Role of c-di-GMP in the riboswitch and antisense RNA-mediated control of cellular processes associated with community behaviour in Clostridium difficile, a bacterium responsible for nosocomial infections – CloSTARn

We study the role of regulatory RNAs associated with c-di-GMP in the infectious cycle control in C. difficile by global and targeted approaches. In particular, we use transcriptome analysis (microarrays and new generation sequencing) to get an overview of the c-di-GMP effects in this pathogen, as well as to identify the function of genes targeted by this control by analyzing the mutant strains inactivated for the corresponding genes. These global approaches are followed by the analysis of the expression of selected genes and phenotypic study to demonstrate their role in the community behavior in C. difficile. To address the molecular mechanisms involved in the regulation process in response to c-di-GMP, we investigate the effects of overexpression and deletion of specific RNA regulators on the expression of target genes and the associated phenotypes including sensitivity to bacteriophages and biofilm formation. We have successfully used a new approach for chromosomal gene deletion in C. difficile and a system of inducible gene expression. To analyze the interactions of C. difficile with bacteriophages we used a high-throughput sequencing approach to identify genome sequences of 10 new bacteriophages isolated by our collaborators in Canada.

We performed a transcriptomic analysis of c-di-GMP effects in C. difficile that shows the global role of this signaling molecule and allows us to identify its targets. We have made progress in the functional characterization of regulatory targets of c-di-GMP-dependent riboswitches. We used a new approach for chromosomal deletion in C. difficile to construct mutant strains for antisense RNAs associated with c-di-GMP-specific riboswitches. This will enable us to advance further in understanding of the mechanisms involved in this control. In addition, we have inactivated the genes encoding the two regulators potentially involved in the control of community behavior in C. difficile. Using global and targeted approaches we study their role in the infectious cycle control in C. difficile. We completed the detailed characterization of the functioning of CRISPR-Cas defense system against foreign DNA in C. difficile and demonstrated the active expression of CRISPR RNA. Through the sequencing of phage genomes and analysis of host range of several new C. difficile phages and plasmid conjugation experiences we provide for the first time evidence of the defensive function of CRISPR-Cas system in C. difficile. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. The results of this collaborative work including 4 laboratories in France, Canada, the United States and Russia are under review in mBio. The PhD student involved in the project participated in the international PhD mobility program in 2015 on the study of CRISPR-Cas system of C. difficile in a heterologous host E. coli in Severinov laboratory in the United States (Rutgers University).

The realization of the CloSTARn project will allow us to better understand the role of new mechanisms based on the action of RNAs in the regulatory network controlling cellular processes crucial for successful development of C. difficile inside the host.
As a long-term perspective our data may be used for development of specific inhibitors for c-di-GMP pathway, of methods to prevent normal C. difficile CRISPR functioning or biofilm formation within gut communities, and of phage therapy approach to limit C. difficile development.

The validation of a new DNA chip including regulatory RNA genes that we recently identified is published in Boudry et al. 2014 J. of Bacteriology. An article describing the use of high throughput sequencing for identification of riboswitches is published in Methods in Enzymology in 2014. Collaborative work on the functional characterization of C. difficile CRISPR defense system against foreign DNA is under review in mBio.

Clostridium difficile-associated diarrhoea is currently the most frequently occurring nosocomial diarrhoea in Europe. Many questions remain unanswered concerning the mechanisms controlling infection cycle of this emergent human pathogen. Our recent deep-sequencing data (RNA-seq) strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. This RNA-seq analysis revealed a large number of cis-acting RNA including 66 “riboswitches” found in 5’-untranslated regions of mRNA that control downstream gene expression by premature termination of transcription or by modulating translation initiation. 16 of them including 3 associated with antisense RNAs might respond to cyclic-di-guanosyl-5’monophosphate (c-di-GMP). This second messenger plays a key role in the control of lifestyle switch from free-living motile state to attached communities in a biofilm, and also in virulence control of several Gram-negative pathogens. Little is known about the functions of cyclic-di-GMP in Gram-positive bacteria. Recent publications and our preliminary results suggest the crucial role of this second messenger in C. difficile. We plan to analyse the role of c-di-GMP in this pathogen in specific riboswitch functioning, in the regulation of gene expression and in c-di-GMP controlled processes including motility, biofilm formation, adhesion and bacteriophage infection resistance. The originality of c-di-GMP-dependent control in C. difficile is the use of RNA molecules as effectors sensing c-di-GMP and an unusual location of some c-di-GMP-dependent riboswitches in antisense orientation to coding sequences. We will analyse in more detail genes induced by high c-di-GMP level in C. difficile through riboswitches that are good candidates for factors important for community behaviour of C. difficile. This includes two regulators and two surface-associated proteins probably involved in biofilm formation. We will also focus on two c-di-GMP riboswitches associated with antisense RNAs potentially controlling the expression of a transcriptional regulator and a CRISPR (clustered regularly interspaced short palindromic repeats) system for defence against foreign DNA invaders like bacteriophages. We will use genome-wide and targeted genetics approaches to investigate the role of these regulatory RNAs in the control of C. difficile infection cycle. A particularly interesting question that will be treated during this project is the possible connection between c-di-GMP signalling and the regulation of CRISPR. The c-di-GMP-dependent induction of a CRISPR system mediated by antisense RNA would be to ensure more efficient defence capacities within bacteriophage-rich gut microbiota. This work will bring important insights into the role of new RNA-based mechanisms in the regulatory network involved in the coordinated control of many processes crucial for successful development of C. difficile inside the host.