Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

Abstract

Clostridium difficile is an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration in C. difficile was recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes, pilA1 (CD3513) and pilB1 (CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions. pilA1 is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-round in vitro transcription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation in C. difficile. The Gram-positive spore-forming bacterium Clostridium difficile is the leading cause of nosocomial diarrhea and antibioticassociated colitis in hospital settings (1). Illness caused by C. difficile infections (CDI) may range from mild diarrhea to lifethreatening, fulminant colitis. Throughout its life cycle, C. difficile has to cope with multiple changing environments. In the early steps of CDI, in order to colonize the colon and produce toxins, C. difficile spores first germinate in the intestine in response to stimuli such as the presence of bile salts (2, 3). Vegetative cells need to reach the colon, likely attaching to and colonizing the gut mucosa, where they proliferate and produce toxins. Hence, C. difficile arguably needs to sense and integrate multiple environmental stimuli in order to coordinate the expression of its colonization and virulence factors during its journey through the gastrointestinal tract. The mechanisms allowing this adaptability in C. difficile have been the focus of many recent studies, including those on the agr quorum-sensing system, alternative sigma factors, two-component systems, the global transcription regulator CodY, and the sigma factor TcdR, which promotes TcdA and TcdB toxin expression (4-9).One way by which bacteria sense and respond to changes in their environment is through signal transduction involving secondary messenger molecules. The bacterial second messenger 3=-5= cyclic diguanosine monophosphate (c-di-GMP) plays multiple key roles in bacterial physiology and virulence (10-12). c-di-GMP has been shown to antagonistically control the...

Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

“…In other bacterial species, TFP mediate surface motility, bacterial adhesion to surfaces and other bacteria, biofilm formation, and/or adherence to host cells (27)(28)(29)(30)(31). In C. difficile, pilus gene expression promotes autoaggregation, a process that has been proposed to contribute to biofilm formation in 630/ 630⌬erm (21,24,25). Consistent with this, C. difficile biofilm formation is also stimulated by elevated c-di-GMP (25).…”

“…Table S2 in the supplemental material lists the plasmids used in this study, and Table S3 in the supplemental material lists the primers. Disruption of the R20291 pilB1 gene was performed with the ClosTron approach, using a targeted group II intron adapted for clostridia essentially as previous described, resulting in an insertion in the sense orientation at position 795 of the R20291 pilB1 gene (24,38,39). The ClosTron plasmid was mated with C. difficile R20291, together with the helper plasmid pR702 (24).…”

“…Regulation occurs via a c-di-GMP-responsive riboswitch that, in the presence of its ligand, promotes transcription elongation of pilA1, which encodes the major pilin subunit (24)(25)(26). In other bacterial species, TFP mediate surface motility, bacterial adhesion to surfaces and other bacteria, biofilm formation, and/or adherence to host cells (27)(28)(29)(30)(31).…”

“…In C. difficile 630/630⌬erm, c-di-GMP promotes the extracellular assembly of type IV pili (TFP) by positively regulating pilus gene expression (24). Regulation occurs via a c-di-GMP-responsive riboswitch that, in the presence of its ligand, promotes transcription elongation of pilA1, which encodes the major pilin subunit (24)(25)(26).…”

CodY-Dependent Regulation of Sporulation in Clostridium difficile

“…Because the toxin and motility phenotypes share some regulatory components and because CodY influences motility in the related organism C. perfringens (36), we asked whether CodY controls motility in the motile 630⌬erm strain (012 ribotype) and UK1 strain (027 ribotype). Motility assays were performed on soft agar plates, and diameters were measured every 24 h over 7 days; a motility-defective sigD mutant was included as a negative control (65). As shown in Fig.…”