Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile

Abstract

The catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (creCD motif), that is, ‘RRGAAAANGTTTTCWW’. Binding of purified CcpA protein to 19 target creCD sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators.

CodY-Dependent Regulation of Sporulation in Clostridium difficile

“…Although higher expression of some sporulation-specific genes was observed in that study, the CodY-regulated sporulation genes that were identified play a role in sporulation only after Spo0A activation. Since that study, additional genes have been found to be associated with entry into sporulation, including sigH (67), ccpA (63,68), the opp and app genes encoding the corresponding permeases (19), rstA (69), and sinRI (19). Of these, only opp and sinRI are recognized as being directly regulated by CodY, as demonstrated by IDAP-seq (37).…”

“…Although there was an increase in the promoter activity in the 630 codY mutant compared to the parent strain, there was no change in activity between log-phase and stationary-phase (T 4 ) cultures of the parent strain. Therefore, alleviation of CodY repression at stationary phase is not sufficient to increase transcription from the sin promoter, most likely because other factors, such as CcpA and SigD, are also involved in regulating sinR transcription (68,72). In addition, alteration of what we predicted to be the CodY binding site (31,37) [PsinR 600(C-290A) ::phoZ] did not result in increased alkaline phosphate activity compared to that seen with the wild-type promoter (Table 4), suggesting either that the mutated base pair is not important for CodY binding or that CodY does not bind to this region of the predicted promoter region.…”

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

“…CcpA can mediate repression of virulence gene expression and carbon catabolism gene expression in Gram-positive bacteria, including some Clostridium spp. For example, CcpA represses xylose utilization by Clostridium acetobutylicum (46) and also represses TcdA and TcdB toxin production by C. difficile (25,26). Similarly, a previous study showed that a C. perfringens mutant deficient in CcpA production exhibits significantly increased phospholipase C (PLC) and perfringolysin O (PFO) production (47).…”

“…CcpA interacts with proteins like phosphorylated HPr, which increases its affinity for certain DNA binding sites and results in repression or activation of gene transcription (20,24). There is accumulating evidence that CcpA can also be involved in the control of virulence gene expression by several Gram-positive pathogens, including C. difficile and S. aureus (25)(26)(27)(28)(29). In C. perfringens, CcpA has been shown to control expression of the enterotoxin gene (cpe) and genes involved in type IV pilus formation and function (30,31).…”

Clostridium difficile Infection

“…Amino acids such as proline, cysteine and certain branched chain amino acids in the local environment of the bacterium repress toxin production through the action of the global transcriptional regulator CodY (known as GTP-sensing transcriptional pleiotropic repressor CodY) 90 . The presence of glucose or other rapidly metabolizable carbon sources in the local environment of the bacterium also inhibits the production of TcdA and TcdB via the carbon catabolite control protein A (CcpA) 91,92 .…”