Albicans NIH B-792 Research Articles

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.

Similarity of Acid Proteinases Secreted by Candida albicans NIH A-207 and NIH B-792 Strains Cultured in BSA-Supplemented Medium

Candida albicans NIH A-207 (serotype A) and NIH B-792 (serotype B) strains secreted one acid proteinases (AP) each in a yeast carbon-based medium supplemented with bovine serum albumin (BSA) as the sole nitrogen source. Isolation of AP from the culture filtrates was achieved by dialysis, followed by DEAE-Sepharose and Biogel P-100 column chromatographies. It was found that both enzymes from the two strains had very similar properties when examined. The molecular weights and isoelectric points were found to be 43 kDa and pH 4.0, respectively. The amino acid components and first 12 N-terminal amino acid sequences were virtually identical in both enzymes. The optimum pH of the enzymes was 3.5-4.0. The enzymes were heat-labile, and decreases in their activities were found above 37 degrees C. The AP activities were completely inhibited by the addition of pepstatin. No other inhibitor among those tested had any effect. The enzymes degraded all proteins examined, especially host defense factors such as immunoglobulin G and the granulocyte colony stimulating factor. The enzymes also caused similar degrees of enhancement of vascular permeability when they were injected into the dorsal skin of guinea pigs.

Characterization of alpha-1,6-mannosyltransferase responsible for the synthesis of branched side chains in Candida albicans mannan.

A particulate insoluble fraction from Candida albicans NIH B-792 (serotype B) strain cells was obtained as the residue after extracting a 105000 x g pellet of cell homogenate with 1% Triton X-100. Incubation of this fraction with a mannopentaose, Man alpha 1-->3Man alpha 1-->2Man alpha 1-->Man alpha 1-->2Man, in the presence of GDP-mannose and Mn2+ at pH 6.0 gave a branched mannohexaose, [sequence: see text] 6 the structure of which was identified by means of sequential off assignment. However, the enzyme fraction obtained from Candida parapsilosis gave Man alpha 1-->2Man alpha 1-->3Man alpha 1-->2Man alpha 1-->2 Man alpha 1-->2Man under the same conditions. These results demonstrate the finding that the structural difference in the mannans of these two species is due to the presence of alpha-1.6-linked branching mannose units in the C. albicans mannan [Shibata, N., Ikuta, K., Imai, T., Satoh, Y., Satoh, R., Suzuki, A., Kojima, C., Kobayashi, H., Hisamichi, K. & Suzuki, S. (1995) J. Biol. Chem. 270, 1113-1122]. The substrate-specificity study of the enzyme indicated that the structural requirement of the alpha-1,6-mannosyltransferase is Man alpha 1-->3Man alpha 1-->. The alpha-1,6-mannosyltransferase also transferred the alpha-1,6-linked branching mannose unit to the mannan of Saccharomyces cerevisiae. The transformation of the mannan was detected by the appearance of antigenic factor 4 using an enzyme-linked immunosorbent assay and two-dimensional homonuclear Hartmann-Hahn spectroscopy.

Existence of Branched Side Chains in the Cell Wall Mannan of Pathogenic Yeast, Candida albicans: STRUCTURE-ANTIGENICITY RELATIONSHIP BETWEEN THE CELL WALL MANNANS OF CANDIDA ALBICANS AND CANDIDA PARAPSILOSIS

Isolation of side chain oligosaccharides from mannans of Candida albicans NIH B-792 (serotype B) and Candida parapsilosis IFO 1396 strains has been conducted by acetolysis under mild conditions. Structural study of these oligosaccharides by 1H and 13C NMR and methylation analyses indicated the presence of novel branched side chains with the following structures in C. albicans mannan. [sequence: see text] It was observed that the H-1 proton chemical shifts of the second and the third mannose units from the reducing terminus in each oligosaccharide are shifted upfield by substitution with an alpha-linked mannose unit at position 6 of the 3-O-substituted mannose unit. An agglutination inhibition assay between factor 4 serum and cells of Candida stellatoidea IFO 1397 lacking the beta-1,2-linked mannose unit, with oligosaccharides obtained from these mannans, indicated that only the branched oligosaccharides were active. This finding suggests that the branched oligosaccharides correspond to the epitope of antigenic factor 4. The presence of the branched structure in other mannans was detected by the characteristic H-1-H-2-correlated cross-peak of the alpha-1,2-linked mannose unit connected with the 3,6-di-O-substituted one by two-dimensional homonuclear Hartmann-Hahn spectroscopy.

Complete Assignment of 1H and 13C Nuclear Magnetic Resonance Chemical Shifts of β-1,2-Linked Mannooligosaccharides Isolated from the Phosphomannan of the Pathogenic Yeast Candida albicans NIH B-792 Strain

To confirm correctness of the previous assignment study of H-i protons of β-1,2-linked mannooligosaccharides provided by Shibata et al. (Biochemistry 31, 5680-5686, 1992), anotber type of nonempirical assignment of the H-1 signals of be same oligosaccharides was achieved by adopting a sequential NMR assignment method that combines two-dimensional 1H-13C correlated spectroscopy, two-dimensional 1H-1H correlated spectroscopy, and two-dimensional heteronuclear multiple-bond connectivity. Based on the above results, all of the 1H and most of the 13C signals in each oligosaccharide could also be assigned. The results of the proton assignment study provided sufficient evidence to confirm the correctness of that obtained in the previous H-1 proton assignment study. Furthermore, the existence of a regularity rule between the order of mannose units and its 1H and 13C chemical shifts has been revealed.

Structural identification of an epitope of antigenic factor 5 in mannans of Candida albicans NIH B-792 (serotype B) and J-1012 (serotype A) as beta-1,2-linked oligomannosyl residues.

In previous articles, we reported the presence of phosphate-bound beta-1,2-linked oligomannosyl residues in the mannans of strains of Candida albicans serotypes A and B and Candida stellatoidea. To identify the antigenic factor corresponding to this type of oligomannosyl residue, a relationship between chemical structure and antigenic specificity in the mannans of C. albicans NIH B-792 (serotype B, B-strain) and C. albicans J-1012 (serotype A, J-strain) was investigated by using a combination of two-dimensional 1H nuclear magnetic resonance spectroscopy of H-1, H-2, and H-5 regions in the mannans and an enzyme-linked immunosorbent assay that employed concanavalin A-coated microtiter plates. It was shown in the present 1H nuclear magnetic resonance study that an examination of chemical shifts not only in the H-1 region but also in the H-5 region was useful for the quantitative determination of the phosphate-bound beta-1,2-linked oligomannosyl residues. In the enzyme-linked immunosorbent assay using concanavalin A-coated plates, it was revealed that, of factor sera 1, 4, and 5, only factor serum 5 showed a reactivity proportional to the densities of the beta-1,2-linked oligomannosyl residues of the mannan subfractions of different phosphate contents that had been prepared from the bulk B-strain mannan by DEAE-Sephadex chromatography. The above results indicate that the phosphate-bound beta-1,2-linked oligomannosyl residues, Manp beta 1----(2Manp beta 1----)n2Man (n = 0-5), correspond to antigenic factor 5.

Sequential nuclear magnetic resonance assignment of beta-1,2-linked mannooligosaccharides isolated from the phosphomannan of the pathogenic yeast Candida albicans NIH B-792 strain.

The H-1 and H-2 signals of beta-1,2-linked mannooligosaccharides isolated from the phosphomannan of Candida albicans NIH B-792 strain by mild acid hydrolysis were assigned by a sequential NMR assignment method that combines two-dimensional 1H-1H correlated spectroscopy (COSY) and two-dimensional nuclear Overhauser enhancement and exchange spectroscopy (NOESY). The results indicated that the H-1 and H-2 of each beta-1,2-linked mannopyranose unit show largely different signals compared with those of the alpha-linked ones and that the correlation between linkages and signals could not be explained by a conventional additivity rule. Furthermore, a regular proportional downfield shift of the H-1 signal was observed in the order of the mannose unit from the reducing terminal except those of the reducing and nonreducing terminal positions. Although the 1H NMR spectra of these oligosaccharides were complicated due to the presence of a large portion of the beta-anomer from the reducing terminal mannose unit, reduction of the oligosaccharides with NaBH4 to the corresponding alcohols gave simple and more readily interpretable 1H NMR spectra. Unexpectedly, however, a shift of H-1 signals by this reduction occurred not only on the second mannose unit but also on the third and fourth mannose units from the modified reducing terminal group of each oligosaccharide alcohol. This result indicates that the reducing terminal mannose unit is able to affect up to the fourth mannose unit from the reducing terminal. The presence of a long-distance interresidue NOE also suggests that the beta-1,2-linked mannooligosaccharides have a compactly folded conformation in solution.

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 m m pyridine-CH 3COOH 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 NaBH 4 by 1H, 13C, and two-dimensional homonuclear Hartmann-Hahn NMR spectroscopies, subsequently, gave results consistent with the structure described below (where Man p represents the mannopyranose unit): ▪ 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 α-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.

Formula omitted][formula omitted] 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 β-1,2-linked mannopyranose unit, although these mannans retained non-reducing terminal α-1,3 linked mannopyranose units, providing a substantiating evidence that the serotype A-specific epitope contains a non-reducing terminal β-1,2-linked mannopyranose unit.

Structure of the d-mannan of Candida stellatoidea IFO 1397 strain. Comparison with that of the phospho- d-mannan of Candida albicans NIH B-792 strain

The structure of the d-mannan of Candida stellatoidea IFO 1397 strain, which has properties identical to those of the phospho- d-mannan of C. albicans serotype B strain, does not contain phosphate groups, and its 1H- and 13C-n.m.r. spectra are quite similar to those of the phospho- d-mannan of C. albicans NIH B-792 strain. However, the 1H-n.m.r. and 1H- 13C-correlation n.m.r. spectra of the products obtained by digestion with a- d-mannosidase of C. stellatoidea d-mannan considerably differed from those of the corresponding digestion products of the C. albicans phospho- d-mannan. Additionally, the enzyme-linked immunosorbent assay, by means of a monoclonal antibody corresponding to (1→2)-linked β- d-oligomannosyl residues, of the phospho- d-mannan of the same C. albicans strain indicated that the C. stellatoidea d-mannan does not contain any (1→2)-linked β- d-oligomannosyl residues. The absence of these residues may be used as one of the criteria of chemotaxonomical identification of C. stellatoidea spp.

Structural study of cell wall phosphomannan of Candida albicans NIH B-792 (serotype B) strain, with special reference to 1H and 13C NMR analyses of acid-labile oligomannosyl residues

Chemical structures of manno-oligosaccharides, from biose to heptaose, released from the phosphomannan of Candida albicans NIH B-792 strain (serotype B) by mild acid hydrolysis were investigated. The results of 1H NMR, 13C NMR, and fast atom bombardment mass spectrometry analyses confirmed that these manno-oli gosaccharides belong to a homologous β-1,2-linked series. Although chemical shifts of 1H NMR patterns of these oligosaccharides were considerably too complicated to be assigned, their 13C NMR patterns were sufficiently simple to be interpreted, exhibiting a regular increase of downfield shift of ppm values of the C-1 atom from each mannopyranose residue in proportion to their molecular weights. In order to determine the whole chemical structure of the parent phosphomannan, the acid-stable domain was subjected to acetolysis and then enzymolysis with the Arthrobacter GJM-1 α-mannosidase and the resultant manno-oligosaccharides were investigated for their chemical structures by 1H NMR spectroscopy. The results of a precipitin-inhibition test using the β-1,2-linked manno-oligosaccharides, from biose to hexaose, in comparison with the corresponding isomers containing α-1,2 linkage with small amounts of α-1,3 linkage, indicated that the haptens possessing the former linkage exhibited much higher inhibitory effects than the corresponding isomers containing the latter linkages did. Based on the present findings, a chemical structure of the phosphomannan of this C. albicans strain was proposed.

Quantitative precipitin reaction and enzyme-linked immunosorbent assay of mannans of Candida albicans NIH A-207 and NIH B-792 strains compared.

We assessed the difference between results by enzyme-linked immunosorbent assay (EIA) in plastic support wells and the quantitative precipitin reaction (QPR) in glass test tubes for antigenic mannans and antibodies of two representative Candida albicans strains, NIH A-207 and NIH B-792. We investigated each of four mannan subfractions, with different phosphate contents, for their reactivities to the homologous polyclonal rabbit antiserum. Each series of mannan subfractions showed a reactivity proportional to their phosphate content in EIA, in a similar manner as observed in QPR. Moreover, in EIA, the cross-reactivities between the bulk mannans of the two C. albicans strains and the polyclonal antiserum of a Saccharomyces cerevisiae wild-type strain containing specific antibodies to the non-reducing terminal alpha-1,3-linked D-mannopyranose unit resembled those of the same antigen-antibody reactions in QPR. However, the mannan of C. albicans NIH A-207 strains, a weak antigen in the cross-QPR system, reacted fairly strongly in EIA in its high concentration range, indicating that EIA can be used to detect such an epitope in these mannans in concentrations undetectable by QPR. We conclude that EIA is a useful technique for immunochemical assay of yeast mannans and their antibodies on a smaller scale than with QPR.

Preparation of monoclonal antibodies reactive with beta-1,2-linked oligomannosyl residues in the phosphomannan-protein complex of Candida albicans NIH B-792 strain.

Hybridomas obtained by fusing the spleen cells of BALB/c female mice hyperimmunized with heat-killed yeast-form cells of Candida albicans NIH B-792 strain and a mouse myeloma cell line, P3X63Ag8.653, produced antibodies to beta-1,2-linked oligomannosyl residues in the phosphomannan-protein complex of the parent cells. Most of these monoclonal antibodies were IgM, but about 10% of the hybridomas produced IgG1 immunoglobulins. Ascites fluid from BALB/c mice inoculated with an IgG1-producing hybridoma showed different precipitability with the phosphomannan-protein complexes of three representative C. albicans strains, with NIH B-792 (serotype B) greater than NIH A-207 (serotype A) greater than J-1012 (serotype A, formerly serotype C). In contrast, a rabbit polyclonal antiserum to C. albicans NIH B-792 cells was unable to distinguish these same complexes. This ascites fluid agglutinated the heat-killed cells of three Candida strains, but not those of three others or of Torulopsis glabrata IFO 0622. The other ascites fluids, containing antibodies of the IgM class, agglutinated cells from three C. albicans strains and also C. tropicalis IFO 0587 cells.

Participation of peptide moieties in adhesive behavior of antigenic mannans of Candida albicans to the plastic microtiter plate in enzyme-linked immunosorbent assay.

We report our studies of the mechanism of the adhesion of mannans of Candida albicans NIH A-207 and C. albicans NIH B-792 strains to the surface of a polystyrene microtiter plate being utilized for enzyme-linked immunosorbent assay (EIA) of antigens of this pathogenic yeast species. This binding was manifested predominantly by the peptide moieties of the mannans forming hydrophobic bonds with the plastic molecule. Eliminating the peptide of each mannan by treating it with a hot aqueous alkaline solution of NaBH4 also eliminated color development in the EIA.

Characterization of phosphomannan-protein complexes isolated from viable cells of yeast and mycelial forms of Candida albicans NIH B-792 strain by the action of Zymolyase-100T

The isolation of phosphomannan-protein complexes from the viable cells of yeast (Y) and mycelial (M) forms of Candida albicans NIH B-792 strain was conducted by treatment with Zymolyase-100T followed by fractional precipitation with cetyltrimethylammonium bromide. The M-form complex was found to contain smaller amount of phosphate (1.3%) than that of the Y-form complex (1.6%). Proton magnetic resonance (PMR) spectra of these complexes indicated that the content of β-1,2-linked oligomannosyl and nonreducing terminal α-1,3-linked mannopyranosyl residues in the M-form complex was lower than that of the Y-form complex. With hot 10 m m HCl, the Y-form complex released a mixture of oligosaccharides ranging from mannose to mannoheptaose, while the M-form complex produced lower oligosaccharides, from mannose to mannotetraose. Upon acetolysis, the acid-modified complex of the M form gave mainly mannotetraose, while that of the Y form produced mainly mannopentaose and mannohexaose in addition to mannotetraose. The average length of branching moieties of the mannan of Y-form cells was therefore longer than that of M-form cells. These results indicate that the Y to M transformation of this C. albicans strain accompanies the suppression of enzyme activity concerning the biosynthesis of mannan such as β-1,2- and α-1,3-mannosyltransferases to synthesize the phosphomannan-protein complex containing mannan moiety with incomplete structure.

Immunochemical study on the mannans of Candida albicans NIH A-207, NIH B-792, and J-1012 strains prepared by fractional precipitation with cetyltrimethylammonium bromide

The mannans of Candida albicans NIH A-207 (A strain, serotype A), C. albicans NIH B-792 (B strain, serotype B), and C. albicans J-1012 (J strain, serotype C) prepared by fractional precipitation with cetyltrimethylammonium bromide (Cetavlon) were investigated for their immunochemical properties. Upon treatment with 10 m m HCl at 100 °C for 60 min, the mannans of A and B strains each released a mixture of manno-oligosaccharides ranging from hexaose to mannose together with (for each one) an acid-modified mannan, while J-strain mannan released lower oligosaccharides, tetraose to mannose. The acid-modified mannan of B strain did not show antibody-precipitating activity against homologous antiserum, whereas acid-modified A- and J-strain mannans retained most of this activity. The acid-released oligosaccharides were assumed to consist of β-1,2-linked d-mannopyranosyl residues from the results of specific rotation and proton magnetic resonance studies.

Relationship between phosphate content and serological activities of the mannans of Candida albicans strains NIH A-207, NIH B-792, and J-1012

The mannans from Candida albicans strains NIH A-207 (serotype A), NIH B-792 (serotype B), and J-1012 (serotype C) were fractionated on a column of diethylaminoethyl-Sephadex into five subfractions containing different amounts of phosphate. Antibody-precipitating activities of the mannan subfractions of strains NIH A-207 and NIH B-792 were proportional to their phosphate content, while those of strain J-1012 did not show regularly proportional precipitin activity. A similar tendency was also observed in the cross-reaction between the mannan su,fractions of strains NIH A-207 and J-1012 and their heterologous antisera. The mannans of strain NIH B-792 showed lower cross-reactivities against antisera of strains NIH A-207 and NIH B-792, i.e., only two subfractions containing larger amounts of phosphate were able to react with these antisera.