Genetic Insights into the Rheological Strength of Candida albicans Biofilms
The principal opportunistic human fungal pathogen Candida albicans forms biofilms resistant to antifungal therapeutics. Biofilms are a class of soft matter with viscoelastic properties and response to flow, but little is known regarding the genes contributing to these rheological phenotypes in fungal biofilms. Here, we identify C. albicans genes with deletion phenotypes of altered biofilm viscoelasticity. We analyzed mutants deleted for genes contributing to cell wall structure or extracellular matrix (ECM) production, and we identified increased elastic moduli, indicative of higher viscoelasticity, in strains singly deleted for PMR1, KRE5, and ALG11. PMR1 encodes a secretory pathway calcium pump. KRE5 encodes a UDP-glucose:glycoprotein glucosyltransferase, and ALG11 encodes alpha-1,2-mannosyltransferase. These mutants form less biofilm ECM by weight relative to wild type when cultured on agar. For these strains, biofilm morphology is smooth, with reduced hyphal formation. The mutants exhibit decreased resistance to the antifungal agent fluconazole relative to wild type biofilm cultures. To identify intracellular changes underlying these altered rheological properties, we globally profiled transcript levels in the respective mutants. Genes encoding membrane proteins were enriched in the set of transcripts differentially abundant in the alg11 deletion mutant. RNA levels are altered for genes associated with translation in the pmr1 deletion mutant and protein catabolism in the kre5 deletion strain. Genes involved in lipid metabolism and filamentous development are differentially expressed in cells from alg11, kre5, and pmr1 deletion mutant biofilms. Collectively, the data indicate C. albicans biofilm rheology as a phenotype affected by ECM production and cell morphology, while identifying genes for the investigation of mechanisms underlying properties of fungal biofilm viscoelasticity. Overall design: Wild-type and deletion mutant biofilms were grown from overnight cultures on polycarbonate membranes (nucleopore 0.8 micrometer pore size) placed on solid nutrient-limited Spider medium supplemented with uridine for seven days at 37 degrees C. Cell pellets were collected by centrifugation, and RNA was extracted from each sample using the RiboPure RNA purification kit according to suggested protocols and published methods. Approximately 10 to the seventh cells were lysed by agitation with cold Zirconia beads in a buffer containing SDS (1% final volume) and phenol:chloroform:isoamyl alcohol. The RNA was purified by ethanol washes and filtration using cartridges in the RiboPure kit. Prior to sequencing, approximately 3 micrograms of each RNA sample was treated with DNase I. The reaction was terminated with EDTA and incubation at 65 degrees C. The samples were subsequently precipitated with ethanol and washed with 70% cold ethanol. For sequencing, messenger RNA was purified from total RNA using poly-T oligonucleotide-attached magnetic beads. cDNA was generated using random hexamer primers for first-strand synthesis, and dUTP-priming for second-strand synthesis. The resulting cDNA library was quantified using fluorometry and real-time PCR. The size distribution of the synthesized cDNA was assessed using a bioanalyzer.