Cloning and characterization of CAD1/AAF1, a gene from Candida albicans that induces adherence to endothelial cells after expression in Saccharomyces cerevisiae

Abstract

Adherence to the endothelial cell lining of the vasculature is probably a critical step in the egress of Candida albicans from the intravascular compartment. To identify potential adhesins that mediate the attachment of this organism to endothelial cells, a genomic library from C. albicans was used to transform a nonadherent strain of Saccharomyces cerevisiae. The population of transformed yeasts was enriched for highly adherent clones by repeated passages over endothelial cells. One clone which exhibited a fivefold increase in endothelial cell adherence, compared with S. cerevisiae transformed with vector alone, was identified. This organism also flocculated. The candidal DNA fragment within this adherent/flocculent organism was found to contain a single 1.8-kb open reading frame, which was designated CAD1. It was found to be identical to AAF1. The predicted protein encoded by CAD1/AAF1 contained features suggestive of a regulatory factor. Consistent with this finding, immunoelectron microscopy revealed that CAD1/AAF1 localized to the cytoplasm and nucleus but not the cell wall or plasma membrane of the transformed yeasts. Because yeasts transformed with CAD1/AAF1 both flocculated and exhibited increased endothelial cell adherence, the relationship between adherence and flocculation was examined. S. cerevisiae expressing either of two flocculation phenotypes, Flo1 or NewFlo, adhered to endothelial cells as avidly as did yeasts expressing CAD1/AAF1. Inhibition studies revealed that the flocculation phenotype induced by CAD1/AAF1 was similar to Flo1. Thus, CAD1/AAF1 probably encodes a regulatory protein that stimulates endothelial cell adherence in S. cerevisiae by inducing a flocculation phenotype. Whether CAD1/AAF1 contributes to the adherence of C. albicans to endothelial cells remains to be determined.

FIG. 1

Effects of p13 and its subclones on the adherence of S. cerevisiae to endothelial cells. The relative sizes of p13 and its subclones are displayed on the left. The graph on the right illustrates the percent adherence to endothelial cells of S. cerevisiae LL-20 transformed with the corresponding plasmids. Results are the mean ± standard deviation for at least three experiments. The hatched rectangle illustrates the location and orientation of the open reading frame of CAD1/AAF1. ∗, P ≤ 0.003 compared to organisms transformed with pE20-H.

FIG. 2

Southern hybridization analysis of genomic DNA from C. albicans ATCC 36082 probed with the CAD1/AAF1-containing XhoI fragment from p13-2x. DNA was digested with XhoI (lane 1), KpnI (lane 2), and BamHI (lane 3). The 2.9-kb XhoI and 2.2-kb KpnI bands were seen during restriction mapping of p13, indicating that the probe bound specifically to CAD1/AAF1. The 6-kb BamHI fragment was not seen during restriction mapping, indicating that a BamHI site exists downstream of the SmaI site in the genome.

FIG. 3

Immunoprecipitation of the epitope-tagged CAD1/AAF1 protein. An anti-HA antibody was used to immunoprecipitate proteins from S. cerevisiae LL-20 transformed with pYef1H (lane 1), pYCADN (lane 2), pYef2H (lane 3), and pYCADC (lane 4). Organisms transformed with pYCADN synthesized CAD1/AAF1 with the HA epitope fused to the N terminus, whereas yeasts transformed with pYCADC synthesized CAD1/AAF1 with the HA epitope attached to the C terminus. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on an 8% gel and then identified by autoradiography. Molecular weight markers (in thousands) are shown on the right.

FIG. 4

Immunogold labeling demonstrated that the HA-CAD1/AAF1 fusion protein localizes to the cytoplasm and nucleus in S. cerevisiae. S. cerevisiae transformed with either vector alone (pYeF1H) (A) or the vector encoding the HA-CAD1/AAF1 fusion protein (pYCADN) (B) is shown. After being fixed, the organisms were sectioned and then the HA-CAD1/AAF1 fusion protein was labeled by exposure to murine anti-HA followed by gold-labeled goat anti-mouse antibodies. The organisms and gold particles were viewed by transmission electron microscopy. Arrows indicate the cell walls of the organisms.