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

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.

Spo0A suppressessinlocus expression inClostridioides difficile

“…C. difficile sin locus suppresses biofilm formation corroborates our finding (22). Further investigation showed that among the two regulators, SinR positively influences sporulation, toxin production, and motility, while SinR’ acts as an antagonist to SinR and control its activity.…”

“…S4). Increased sigD expression can lead to increased flagellar, toxin production and a reduced biofilm formation in the spo0A mutant (22, 32) (Fig. S3BC).…”

Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in Clostridium difficile

“…Ongoing studies examining mutants in these genes, individually and in combination, will determine the roles of these loci in the surface behaviors of C. difficile . Several studies have described C. difficile biofilm development in vitro ( 50 – 54 ), and a few microbial factors that contribute to this process have been reported, including cell surface proteins, their regulators, and more ( 31 , 35 , 55 – 62 ). Some of the factors involved in biofilm formation include surface proteins regulated by c-di-GMP, including TFP, flagellin, CD630_28310, and CD630_32460 ( 31 , 55 , 56 , 63 ).…”

Redefining theClostridioides difficileσBregulon: σBactivates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

“…These genes were similarly positively (CD0583, CD0584, both GGDEF domain containing proteins (25); CD2214, CD2215, both potential transcriptional regulators (26)) and negatively (CD1616, an EAL domain protein (25)) regulated in all hybridizations, including those where sigB expression was not induced. These results suggest that the basis for the observed differential expression of these genes was vector specific, but not dependent on σ B induction.…”