Global Identification of Biofilm-Specific Proteolysis in Candida albicans

Abstract

Candida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans. Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation.

Assessment and Optimizations of Candida albicans In Vitro Biofilm Assays

“…We performed 384-well standard optical density assays for four deletion strains (strains with the efg1, bcr1, als1, and sap6 deletions) that had previously been reported to have various but significant defects in biofilm formation (Fig. 1A) (13,15,35,40). Three of the mutants (the efg1, bcr1, and sap6 mutants) differed significantly from the isogenic wild-type strain under both medium conditions tested (RPMI medium and Spider medium).…”

“…The cell adhesion assay is based on the protocol initially reported by Winter and colleagues (35) and mimics the adherence step of a 96-well biofilm assay (e.g., the standard optical density assay). Cells are allowed to adhere under standard biofilm-forming conditions (OD 600 ϭ 0.…”

“…Specialized assays have also been developed to examine specific stages of biofilm development; for example, the number of cells dispersed from an established biofilm can be monitored by CFU counts, hemocytometer counts, and/or optical density measurements (32,33). Other specialized assays examine factors like growth in a microfluidic chamber (18,34,35), active attachment to a surface other than the bottom of a polystyrene plate (34), or the ability of drugs to penetrate into an established biofilm (36).…”

“…Third, we developed two types of assays (called the standard dispersal assay and the sustained dispersal assay) to quantify cell dispersion at different stages of biofilm development, using only a standard plate reader for analysis (33). Finally, we developed an assay to observe biofilm formation under linear flow in a microfluidic device where adherence and cellular morphology can be visually and temporally tracked using time-lapse microscopy throughout the process of biofilm development (35). We refer to this assay as the microfluidic assay.…”

“…First, we established an assay based on the OD at 600 nm (OD 600 ) to rapidly screen large numbers of conditions and to track the same condition over multiple time points (17,33,35). Second, we also developed a new assay to quantitatively determine the number of cells adhering to a surface in the first stage of biofilm development (35).…”