Function of Candida albicans adhesin Hwp1 in biofilm formation

Abstract

Hwp1 is a well-characterized Candida albicans cell surface protein, expressed only on hyphae, that mediates tight binding to oral epithelial cells. Prior studies indicate that HWP1 expression is dependent upon Bcr1, a key regulator of biofilm formation. Here we test the hypothesis that Hwp1 is required for biofilm formation. In an in vitro model, the hwp1/hwp1 mutant produces a thin biofilm that lacks much of the hyphal mass found in the hwp1/HWP1 reconstituted strain. In a biofilm cell retention assay, we find that the hwp1/hwp1 mutant is defective in retention of nonadherent bcr1/bcr1 mutant cells. In an in vivo rat venous catheter model, the hwp1/hwp1 mutant has a severe biofilm defect, yielding only yeast microcolonies in the catheter lumen. These properties of the hwp1/hwp1 mutant are consistent with its role as a hypha-specific adhesin and indicate that it is required for normal biofilm formation. Overexpression of HWP1 in a bcr1/bcr1 mutant background improves adherence in the in vivo catheter model. This finding provides additional support for the model that Hwp1 is critical for biofilm adhesion. Hwp1 is the first cell surface protein known to be required for C. albicans biofilm formation in vivo and is thus an excellent therapeutic target.

FIG. 1.

In vitro biofilm formation. Biofilms were grown under our standard conditions (13) in Spider medium and stained with concanavalin A for CSLM visualization. Artificially colored CSLM depth views, in which blue color represents cells closest to the silicone and red color represents cells farthest from the silicone, are shown in panels A and B, in which blue represents 0 μm and red represents 300 μm (panel A) or 500 μm (panel B). CSLM side views are shown in lower panels C and D, in which the scale bars represent 50 μm. Cells in the surrounding medium of the hwp1/hwp1 biofilm were visualized through phase-contrast microscopy at ×400 magnification (panel E).

FIG. 2.

Requirement for Hwp1 in biofilm cell retention in vitro. Biofilm cultures were inoculated with mixtures of biofilm-defective bcr1/bcr1 mutant and the test strains indicated. The ratio of bcr1/bcr1 mutant strain to test strain in each well was 0%, 50%, 62.5%, or 87.5%, as specified on the x axis. A 100% bcr1/bcr1 biofilm culture was included as a control. Cell retention was measured after 48 h growth by measuring the OD₆₀₀ of the surrounding medium. The results of triplicate assays are shown, with error bars indicating standard deviations.

FIG. 3.

Roles of Bcr1 target genes in biofilm cell retention in vitro. Biofilm cultures were inoculated with mixtures of biofilm-defective bcr1/bcr1 mutant and the test strains indicated. The ratio of bcr1/bcr1 mutant strain to test strain in each well was 0%, 50%, 62.5%, or 87.5%, as indicated by the shaded wedge. Cell retention was measured after 48 h growth by measuring the OD₆₀₀ of the surrounding medium. The results of triplicate assays are shown, with error bars indicating standard deviations.

FIG. 4.

Hwp1 requirement for biofilm formation in vivo. Central venous catheters were introduced into rats, inoculated with C. albicans strain CAH7-1A1E2 (hwp1/hwp1) (A), CAHR3 (hwp1/HWP1) (B), or CJN1222 (bcr1/bcr1 TEF1-HWP1/HWP1) (C), and then flushed and incubated (1). Catheters were then removed, and their contents were visualized by scanning electron microscopy after 24 h.