The cell wall lipoprotein CD1687 acts as a DNA binding protein during deoxycholate-induced biofilm formation inClostridioides difficile

Abstract

The ability of bacterial pathogens to establish recurrent and persistent infections is frequently associated with their ability to form biofilms. Clostridioides difficile infections have a high rate of recurrence and relapses and it is hypothesised that biofilms are involved in its pathogenicity and persistence. Biofilm formation by C. difficile is still poorly understood. It has been shown that specific molecules such as deoxycholate (DCA) or metronidazole induce biofilm formation, but the mechanisms involved remain elusive. In this study, we describe the role of the C. difficile lipoprotein CD1687 during DCA-induced biofilm formation. We showed that the expression of CD1687, which is part of an operon within the CD1685-CD1689 gene cluster, is controlled by multiple transcription starting sites and some are induced in response to DCA. Only CD1687 is required for biofilm formation and the overexpression of CD1687 is sufficient to induce biofilm formation. Using RNAseq analysis, we showed that CD1687 affects the expression of transporters and metabolic pathways and we identified several potential binding partners by pull-down assay, including transport-associated extracellular proteins. We then demonstrated that CD1687 is surface exposed in C. difficile, and that this localization is required for DCA-induced biofilm formation. Given this localization and the fact that C. difficile forms eDNA-rich biofilms, we confirmed that CD1687 binds DNA in a non-specific manner. We thus hypothesize that CD1687 is a component of the downstream response to DCA leading to biofilm formation by promoting interaction between the cells and the biofilm matrix by binding eDNA.

Extracellular succinate induces spatially organized biofilm formation inClostridioides difficile

“…In addition, several genes encoding putative membrane and cell wall proteins of unknown function as well as lipoproteins are up-regulated in the presence of succinate (Table S2). We noted that the lipoprotein CD1687 recently demonstrated to be essential in the DCA-induced biofilm (10,45) is not regulated by succinate and confirmed that a CD1687 mutant is not affected in biofilm formation in the presence of this inducer (Figure 4a).…”

“…On the other hand, lipoproteins which are involved among other things in nutrient uptake and signalling systems (52), may be able to detect extracellular succinate. We recently demonstrated that the lipoprotein CD1687 of the 630Δ erm strain mediates in part the metabolic reorganization occurring in DCA-induced biofilm (10,45). Since CD1687 is not involved in succinate-induced biofilm formation, we cannot exclude that lipoproteins up-regulated in the presence of succinate (Table S2), could sense succinate and/or induce metabolic pathways involved in biofilm formation.…”