Phase-variable expression of pdcB, a phosphodiesterase, influences sporulation in Clostridioides difficile.

Clostridioides difficile is the causative agent of antibiotic-associated diarrhea and is the leading cause of nosocomial infection in developed countries. An increasing number of C. difficile infections are attributed to epidemic strains that produce more toxins and spores. C. difficile spores are the major factor for the transmission and persistence of the organism. Previous studies have identified global regulators that influence sporulation in C. difficile. This study discovers that PdcB, a phosphodiesterase, enhances sporulation in C. difficile strain UK1. Through genetic and biochemical assays, we show that phase-variable expression of pdcB results in hypo- and hyper-sporulation phenotypes. In the "ON" orientation, the identified promotor is in the right orientation to drive the expression of pdcB. Production of the PdcB phosphodiesterase reduces the intracellular cyclic-di-GMP (c-di-GMP) concentration, resulting in a hyper-sporulation phenotype. Loss of PdcB due to the pdcB promoter being in the OFF orientation or mutation of pdcB results in increased c-di-GMP levels and a hypo-sporulation phenotype. Additionally, we demonstrate that CodY binds to the upstream region of pdcB. DNA inversion reorients the CodY binding site so that in the OFF orientation, CodY binds a site that is upstream of the pdcB promoter and can further repress gene expression.

Characterization of an operon required for growth on cellobiose in Clostridioides difficile.

Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of Clostridiodes difficile to investigate the role of cellobiose metabolism in C. difficile pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into C. difficile and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pulldown experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in C. difficile physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to in vitro study findings, our in vivo hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model - substantiating the role of cellobiose metabolism in C. difficile pathogenesis.

Spo0A Suppresses sin Locus Expression in Clostridioides difficile.

Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR' (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficileIMPORTANCEClostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

Clostridioides difficile SinR' regulates toxin, sporulation and motility through protein-protein interaction with SinR.

Clostridioides difficile is a Gram-positive, anaerobic bacterium. It is known that C. difficile is one of the major causes of antibiotic associated diarrhea. The enhanced antibiotic resistance observed in C. difficile is the result of highly resistant spores produced by the bacterium. In Bacillus subtilis, the sin operon is involved in sporulation inhibition. Two proteins coded within this operon, SinR and SinI, have an antagonistic relationship; SinR acts as an inhibitor to sporulation whereas SinI represses the activity of SinR, thus allowing the bacterium to sporulate. In a previous study, we examined the sin locus in C. difficile and named the two genes associated with this operon sinR and sinR', analogous to sinR and sinI in B. subtilis, respectively. We have shown that SinR and SinR' have pleiotropic roles in pathogenesis pathways and interact antagonistically with each other. Unlike B. subtilis SinI, SinR' in C. difficile carries two domains: the HTH domain and the Multimerization Domain (MD). In this study, we first performed a GST Pull-down experiment to determine the domain within SinR' that interacts with SinR. Second, the effect of these two domains on three phenotypes; sporulation, motility, and toxin production was examined. The findings of this study confirmed the prediction that the Multimerization Domain (MD) of SinR' is responsible for the interaction between SinR and SinR'. It was also discovered that SinR' regulates sporulation, toxin production and motility primarily by inhibiting SinR activity through the Multimerization Domain (MD).