Proteomic Analysis to Identify Oral Epithelial Cell-Antifungal Effector Protein Candidates

Objective: Oropharyngeal candidiasis (OPC) caused by the mucosal commensal organism, Candida albicans, remains a significant problem in immunocompromised individuals. Innate and adaptive immunity are considered to be critical to protection against OPC. Among innate anti-Candida mechanisms, it has been demonstrated that oral epithelial cells significantly inhibit the growth of Candida in vitro. The mechanism of action, involving a protein moiety, is fungistatic, acid-labile, and includes a strict requirement for cell contact with no role for soluble factors by intact, but not necessarily live, epithelial cells. In accordance, extracted epithelial cell surface proteins have similar antifungal activity. The purpose of the present study was to identify candidates for the antifungal effector protein using the acid-labile property and proteomic analysis. Methods: Primary oral epithelial cells harvested from human saliva were treated with periodic acid (PA) or phosphate-buffered saline (PBS). Surface membrane proteins were biotinylated and extracted with octyl-â-glucopyranoside. Biotinylated extracted proteins were bound to and then eluted from both yeast and hyphal forms of Candida. Extracted and eluted proteins were examined by Western blot for unique bands between acid- and buffer-treated cells. Similar SDS-PAGE gels using extracted proteins were prepared for proteomics. Bands of interest were excised and analyzed by mass spectrometry. Results: When compared to proteins extracted from PA-treated cells, proteins from PBS-treated cells and eluted from Candida showed two unique bands of approximately 33 and 45 kDa by Western blot. The full compliment of extracted proteins visualized by Western blot or SDS-PAGE showed similar results. Proteomic analysis revealed the 33 kDa and 45 kDa proteins of interest to be Annexin-A1 and Actin, respectively. Conclusion: These results suggest that Annexin-A1, known to play a role in inhibition of cell growth, is a strong candidate as the antifungal effector protein on oral epithelial cells. This work was supported by NIH grant DE12178.