Candida albicans biofilm-induced vesicles confer drug resistance through matrix biogenesis

Abstract

Cells from all kingdoms of life produce extracellular vesicles (EVs). Their cargo is protected from the environment by the surrounding lipid bilayer. EVs from many organisms have been shown to function in cell-cell communication, relaying signals that impact metazoan development, microbial quorum sensing, and pathogenic host-microbe interactions. Here, we have investigated the production and functional activities of EVs in a surface-associated microbial community or biofilm of the fungal pathogen Candida albicans. Crowded communities like biofilms are a context in which EVs are likely to function. Biofilms are noteworthy because they are encased in an extracellular polymeric matrix and because biofilm cells exhibit extreme tolerance to antimicrobial compounds. We found that biofilm EVs are distinct from those produced by free-living planktonic cells and display strong parallels in composition to biofilm matrix material. The functions of biofilm EVs were delineated with a panel of mutants defective in orthologs of endosomal sorting complexes required for transport (ESCRT) subunits, which are required for normal EV production in diverse eukaryotes. Most ESCRT-defective mutations caused reduced biofilm EV production, reduced matrix polysaccharide levels, and greatly increased sensitivity to the antifungal drug fluconazole. Matrix accumulation and drug hypersensitivity of ESCRT mutants were reversed by addition of wild-type (WT) biofilm EVs. Vesicle complementation showed that biofilm EV function derives from specific cargo proteins. Our studies indicate that C. albicans biofilm EVs have a pivotal role in matrix production and biofilm drug resistance. Biofilm matrix synthesis is a community enterprise; prior studies of mixed cell biofilms have demonstrated extracellular complementation. Therefore, EVs function not only in cell-cell communication but also in the sharing of microbial community resources.

Fig 1. C. albicans biofilms secrete unique EVs.

(a) A SEM of EV-like structures on the surface of C. albicans growing in a biofilm. Scale bar indicates 0.6 μm. (b) A SEM of EV-like structures within deposits of the extracellular matrix in biofilms. Scale bar indicates 0.5 μm. (c) A cryoTEM of Candida biofilm-derived EVs are surrounded by a 7-nm-thick lipid bilayer. Scale bar indicates 100 nm. (d) Quantitative analysis of EVs in C. albicans biofilms measured at various culture growth time points using imaging flow cytometry. Biofilm cell number was quantified by dry weight. The measurements were done in triplicate. (e) Size distribution of C. albicans planktonic EVs evaluated by dynamic light scattering. (f) Size distribution of C. albicans biofilm EVs evaluated by dynamic light scattering. Underlying data can be found in S1 Data. EV, extracellular vesicle; SEM, scanning electron micrograph.

Fig 2. Unique C. albicans biofilm EV protein, lipid, and carbohydrate cargo delivers biofilm extracellular matrix components.

Biofilm EVs proteomes are shown in orange versus planktonic EVs shown in blue (a, b, c), whereas ECM is shown in green (d, e, f). Smallest regions (a, b, d, e) represent identified proteins and are arranged inside higher level regions according to their KEGG functional category and pathway assignment by using a Voronoi treemap layout. (a, b) Log2 biofilm EVs/planktonic EVs ratios of proteins relative abundances were mapped to a color ramp starting with orange (more protein in biofilm EVs), passing grey (similar protein proportions in biofilm EVs as well as planktonic EVs), and reaching blue (more protein in planktonic cells). (c) The number of exclusive (blue and orange) and common (white) biofilm EVs and planktonic EVs proteins are illustrated by using a Venn diagram. (d, e, f) Accordingly, the proteome comparisons of biofilm EVs (orange) and ECM (green) are shown. (I) Lipidomics profiles in biofilm EVs and the ECM. The treemaps reflect relative amounts of individual lipid species present in EVs or ECM. The coloration of individual clusters based on their classification showing phospholipids in orange, neutral lipids in red, and sphingolipids in blue. Quantitative differences of biofilm EVs lipids and extracellular matrix lipids are given by using z-scores. Blue illustrates lipids with higher concentrations in biofilm EVs, whereas orange reflects lipids more abundant in ECM. ECM, extracellular matrix; EV, extracellular vesicle; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Fig 3. ESCRT driven biofilm EVs are responsible for drug resistance due to delivery of macromolecules to the C. albicans extracellular matrix.

(a) A diagram of the ESCRT machinery involved in sorting cargo via EVs. (b) Quantitative analysis of biofilm EVs in the C. albicans WT strain and the ESCRT null mutants assessed by the imaging flow cytometry system. The experiment and assays were done in triplicate and data presented as particles per ml. Bars indicate standard deviation of the median. (c) The percent of reduction in biofilm formation following 48-h treatment with fluconazole (1 mg/ml) compared with untreated biofilms, as quantified using the 96-well XTT assay. The null deletions and corresponding complemented strains are shown for mutants with fluconazole susceptibility phenotype. The experiments and assays were performed in triplicate. Asterisks indicate values significantly different from the reference strain based on one-way ANOVA with the posthoc Tukey HSD test. Bars indicate standard deviation of the median. (d) Quantification of in vivo biofilms using a rat central venous catheter model. Individual fluconazole-susceptible ESCRT null mutants were treated either with fluconazole 250 μg/ml or 0.9 M NaCl followed by the CFU analysis. Three animal and culture replicates per condition. (e) Sequestration of ³H-labelled fluconazole by intact biofilms grown from the reference and ESCRT mutant strains. Biofilms were exposed to the radiolabeled drug, washed, and harvested. Scintillation counting was performed in triplicate to determine the fluconazole content in the intact biofilms and the isolated matrix. (f) The percent reduction of mannan and glucan concentration in biofilm matrices of fluconazole-susceptible ESCRT null mutants measured by gas chromatography. Three biological and assay replicates per data point. (g) Impact of ESCRT null mutants on biofilm architecture and extracellular matrix based upon SEM imaging of mature (24 h) in vitro biofilms. Underlying data can be found in S1 Data. CFU, colony-forming unit; ESCRT, endosomal sorting complexes required for transport; EV, extracellular vesicle; H, hydrogen; HSD, honestly significant difference; NaCl, sodium chloride; SEM, scanning electron micrograph; WT, wild-type.

Fig 4. Exogenous delivery of WT vesicles restores the biofilm drug-resistant phenotype and matrix composition.

(a) A diagram depicting the addition of purified WT EVs from C. albicans biofilm cultures to mutant biofilms. (b) Effect of exogenous WT biofilm EVs on biofilm fluconazole susceptibility for select ESCRT null mutants as measured by the 96-well XTT assay. Biofilm cultures of fluconazole-sensitive mutant strains amended with WT EVs (21,804 ± 1,711 EVs/ml) regain their ability to grow in the presence of fluconazole. Each experiment and assay was performed in triplicate. (c) Exogenous WT EVs rescue matrix production in ESCRT mutant biofilms. The fluconazole-susceptible biofilm of HSE1 null mutant does not produce extracellular matrix (upper SEM). The addition of exogenous vesicles restores the mutant’s ability to produce the extracellular matrix (lower SEM). Scale bars indicate 11 μm. (d) Exogenous EVs restore mannan and glucan concentrations in the biofilm matrix of HSE1 null mutant as measured by gas chromatography. Each experiment and assay was performed in triplicate. (e) Effect of exogenous biofilm EVs on drug susceptibility of select C. albicans matrix glucan-modification null mutants as measured by the 96-well XTT assay. The vesicle cargo mutants (PHR1, SUN41) regain their ability to grow in the presence of fluconazole after the addition of exogenous WT EVs. Each experiment and assay was performed in triplicate. Underlying data can be found in S1 Data. ESCRT, endosomal sorting complexes required for transport; EV, extracellular vesicle; SEM, scanning electron micrograph; SUN41, putative endo-beta-D-glucosidase; PHR1, putative glycanosyltransferase; WT, wild-type.