Shotgun Proteomics elucidates the regulatory pathway of Candida biofilms

Objectives: Biofilm formation and filamentation are major virulent attributes of the fungal pathogen Candida albicans. Mitogen Activated Proteins Kinase (MAPK) and cAMP/Protein Kinase (PKA) pathways are the two major biological pathways that regulate these attributes. Therefore, aim of the present study was to elucidate the role of MAPK, PKA pathways using Candida mutants lacking CPH1 and EFG1; major regulators of MAPK and PKA pathways, respectively. Methods: Initially, biofilm formation, anti-oxidative capacities and antifungal susceptibility of Candida mutants were studied using standard methodology. Thereafter, protein lysates of respective biofilms were quantified, denatured, and cysteines blocked. Each sample was then digested with trypsin and labeled with the iTRAQ tags. Differentially labeled digests were subjected to LC-MALDI-TOF/TOF analysis. The MS/MS peptides and phosphorylation patterns were identified by bioinformatics tools and further validated using Candida Genome Database (CGD). Results: The mutant lacking EFG1 was devoid of filaments and unable to form mature biofilms, and more susceptible to oxidative stress and antifungals, compared to the wild type strain. Totally, 861 proteins over 95% C.I. which are related to MAPK and PKA pathways were identified. In particular out of 452 known and characterized proteins, 133 related to PKA pathway were down-regulated more than two folds compared to 20 proteins down-regulated in MAPK pathway. Of these down-regulated proteins a considerable number was related to the stress response and drug resistance. Of the characterized proteins, 317 were phosphorylated and 24 were fungal specific. Further, 409 yet uncharacterized proteins were also identified. . Conclusion: cAMP/PKA pathway, but not MAPK pathway governs the filamentation and biofilm formation of Candida albicans. PKA related stress response proteins may potentially mediate the higher antifungal resistance seen in Candida biofilms. Fungal specific, PKA pathway biomarkers, and their phosphorylation profiles are likely to be of use in the search for the novel antifungals. (RGC-Grant HKU-7624/06M