Polysaccharide Deacetylase Research Articles

Streptococcus mutans SMU.623c Codes for a Functional, Metal-Dependent Polysaccharide Deacetylase That Modulates Interactions with Salivary Agglutinin

The genome sequence of the oral pathogen Streptococcus mutans predicts the presence of two putative polysaccharide deacetylases. The first, designated PgdA in this paper, shows homology to the catalytic domains of peptidoglycan deacetylases from Streptococcus pneumoniae and Listeria monocytogenes, which are both thought to be involved in the bacterial defense mechanism against human mucosal lysozyme and are part of the CAZY family 4 carbohydrate esterases. S. mutans cells in which the pgdA gene was deleted displayed a different colony texture and a slightly increased cell surface hydrophobicity and yet did not become hypersensitive to lysozyme as shown previously for S. pneumoniae. To understand this apparent lack of activity, the high-resolution X-ray structure of S. mutans PgdA was determined; it showed the typical carbohydrate esterase 4 fold, with metal bound in a His-His-Asp triad. Analysis of the protein surface showed that an extended groove lined with aromatic residues is orientated toward the active-site residues. The protein exhibited metal-dependent de-N-acetylase activity toward a hexamer of N-acetylglucosamine. No activity was observed toward shorter chitooligosaccharides or a synthetic peptidoglycan tetrasaccharide. In agreement with the lysozyme data this would suggest that S. mutans PgdA does not act on peptidoglycan but on an as-yet-unidentified polysaccharide within the bacterial cell surface. Strikingly, the pgdA-knockout strain showed a significant increase in aggregation/agglutination by salivary agglutinin, in agreement with this gene acting as a deacetylase of a cell surface glycan.

Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps

Caulobacter crescentus cells adhere to surfaces by using an extremely strong polar adhesin called the holdfast. The polysaccharide component of the holdfast is comprised in part of oligomers of N-acetylglucosamine. The genes involved in the export of the holdfast polysaccharide and the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated that these genes are involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-sugar lipid-carrier transferase, the glycosyltransferase that catalyzes the first step in polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-frame hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE, two of which are redundant to hfsE for holdfast synthesis. We also identified a redundant paralog to the hfsC gene, encoding the putative polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs, together with the hfs genes, are absolutely required for proper holdfast synthesis.

Logical Identification of an Allantoinase Analog (puuE) Recruited from Polysaccharide Deacetylases

The hydrolytic cleavage of the hydantoin ring of allantoin, catalyzed by allantoinase, is required for the utilization of the nitrogen present in purine-derived compounds. The allantoinase gene (DAL1), however, is missing in many completely sequenced organisms able to use allantoin as a nitrogen source. Here we show that an alternative allantoinase gene (puuE) can be precisely identified by analyzing its logic relationship with three other genes of the pathway. The novel allantoinase is annotated in structure and sequence data bases as polysaccharide deacetylase for its homology with enzymes that catalyze hydrolytic reactions on chitin or peptidoglycan substrates. The recombinant PuuE protein from Pseudomonas fluorescens exhibits metal-independent allantoinase activity and stereospecificity for the S enantiomer of allantoin. The crystal structures of the protein and of protein-inhibitor complexes reveal an overall similarity with the polysaccharide deacetylase beta/alpha barrel and remarkable differences in oligomeric assembly and active site geometry. The conserved Asp-His-His metal-binding triad is replaced by Glu-His-Trp, a configuration that is distinctive of PuuE proteins within the protein family. An extra domain at the top of the barrel offers a scaffold for protein tetramerization and forms a small substrate-binding cleft by hiding the large binding groove of polysaccharide deacetylases. Substrate positioning at the active site suggests an acid/base mechanism of catalysis in which only one member of the catalytic pair of polysaccharide deacetylases has been conserved. These data provide a structural rationale for the shifting of substrate specificity that occurred during evolution.

A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix

Despite the proclamation of Lowenstam and Weiner that crustaceans are the "champions of mineral mobilization and deposition of the animal kingdom," relatively few proteins from the two main calcification sites in these animals, i.e., the exoskeleton and the transient calcium storage organs, have been identified, sequenced, and their roles elucidated. Here, a 65-kDa protein (GAP 65) from the gastrolith of the crayfish, Cherax quadricarinatus, is fully characterized and its function in the mineralization of amorphous calcium carbonate (ACC) of the extracellular matrix is demonstrated. GAP 65 is a negatively charged glycoprotein that possesses three predicted domains: a chitin-binding domain 2, a low-density lipoprotein receptor class A domain, and a polysaccharide deacetylase domain. Expression of GAP 65 was localized to columnar epithelial cells of the gastrolith disk during premolt. In vivo administration of GAP 65 dsRNA resulted in a significant reduction of GAP 65 transcript levels in the gastrolith disk. Such gene silencing also caused dramatic structural and morphological deformities in the chitinous-ACC extracellular matrix structure. ACC deposited in these gastroliths appeared to be sparsely packed with large elongated cavities compared with the normal gastrolith, where ACC is densely compacted. ACC spherules deposited in these gastroliths are significantly larger than normal. GAP 65, moreover, inhibited calcium carbonate crystallization in vitro and stabilized synthetic ACC. Thus, GAP 65 is the first protein shown to have dual function, involved both in extracellular matrix formation and in mineral deposition during biomineralization.

Characterization and cloning of chitin deacetylases from Rhizopus circinans

Chitin deacetylase catalyzes hydrolysis of the acetamido groups of N-acetylglucosamine of chitin in fungal cell walls. Here a chitin deacetylase secreted by Rhizopus circinans was purified to homogeneity and partially characterized. The enzyme exhibits an apparent molecular weight of ∼75kDa. At 37°C it shows optimal activity at pH 5.5–6. Its pH stability and thermal stability are good. Mn2+ and Mg2+ slightly enhance the activity of the enzyme and Cu2+ strongly inhibits it. An R. circinans cDNA library was constructed and screened with a homologous probe synthesized by RT-PCR or with synthetic primers derived from the N-terminal amino-acid sequence of the native purified chitin deacetylase. Three chitin deacetylase cDNAs (RC, D2, and I3/2) were isolated from the cDNA library and sequenced. These cDNAs exhibit features characteristic of chitin deacetylase sequences: the presence of a polysaccharide deacetylase domain, a metal-binding triad, the conserved catalytic residues, and high homology with various chitin deacetylase genes. The cDNAs were cloned in a Pichia pastoris expression system and produced as polyhistidine-tagged proteins. Only one recombinant enzyme (called RC) was active under the tested conditions. It was purified to homogeneity in a single step and further characterized. The protein showed an apparent molecular mass of ∼75kDa and, like the native enzyme, showed optimal activity at pH 5.5–6 at 37°C. It was strongly inhibited by Cu2+. The isolation of several chitin deacetylase cDNAs from the same microorganism is discussed.

Cloning and sequence analysis of complete cDNA of chitin deacetylase from Mucor racemosus

AbstractA complete chitin deacetylase (CDA) complementary DNA (cDNA) from Mucor racemosus was cloned and sequenced by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification cDNA end (RACE) with gene special conserved primers. The cDNA sequence was submitted to GenBank (DQ538514). The complete cDNA with full-length of 1506 bp contained a 67 bp 5′-untranslated region, an open reading frame of 1344 bp and 95 bp 3′-untranslated region including tailing site AATAAA. The gene encoded a sequence of 448 amino acid residues and consisted of core nucleotides encoding a polysaccharide deacetylase domain covering 32% of the entire sequence. The CDA gene shared sequence homology with those of several fungi. The corresponding homology of the deduced amino acid sequences varied from 21 to 69%. Phylogenetic analysis according to the deduced amino acid sequences matched the classical fungi taxonomy. The three-dimensional structure of this protein was predicted. The protein had a whole CDA functional domain and a polysaccharide deacetylase domain.

Direct analysis of the extracellular proteome from two strains of Helicobacter pylori

Helicobacter pylori extracellular proteins are of interest because of possible roles in pathogenesis, host recognition, and vaccine development. We utilized a unique approach by growing two strains (including one nonsequenced strain) in a defined serum-free medium and directly analyzing the proteins present in the culture supernatants by LC-MS/MS. Over 125 proteins were identified in the extracellular proteomes of two H. pylori strains. Forty-five of these proteins were enriched in the extracellular fraction when compared to soluble cell-associated protein samples. Our analysis confirmed and expanded on the previously reported H. pylori extracellular proteome. Extracellular proteins of interest identified here included cag pathogenicity island protein Cag24 (CagD); proteases HP0657 and HP1012; a polysaccharide deacetylase, HP0310, possibly involved in the hydrolysis of acetyl groups from host N-acetylglucosamine residues or from residues on the cell surface; and HP0953, an uncharacterized protein that appears to be restricted to Helicobacter species that colonize the gastric mucosa. In addition, our analysis found eight previously unidentified outer membrane proteins and two lipoproteins that could be important cell surface proteins.

Xanthomonascaspase displays an inherent PARP-like activity

In an earlier study, intracellular accumulation of metabolites such as pyruvate and citrate in Xanthomonas campestris pv. glycines (Xcg) was found to result in a caspase dependent stationary phase rapid cell death (RCD). In the present study, the presence of poly ADP-ribose polymerase (PARP)-like activity associated with caspase-3-like protein of Xcg is reported. This activity was found to be responsible for depletion of cellular NAD(+) levels in RCD-promoting media such as Luria-Bertani medium and starch medium fortified with citrate. Addition of PARP-specific inhibitors such as 3-aminobenzamide to RCD-promoting media restored the intracellular NAD(+) levels and thereby prevented RCD. The inherent association of PARP-like activity with the caspase protein was demonstrated by PARP cellular assay, immuno-precipitation and Western analysis. A truncated polysaccharide deacetylase gene having a caspase-like domain was cloned. The expressed protein though found to be inactive, cross-reacted with human caspase and PARP antibodies. This is the first report demonstrating the presence of a PARP-like activity in a prokaryote and its involvement in cell death.

Identification of critical amino acid residues in the plague biofilm Hms proteins

Yersinia pestis biofilm formation causes massive adsorption of haemin or Congo red in vitro as well as colonization and eventual blockage of the flea proventriculus in vivo. This blockage allows effective transmission of plague from some fleas, like the oriental rat flea, to mammals. Four Hms proteins, HmsH, HmsF, HmsR and HmsS, are essential for biofilm formation, with HmsT and HmsP acting as positive and negative regulators, respectively. HmsH has a beta-barrel structure with a large periplasmic domain while HmsF possesses polysaccharide deacetylase and COG1649 domains. HmsR is a putative glycosyltransferase while HmsS has no recognized domains. In this study, specific amino acids within conserved domains or within regions of high similarity in HmsH, HmsF, HmsR and HmsS proteins were selected for site-directed mutagenesis. Some but not all of the substitutions in HmsS and within the periplasmic domain of HmsH were critical for protein function. Substitutions within the glycosyltransferase domain of HmsR and the deacetylase domain of HmsF abolished biofilm formation in Y. pestis. Surprisingly, substitution of highly conserved residues within COG1649 did not affect HmsF function.

Identification and characterization of a polysaccharide deacetylase gene from Bacillus thuringiensis

One polysaccharide deacetylase gene was cloned from Bacillus thuringiensis and designated pdaA. Disruption of pdaA did not affect vegetative growth and sporulation but obviously affected spore germination. When L-alanine was added into the spore suspension, the spores of the pdaA disruption mutant showed a slow and partial reduction in absorbance at OD600 and became phase pale gray compared with phase dark of the wild-type strain. In contrast with the outgrowing of wild-type spores after germination, the pdaA mutant spores were blocked at the stage of spore germination. Transmission electron micrographs revealed a significant difference between the pdaA mutant and the wild-type strain in the spore cortex. Introduction of the pdaA gene into the pdaA disruption mutant complemented the germination-negative phenotype. Reverse transcription--polymerase chain reaction showed that pdaA was transcribed after incubation for 10 h in CCY medium.

Xyn11A, a multidomain multicatalytic enzyme fromPseudobutyrivibrio xylanivorans Mz5T

The rumen bacterium Pseudobutyrivibrio xylanivorans Mz5T has a potent xylanolytic enzyme system. A small native peptide (approximately 30-kDa, designated Xyn11A) from the bacterium was first isolated and characterized by Edman degradation. The gene coding for Xyn11A was identified using PCR amplification with consensus primers. It was then fully sequenced to reveal an open reading frame of 1809 bp. The predicted N-terminal domain exhibited xylanolytic activity and was classed to the family 11 of glycosyl hydrolases; it is followed by a region with homology to a family 6 cellulose binding module. The C-terminal domain codes for a putative NodB-like polysaccharide deacetylase which is predicted to be an acetyl esterase implicated in debranching activity in the xylan backbone. As similar domain organization was also found in several other xylanases from a diverse range of bacteria, a common ancestor of such a xylanase is considered to be present and spread, possibly by horizontal gene transfer, to other microorganisms from different ecological niches.

Purification and molecular characterization of chitin deacetylase from Rhizopus nigricans

Chitin deacetylase is an enzyme, which catalyses the hydrolysis of N-acetamido bonds of chitin, converting it to chitosan. In the present report we purified the chitin deacetylase from mycelial extracts of a zygomycete Rhizopus nigricans by sequential ammonium sulfate precipitation, CM Sepharose chromatography and DEAE-cellulose chromatography. The progress of enzyme purification was followed by measurement of enzyme activity using partially O-hydroxyethylated chitin (glycol chitin) radiolabelled in N-acetyl groups as a substrate. The apparent molecular mass of chitin deacetylase obtained by SDS-PAGE was approximately 100 kDa. A cDNA library containing a chitin deacetylase gene from R. nigricans was constructed and the complete gene was sequenced. The complete gene contains an open reading frame of 1341 nucleotides, which encodes a sequence of 447 amino acid residues. The estimated molecular mass is 47 kDa, suggesting that carbohydrate content is 53% by weight of the protein. The gene sequence consists of nucleotides encoding a conserved polysaccharide deacetylase domain located in the middle, covering 34% of the entire sequence. Overall, there were eight possible N-linked glycosylation sites. The deduced amino acid sequence shows the highest identity with chitin deacetylases from other zygomycetes Phycomyces blakesleeanus, Gongronella butleri, Rhizopus oryzae and Mucor rouxii (55%, 48%, 43% and 41% identity, respectively).

Septate-Junction-Dependent Luminal Deposition of Chitin Deacetylases Restricts Tube Elongation in the Drosophila Trachea

The function of tubular epithelial organs like the kidney and lung is critically dependent on the length and diameter of their constituting branches. Genetic analysis of tube size control during Drosophila tracheal development has revealed that epithelial septate junction (SJ) components and the dynamic chitinous luminal matrix coordinate tube growth. However, the underlying molecular mechanisms controlling tube expansion so far remained elusive. Here, we present the analysis of two luminal chitin binding proteins with predicted polysaccharide deacetylase activities (ChLDs). ChLDs are required to assemble the cable-like extracellular matrix (ECM) and restrict tracheal tube elongation. Overexpression of native, but not of mutated, ChLD versions also interferes with the structural integrity of the intraluminal ECM and causes aberrant tube elongation. Whereas ChLD mutants have normal SJ structure and function, the luminal deposition of the ChLD requires intact cellular SJs. This identifies a new molecular function for SJs in the apical secretion of ChLD and positions ChLD downstream of the SJs in tube length control. The deposition of the chitin luminal matrix first promotes and coordinates radial tube expansion. We propose that the subsequent structural modification of chitin by chitin binding deacetylases selectively instructs the termination of tube elongation to the underlying epithelium.

Characterization of Two Novel Cryptococcal Mannoproteins Recognized by Immune Sera

Host defenses against the encapsulated yeast Cryptococcus neoformans involve both humoral and cell-mediated immunity. Mannoproteins (MPs) are a heterogeneous class of immunodominant glycoproteins which have been only incompletely characterized. In this study, we report on the molecular features of two novel MPs that are recognized by serum antibodies during cryptococcosis. After fractionation of extracellular cryptococcal products, MPs reacted more strongly than other components with sera from C. neoformans-infected AIDS patients. Further fractionation and Western blot analysis of MPs evidenced the presence of highly reactive bands with molecular masses of 250, 125, 115, and 84 kDa. The 115- and 84-kDa bands contained significant amounts of N-linked oligosaccharides, as shown by decreased molecular mass after peptide-N-glycosidase F treatment. N-terminal amino acid sequences of the two bands were used to search C. neoformans nucleotide databases. Homologous genomic sequences were used to synthesize DNA probes and isolate cDNA clones containing the full-length genes, which were designated MP84 and MP115. Both genes showed the presence of a serine/threonine-rich region, a potential site for heavy glycosylation. MP84 and MP115 showed homology with, respectively, polysaccharide deacetylases and carboxylesterases from other organisms. Recombinant, deglycosylated proteins expressed in Escherichia coli still reacted with sera from patients, albeit more weakly than natural MPs, indicating that at least some of the reactive epitopes were retained in the recombinant forms. In conclusion, we identified two novel MPs that are important targets of antibody responses during cryptococcosis. These data may be useful to devise alternative immunity-based strategies to control the disease.

Peptidoglycan N-Acetylglucosamine Deacetylases from Bacillus cereus, Highly Conserved Proteins in Bacillus anthracis

The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain 10 polysaccharide deacetylase homologues. Six of these homologues have been proposed to be peptidoglycan N-acetylglucosamine deacetylases. Two of these genes, namely bc1960 and bc3618, have been cloned and expressed in Escherichia coli, and the recombinant enzymes have been purified to homogeneity and further characterized. Both enzymes were effective in deacetylating cell wall peptidoglycan from the Gram(+) Bacillus cereus and Bacillus subtilis and the Gram(-) Helicobacter pylori as well as soluble chitin substrates and N-acetylchitooligomers. However, the enzymes were not active on acetylated xylan. These results provide insight into the substrate specificity of carbohydrate esterase family 4 enzymes. It was revealed that both enzymes deacetylated only the GlcNAc residue of the synthetic muropeptide N-acetyl-D-glucosamine-(beta-1,4)-N-acetylmuramyl-L-alanine-D-isoglutamine. Analysis of the constituent muropeptides of peptidoglycan from B. subtilis and H. pylori resulting from incubation of the enzymes BC1960 and BC3618 with these polymers and subsequent hydrolysis by Cellosyl and mutanolysin, respectively, similarly revealed that both enzymes deacetylate GlcNAc residues of peptidoglycan. Kinetic analysis toward GlcNAc(2-6) revealed that GlcNAc4 was the favorable substrate for both enzymes. Identification of the sequence of N-acetychitooligosaccharides (GlcNAc(2-4)) following enzymatic deacetylation by using 1H NMR revealed that both enzymes deacetylate all GlcNAc residues of the oligomers except the reducing end ones. Enzymatic deacetylation of chemically acetylated vegetative peptidoglycan from B. cereus by BC1960 and BC3618 resulted in increased resistance to lysozyme digestion. This is the first biochemical study of bacterial peptidoglycan N-acetylglucosamine deacetylases.

The putative chitin deacetylase of Encephalitozoon cuniculi: A surface protein implicated in microsporidian spore-wall formation

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide deacetylase family with a suspected chitin deacetylase activity ( EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.

Proteomics of the Microsporidia Encephalitozoon cuniculi: from Proteins of unknown Function to New Cell Wall Ones

The microsporidia Encephalitozoon cuniculi, a human opportunist pathogen, is an obligate intracellular parasite, the nuclear genome of which has been fully sequenced. Our descriptive proteomic analysis of the late developmental stages showed that one‐third of the most abundant proteins are of unknown function. The spore, stage of resistance and dissemination, is protected by a wall, which plays the major role for survival in the environment. In order to find new spore wall candidates among the unknown proteins, a bioinformatic screening on the basis of the prediction of secretion, membrane anchoring and eventually of some domain homology was performed. In a first validation test, we used proteomics data to identify the target of a polyclonal antibody directed against the cell wall. The identified protein matched with criteria of selection and presented homologies with the polysaccharide deacetylase family, suggesting its implication in cell wall biogenesis. Its localization and the timing of its expression were studied by TEM immunocytochemistry. Screening was then applied to 44 expressed proteins of unknown function. Four of the nine potential cell wall components were demonstrated as being either endospore or exospore proteins, using polyclonal antibodies directed against GST recombinant proteins. The analysis of these new wall proteins should facilitate further bioinformatic screenings and should help in the better understanding of the structure and function of the microsporidian spore wall.

A Polysaccharide Deacetylase Homologue, PdaA, inBacillus subtilisActs as anN-Acetylmuramic Acid Deacetylase In Vitro

A polysaccharide deacetylase homologue, PdaA, was determined to act as an N-acetylmuramic acid deacetylase in vitro. Histidine-tagged truncated PdaA (with the putative signal sequence removed) was overexpressed in Escherichia coli cells and purified. Measurement of deacetylase activity showed that PdaA could deacetylate peptidoglycan treated with N-acetylmuramoyl-L-alanine amidase CwlH but could not deacetylate peptidoglycan treated with or without DL-endopeptidase LytF (CwlE). Reverse-phase high-performance liquid chromatography and mass spectrometry (MS) and MS-MS analyses indicated that PdaA could deacetylate the N-acetylmuramic acid residues of purified glycan strands derived from Bacillus subtilis peptidoglycan.

Characterization of a Polysaccharide Deacetylase Gene Homologue (pdaB) on Sporulation of Bacillus subtilis

The predicted amino acid sequence of Bacillus subtilis ybaN (renamed pdaB) exhibits high similarity to those of several polysaccharide deacetylases. Northern hybridization analysis with sporulation sigma mutants indicated that the pdaB gene is transcribed by EsigmaE RNA polymerase and negatively regulated by SpoIIID. The pdaB mutant was deficient in spore formation. Phase- and electron microscopic observation showed morphological changes of spores in late sporulation periods. The pdaB spores that had lost their viability were empty. Moreover, GFP driven by the promoter of the sspE gene was localized in the forespore compartment for the wild type, but was localized in both the mother cell and forespore compartments for phase-gray/dark forespores of the pdaB mutant. This indicates that GFP expressed in the forespores of the mutant leaks into the mother cells. Therefore, PdaB is necessary to maintain spores after the late stage of sporulation.

Induction of T helper type 1 responses by a polysaccharide deacetylase from Cryptococcus neoformans.

A 25-kDa cryptococcal deacetylase (d25) was found here to induce cell proliferation, as well as secretion of interleukin 2 and gamma interferon, but not interleukin 4, in spleen cells from d25-immunized or Cryptococcus neoformans-infected mice. The gamma interferon, but not the interleukin 2, response was required for the protective activities of d25 immunization in a murine cryptococcosis model.