The current riboswitch landscape in Clostridioides difficile

Abstract

Riboswitches are 5' RNA regulatory elements that are capable of binding to various ligands, such as small metabolites, ions and tRNAs, leading to conformational changes and affecting gene transcription or translation. They are widespread in bacteria and frequently control genes that are essential for the survival or virulence of major pathogens. As a result, they represent promising targets for the development of new antimicrobial treatments. Clostridioides difficile, a leading cause of antibiotic-associated nosocomial diarrhoea in adults, possesses numerous riboswitches in its genome. Accumulating knowledge of riboswitch-based regulatory mechanisms provides insights into the potential therapeutic targets for treating C. difficile infections. This review offers an in-depth examination of the current state of knowledge regarding riboswitch-mediated regulation in C. difficile, highlighting their importance in bacterial adaptability and pathogenicity. Particular attention is given to the ligand specificity and function of known riboswitches in this bacterium. The review also discusses the recent progress that has been made in the development of riboswitch-targeting compounds as potential treatments for C. difficile infections. Future research directions are proposed, emphasizing the need for detailed structural and functional analyses of riboswitches to fully harness their regulatory capabilities for developing new antimicrobial strategies.

Keywords: C. difficile; pathogen; riboswitch; therapeutics.

Fig. 1.. General mechanism of riboswitch action at the transcriptional or translational levels. (a) Regulation mechanism of riboswitches controlling the transcription termination. The binding of the ligand to the riboswitch aptamer induces a conformational change. In the case of an ‘OFF’ switch, it results in the formation of a terminator triggering the premature termination of the transcription and preventing the gene expression. In the case of an ‘ON’ switch, an antiterminator is formed, leading to the transcription of the downstream ORF. (b) Regulation mechanism of riboswitches controlling the translation initiation. Ligand binding induces a conformational change resulting either in the sequestration of the RBS, which prevents the translation (‘OFF’ switch), or in the formation of an anti-anti-sequestrator structure releasing the RBS to allow ribosome binding and translation. Figure created with BioRender.com.

Fig. 2.. Diversity of riboswitches in C. difficile. (a) Forty-nine metabolites or ion-binding riboswitches have been identified in the genome of C. difficile so far. The different riboswitch ligands and the biochemical category they belong to are indicated. (b) C. difficile harbours 26 T-box riboswitches. The functional categories of the genes downstream the T-box riboswitches are indicated.

Fig. 3.. Control of the purine metabolism by riboswitches in C. difficile. Four guanine riboswitches acting as ‘OFF’ switches and controlling the expression of genes involved in the purine metabolism (guaA and xpt) or the transport of purine derivatives (CD2107 and CD2704) are present in C. difficile. IMP, inosine 5′-monophosphate; XMP, xanthosine 5′-monophosphate; GMP, guanosine 5′-monophosphate. Figure created with BioRender.com.

Fig. 4.. Control of the PreQ1 salvage pathway by riboswitches in C. difficile. A PreQ1-I riboswitch is located upstream of a three-gene operon-encoding QueK, QrtT and QueL, which are involved in PreQ1 salvage. QrtT is the substrate-specific component of a queuosine ECF transporter and interacts with the core components (ECF core) of this transporter. Figure created with BioRender.com.

Fig. 5.. Regulation network of riboswitches responding to c-di-GMP in C. difficile. (a) General regulation mechanisms of c-di-GMP riboswitches and controlled pathways. Two types of c-di-GMP riboswitches are present in C. difficile. Class I riboswitches act as transcriptional OFF switches, while class II riboswitches are transcriptional ON switches with the exception of riboswitch Cdi2_1, which controls the translation initiation (not depicted). Riboswitches responding to c-di-GMP control the expression of genes in numerous cellular processes including flagellar motility, type IV pilus formation and surface exposure of adhesins. (b) Low c-di-GMP concentrations promote swimming motility through the induction of flagellar biosynthesis and toxin production, which could enhance the likelihood of adhesion and propagation of the bacteria in inflamed intestinal tissues. An elevation of the c-di-GMP levels results in a switch from free-living planktonic to a sessile biofilm-associated lifestyle. High c-di-GMP concentrations stimulate type IV pilus formation and production of the CD2831 and CD3246 adhesins to promote cell aggregation, biofilm formation and adherence to the intestinal epithelium. When c-di-GMP concentrations decrease again, the metalloprotease PPEP-1 is secreted and specifically cleaves the two adhesins, resulting in the detachment of C. difficile cells from the epithelium. Flagella are concomitantly restored allowing C. difficile to spread. Figure created with BioRender.com.

Fig. 6.. General mechanism of T-box action at the transcriptional or translational levels. (a) Regulation mechanism of T-boxes controlling the transcription termination. Uncharged tRNA binds through base pairing the specifier loop of a cognate T-box and a specific sequence leading to the stabilization of an antiterminator and the transcription of the downstream ORF. Aminoacylated tRNA binds the specifier loop, but the amino acid prevents the binding to the specific sequence, resulting in the formation of a terminator triggering premature termination of the transcription and halting gene expression. (b) Regulation mechanism of T-boxes controlling the translation initiation. Uncharged tRNA binds through base pairing the specifier loop of a cognate T-box and a specific sequence leading to the formation of an anti-anti-sequestrator structure, releasing the RBS to allow ribosome binding and translation. Aminoacylated tRNA binds the specifier loop, but the amino acid prevents the binding to the specific sequence, resulting in the sequestration of the RBS, which prevents the translation. Figure created in BioRender.com.