Cyclic Diguanylate Inversely Regulates Motility and Aggregation in Clostridium difficile

Abstract

c Clostridium difficile-associated disease is increasing in incidence and is costly to treat. Our understanding of how this organism senses its entry into the host and adapts for growth in the large bowel is limited. The small-molecule second messenger cyclic diguanylate (c-di-GMP) has been extensively studied in Gram-negative bacteria and has been shown to modulate motility, biofilm formation, and other processes in response to environmental signals, yet little is known about the functions of this signaling molecule in Gram-positive bacteria or in C. difficile specifically. In the current study, we investigated the function of the second messenger c-di-GMP in C. difficile. To determine the role of c-di-GMP in C. difficile, we ectopically expressed genes encoding a diguanylate cyclase enzyme, which synthesizes c-di-GMP, or a phosphodiesterase enzyme, which degrades c-di-GMP. This strategy allowed us to artificially elevate or deplete intracellular c-di-GMP, respectively, and determine that c-di-GMP represses motility in C. difficile, consistent with previous studies in Gram-negative bacteria, in which c-di-GMP has a negative effect on myriad modes of bacterial motility. Elevated c-di-GMP levels also induced clumping of C. difficile cells, which may signify that C. difficile is capable of forming biofilms in the host. In addition, we directly quantified, for the first time, c-di-GMP production in a Gram-positive bacterium. This work demonstrates the effect of c-di-GMP on the motility of a Gram-positive bacterium and on aggregation of C. difficile, which may be relevant to the function of this signaling molecule during infection. C yclic diguanylate (3=,5=-cyclic diguanylic acid) (c-di-GMP) is a second messenger utilized exclusively by prokaryotes to effect global changes in cellular physiology (45). c-di-GMP has been implicated in changes to the cellular envelope in multiple species and mediates the transition from planktonic growth to biofilm formation in many Gram-negative bacteria. c-di-GMP augments biofilm formation by increasing the production of adhesins and extracellular matrix components through altered gene regulation (21,36,37,56,67,68) and posttranslational modifications (10, 73). Conversely, c-di-GMP inhibits motility by reducing the transcription or translation of flagellar genes (1,2,26,29,35), impeding pilus assembly (24, 28), or inhibiting the rotation of assembled flagella (4,8,40,49). In addition to regulating biofilm formation and motility, c-di-GMP coordinates cell-wide responses to lifestyle transitions such as entry into stationary phase (60), expression of virulence genes (22,30,64,69), resistance to antibiotics and host immune responses (22, 33), and cell developmental shifts (13,41,70).The level of c-di-GMP in the cell is controlled by three types of enzymes. c-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs) containing a GGDEF domain, named for a conserved motif in the active site (46,50,56). c-di-GMP is hydrolyzed by two distinct families of phosphodiestera...

Two DHH Subfamily 1 Proteins in Streptococcus pneumoniae Possess Cyclic Di-AMP Phosphodiesterase Activity and Affect Bacterial Growth and Virulence

“…The majority of diguanylate cyclases have been identified in Gram-negative bacteria, whereas diadenylate cyclases have been reported mostly in Gram-positive bacteria. The presence of c-di-GMP signaling in Gram-positive bacteria has been directly demonstrated only in B. subtilis (55,56) and the spore-forming, obligate anaerobe Clostridium difficile (57). We were unable to find any GGDEF motif in S. pneumoniae proteins.…”

CodY-Dependent Regulation of Sporulation in Clostridium difficile

“…Escherichia coli strains were cultured at 37°C in LB (43) or BHIS medium supplemented with 20 g chloramphenicol ml Ϫ1 and 100 g ampicillin ml Ϫ1 as needed to maintain plasmid selection. To counterselect against E. coli following conjugation, media were supplemented with 50 g kanamycin ml Ϫ1 (44,45). B. subtilis strains were cultured at 37°C in LB (43) or BHIS medium supplemented with 5 g chloramphenicol ml…”

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…Importantly, the c-di-GMP-dependent motility-to-sessility transition occurs not only when a swimming cell approaches a surface for settling down but also upon building of three-dimensional (3D) biofilms and during biofilm dispersal. In addition to swimming, c-di-GMP regulates swarming, twitching, and gliding motility on colonized surfaces (174,(199)(200)(201), not only in diverse Proteobacteria but also in the Spirochetes, Firmicutes, and Cyanobacteria (128,187,202). There- fore, the motility-to-sessility transition is a very common, possibly universal phenomenon controlled by c-di-GMP.…”