What’s a Biofilm?—How the Choice of the Biofilm Model Impacts the Protein Inventory of Clostridioides difficile

Abstract

The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile’s V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation.

Wolf in Sheep’s Clothing: Clostridioides difficile Biofilm as a Reservoir for Recurrent Infections

“…All of these results are consistent with transcriptional data performed during biofilm formation which show that the genes encoding the flagella components are less expressed, while those of the T4P are more expressed [184,185,192]. Moreover, a recent systematic proteomics study showed that flagella and pili proteins were more abundant in biofilm formed in liquid cultures than in biofilm formed on agar [189]. This confirms that pili and flagella are expressed and produced under biofilm forming conditions but are dependent on the model and conditions used.…”

“…Several regulation pathways and cascades are involved in C. difficile biofilm formation, and those involved vary according to the growth conditions. Every study using biofilms from liquid cultures has identified the transcriptional regulators CcpA, CodY, Spo0A and SigL/RpoN as key transcriptional factors involved in biofilm formation [184,185,189,192,219]. CcpA, CodY and SigL/RpoN are global regulators of the metabolism and support the idea that biofilm formation is dependent on a metabolic shift in planktonic cells.…”

“…These latter two methods have the advantage of incorporating physiologically relevant conditions of a CDI; however, these types of models can be difficult to set up and require specialized material when compared to their closed systems counterparts. Colony model biofilms are also used to study and image C. difficile biofilms [189][190][191]. As a recent study demonstrated, biofilms grown on agar plates had different characteristics in terms of cell-surface protein expression, metabolism, and regulations than biofilms obtained from liquid culture [189].…”

“…Colony model biofilms are also used to study and image C. difficile biofilms [189][190][191]. As a recent study demonstrated, biofilms grown on agar plates had different characteristics in terms of cell-surface protein expression, metabolism, and regulations than biofilms obtained from liquid culture [189].…”

“…Finally, current evidence strongly supports the idea that autolytic enzymes are probably the main mechanism contributing to eDNA. Indeed, transglycosylases involved in autolysis, such as Cwp19, [210] are more expressed in biofilms than in planktonic cells, [184,189] and inactivation of cwp19 in the strain 630∆erm inhibited biofilm formation in the presence of DOC [184]. Cwp19 requires glucose for its activity [211] and is dispensable for biofilm formation when a different sugar is used (Tremblay and Dupuy, unpublished data).…”