Discovery of new type I toxin–antitoxin systems adjacent to CRISPR arrays in Clostridium difficile

Abstract

Clostridium difficile, a major human enteropathogen, must cope with foreign DNA invaders and multiple stress factors inside the host. We have recently provided an experimental evidence of defensive function of the C. difficile CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system important for its survival within phage-rich gut communities. Here, we describe the identification of type I toxin–antitoxin (TA) systems with the first functional antisense RNAs in this pathogen. Through the analysis of deep-sequencing data, we demonstrate the general co-localization with CRISPR arrays for the majority of sequenced C. difficile strains. We provide a detailed characterization of the overlapping convergent transcripts for three selected TA pairs. The toxic nature of small membrane proteins is demonstrated by the growth arrest induced by their overexpression. The co-expression of antisense RNA acting as an antitoxin prevented this growth defect. Co-regulation of CRISPR-Cas and type I TA genes by the general stress response Sigma B and biofilm-related factors further suggests a possible link between these systems with a role in recurrent C. difficile infections. Our results provide the first description of genomic links between CRISPR and type I TA systems within defense islands in line with recently emerged concept of functional coupling of immunity and cell dormancy systems in prokaryotes.

Type I toxin-antitoxin systems contribute to mobile genetic elements maintenance inClostridioides difficileand can be used as a counter-selectable marker for chromosomal manipulation

“…S2B). Similarly to previous observations with other C. difficile type I TA modules 21 , the analysis of liquid cultures by light microscopy showed that toxin overexpression was accompanied by an increase in cell length in about 10% of the cells (Fig. S2C).…”

“…S8A). We demonstrated the functionality of RCd8- CD2517.1 type I TA module in C. difficile in a previous study 21 . In our new vector, designated pMSR, the inducible toxic expression of CD2517.1 is used as a counter-selection marker to screen for plasmid excision and loss (see Materials and Methods) and this greatly facilitates the isolation of C. difficile deletion mutant generated by double cross-over allele exchange (Fig.…”

“…Replacement of RCd11 with RCd12 led to the same result. By contrast, expression of the more divergent RCd10 (the antitoxin of CD0956.2 toxin from a previously characterized TA module lying within phiCD630-1 21 ) in trans failed to revert the growth defect induced by CD0977.1 expression (Fig. S5B and 3B).…”

“…We recently reported the identification of the first type I TA systems associated with CRISPR arrays in C. difficile genomes 21 . The co-localization and co-regulation by the general stress response Sigma B factor and biofilm-related factors suggested a possible genomic link between these cell dormancy and adaptive immunity systems.…”

In vivocommensal control ofClostridioides difficilevirulence

“…Mutants were created using a newly developed Allele-Coupled Exchange (ACE) vector, derived from pMTL-SC7215, a codA-based "pseudosuicide" plasmid that replicates in the cells at a rate lower than that of the host chromosome (52). The codA cassette was removed by inverse PCR and replaced with the RCd8-CD2517.1 type I toxin-antitoxin module from C. difficile 630 using NEBuilder HiFi DNA Assembly (NEB), yielding pMSR0 (53). In this vector, the CD2517.1 toxin gene was placed under control of the Ptet inducible promoter and the Rcd8 antitoxin was expressed from its own promoter.…”