Candida albicans (CA) is the most commonly isolated fungus associated with biofilms [extracellular matrix (ECM)-encased microbial communities formed on indwelling medical devices which are resistant against commonly used antifungals. The carbohydrate-rich ECM is believe to play critical role in the biofilm forming ability of different bacteria, but its role in fungal biofilm formation and antifungal resistance has not been investigated. Therefore, obvious knowledge gaps exist in the study of fungal biofilm ECM. In Preliminary Studies, we: (1) standardized an EDTA-treatment based method to isolate ECM associated proteins from CA biofilm, (2) demonstrated that the levels of total carbohydrates (TC), total protein (TP), and extracellular DNA (eDNA) increase during biofilm development, (3) optimized a gel filtration method to isolate ECM proteins, (4) used traditional two-dimensional gel electrophoresis (2DGE) to identify 15 proteins differentially expressed in biofilm ECM, (5) used state-of-the-art technique of 2D difference in gel electrophoresis (DIGE) proteomics to demonstrate that 231 ECM proteins were differentially expressed in ECM of biofilm and planktonic CA. These analyses also showed 10 unique proteins to be expressed in ECM of biofilms eight in mature biofilms, and two in early phase biofilms. (6) Used preliminary mass spectrometry (MS) analyses to identify five differentially expressed proteins. Based on these Preliminary Data, we hypothesize that CA biofilm-specific ECM proteins play critical roles in biofilm formation and antifungal resistance. To test this hypothesis, in this grant application, we propose studies based on the following specific aims:
Aim I. Determine the identity of proteins uniquely expressed in matrix of mature biofilms, using 2D-DIGE proteomics.
Aim II. Construct isogenic mutant strains of C. albicans lacking the biofilm matrix-specific proteins.
Aim III. Determine the effect of: (A) EDTA treatment of biofilms on their antifungal susceptibility, and (B) Targeted gene deletions on the ability of CA to form biofilms and the morphology, architecture, and antifungal susceptibility of these biofilms. The studies described in this proposal will allow us to identify biofilm-specific ECM proteins that are critical to Candida biofilm formation in vitro and in vivo, which may represent novel anti-biofilm therapeutic targets and/or diagnostic markers. Candida albicans (CA) is the most commonly isolated fungus associated with biofilms [extracellular matrix (ECM)-encased microbial communities formed on indwelling medical devices, which are resistant against commonly used antifungals. The studies described in this proposal will allow us to identify biofilm-specific ECM proteins that are critical to Candida biofilm formation in vitro, which may represent novel anti-biofilm therapeutic targets and/or diagnostic markers.
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