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.
Highlights
Clostridiodes difficile (Clostridium difficile), an anaerobic, opportunistic pathogen, is the causative agent of C. difficile infection (CDI), a debilitating condition with symptoms ranging from mild diarrhoea to severe pseudomembranous colitis. ~453,000 cases of CDI were reported in the United States in 20111 and there have been increasing reports of CDI from different parts of the world[2,3,4]
We previously reported that a R20291 C. difficile luxS mutant was defective in biofilm formation as measured by crystal violet (CV) staining[27]
In our subsequent studies, we see a reduction in biofilms at 24 h, we observe a high variability in the wild type (WT) biofilms formed at 24 h and in the reduction in the luxS mutant between experiments (Fig. 1A)
Summary
Clostridiodes difficile (Clostridium difficile), an anaerobic, opportunistic pathogen, is the causative agent of C. difficile infection (CDI), a debilitating condition with symptoms ranging from mild diarrhoea to severe pseudomembranous colitis. ~453,000 cases of CDI were reported in the United States in 20111 and there have been increasing reports of CDI from different parts of the world[2,3,4]. CDI is primarily a hospital-acquired infection with the elderly being at highest risk[6] and has been associated with the disruption of the gut microbiota as a result of the use of broad-spectrum antibiotics. Colonisation of C. difficile and development of CDI is influenced by composition of the native gut microbiota. C. difficile produces biofilms that confer increased resistance to antibiotics[27,28,29] and have recently shown to be associated with C. difficile infection in vivo, in close association with other commensal gut species[30]. DPD is an unstable compound that spontaneously cyclises into several different forms These ligands are collectively known as autoinducer-2 (AI-2), a group of potent, cross-species QS signalling molecules[33]. The precise mode of action for LuxS in C. difficile has remained elusive as a result of conflicting studies and the lack of a clear receptor for AI-234,40,41
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