Yoshio Okawa

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.

Protective effect of N-acetyl chitohexaose on Listeria monocytogenes infection in mice

A water‐soluble oligosaccharide, N‐acetyl chitohexaose (NACOS‐6) was able to enhance the protecting effect of BALB/c male mice against Listeria monocytogenes infection, when administered intraperitoneally 24 hr before the challenge with this microbe. Significant decrease in number of microbes within the peritoneal cavity, spleen, and liver from the mice of NACOS‐6‐administered group was not observed 1 day after the infection but 4 days after the infection. Administration of NACOS‐6 enhanced the delayed‐type hypersensitivity response against sheep red blood cells (SRBC) or heat‐killed L. monocytogenes. Splenic T lymphocytes from mice administered NACOS‐6 released macrophage activating factor (MAF). These results suggested that NACOS‐6 was also able to elevate the function of cellular immunity. Macrophages treated with a combination of NACOS‐6 and the culture supernatant of splenic T lymphocytes from mice administered NACOS‐6, “NACOS‐6 sup,” were found to exert a fairly strong growth‐inhibitory effect on L. monocytogenes. Interferon‐γ (IFN‐γ) and interleukin 2 (IL‐2) were able to enhance the growth‐inhibitory effect on L. monocytogenes by the NACOS‐6‐treated macrophages.

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.

Protecting Effect of Chitin and Chitosan on Experimentally Induced Murine Candidiasis

Chitin and chitosan were found to exhibit a protective effect on mice administered these polysaccharides intraperitoneally against infection of the viable cells of Candida albicans NIH A‐207 strain. A significant difference was observed between the protective effects of chitin and chitosan, i.e., chitin was much more effective than chitosan when the C. albicans cells were challenged via the intravenous route. In intraperitoneal inoculations of C. albicans cells, however, chitosan provided stronger resistance for mice than chitin.

Effect of N-Acetylchito-Oligosaccharides on Activation of Phagocytes

Four N‐acetylchito‐oligosaccharides, from tetra‐N‐acetylchitotetraose (NACOS‐4) to hepta‐N‐acetylchitoheptaose (NACOS‐7), were found to increase the number of peritoneal exudate cells (PEC) in male BALB/c mice after 3 hr intraperitoneal administration of 50 mg/kg of each oligosaccharide. The number of attracted cells, consisting largely of polymorphonuclear leukocytes (PMN), was proportional to the molecular weights of the administered oligosaccharides, except for NACOS‐7 which displayed the same activity as NACOS‐6. In an in vitro chemotaxis assay using normal mouse leukocytes, it was found that NACOS‐6 displayed stronger effects than muramyl dipeptide. The PEC from NACOS‐6 treated mice showed a higher active oxygen‐generating activity. PMN from normal mouse peripheral blood were also shown to have enhanced active oxygen‐generating activity in vitro. PEC from NACOS‐6 treated mice were shown to possess strong candidacidal activity in vitro.

Candidaalbicans serotype a strains grow in yeast extract-added sabouraud liquid medium at pH 2.0, elaborating mannans without β-1,2 linkage and phosphate group

Cultivation of three Candida albicans strains, NIH A-207, J-1012, and NIH B-792, abbreviated as A-, J-, and B-strains, respectively, in yeast extract-enrich Sabouraud liquid medium at pH 2.0 provided the following findings, i.e., the two former strains belonging to serotype A were able to grow in this medium in almost the same rates as those in the same medium of pH 5.9, while B-strain cells did not proliferate under the former condition. The cells of A- and J-strains cultivated at pH 2.0 did not undergo agglutination with the factor serum 6 in a commercially available factor serum kit, Candida Check, corresponding to C. albicans serotype A-specific epitope. It was also revealed by 1H-13C correlation spectra of the mannans isolated from the cells of A- and J-strains contained neither phosphate group nor beta-1,2-linked mannopyranose unit, although these mannans retained non-reducing terminal alpha-1,3 linked mannopyranose units, providing a substantiating evidence that the serotype A-specific epitope contains a non-reducing terminal beta-1,2-linked mannopyranose unit.

Temperature-dependent change of serological specificity of Candida albicans NIH A-207 cells cultured in yeast extract-added Sabouraud liquid medium: disappearance of surface antigenic factors 4, 5, and 6 at high temperature

The cells of Candida albicans NIH A‐207 strain (A‐strain) cultivated in YSLM at high temperatures (37 and 40°C) did not undergo agglutination with the factor sera 4, 5, and 6 in a commercially available factor serum kit, ‘Candida Check’, and formed a grape‐like shape. The mannans isolated from the cells had lost their reactivity against the factor sera in ELISA. It was also revealed by 1H NMR analysis that the mannans contained neither a phosphate group nor a β‐1,2‐linked mannopyranose unit, although these mannans increased the non‐reducing terminal α‐1,3‐linked mannopyranose unit. The cells and the mannans prepared by cultivation at such high temperatures followed by 27°C in the same medium entirely recovered the reactivity with the factor sera.

Detection of β-1,2-mannosyltransferase in Candida albicans cells

A particulate insoluble fraction from Candida albicans J‐1012 (serotype A) strain cells was obtained as the residue after extracting a 105,000 × g pellet of cell homogenate with 1% Triton X‐100. Incubation of this fraction with a mannopentaose, Manβ1 → 2Manα1 → (2Manα1 →)22Man (αβMan5), in the presence of GDP‐mannose followed by high performance liquid chromatography showed the formation of a mannohexaose. Analysis of the product by 1H NMR indicates that αβMan5 was changed to Manβ1 → 2Manβ1 → 2Manα1 → (2Manα1 →)22Man (αβMan6). This β‐1,2‐mannosyltransferase (ManTase) II activity was completely inhibited by Zn2+ and was not restored by the addition of EDTA. The corresponding enzyme fraction from C. albicans NIH B‐792 (serotype B) strain cells, the mannan of which does not possess both the αβMan5 and αβMan6 side chains, also exhibited the same β‐1,2‐ManTase II activity.

Structural study on a phosphorylated mannotetraose obtained from the phosphomannan of Candida albicans NIH B-792 strain by acetolysis

A mixture of phosphorylated manno-oligosaccharides was isolated from the acid-stable domain of phosphomannan of Candida albicans NIH B-792 strain (serotype B) by acetolysis and was fractionated on a column of Bio-Gel P-2 equilibrated with 50 mM pyridine-CH3COOH buffer, pH 5.0. A monophosphorylated mannotetraose was isolated as the major constituent. Structural analyses of this phosphate-containing tetraose and its reduction product with NaBH4 by 1H, 13C, and two-dimensional homonuclear Hartmann-Hahn NMR spectroscopies, subsequently, gave results consistent with the structure described below (where Manp represents the mannopyranose unit): [formula: see text] It was unexpected that the major phosphorylated branch in the acid-stable domain of the parent phosphomannan of this C. albicans strain is a relatively short mannotetraosyl residue containing solely alpha-1,2-linked mannopyranose units, and a phosphate group as a 6-O-ester on the intermediary unit adjacent to the nonreducing terminal group. These findings indicate that the size of the major phosphorylated branch of this phosphomannan is the same as that of Saccharomyces cerevisiae.

Candidacidal Effect of Peritoneal Exudate Cells in Mice Administered with Chitin or Chitosan

Recently, several papers have been published on the mechanism of the protective effect of microbial infection and on the role of reactive oxygen intermediates (ROI) and myeloperoxidase (MPO) (2-4, 6-8), demonstrating that polymorphonuclear leukocytes (PMN) play an important role in the killing of Candida albicans by generating large amounts of ROI (2), while macrophages (MO) were able to kill the yeast form cells of C. albicans by releasing a cationic protein (4). However, the role of these phagocytes in the killing process of C. albicans cells has not been fully elucidated . In the preceding paper of this series, we reported that both chitin (poly-Nacetyl-D-glucosamine) and chitosan (de-N-acetylated product of chitin) showed