Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture.

Isabelle Poquet,Alexis Canette,Bruno Dupuy,Isabelle Martin-Verstraete,Jean-Marc Ghigo,Laure Saujet,Olga Soutourina,Romain Briandet,Marc Monot,Jovanna Mihajlovic

doi:10.3389/fmicb.2018.02084

Abstract

Clostridium difficile is an opportunistic entero-pathogen causing post-antibiotic and nosocomial diarrhea upon microbiota dysbiosis. Although biofilms could contribute to colonization, little is known about their development and physiology. Strain 630Δerm is able to form, in continuous-flow micro-fermentors, macro-colonies and submersed biofilms loosely adhesive to glass. According to gene expression data, in biofilm/planktonic cells, central metabolism is active and fuels fatty acid biosynthesis rather than fermentations. Consistently, succinate is consumed and butyrate production is reduced. Toxin A expression, which is coordinated to metabolism, is down-regulated, while surface proteins, like adhesins and the primary Type IV pili subunits, are over-expressed. C-di-GMP level is probably tightly controlled through the expression of both diguanylate cyclase-encoding genes, like dccA, and phosphodiesterase-encoding genes. The coordinated expression of genes controlled by c-di-GMP and encoding the putative surface adhesin CD2831 and the major Type IV pilin PilA1, suggests that c-di-GMP could be high in biofilm cells. A Bacillus subtilis SinR-like regulator, CD2214, and/or CD2215, another regulator co-encoded in the same operon as CD2214, control many genes differentially expressed in biofilm, and in particular dccA, CD2831 and pilA1 in a positive way. After growth in micro-titer plates and disruption, the biofilm is composed of robust aggregated structures where cells are embedded into a polymorphic material. The intact biofilm observed in situ displays a sparse, heterogeneous and high 3D architecture made of rods and micro-aggregates. The biofilm is denser in a mutant of both CD2214 and CD2215 genes, but it is not affected by the inactivation of neither CD2831 nor pilA1. dccA, when over-expressed, not only increases the biofilm but also triggers its architecture to become homogeneous and highly aggregated, in a way independent of CD2831 and barely dependent of pilA1. Cell micro-aggregation is shown to play a major role in biofilm formation and architecture. This thorough analysis of gene expression reprogramming and architecture remodeling in biofilm lays the foundation for a deeper understanding of this lifestyle and could lead to novel strategies to limit C. difficile spread.

Highlights

C. difficile biofilm was held together as a whole without dissociating even after having been vortexed. All these results indicate that, under these conditions, C. difficile is able to efficiently form macro-colonies and submersed biofilms characterized by inward cohesion despite loose adhesion to the surface
CD2214–CD2215 Regulon in Strain 630 erm We previously identified C. difficile putative DNA-binding protein CD2214 as a SinR-like protein (Saujet et al, 2011): it is the best homolog of SinR, the B. subtilis repressor of biofilm formation, sporulation, motility and autolysis (Cairns et al, 2014), even though their similarity is low and essentially restricted to the HTH domain
We showed here that C. difficile is able to grow, in TYt medium, as original biofilms compared to most previously described biofilms grown under different conditions

Summary

Introduction

Clostridium difficile is a Gram-positive, spore-forming obligate anaerobe and an opportunistic entero-pathogen (Leffler and Lamont, 2015; Abt et al, 2016; Smits et al, 2016). A healthy gut microbiota plays a major role in resistance to colonization and control of C. difficile infections (Theriot and Young, 2015; Abt et al, 2016). Metabolism is modified and favors spore germination and vegetative cell expansion (Theriot and Young, 2015; Abt et al, 2016). Infection culminates with the tightly controlled production of Tcd toxins (Martin-Verstraete et al, 2016) These virulence factors are glycosyl-transferases inactivating Rho/Ras/Rac GTP-binding proteins, leading to colonic epithelium damage and pain (Abt et al, 2016). Cell sporulation concludes the cycle, allowing dissemination and spread of highly resistant infectious forms (Smits et al, 2016)

Methods

Results

Conclusion