MNN5, 1,6 mannosyltransferase gene, OCH1, protein O chemical compound library mannosyltransferase gene, PMT, or 1,2 mannosyltransferase gene for the O linked oligosaccharide synthesis, MNT, which are responsible for the synthesis of the mannoprotein, markedly reduces the virulence of C. albicans in mice. Furthermore, the deletion of CaMIT1, which encodes the GDP mannose:inositol phospho ceramide mannose transferase responsible for the synthesis of phospholipomannan, also reduces the virulence in mice. Therefore, we are interested in the correlation between the structure of the cell wall mannans and the pathogenicity of the cells or susceptibility to antifungal agents. In this report, we show the presence of a significant difference in the structure of the cell wall mannans from three C. glabrata strains. We also show the existence of a difference in the susceptibility to some antifungal drugs in these strains correlated to the structural difference in the cell wall mannans. The C. glabrata NBRC 0005, NBRC 0622, and NBRC 103857 strains were obtained from the NITE Biological Resource Center of the National Institute of Technology and Evaluation. The cells were grown at 28 with shaking in YPD liquid medium for 2 days. Materials The 2,3 mannosidase was obtained from New England BioLabs Japan, Inc. The mannans of Saccharomyces cerevisiae X2180 1A, Saccharomyces kluyveri NBRC 1685 and its 1,6 branched mannopentaose were the same specimens used in a preceding study. Preparation of cell wall mannoprotein The cells were treated overnight with 4% formaldehyde, washed with deionized water, and dehydrated with acetone. The cell wall mannoprotein was extracted from the acetone dried cells by deionized water at 120 for 2 h. The extract was dialyzed against water and then lyophilized. The cell wall extract was fractionated by hexadecyltrimethylammonium bromide followed by the method of Lloyd et al.
A Cetavlon fraction precipitated at pH 9.0 in the presence of borate was recovered as the mannan fraction. Release of O linked oligosaccharides from the mannoprotein by elimination The mannoprotein was dissolved in 0.5 M NaBH4/0.1 M NaOH and incubated for 18 h at 25. After incubation, the reaction mixture was neutralized with 50% acetic acid, and the solution was applied onto a column Silodosin of Bio Gel P 2 and eluted with water. The liberated oligosaccharide alditol fractions were separately collected and the N linked polysaccharide was recovered in the void volume. The acetolysis selectively cleaves the 1,6 linkages. The mannoprotein was dissolved in formamide and acetylated by adding pyridine/acetic anhydride and incubated at 40 for 12 h. The reaction mixture was then evaporated to dryness in vacuo. The O acetylated derivative was dissolved in acetic anhydride/acetic acid/sulfuric acid, and acetolysis was carried out at 40 for 36 h as the mild conditions. The resultant acetylated fragmented oligosaccharides were extracted into chloroform and washed with water. After deacetylation of the oligosaccharides using 1 M sodium methoxide, the mixture was neutralized with 50% acetic acid, deionized, and lyophilized. Monosaccharide linkage analysis The oligosaccharide was dissolved in a NaOH/DMSO suspension prepared using powdered NaOH. After stirring for 30 min, methyl iodide was added.