Detection of Als proteins on the cell wall of Candida albicans in murine tissues

Abstract

A murine model of disseminated candidiasis was utilized to determine whether Candida albicans Als proteins are produced in vivo. The kidneys, spleen, heart, liver, and lungs were collected from mice inoculated with one of three C. albicans strains (SC5314, B311, or WO-1). Immunohistochemical analysis of murine tissues by using a rabbit polyclonal anti-Als serum indicated that Als proteins were produced by each C. albicans cell in the tissues examined. Patterns of staining with the anti-Als serum were similar among the C. albicans strains tested. These data indicated that Als protein production was widespread in disseminated candidiasis and that, despite strain differences in ALS gene expression previously noted in vitro, Als protein production in vivo was similar among C. albicans strains. The extensive production of Als proteins in vivo and their presence on the C. albicans cell wall position these proteins well for a role in host-pathogen interaction.

FIG. 1

Light micrographs of serial kidney sections from mouse 7 stained with preimmune serum (left) or anti-Als serum (right). C. albicans hyphal cells that failed to stain with the preimmune serum appear light purple, while cells staining with the DAB substrate are brown.

FIG. 2

Light micrographs of serial kidney sections from mouse 7 stained with the GMS method (left) or with the anti-Als serum (right). The full thickness of the fungal cell wall is stained by each method, supporting the conclusion that Als proteins are localized in the C. albicans cell wall.

FIG. 3

Amino acid sequences of the four Als1p 10-mer peptides used to raise anti-Als serum aligned with the corresponding sequences predicted from other characterized ALS genes (–10). Amino acids that differ from the Als1p sequence are boxed. Coordinates of peptides selected from Als1p are Peptide 1 (amino acids 53 to 62), Peptide 2 (amino acids 98 to 107), Peptide 3 (amino acids 139 to 148), and Peptide 4 (amino acids 156 to 165). The Als5p sequence shown was originally designated Ala1p (5).

FIG. 4

Analysis of the specificity of the anti-Als serum. An N-terminal, 433-amino-acid fragment of each Als protein was heterologously produced in S. cerevisiae and run in duplicate sets of lanes on a sodium dodecyl sulfate-polyacrylamide gel. One-half of the gel was silver stained (left panel) and the other half Western blotted with the anti-Als serum (right panel). The variability in migration of each protein is attributable to differential glycosylation of the Als proteins (7). Molecular size (in kilodaltons) is shown at the left of the figure.