Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans

Abstract

Polymicrobial biofilms are of large medical importance, but relatively little is known about the role of interspecies interactions for their physiology and virulence. Here, we studied two human pathogens co-occuring in the oral cavity, the opportunistic fungus Candida albicans and the caries-promoting bacterium Streptococcus mutans. Dual-species biofilms reached higher biomass and cell numbers than mono-species biofilms, and the production of extracellular polymeric substances (EPSs) by S. mutans was strongly suppressed, which was confirmed by scanning electron microscopy, gas chromatography-mass spectrometry and transcriptome analysis. To detect interkingdom communication, C. albicans was co-cultivated with a strain of S. mutans carrying a transcriptional fusion between a green fluorescent protein-encoding gene and the promoter for sigX, the alternative sigma factor of S. mutans, which is induced by quorum sensing signals. Strong induction of sigX was observed in dual-species biofilms, but not in single-species biofilms. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion of comS encoding the synthesis of the sigX-inducing peptide precursor abolished this activity, whereas deletion of comC encoding the competence-stimulating peptide precursor had no effect. Transcriptome analysis of S. mutans confirmed induction of comS, sigX, bacteriocins and the downstream late competence genes, including fratricins, in dual-species biofilms. We show here for the first time the stimulation of the complete quorum sensing system of S. mutans by a species from another kingdom, namely the fungus C. albicans, resulting in fundamentally changed virulence properties of the caries pathogen.

Figure 1

Growth and morphology of S. mutans and C. albicans in single- and dual-species biofilms. (a) Phase contrast micrograph of a dual-species biofilm. C albicans mainly grows in the hyphal form; some cells growing in the yeast form are also visible. S. mutans attaches to the hyphae of C. albicans. (b) Biofilm mass determined by crystal violet staining (mean and s.d. from three independent experiments). (c) Cell numbers of S. mutans (c) and C. albicans (d) determined by quantitative PCR of the 16S rRNA gene and the 18S rRNA gene, respectively. Mean and s.d. of three independent experiments with two technical replicates each are shown. (e and f) Scanning electron micrographs of 10-h biofilms of S. mutans (e), dual-species biofilm with C. albicans (f) and C. albicans (g). Scale bar (e–g) 4 μm.

Figure 2

EPS matrix in single- and dual-species biofilms. Biofilms (10-h old) were stained with two fluorescent dyes: The lectin concanavalin A labelled with AlexaFluor 488 binds specifically to termical sugar moieties of glycans and fluoresces green. The dye DAPI (4′,6-diamidino-2-phenylindole) binds to DNA and fluoresces blue. Green and blue fluorescence are shown separately (left and middle panel) and overlaid. Scale bar (upper two panels) 50 μm, (bottom panel) 10 μm.

Figure 3

Induction of the alternative sigma factor SigX of S. mutans in dual-species biofilms. (a) Fluorescence intensity of SMP_sigXGFP, a gfp-reporter for sigX expression in S. mutans, grown as a single-species biofilm (grey bars) or together with C. albicans as a dual-species biofilm (black bars) quantified using the VictorWallac 1420 fluorescence plate reader. (b) Quantitative RT-PCR of sigX expression in S. mutans wild-type biofilms grown alone (grey bars) or with C. albicans (black bars). The data were normalized to sigX expression in a S. mutans biofilm after 6 h. Mean and s.d. from four independent experiments are shown. (c) Fluorescence microscopy of single- and dual-species biofilms after 10 h of growth. Phase contrast and gfp channel are overlaid. Scale bars (c, left) 50 μm, (c, right) 20 μm.

Figure 4

Activation of sigX-gfp by culture supernatants and pheromones and role of the autoinducer synthases ComC and ComS. (a) Culture supernatants were obtained from biofilms of S. mutans and C. albicans cultivated separately or together for 4–24 h and added to 6, 10 and 24 h-old-test biofilms of the reporter strain SMP_sigXGFP. Fluorescence intensity was determined after 2 h of incubation. (b) Activation of sigX-gfp in reporter strain biofilms of S. mutans by the quorum sensing pheromones CSP (competence-stimulating peptide) and XIP (sigX-inducing peptide) produced by S. mutans and by farnesol produced by C. albicans. The autoinducers were added as pure compounds at the indicated concentrations. Fluorescence was determined after 2–24 h of biofilm growth. (c) Same experiment as in (a), except that culture supernatants from deletion mutants for comC and comS of S. mutans were tested. comC encodes the synthesis of the CSP precursor, whereas comS encodes the synthesis of the XIP precursor. Mean and s.d. of four experiments are shown in all cases.

Figure 5

EPS formation of S. mutans biofilms in conditioned media. S. mutans biofilms were cultivated in conditioned media from 10-h-old single- (left and right panels) and dual-species biofilms (middle panel) for 10 h and analysed by scanning electron microscopy. Scale bar, 5 μm.

Figure 6

Induction of the quorum sensing regulon and late competence genes in dual-species biofilms. Upper panel: schematic view of the quorum sensing regulon of S. mutans (modified from (Perry et al., 2009; Lemme et al., 2011)) and differential gene expression after 6, 10 and 24 h of biofilm growth in dual-species biofilms with C. albicans compared with single-species biofilms of S. mutans alone. The ComRS system is shown in green, the ComCDE system is shown in blue. Black and red arrows correspond to processing of the signalling peptide and transcriptional regulation by ComR/E, respectively. Pictograms are explained below the scheme. Lower panel: differential gene expression of the late competence genes, genes related to DNA metabolism and repair and mutacins of S. mutans in dual-species biofilms.

Figure 7

Uptake of DNA in dual-species biofilms of C. albicans and S. mutans. The reporter strain SMP_sigXGFP was cultivated for 10 h alone (top panel) or together with C. albicans (middle and bottom panel). DNA labelled with Cy3 was added, and after incubation for 30 min excess DNA was removed by DNAse treatment. See Methods for experimental details. The four rows show (from left to right) the green channel for GPF, the red channel for Cy3, phase contrast and the overlay of red and green channels. Scale bar, 20 μm (top and middle panels) and 5 μm (bottom panel).

Figure 8

Transcriptional profiling of genes related to sugar metabolism and oxidative stress in dual-species biofilms. Gene expression after 6, 10 and 24 h of biofilm growth of S. mutans in dual-species biofilms with C. albicans compared with expression in single-species biofilms of S. mutans alone.

Figure 9

Sugar composition of the cultivation medium after 10 h of biofilm growth. Biofilm supernatants were sterile filtered and analysed by gas chromatography–mass spectrometry. (a) Cultivation medium, (b) S. mutans biofilm supernatant, (c) C. albicans biofilm supernatant and (d) spent medium from dual-species biofilm of S. mutans and C. albicans. The following peaks were identified: 1, sucrose; 2, N-acetylglucosamine; 3 and 4, glucose; 5 and 6, fructose; 7 possibly C5 sugars (pentose). Peak height shows the maximum ion count for this specific mass (arbitrary units). Note the 10-fold enlargement of the y-axis in the left part of the chromatogram.

Figure 10

Working hypothesis for cross-feeding and interkingdom communication in dual-species biofilms of S. mutans and C. albicans. S. mutans growing in single culture in a biofilm (a) forms EPS from sucrose due to the glucosyltransferase exoenzymes. The quorum sensing genes are not activated. In the presence of C. albicans (b) sucrose is taken up by C. albicans and no EPS is formed by S. mutans. Extracellular proteases of C. albicans could degrade S. mutans proteins resulting in the production of the heptamer XIP, which is the main quorum sensing signal of S. mutans. XIP is internalized by the Opp transporter and activates the transcriptional regulator comR, thereby triggering activation of the quorum sensing signalling cascade. ComR induces transcription of comS, the precursor of XIP, which is processed and exported to yield active extracellular XIP, resulting in a positive feedback loop. ComR induces expression of the alternative sigma factor sigX, resulting in transcription of the transformasome genes and genetic competence of the cell. Bacteriocin synthesis is induced either through the response regulator ComE or through ComR. Note that the positive feedback loop established through comRS is essential for the observed induction of competence in co-culture, which was not obtained if the comS gene was knocked out in S. mutans.