Investigating the Proteolytic Activity of Secreted Proteins from Candida albicans Biofilms

Hospital-acquired infections can be life-threatening to immunocompromised patients and the most common hospital-acquired fungal infections are caused by Candida albicans (C. albicans). Part of the difficulty in treating C. albicans infections is that cells frequently group together to form a protective polymeric matrix known as a biofilm. Antifungal treatments are unable to penetrate the biofilm to treat the infection, resulting in long-term, high dose antifungal therapy. Proteases, degradative enzymes secreted by fungi, provide an essential source of nutrients to the organism in both the free-floating (planktonic) and biofilm stages.  Little is known about the substrate specificity of secreted proteases from the biofilm compared to cells in the free flowing or planktonic state. We hypothesize that there will be a difference in cleavage sites among the biofilm and inhibiting the biofilm proteases will decrease the viability of cells protected by the biofilm. We have profiled the global activity of secreted proteases from planktonic cells and the biofilm using a physicochemically diverse peptide library. Liquid-Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis was used to detect changes in the peptide sequence. We have determined the preferred amino acid sequences of the biofilm and planktonic. The biofilm had a greater amount of unique cleavage sites than the planktonic stage. The information gathered on substrate specificity can then be used to design selective inhibitors that target biofilm proteases. Learning about the substrate specificity of proteases secreted from the biofilm and planktonic C. albicans can lead to the development of technologies for diagnosis and therapy.