Investigating the Transcriptome of Candida albicans in a Dual-Species Staphylococcus aureus Biofilm Model

Abstract

Candida albicans is an opportunistic pathogen found throughout multiple body sites and is frequently co-isolated from infections of the respiratory tract and oral cavity with Staphylococcus aureus. Herein we present the first report of the effects that S. aureus elicits on the C. albicans transcriptome. Dual-species biofilms containing S. aureus and C. albicans mutants defective in ALS3 or ECE1 were optimised and characterised, followed by transcriptional profiling of C. albicans by RNA-sequencing (RNA-seq). Altered phenotypes in C. albicans mutants revealed specific interaction profiles between fungus and bacteria. The major adhesion and virulence proteins Als3 and Ece1, respectively, were found to have substantial effects on the Candida transcriptome in early and mature biofilms. Despite this, deletion of ECE1 did not adversely affect biofilm formation or the ability of S. aureus to interact with C. albicans hyphae. Upregulated genes in dual-species biofilms corresponded to multiple gene ontology terms, including those attributed to virulence, biofilm formation and protein binding such as ACE2 and multiple heat-shock protein genes. This shows that S. aureus pushes C. albicans towards a more virulent genotype, helping us to understand the driving forces behind the increased severity of C. albicans-S. aureus infections.

Keywords: Candida; Staphylococcus; biofilm; interkingdom interactions; transcriptomics.

Figure 1

Als3 is responsible for Candida-Staphylococcus interactions. Single and multi-species biofilms containing C. albicans and S. aureus were grown for (A) 4 h and (B) 24 h; (C) single species S. aureus biofilm only. Biomass was quantified by staining using 0.05% crystal violet. Experiments were performed on three separate occasions and error bars represent standard deviation of the mean (*; P < 0.05, ***; P < 0.001, ****; P < 0.0001).

Figure 2

Staphylococcus aureus does not influence the total number of fungal cells in a dual-species biofilm. Biofilm biomass was removed via sonication, DNA was extracted and the total number of fungal cells in each biofilm was quantified using qPCR. The total number of C. albicans cells of (A) 4 h and (B) 24 h biofilms are presented as colony forming equivalents per mL (CFE/mL). Experiments were repeated three times on three separate occasions. Data points represent individual biofilms. CFEs of C. albicans SC5314 and ece1Δ/Δ dual-species biofilms were compared to that of als3Δ/Δ (*, P < 0.05).

Figure 3

Visualising cell-cell interactions between Candida mutants and S. aureus. C albicans biofilms of (A) wild type SC5314 and (B) ALS3 and (C) ECE1 deletion mutants were grown for 2 h before adding 5x10⁷ cells/mL of S. aureus (which had been pre-stained with hexidium iodide for 1 h prior) for a further hour. Calcofluor white was added to the bacterial inoculum to stain the fungal biofilm before washing the biofilm to remove any non-adherent cells before imaging. Scale bars represent 100μm.

Figure 4

Presence of Staphylococcus aureus determines mature biofilm transcriptome. (A) Principal component analysis plot shows distinct grouping of 24 h samples with the variable of largest variance along PC1 (65%) and second largest along PC2 (13%). Venn diagrams show the number of genes upregulated in 24 h (B) single (C. albicans only) and (C) dual-species (C. albicans and S. aureus) biofilms.

Figure 5

Staphylococcus aureus induces significant upregulation of biofilm-associated genes when binding to Als3. Gene networks show interactions between upregulated genes specific to (A) WT, (B) ALS3 and (C) ECE1 null mutant dual-species, mature biofilms. Genes of similar function are grouped together to form nodes (circles) and nodes with similar functions are linked by edges (lines). Nodes are coloured by levels of significance and node size increases with the number of genes involved in each function. Networks were created using ClueGO.

Figure 6

Identification of key genes with increased expression in dual-species biofilms. Log₂ fold change of key genes in 24 h dual-species biofilms of (A) WT, (B) als3Δ/Δ and (C) ece1Δ/Δ C albicans with S. aureus.