Significant structural change in both O- and N-linked carbohydrate moieties of the antigenic galactomannan from Aspergillus fumigatus grown under different culture conditions.

Invasive aspergillosis is an important cause of morbidity and mortality in immunocompromised patients. Diagnosis of this infection frequently employs detection of the circulating galactomannan in the patient serum using enzyme immunoassay (EIA), a highly sensitive and specific system. Although there are many structural studies of the galactomannan of Aspergillus fumigatus, some inconsistencies are present in these results. In this study, to clarify the relationship between the growth conditions and structure of the galactomannans, we cultured A. fumigatus using two distinct yeast/fungal cultivation media, i.e. the yeast extract-peptone-dextrose (YPD) medium and yeast nitrogen base (YNB) medium. Galactomannans prepared from the resulting culture supernatants were structurally characterized by (1)H and (13)C nuclear magnetic resonance, methylation analysis, acetolysis and α-mannosidase degradation. These assays revealed that the galactomannan from the YPD cultivation had short ß-1,5-linked galactofuranose (Galf) oligosaccharide chains in both the O- and N-linked carbohydrate moieties, while the galactomannan from the YNB cultivation incorporated long Galf oligosaccharide chains. The galactomannans derived from the two culture conditions significantly differed in reactivity based on the EIA diagnostic system. We also demonstrated the presence of a novel Galf-containing branched oligosaccharide in the O-linked moiety.

Identification of distinct ligands for the C-type lectin receptors Mincle and Dectin-2 in the pathogenic fungus Malassezia.

Various C-type lectin receptors (CLRs), including Mincle and Dectin-2, function as pattern recognition receptors and play a central role in immunity to fungal pathogens. However, the precise structures of the CLR ligands in various pathogenic fungi have yet to be completely defined. Here we report that Malassezia, an opportunistic skin fungal pathogen, is cooperatively recognized by Mincle and Dectin-2 through distinct ligands. Solvent-based fractionation revealed that Mincle and Dectin-2 recognize lipophilic and hydrophilic components of Malassezia, respectively. Mass spectrometry and nuclear magnetic resonance (NMR) revealed glyceroglycolipid and unique mannosyl fatty acids linked to mannitol as two Mincle ligands. An O-linked mannobiose-rich glycoprotein was identified as a Malassezia ligand for Dectin-2. Cytokine production in response to the Mincle ligands and the Dectin-2 ligand was abrogated in Mincle(-/-) and Dectin-2(-/-) dendritic cells, respectively. These results demonstrate that Mincle and Dectin-2 recognize distinct ligands in Malassezia to induce host immune responses.

Significant differences in the cell-wall mannans from three Candida glabrata strains correlate with antifungal drug sensitivity.

Candida glabrata is often the second or third most common cause of candidiasis after Candida albicans. C. glabrata infections are difficult to treat, often resistant to many azole antifungal agents and are associated with a high mortality rate in compromised patients. We determined the antigenic structure of the cell-wall mannoproteins from three C. glabrata strains, NBRC 0005, NBRC 0622 and NBRC 103857. (1)H NMR and methylation analyses of the acetolysis products of these mannoproteins showed a significant difference in the amount of the ß-1,2-linked mannose residue and side-chain structure. The C. glabrata NBRC 103857 strain contained up to the triose side chains and the nonreducing terminal of the triose was predominantly the ß-1,2-linked mannose residue. By contrast, the mannans of the two former strains possessed up to the tetraose side chains and the amount of the ß-1,2-linked mannose residue was very low. Larger oligosaccharides than tetraose in the acetolysis products of these mannans were identified as incomplete cleavage fragments by analyzing methylation, (1)H NMR spectra and the α1-2,3 mannosidase degradation reaction. Resistance to the antifungal drugs itraconazole and micafungin was significantly different in these strains. Interestingly, the NBRC 103857 strain, which involved a large amount of the ß-1,2-linked mannose residues, exhibited significant sensitivity to these antifungal drugs.

Difference in fine specificity to polysaccharides of Candida albicans mannoprotein between mouse SIGNR1 and human DC-SIGN.

C-type lectin SIGNR1 directly recognizes Candida albicans and zymosan and has been considered to share properties of polysaccharide recognition with human DC-SIGN (hDC-SIGN). However, the precise specificity of SIGNR1 and the difference from that of hDC-SIGN remain to be elucidated. We prepared soluble forms of SIGNR1 and hDC-SIGN and conducted experiments to examine their respective specificities. Soluble SIGNR1 (sSIGNR1) bound several types of live C. albicans clinical isolate strains in an EDTA-sensitive manner. Inhibition analyses of sSIGNR1 binding by glycans from various yeast strains demonstrated that SIGNR1 preferentially recognizes N-glycan α-mannose side chains in Candida mannoproteins, as reported in hDC-SIGN. Unlike shDC-SIGN, however, sSIGNR1 recognized not only Saccharomyces cerevisiae, but also C. albicans J-1012 glycan, even after α-mannosidase treatment that leaves only ß1,2-mannose-capped α-mannose side chains. In addition, glycomicroarray analyses showed that sSIGNR1 binds mannans from C. albicans and S. cerevisiae but does not recognize Lewis(a/b/x/y) antigen polysaccharides as in shDC-SIGN. Consistent with these results, RAW264.7 cells expressing hDC-SIGN in which the carbohydrate recognition domain (CRD) was replaced with that of SIGNR1 (RAW-chimera) produced comparable amounts of interleukin 10 (IL-10) in response to glycans from C. albicans and S. cerevisiae, but those expressing hDC-SIGN produced less IL-10 in response to S. cerevisiae than C. albicans. Furthermore, RAW-hDC-SIGN cells remarkably reduced IL-10 production after α-mannosidase treatment compared with RAW-chimera cells. These results indicate that SIGNR1 recognizes C. albicans/yeast through a specificity partly distinct from that of its homologue hDC-SIGN.

Chemical structure of beta-galactofuranose-containing polysaccharide and O-linked oligosaccharides obtained from the cell wall of pathogenic dematiaceous fungus Fonsecaea pedrosoi.

Fonsecaea pedrosoi is the main etiologic agent of chromoblastomycosis, usually occurring in tropical and subtropical areas. The cell wall components of pathogenic microorganisms behave as an antigen and/or ligand of the innate immune response. The cells of F. pedrosoi reacted with the α-galactopyranose-binding lectin (Griffonia simplicifolia lectin 1B4 isolectin, GSL 1B4), as well as the α-mannose-binding lectin, concanavalin A. The cell wall glycoprotein was isolated from conidial cells of F. pedrosoi, and its structure was analyzed by (1)H-nuclear magnetic resonance (NMR) and (13)C-NMR experiments. The N-linked polysaccharide moiety consists of a backbone ß-1,6-linked galactofuranose and α-1,6-linked mannose polymers, both of which are substituted with α-1,2-linked glucose side-chains. Furthermore, the glycoprotein contained a large amount of O-linked oligosaccharides, especially a hexaose that constituted approximately 20% of the glycoprotein. Unexpectedly, the hexaose had a highly branched structure which consisted of galactofuranose, galactopyranose, glucose and mannose residues as follows: An anti-F. pedrosoi antibody specifically reacted with the cells of F. pedrosoi, whereas other fungal cells that contain galactofuranose residues did not react. The reactivity of the antibody was strongly inhibited by the branched hexaose, suggesting that the characteristic structure of the O-linked hexaose involves the antigenic specificity of the cells.

Conformational analysis of ß-1,2-linked mannobiose to mannoheptaose, specific antigen of pathogenic yeast Candida albicans.

Candida albicans contains characteristic ß-1,2-linked oligomannosyl moieties in the cell wall mannan. Reduction of the reducing termini of these oligosaccharides by NaBH(4) causes a significant downfield shift in the NMR signals for the second and third mannose units and upfield shift of the fourth mannose unit. To show the correlation between the shift in the NMR signals and the conformations of the ß-1,2-linked mannooligosaccharides, we performed molecular mechanics calculations. Six energy minima of the ß-1,2-linked mannobiose were found in the relaxed map computed using the AMBER force field. Five of the six energy minima could also be generated by a simulated annealing from a 900 K molecular dynamics. Similarly, the solution conformation of the ß-1,2-linked mannotriose to mannoheptaose was identified by the high temperature-simulated annealing. In the mannotetraose, the nonreducing terminal mannose unit was located near the reducing terminal one and formed a folded conformation. This result suggests that a mannose unit affects the H-1 chemical shifts of not only the second mannose unit, but also the third and fourth mannose units.

Enzymatic synthesis of new oligosaccharides using mannosyltransferases from Candida species and their NMR assignments.

The outer layer of the cell wall of pathogenic fungi, Candida species, consists of mannan, which plays an important role in infection. In this study, we synthesized several oligosaccharides using mannosyltransferases obtained from Candida parapsilosis and Candida albicans. Namely, we synthesized mannotetraoses [Manalpha1--> 2Manalpha1-->3Manalpha1-->2Man and Manalpha1-->3(Manalpha1-->6)Manalpha1-->2Man] from mannotriose, Manalpha1-->3Manalpha1--> 2Man, and mannohexaoses [Manalpha1-->2Manalpha1-->3Manalpha1-->3Manalpha1-->2Manalpha1-->2Man and Manalpha1-->3(Manalpha1-->6)Manalpha1-->3Manalpha1-->2Manalpha1-->2Man] from mannopentaose, Manalpha1-->3Manalpha1-->3Manalpha1-->2Manalpha1-->2Man. The linkage sequence of these oligosaccharides was identified by a sequential (1)H-NMR assignment method combined with rotating frame nuclear Overhauser enhancement spectroscopy and relayed coherence transfer spectroscopy. The steric effect by the alpha-1,6-linked branching mannose residue to the H-1 proton chemical shift of the neighboring 3-O-substituted mannose residue was different from that of the 2-O-substituted mannose residue. These oligosaccharides having novel structures seem to be useful as the substrate or ligand for glycomics.

Some properties of beta-1,2-mannosyltransferases related to the biosynthesis of the acid-labile oligomannosyl side chains in Candida albicans NIH B-792 strain cells.

We detected the beta-1,2-mannosyltransferases (beta-1,2-MTs), which participate in the biosynthesis of oligomannosyl side chains in the mannan acid-labile fraction, in a particulate insoluble fractions prepared from Candida albicans NIH B-792 strain cells grown at 27 degrees C and at 37 degrees C in a yeast extract-added Sabouraud liquid medium (YSLM). The beta-1,2-MT VI-6 prepared from the cells grown at 27 degrees C exhibited the maximum activity at pH 7.0 and at 30 degrees C. The beta-1,2-MT VI-6 activity was only slightly affected by Mn2+, Mg2+, Ca2+, and ethylenediaminetetraacetic acid, but completely inhibited by Zn2+ and Ni2+. The beta-1,2-MT activities from the cells grown at 37 degrees C were lower than that from the cells grown at 27 degrees C, especially on the longer beta-1,2-mannooligosaccharides than tetraose.

Influence of oxidative and osmotic stresses on the structure of the cell wall mannan of Candida albicans serotype A.

In this study, we investigated the structural changes in the cell wall mannan of Candida albicans serotype A strain cells cultured under various stress conditions, that is, oxidative stress of 3.5 mM H(2)O(2), osmotic stress of 1.5M NaCl, and heat stress at 37 degrees C, compared with the normal condition of 30 degrees C in yeast extract-added Sabouraud liquid medium (YSLM). Based on the (1)H nuclear magnetic resonance (NMR) and fluorophore-assisted carbohydrate electrophoresis (FACE) analyses of the mannans, we showed that the proportion of the terminal beta-1,2-linked mannose side chain unit in the mannan increased in the cell proliferation process under both the normal condition and the oxidative stress condition. The osmotic stress induced a slight decrease in the proportion of the beta-1,2-linked mannose unit in the acid-labile fraction. The heat stress induced a significant decrease in the proportions of the beta-1,2-linked mannose unit in both the acid-labile and acid-stable fractions. Based on these results, we propose that C. albicans significantly changes the mannan structures under various stress conditions and that sufficient attention to the cultural conditions is needed to perform an accurate diagnosis of candidiasis.

The cell wall galactomannan antigen from Malassezia furfur and Malassezia pachydermatis contains beta-1,6-linked linear galactofuranosyl residues and its detection has diagnostic potential.

Lipophilic yeasts of the genus Malassezia are associated with several skin diseases, such as pityriasis versicolor, Malassezia folliculitis, seborrhoeic dermatitis and atopic dermatitis, and are also increasingly associated with catheter-related fungaemia. The cell wall components of pathogenic micro-organisms behave as an antigen and/or ligand of the innate immune response. Live cells of Malassezia furfur and Malassezia pachydermatis did not react with an anti-alpha-1,2-mannoside antibody. However, they showed a strong hydrophobicity and reactivity with an anti-beta-1,3-glucan antibody compared to those of C. albicans. The cell wall polysaccharides of M. furfur and M. pachydermatis were isolated and their structures analysed by (1)H and (13)C NMR experiments. Both polysaccharides were shown to be beta-1,6-linked linear galactofuranosyl polymers with a small amount of mannan. The presence of galactomannan on cells of Malassezia species has not been described previously. The galactomannan did not react with an anti-Aspergillus fumigatus monoclonal antibody which has specificity for beta-1,5-linked galactofuranosyl residues. An anti-M. furfur antibody strongly reacted with the galactomannans of M. furfur and M. pachydermatis, but did not react with the galactomannans of Trichophyton rubrum, A. fumigatus or Fonsecaea pedrosoi. The characteristics of the anti-M. furfur antibody suggest that there is potential for diagnosis of Malassezia infections by antigen detection.

Comparison of pathogenicity of various Candida tropicalis strains.

To clarify the pathogenicity and the pathogenic factors of Candida tropicalis strains, five strains, IFO 0199, IFO 0587, IFO 0589, IFO 1400, and IFO 1647, of C. tropicalis were tested for their lethality to mice, adherence to Hela cells, hydrophobicity, cell growth under acidic conditions (pH 2.0-5.9), and sucrose assimilation using C. albicans NIH A-207 strain as reference. The pathogenicity for mice of all strains was observed in the increasing order IFO 1400=IFO 0589, IFO 0587, IFO 1647=NIH A-207, and IFO 0199. The pathogenicity for mice by all the tested C. tropicalis strains was not correlated with the adherence, the hydrophobicity, or the cell growth. On the other hand, the pathogenicity correlated well with the sucrose assimilation ability. These results show that the pathogenic mechanisms of the C. tropicalis strains were different from those of the C. albicans strains.

Activity and stability of alpha- and beta-mannosyltransferases in Candida albicans cells cultured at high temperature and at low pH.

We measured the activity and stability of three mannosyltransferases to ascertain the mechanisms of changes in the antigenicity and the mannan structure of Candida albicans cells cultured at high temperature (37 degrees C) under acidic (pH 2.0) conditions in a liquid medium. The alpha-1,6-mannosyltransferase (alpha-1,6-MT) activity of the particulate-insoluble enzyme fractions prepared from C. albicans J-1012 (J-1012) cells cultured at 37 degrees C was retained compared to those at 27 degrees C, whereas beta-1,2-mannosyltransferase II (beta-1,2-MT II) activity was detected in the 27 degrees C fraction but not in the 37 degrees C fraction. Similar results were obtained in the fraction prepared from J-1012 cells cultured at pH 2.0. The alpha-1,6-MT activities of fractions prepared from C. albicans NIH B-792 (B-792) strain cells cultured at 37 degrees C were retained compared to those at 27 degrees C, whereas beta-1,2-mannosyltransferase VI-6 (beta-1,2-MT VI-6) activity was detected in the fraction of B-792 cells cultured at 27 degrees C but not detected in the 37 degrees C fraction. We also found that the beta-1,2-MT II and beta-1,2-MT VI-6 activity of C. albicans cells was more sensitive to both high temperature and low pH compared with alpha-1,6-MT activity.

Primary structure and function of a cytotoxic outer-membrane protein (ComP) of Plesiomonas shigelloides.

We previously isolated and characterized a 40-kDa cytotoxic outer-membrane protein (ComP) produced by Plesiomonas shigelloides strain P-1 (P-1). Sequence analysis of the comP gene revealed a coding region of 1068 bp, with a predicted mature protein composed of 335 amino acids and a molecular mass of 38.597 kDa. Three-dimensional structural modeling of ComP suggests that it has a beta-barrel structure with 16 transmembrane strands, eight short periplasmic turns and eight external loops. blast search results and protein modeling suggest that ComP may be a novel porin protein of P. shigelloides. In order to understand the role of ComP during P. shigelloides infection, we constructed a deletion mutant strain (P. shigelloides DeltacomP; P-1201), and compared the pathogenicity of P-1201 vs. the wild-type strain P-1 in Caco-2 cells. Unlike P-1, the deletion strain P-1201 was not cytotoxic to Caco-2 cells and did not lead to apoptosis. These data indicate that ComP may be the predominant virulence factor that triggers cell death in the host cells following infection.

Structural analysis of cell wall mannan of Candida sojae, a new yeast species isolated from defatted soybean flakes.

We investigated the structural and immunochemical characteristics of cell wall mannan obtained from Candida sojae JCM 1644, which is a new yeast species isolated from defatted soybean flakes. The results of a slide-agglutination test and of an enzyme-linked immunosorbent assay using anti-factor sera to the pathogenic Candida species indicated that the cells and the C. sojae mannan were cross-reactive to the specific anti-factor sera against Candida albicans serotype A (FAb 6) and Candida guilliermondii (FAb 9). Two-dimensional homonuclear Hartmann-Hahn analysis indicated that the mannan consisted of various linked oligomannosyl side chains containing alpha-1,2-, alpha-1,3-, alpha-1,6- and beta-1,2-linked mannose residues. However, although the determinants of antigenic factors 6 and 9 could be not found in this mannan, branched side chains, Manbeta1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)n2Man and a linear alpha-1,6-linked polymannosyl backbone, which are cross-reacted by FAbs 6 and 9, respectively, were identified. The mannan was subjected to acetolysis in order to determine the polymerization length of the alpha-1,2-linked oligomannosyl residue in the side chains. The result of (1)H-nuclear magnetic resonance analysis of the released oligosaccharides showed that the remarkable regularity in the length of alpha-1,2-linked oligomannosyl side chains, which were previously found in mannans of other Candida species, is not observed in this mannan.

Comparison of pathogenicity of various Candida albicans and C. stellatoidea strains.

In order to clarify the pathogenicity and the pathogenic factors of various Candida species strains, three strains, NIH A-207 and J-1012 (serotype A), and NIH B-792 (serotype B) of Candida albicans and two strains, ATCC 20408 (karyotype II) and ATCC 36232 (karyotype I) of C. stellatoidea, a synonym for C. albicans, were tested for their lethality to mice, adherence to Hela cells, hydrophobicity, and cell growth under acidic conditions, pH 2.0-5.9. The pathogenicity for mice of all the strains was observed in the order NIH B-792, ATCC 36232, J-1012, NIH A-207, and ATCC 20408. The pathogenicity for mice by all the strains used was well correlated with adherence to the Hela cells, the hydrophobicity, and the cell growth under the acidic condition, pH 2.0. These results emphasize that these specific properties of the C. albicans and C. stellatoidea strains play an important role in the pathogenesis of candidosis.

Chemical structure of the cell-wall mannan of Candida albicans serotype A and its difference in yeast and hyphal forms.

The structure of the cell-wall mannan from the J-1012 (serotype A) strain of the polymorphic yeast Candida albicans was determined by acetolysis under mild conditions followed by HPLC and sequential NMR experiments. The serotype A mannan contained beta-1,2-linked mannose residues attached to alpha-1,3-linked mannose residues and alpha-1,6-linked branching mannose residues. Using a beta-1,2-mannosyltransferase, we synthesized a three-beta-1,2-linkage-containing mannoheptaose and used it as a reference oligosaccharide for 1H-NMR assignment. On the basis of the results obtained, we derived an additivity rule for the 1H-NMR chemical shifts of the beta-1,2-linked mannose residues. The morphological transformation of Candida cells from the yeast form to the hyphal form induced a significant decrease in the phosphodiesterified acid-labile beta-1,2-linked manno-oligosaccharides, whereas the amount of acid-stable beta-1,2 linkage-containing side chains did not change. These results suggest that the Candida mannan in candidiasis patients contains beta-1,2-linked mannose residues and that they behave as a target of the immune system.

Adhesion of Candida albicans to HeLa cells: studies using polystyrene beads.

The adherence to HeLa cells by the yeast-type cells of the Candida albicans NIH A-207 strain cultivated for 2 d at 27 degrees C in the yeast extract-added Sabouraud liquid medium (YSLM) and the 500 mM galactose-added yeast nitrogen base medium (YNB-Gal) was compared. The yeast cells cultured in the YNB-Gal clearly increased the adherence to the HeLa cells compared to the YSLM. The adherence drastically decreased by pronase treatment of the yeast cells. Next, to define the sugar receptors of the yeast cells, lactosylceramide (LC)-, the H type 1 antigen (HA)-, Lewis(a) antigen (Le(a))-, mannan- and BSA-coated polystyrene beads were used for the adherence to the yeast cells. Only the LC- and HA-beads were bound to the yeast cells. The adherence of the two beads to the yeast cells cultured in the YNB-Gal was higher than that to the yeast cells cultured in the YSLM. The yeast cell wall mannan-coated polystyrene beads did not adhere at all to the Hela cells. Based on these results, it is evident that the protein parts involving the LC and HA receptors on the surface of the yeast cells correlate with the adherence to the HeLa cells.

Cell adherence-promoted activity of Plesiomonas shigelloides groEL.

Previously, it has been demonstrated that the invasion of Caco-2 cells by Plesiomonas shigelloides induces apoptotic cell death. Therefore, the attachment to and colonization of eukaryotic intestinal host cells by P. shigelloides are important steps in causing pathogenicity. In this study, the participation of P. shigelloides GroEL in the attachment of P. shigelloides was examined. The groESL operon of P. shigelloides was isolated by PCR. The nucleotide sequence of the groESL operon of P. shigelloides revealed two ORFs of 294 nucleotides for groES and 1647 nucleotides for groEL. Cell fractionation and immunostaining experiments suggested that the GroEL of P. shigelloides was associated with the bacterial cell surface. The expression of the groEL gene was upregulated during the attachment and apoptosis-induction stages, and the expression of the protein was also induced during the attachment stage. Furthermore, GroEL efficiently promoted the attachment of P. shigelloides to Caco-2 cells, as measured by a FACSCalibur flow cytometer. These results demonstrated that GroEL has a positive influence on the attachment of P. shigelloides to Caco-2 cells.

Antigenicity of cell wall mannans of Candida albicans and Candida stellatoidea cultured at high temperatures in BACTEC medium.

The study of the antigenicity of pathogenic Candida albicans and Candida stellatoidea cells grown in BACTEC fungal medium (BFM) is useful for clinical analysis so as accurately to diagnose candidiasis. When C. albicans NIH A-207 was grown in BFM and fetal bovine serum-added BFM at the high temperatures of 36 and 40 degrees C, the cell density increased, with a mixture of yeast cells, pseudohyphae, and hyphae and with full hyphal development in the cultures compared with cultivation (mostly cells in yeast form) at 27 degrees C in both media. The mannans produced when cells were grown at these high temperatures were less reactive by enzyme-linked immunosorbent assay with factor sera 4, 5, and 6 in the commercially available kit 'Candida Check' than were the mannans obtained following growth at 27 degrees C. Based on 1H-nuclear magnetic resonance analysis, the mannans from cells grown at high temperatures had lost a phosphate group and a beta-1,2-linked mannopyranose unit, and had increased the number of non-reducing terminal alpha-1,3-linked mannopyranose units. We obtained similar results for mannans produced by C. albicans J-1012, C. albicans NIH B-792, C. albicans JCM 9061, C. stellatoidea ATCC 20408, and C. stellatoidea ATCC 36232 strains cultivated in BFM at 36 degrees C. These results suggest that both C. albicans and C. stellatoidea cells cultured at high temperatures, irrespective of the medium and shape of the cells, alter their antigenicity and chemical structure of cell wall mannans.

[Structure of fungal cell wall polysaccharides].

Candida albicans mannan consists of the alpha-1,6-linked backbone moiety and the alpha-1,2- and alpha-1,3-linked side chains. It also contains alpha-1,6-branched mannose units, beta-1,2-linked mannose units, and phosphate groups. The cell wall mannans of the genus Candida possess three types of beta-1,2 linked mannose units. One is linked via the phosphodiester linkage, the second type is connected to an alpha-1,2-linked mannose unit, and the third type is attached to an alpha-1,3-linked mannose unit. These beta-1,2-linked mannose units showed a strong antigenicity and produce the characteristic NMR chemical shifts. Using two-dimensional NMR techniques, we will practically determine the structure of these polysaccharides in a nondestructive manner.