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. 2019 Jul 9;9(1):9903.
doi: 10.1038/s41598-019-46143-6.

Clostridioides difficile LuxS mediates inter-bacterial interactions within biofilms

Ross T Slater  1 , Lucy R Frost  1 , Sian E Jossi  1 , Andrew D Millard  2 , Meera Unnikrishnan  3
Affiliations

Clostridioides difficile LuxS mediates inter-bacterial interactions within biofilms

Ross T Slater et al. Sci Rep. .

Abstract

The anaerobic gut pathogen, Clostridioides difficile, forms adherent biofilms that may play an important role in recurrent C. difficile infections. The mechanisms underlying C. difficile community formation and inter-bacterial interactions are nevertheless poorly understood. C. difficile produces AI-2, a quorum sensing molecule that modulates biofilm formation across many bacterial species. We found that a strain defective in LuxS, the enzyme that mediates AI-2 production, is defective in biofilm development in vitro. Transcriptomic analyses of biofilms formed by wild type (WT) and luxS mutant (luxS) strains revealed a downregulation of prophage loci in the luxS mutant biofilms compared to the WT. Detection of phages and eDNA within biofilms may suggest that DNA release by phage-mediated cell lysis contributes to C. difficile biofilm formation. In order to understand if LuxS mediates C. difficile crosstalk with other gut species, C. difficile interactions with a common gut bacterium, Bacteroides fragilis, were studied. We demonstrate that C. difficile growth is significantly reduced when co-cultured with B. fragilis in mixed biofilms. Interestingly, the absence of C. difficile LuxS alleviates the B. fragilis-mediated growth inhibition. Dual species RNA-sequencing analyses from single and mixed biofilms revealed differential modulation of distinct metabolic pathways for C. difficile WT, luxS and B. fragilis upon co-culture, indicating that AI-2 may be involved in induction of selective metabolic responses in B. fragilis. Overall, our data suggest that C. difficile LuxS/AI-2 utilises different mechanisms to mediate formation of single and mixed species communities.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
LuxS biofilm defect is reversed by addition of DPD. (A) WT and LuxS biofilms were grown for 24 h or 72 h and stained with 0.2% CV, followed by measuring OD570, N = 5. (B) Representative pictures of crystal violet stained C. difficile WT and luxS biofilms after 72 h. (C) Colony counts (vegetative cells) from biofilms (N = 7) after 24 h and 72 h. (D) The AI-2 precursor, DPD, was exogenously supplemented to LuxS at a concentration of 100 nM, followed by biofilm staining and quantitation with 0.2% CV after 72 h (N = 4). Error bars indicate SD, *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Student’s t-test or by Mann-Whitney U test, ns- not significant.
Figure 2
Figure 2
Down-regulation of prophage genes in the C. difficile luxS mutant. (A) Pairwise analysis identified 21 differentially expressed genes in luxS (red points). All 18 down-regulated genes clustered into two regions. (B) Three prophage regions are identified in the C. difficile genome using Phaster. Regions 2 and 3 were down regulated in luxS. (C) Heat map representation of the genes that were differentially expressed in luxS, red and green indicate down- and up-regulation respectively when compared to WT. 18 prophage genes were found to be down-regulated in luxS relative to WT, whilst two genes involved in trehalose metabolism were up-regulated in luxS relative to WT. Data shown is the mean of 3 independent experiments in triplicates. Differential expression was defined as ≥1.6-fold change relative to WT with an adjusted p-value ≤ 0.05.
Figure 3
Figure 3
Presence of phage and eDNA in C. difficile biofilms. The phage origin of DNA isolated from WT biofilms was confirmed by PCR, using primers for 16S (A) and two phage genes (CDR20291_1436 and CDR20291_1208) (B). The negative controls were run on a different part of the same gel. The gel pictures were trimmed with no adjustment to the intensities. WT-1–3 are three biological replicates. (C) Total eDNA extracted from the WT and luxS mutant biofilms after 24 h and 72 h, normalised to the biofilm biomass. N = 3, ****p < 0.0001 as determined by Mann-Whitney U test.
Figure 4
Figure 4
B. fragilis mediated inhibition of C. difficile in mixed biofilms. (A) Biofilm of C. difficile, B. fragilis and both species co-cultured (mixed) were grown for 24 h and stained with 0.2% CV, followed by measuring OD570. (B) Colony counts for both C. difficile (vegetative cells) and B. fragilis from mono and co-culture biofilms after 24 h. (C) Colony counts for both C. difficile (vegetative cells) and B. fragilis from mono and co-culture during planktonic growth. Data shown is the mean of 3 independent experiments in triplicates and error bars indicate SD, **p < 0.005, ****p < 0.0001 as determined by one-way ANOVA, Tukey’s multiple comparison test, ns -not significant (significant differences were determined for C. difficile or B. fragilis mean CFU counts between single and mixed biofilms).
Figure 5
Figure 5
B. fragilis-mediated inhibition of C. difficile is more prominent for WT than LuxS. (A) Biofilms for mono and co-cultures of C. difficile WT and luxS with B. fragilis were grown for 24 h and stained with 0.2% CV and were quantified using a spectrophotometer OD570. (B) Colony counts for C. difficile WT, C. difficile luxS (vegetative cells) during co-culture with B. fragilis were performed at 24 h. Data shown is the mean of 3 independent experiments in triplicates and error bars indicate SD, **p < 0.01, ***p < 0.001 as determined by one-way ANOVA, Tukeys multiple comparison test (significant differences were determined for C. difficile or B. fragilis mean CFU counts between single and mixed biofilms).
Figure 6
Figure 6
Dual species RNA-seq shows modulation of metabolic pathways in C. difficile WT, luxS and B. fragilis. Heat maps showing clustering of up- and down-regulated genes in (A) C. difficile WT and luxS co-cultured with B. fragilis compared to C. difficile WT mono-culture, and in (B) B. fragilis co-cultured with C. difficile WT and luxS compared to B. fragilis mono-culture. Red indicates genes that are down-regulated, whilst green indicates genes that are up-regulated.
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