Clostridioides difficileLuxS mediates inter-bacterial interactions within biofilms

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 RNAsequencing 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.

Removal of mobile genetic elements from the genome of Clostridioides difficile and the implications for the organism’s biology

“…Lysogeny is frequently observed in C. difficile (Sebaihia et al, 2006;Ramirez-Vargas et al, 2018), with prophages most commonly belonging to the order Siphoviridae and Myoviridae, and most commonly identified with ϕC2 , ϕMMP04 (Meessen-Pinard et al, 2012), ϕCD119 (Govind et al, 2006), ϕCDHM1 (Hargreaves et al, 2014), ϕCD38-2 (Fortier andMoineau, 2007), and ϕCD27 (Mayer et al, 2008), ranging in size from 31 to 56 kb with a GC content similar to that of the C. difficile genome (28-30%) (Knight et al, 2015). C. difficile prophages can influence host toxin regulation Govind et al, 2009Govind et al, , 2011Sekulovic et al, 2011;Riedel et al, 2017), quorum sensing (Hargreaves et al, 2014), biofilm formation (Slater et al, 2019) and fitness including transduction (Goh et al, 2013), phage immunity (Boudry et al, 2015;Li et al, 2020), and plasmid/ prophage maintenance (Peltier et al, 2020). Some of these studies were carried out by infecting C. difficile with a phage of interest and examining changes to the transcriptome or selected phenotype.…”