Phosphoproteomics of candida biofilms

Abstract

OBJECTIVES: Biofilm formation is a major virulent attribute of Candida species, which is directly related to therapeutic failure. Protein phosphorylation is a key reversible modification that regulates signaling networks. Traditionally protein phosphorylation has been studied by small number of proteins. However, recent advent in phosphoproteomics technologies has opened a new avenue for researchers to identify and quantify the phosphorylation of proteome in global scale. This study aimed to analyze the phosphoproteomics of different species of Candida under biofilm mode of growth. METHODS: Initially, biofilm formation of C. albicans, C. dubliniensis, C. krusei and C. tropicalis were studied for their growth kinetics and antifungal susceptibility. Next, proteins lysate was extracted from 48 h Candida biofilms using optimized extraction method. Proteins were separated by two-dimensional gel electrophoresis and phosphorylated proteins were stained with ProQ-Diamond stain, followed by Typhoon scanning. Next, total proteome maps were obtained with staining quantitative SYPRO Ruby fluorescent die followed by silver staining. Phophoyrylated proteins were identified by tandem mass spectrometry (MS/MS) using denovo sequencing of peptides. Phosphorylation sites were confirmed by spectral analysis. RESULTS: Differential proteomics and phosphor-proteomics maps were obtained for all Candida species, which showed both generic and specific patterns. There were 20, 17, 14, 14 phosphorylated proteins in the C. albicans, C. dubliniensis, C. krusei and C. tropicalis, respectively. Pil1p was heavily phosphorylated indicating its active role in the biofilm mode. CONCLUSION: We unraveled for the first time the phosphoproteomics of Candida biofilms with the generic and specific patterns among different Candida species, which facilitate developing targeted anti-fungal strategies against Candida biofilms (Supported by HKU funding 200907176121 for CJS and 20080717617 for LPS).