [Cdas]) that deacetylate chitin, converting it to chitosan. The functional role of chitosan in the fungal cell wall is not well defined, but it is an important virulence determinant of C. neoformans Mutant strains deficient in chitosan are completely avirulent in a mouse pulmonary infection model. C. neoformans carries genes that encode three Cdas (Cda1, Cda2, and Cda3) that appear to be functionally redundant in cells grown under vegetative conditions. Here we report that C. neoformans Cda1 is the principal Cda responsible for fungal pathogenesis. Point mutations were introduced in the active site of Cda1 to generate strains in which the enzyme activity of Cda1 was abolished without perturbing either its stability or localization. When used to infect CBA/J mice, Cda1 mutant strains produced less chitosan and were attenuated for virulence. We further demonstrate that C. neoformans Cda genes are transcribed differently during a murine infection from what has been measured in vitroIMPORTANCECryptococcus neoformans is unique among fungal pathogens that cause disease in a mammalian host, as it secretes a polysaccharide capsule that hinders recognition by the host to facilitate its survival and proliferation. Even though it causes serious infections in immunocompromised hosts, reports of infection in hosts that are immunocompetent are on the rise. The cell wall of a fungal pathogen, its synthesis, composition, and pathways of remodelling are attractive therapeutic targets for the development of fungicides. Chitosan, a polysaccharide in the cell wall of C. neoformans is one such target, as it is critical for pathogenesis and absent in the host. The results we present shed light on the importance of one of the chitin deacetylases that synthesize chitosan during infection and further implicates chitosan as being a critical factor for the pathogenesis of C. neoformans.
| S-EPMC6247093 | biostudies-literature
Project description:The polysaccharide capsule is known to be the major factor required for the virulence of Cryptococcus neoformans. We have cloned and characterized a gene, designated CPS1, that encodes a protein containing a glycosyltransferase moiety and shares similarity with the type 3 polysaccharide synthase encoded by the cap3B gene of Streptococcus pneumoniae. Cps1p also shares similarity with hyaluronan synthase of higher eukaryotes. Deletion of the CPS1 gene from a serotype D strain of C. neoformans resulted in a slight reduction of the capsule size as observed by using an India ink preparation. The growth at 37 degrees C was impaired, and the ability to associate with human brain endothelial cells in vitro was also significantly reduced by the deletion of CPS1. Using site-specific mutagenesis, we showed that the conserved glycosyltransferase domains are critical for the ability of the strain to grow at elevated temperatures. A hyaluronan enzyme-linked immunosorbent assay method demonstrated that CPS1 is important for the synthesis of hyaluronan or its related polysaccharides in C. neoformans. Comparisons between the wild-type and the cps1Delta strains, using three different transmission electron microscopic methods, indicated that the CPS1 gene product is involved in the composition or maintenance of an electron-dense layer between the outer cell wall and the capsule. These and the virulence studies in a mouse model suggested that the CPS1 gene is important in the pathobiology of C. neoformans.
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Project description: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 (15)N-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.
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Project description:Wild type strain H99 compared to independent sir2 deletion mutants grown to mid-log (0.6 OD600) in rich medium (YPD) shaking at 250rpm in baffled flasks.
2022-04-01 | GSE173209 | GEO
Project description: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.
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