Production and Isolation of the Candida Species Biofilm Extracellular Matrix.

Abstract

The extracellular matrix (ECM) is a dynamic structure comprising of all four classes of macromolecules. In the biofilm setting, this matrix is key to the survival of microbial communities by conferring to biofilms both structural integrity and protection against diverse environmental insults. In Candida spp., this matrix contributes to pathogenesis by conferring to biofilms both drug resistance and protection against immune attack. Understanding the biochemical nature of the matrix and its individual components is critical to the development of novel diagnostics and antifungal strategies against persistent Candida biofilm infections. Therefore, efficient methods for ECM isolation are required. The two matrix isolation protocols described herein are adapted for both small- and large-scale isolation of biofilm matrix. Both procedures involve seeding of biofilms in either 6-well plates or large-surface-area roller bottles, followed by cell adhesion and biofilm maturation for 2days with continuous motion. In both cases, the matrix is separated from the biomass via sonication, a step which gently and effectively removes the matrix without disturbing the fungal cell wall. The large-scale protocol includes additional filtration, lyophilization, and dialysis steps to yield purified matrix material sufficient for numerous biochemical, structural, and functional assays. Small-scale isolation yields enough matrix for gas chromatography (GC), total carbohydrate quantification via the phenol-sulfuric acid method, and total protein quantification via the bicinchoninic acid (BCA) assay. Large-scale isolation yields enough matrix to perform NMR spectroscopy, liquid chromatography, mass spectrometry, and nucleic acid sequencing. These protocols have been adapted for use in Candida species but may be adapted for other biofilm-forming fungal species and bacteria.