Localization studies of the moonlighting protein Tsa1p in C. albicans

The cell wall is the first contact site between host and pathogen and is thus critical for colonization and infection of the host. We have identified Tsa1p (Thiol-specific antioxidant-like protein) as part of the cell wall and within the cytoplasm of C. albicans. Tsa1p has been shown to be responsible for several distinct functions, including functions in oxidative stress and genome stability. It does not contain a typical signal sequence for entry into the secretory pathway therefore the mechanism by which Tsa1p is released to the cell surface is unknown. We could show that localization of Tsa1p to the cell wall is determined by at least two different parameters. In previous experiments Tsa1p could only be detected at the cell surface in hyphaeinducing media indicating a morphology-dependent localization of Tsa1p to the cell surface. In addition, time course experiments showed that transfer to fresh media components also induces a temporary translocation of Tsa1p to the cell surface in yeast form cells. This indicates a connection with quorum sensing. Indeed, addition of farnesol to YPD medium results in stronger and longer lasting accumulation of Tsa1p to the cell surface. To check on regions within TSA1 that are required for localization, we deleted the C-terminal 12 amino acids of CaTSA1. From its human homologue it is known that a signal for membrane localization resides in the C-terminal part. Additionally, the cysteines of the two active sites of Tsa1p were substituted by serines to check their role in Tsa1p function and localization. All mutants showed sensitivity to oxidative stress (H2O2) like the delta-TSA1 strain. Cell surface localization of Tsa1p in the active site mutants is strongly reduced compared to the wildtype indicating that an active form of Tsa1p is needed for localization of Tsa1p to the cell surface. However, in the mutant strain containing the TSA1 copy with deleted C-terminus Tsa1p is still able to localize to the cell surface, indicating that the C-terminus is not responsible for its localization in C. albicans. In addition, all mutants show a reduced survival rate when exposed to human neutrophils and also have a higher beta-glucan exposure at the cell surface of blastospores compared to the wildtype. This confirms that the cell wall composition in these mutants is altered and that Tsa1p has an important role in maintaining the cell wall composition.