Proteomics and Pathway Mapping Analyses Reveal Phase-Dependent Over-Expression of Proteins Associated with Carbohydrate Metabolic Pathways in Candida albicans Biofilms

Candida albicans is the most commonly isolated fungus associated with biofilms, which are extracellular matrix (ECM)-encased, drug-resistant microbial communities formed on indwelling medical devices. Protein profiles of fungal biofilms have not been investigated in detail, although such profiles are believed to play critical roles in fungal biofilm formation. In this study, we used two-dimensional difference-in-gel electrophoresis (DIGE)-based proteomics to identify differentially expressed proteins in C. albicans biofilms grown to early and mature phases, compared to planktonic cells. The resulting proteomic data set was subjected to pathway mapping to reveal phase-specific pathways that were differentially expressed in biofilm cell walls and extracellular matrix (ECM). Our analyses showed 107 proteins to be differentially expressed in ECM, while 44 were differentially expressed in cell walls during biofilm formation, compared to planktonic controls. Furthermore, 95% (102/107) and 68% (30/44) of these differentially expressed proteins were upregulated in ECM and cell walls of biofilms, respectively. These proteins were mapped to cellular pathways, which revealed that these differentially expressed proteins were associated with several metabolic pathways, in a phase-dependent manner. For example, among ECM-associated proteins, proteins within 18 pathways were differentially expressed, with two pathways (glutamate and nitrogen metabolism) unique to early phase, and four pathways (purine, Gly/Ser/Thr, and inositol metabolism, and carbon fixation) unique to mature phase biofilms. Such differences were also observed in cell wallassociated proteins, where proteins associated with 14 specific pathways were differentially regulated. We also found glycolytic enzymes including the key enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were overexpressed in biofilms at both early and mature phases, compared to planktonic controls. Iodoacetate-mediated inhibition of this enzyme completely abrogated the ability of C. albicans to form biofilms, indicating the role of glycolysis/gluconeogenesis pathways in biofilm formation. Taken together, we demonstrate that ECM and cell walls of C. albicans biofilms express increased levels of specific proteins within pathways in a phase-dependent manner, suggesting that these pathways, especially glycolysis/gluconeogenesis, play critical roles in fungal biofilm formation and maintenance.