Contribution of phospholipomannan to the surface expression of beta-1,2-oligomannosides in Candida albicans and its presence in cell wall extracts

Abstract

beta-1,2-Oligomannosides (beta-1,2-Man) derived from Candida albicans mannan have been shown to act as adhesins and to induce protective antibodies. We used monoclonal antibodies specific for beta-1,2-Man in electron, confocal, and fluorescence microscopy to study the surface expression of beta-1,2-Man epitopes. These monoclonal antibodies were also used for Western blotting of cell surface extracts to study the nature of the molecules expressing the beta-Man epitopes. Evidence was obtained for the contribution of a glycolipid, phospholipomannan (PLM), to the complex expression of beta-1,2-Man epitopes at the cell wall surfaces of yeasts grown on solid media. PLM was present in intercellular matrixes of colonies grown on agar and was detected as a contaminant in mannan batches prepared by conventional methods.

FIG. 1.

Ultrathin sections of C. albicans cells grown on agar, fixed by cryosubstitution, and labeled with gold particles reveal the distribution of β-1,2-oligomannoside epitopes (MAb AF1 reactive). (a) Low-magnification micrograph showing C. albicans organelles and distribution of epitopes at the cell periphery, inside, and at the cell wall surface. (b) Details of the peripheral location of β-1,2-oligomannoside epitopes in the haloplasm or associated with a vesicle located close to the plasmalemma, where they are distributed either inside or on the membrane. (c) β-1,2-Oligomannoside epitopes cover the fibrils of adjacent cells up to the point where fibrils merge (arrows). Bars, 1 μm.

FIG. 2.

Confocal microscopy of C. albicans cells grown on agar. β-1,2-Oligomannoside epitopes are not homogeneously distributed at the cell surface, and some cells may be negative (see mother and daughter cells in panel a); others exhibit a patchy distribution (a and b) or double polarity in the secretion process (c). Bars, 5 μm

FIG. 3.

Fluorescent staining of a colony print made on a microscopic slide after extensive washing. (a) Staining with ConA-FITC. A few cells which remain on the slide have their surfaces homogeneously stained by ConA, but this lectin does not bind to any amorphous material remaining on the slide. (b and c) Staining with MAb AF1, specific for β-1,2-oligomannoside epitopes. In contrast, this staining reveals material still adhering to the slide whose distribution corresponds to the spaces between cells that have been removed by washing. Bars, 5 μm

FIG. 4.

Comparative Western blot analysis of protein patterns and glycoconjugates expressing α-linked oligomannoside and/or β-1,2-oligomannoside epitopes in AERC extracts (A through C, lanes 1) and β-mercaptoethanol extracts (A through C, lanes 2; D, lanes 1 and 2) prepared from C. albicans VW32 grown at 37°C on SDA. To control the efficiency of transfer onto the membranes, proteins were stained with Ponceau S prior to immunostaining (A). ConA-peroxidase (B, lanes 1 and 2; D, lane 2) and MAb AF1 (C, lanes 1 and 2; D, lane 1) were used to detect α-linked oligomannoside and β-1,2-oligomannoside epitopes, respectively. (D) A selective chloroform-methanol-water extraction performed on the β-mercaptoethanol extract confirmed the glycolipid nature of PLM, which expressed β-1,2-oligomannoside epitopes (lane 1) in the absence of α-linked oligomannoside epitopes (lane 2).

FIG. 5.

(A and B) Western blot analysis of the distribution of protein patterns and β-1,2-oligomannoside epitopes on glycoconjugates in an AERC extract (lane 1), a 4% SDS extract (lane 2), and a water extract (lanes 3) prepared from C. albicans VW32 grown at 37°C. (C) Results of chloroform-methanol-water extraction performed on the 4% SDS (lane 1) and water extracts (lane 2). Staining was performed with Ponceau red (A) or MAb AF1 revealed with a phosphatase-labeled anti-mouse IgM antibody (B and C).

FIG. 6.

Western blots of different mannan batches extracted from strain VW32, serotype A (A), and strain NIH A207 (B and C). Mannans in panels A and B were prepared in our laboratory, and the mannan analyzed in panel C was prepared in S. Suzuki's laboratory in Sendai, Japan. Lanes 1 were revealed with MAb EB-CA1, specific for α-linked mannose residues. Lane A2 was stained with ConA-peroxidase, which is also specific for α-linked mannose residues. MAb AF1 was used to reveal β-1,2-linked oligomannosides (lanes A3, B2, and C2). PLM was always present in the mannan extracts, irrespective of the strain or preparation procedure, but never expressed α-linked oligomannoside epitopes.