Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in Clostridioides difficile.

IMPORTANCE: Clostridioides difficile is the widespread anaerobic spore-forming bacterium that is a major cause of potentially lethal nosocomial infections associated with antibiotic therapy worldwide. Due to the increase in severe forms associated with a strong inflammatory response and higher recurrence rates, a current imperative is to develop synergistic and alternative treatments for C. difficile infections. In particular, phage therapy is regarded as a potential substitute for existing antimicrobial treatments. However, it faces challenges because C. difficile has highly active CRISPR-Cas immunity, which may be a specific adaptation to phage-rich and highly crowded gut environment. To overcome this defense, C. difficile phages must employ anti-CRISPR mechanisms. Here, we present the first anti-CRISPR protein that inhibits the CRISPR-Cas defense system in this pathogen. Our work offers insights into the interactions between C. difficile and its phages, paving the way for future CRISPR-based applications and development of effective phage therapy strategies combined with the engineering of virulent C. difficile infecting phages.

Protospacer-Adjacent Motif Specificity during Clostridioides difficile Type I-B CRISPR-Cas Interference and Adaptation.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C. difficile genome where generation of free DNA termini is expected. Results from protospacer-adjacent motif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR-Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.

Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile.

Toxin-antitoxin (TA) systems are widespread on mobile genetic elements and in bacterial chromosomes. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA. The first type I TA were recently identified in the human enteropathogen Clostridioides difficile. Here we report the characterization of five additional type I TA within phiCD630-1 (CD0977.1-RCd11, CD0904.1-RCd13 and CD0956.3-RCd14) and phiCD630-2 (CD2889-RCd12 and CD2907.2-RCd15) prophages of C. difficile strain 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest that is neutralized by antitoxin RNA co-expression. We show that type I TA located within the phiCD630-1 prophage contribute to its stability and heritability. We have made use of a type I TA toxin gene to generate an efficient mutagenesis tool for this bacterium that allowed investigation of the role of these widespread TA in prophage maintenance.

Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile.

The human enteropathogen Clostridium difficile constitutes a key public health issue in industrialized countries. Many aspects of C. difficile pathophysiology and adaptation inside the host remain poorly understood. We have recently reported that this bacterium possesses an active CRISPR-Cas system of subtype I-B for defense against phages and other mobile genetic elements that could contribute to its success during infection. In this paper, we demonstrate that redirecting this endogenous CRISPR-Cas system toward autoimmunity allows efficient genome editing in C. difficile We provide a detailed description of this newly developed approach and show, as a proof of principle, its efficient application for deletion of a specific gene in reference strain 630Δerm and in epidemic C. difficile strain R20291. The new method expands the arsenal of the currently limiting set of gene engineering tools available for investigation of C. difficile and may serve as the basis for new strategies to control C. difficile infections.IMPORTANCEClostridium difficile represents today a real danger for human and animal health. It is the leading cause of diarrhea associated with health care in adults in industrialized countries. The incidence of these infections continues to increase, and this trend is accentuated by the general aging of the population. Many questions about the mechanisms contributing to C. difficile's success inside the host remain unanswered. The set of genetic tools available for this pathogen is limited, and new developments are badly needed. C. difficile has developed efficient defense systems that are directed against foreign DNA and that could contribute to its survival in phage-rich gut communities. We show how one such defense system, named CRISPR-Cas, can be hijacked for C. difficile genome editing. Our results also show a great potential for the use of the CRISPR-Cas system for the development of new therapeutic strategies against C. difficile infections.

New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System.

Over the last decades the enteric bacterium Clostridium difficile (novel name Clostridioides difficile) - has emerged as an important human nosocomial pathogen. It is a leading cause of hospital-acquired diarrhea and represents a major challenge for healthcare providers. Many aspects of C. difficile pathogenesis and its evolution remain poorly understood. Efficient defense systems against phages and other genetic elements could have contributed to the success of this enteropathogen in the phage-rich gut communities. Recent studies demonstrated the presence of an active CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) subtype I-B system in C. difficile. In this mini-review, we will discuss the recent advances in characterization of original features of the C. difficile CRISPR-Cas system in laboratory and clinical strains, as well as interesting perspectives for our understanding of this defense system function and regulation in this important enteropathogen. This knowledge will pave the way for the development of promising biotechnological and therapeutic tools in the future. Possible applications for the C. difficile strain monitoring and genotyping, as well as for CRISPR-based genome editing and antimicrobials are also discussed.

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

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.

The HSP70 chaperone machines of Chlamydomonas are induced by cold stress.

The responses of Chlamydomonas reinhardtii cells to low temperatures have not been extensively studied compared with other stresses. Like other organisms, this green alga has heat shock protein 70s (HSP70s) that are located in chloroplast, mitochondrion and cytosol. To test whether temperature downshifts affected HSP70s synthesis, we used real-time PCR and protein gel blot analysis. C. reinhardtii cells exposed to cold stress show increased HSP70s mRNA levels. Genes encoding other components of HSP70 chaperone machines (e.g. CGE1, CDJ1, HSP90C and HSP90A) are also up-regulated in response to decreased temperature. We demonstrated that the accumulation of all analyzed mRNA occur more slowly and with reduced amplitude in cells exposed to cold than in cells treated with heat. Furthermore, C. reinhardtii cells display the splicing of the CGE1 transcript that was dependent on low temperature. Finally, the transcription regulator of C. reinhardtii HSF1 is also cold-responsive, suggesting its role in the transcriptional regulation of HSP genes at low temperature.

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system.

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.