Rbt1 Protein Domains Analysis in Candida albicans Brings Insights into Hyphal Surface Modifications and Rbt1 Potential Role during Adhesion and Biofilm Formation

Abstract

Cell wall proteins are central to the virulence of Candida albicans. Hwp1, Hwp2 and Rbt1 form a family of hypha-associated cell surface proteins. Hwp1 and Hwp2 have been involved in adhesion and other virulence traits but Rbt1 is still poorly characterized. To assess the role of Rbt1 in the interaction of C. albicans with biotic and abiotic surfaces independently of its morphological state, heterologous expression and promoter swap strategies were applied. The N-terminal domain with features typical of the Flo11 superfamily was found to be essential for adhesiveness to polystyrene through an increase in cell surface hydrophobicity. A 42 amino acid-long domain localized in the central part of the protein was shown to enhance the aggregation function. We demonstrated that a VTTGVVVVT motif within the 42 amino acid domain displayed a high β-aggregation potential and was responsible for cell-to-cell interactions by promoting the aggregation of hyphae. Finally, we showed through constitutive expression that while Rbt1 was directly accessible to antibodies in hyphae, it was not so in yeast. Similar results were obtained for another cell wall protein, namely Iff8, and suggested that modification of the cell wall structure between yeast and hyphae can regulate the extracellular accessibility of cell wall proteins independently of gene regulation.

Candida albicans Biofilm Development and Its Genetic Control

“…The GPI anchor is then cleaved; the protein and anchor remnant are transferred to β-1,6-glucan and remain attached to the cell wall (32). Adhesins of this class include members of the Als ( A gglutinin L ike S equence) family (33), Eap1 ( E nhanced A dherence to P olystyrene 1) (34), Hwp1 ( H yphal W all P rotein 1) (35,36), and Rbt1 ( R epressed B y T UP1) (37), all of which are expressed at much higher levels in hyphal cells than in yeast cells. There is an adhesin-like protein expressed at highest levels in yeast cells, Ywp1 ( Y east W all P rotein 1), but it seems to function as an anti-adhesin (38).…”

“…Rbt1 is in the same adhesin family as Hwp1 (37,53). An rbt1Δ/Δ mutant has a mild biofilm defect in vitro , but shows additive effects with mutations in family members HWP1 and HWP2 (53).…”

“…An rbt1Δ/Δ mutant has a mild biofilm defect in vitro , but shows additive effects with mutations in family members HWP1 and HWP2 (53). Its N-terminal region promotes surface hydrophobicity and mediates adherence to polystyrene (37). A central domain is predicted to have high aggregation potential, and amyloid-inhibitor experiments similar to those carried out with Als5 support such a function (37).…”

“…Its N-terminal region promotes surface hydrophobicity and mediates adherence to polystyrene (37). A central domain is predicted to have high aggregation potential, and amyloid-inhibitor experiments similar to those carried out with Als5 support such a function (37). Although Rbt1 is normally expressed only on hyphal cells, Monniot et al could create a constitutive RBT1 allele through fusion to the TEF1 promoter.…”

“…Interestingly, this consitutively expressed protein could be recognized by anti-epitope antibodies only on hyphal cell surfaces. Recognition on yeast cell surfaces required mild digestion of the cell wall with zymolyase (37). These observations suggest that there is a fundamental structural difference between yeast and hyphal cell walls that affects the exposure of Rbt1 and, potentially, many other adhesins.…”