A Novel Bacteriophage with Broad Host Range against Clostridioides difficile Ribotype 078 Supports SlpA as the Likely Phage Receptor.

Bacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ΦCD08011, ΦCD418, ΦCD1801, and ΦCD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ΦCD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ΦCD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI. IMPORTANCE Research into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridioides difficile infection (CDI) is in its infancy, where an optimal "one size fits all" phage cocktail is yet to be derived. The pursuit thus far has aimed to find phages with the broadest possible host range. However, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad host range activity are yet to be discovered. In this study, we isolate four novel myoviruses, including ΦCD1801, which exerts the broadest host range activity toward RT 078 reported in the literature. Through the application of ΦCD1801 to phage-binding assays, we provide data to support the prior notion that SlpA represents the likely phage receptor on the bacterial cell surface. Our finding directs research attention toward the isolation of phages with activity toward strains possessing defined S-layer cassette types.

Phosphorylation and functionality of CdtR in Clostridium difficile.

The production of TcdA, TcdB and CDT in Clostridium difficile PCR ribotype 027, is regulated by the two-component system response regulator CdtR. Despite this, little is known about the signal transduction pathway leading to the activation of CdtR. In this study, we generated R20291ΔPalocΔcdtR model strains expressing CdtR phospho-variants in which our predicted phospho-accepting Asp, Asp61 was mutated for Ala or Glu. The constructs were assessed for their ability to restore CDT production. Dephospho-CdtR-Asp61Ala was completely non-functional and mirrored the cdtR-deletion mutant, whilst phospho-CdtR-Asp61Glu was functional, possessing 38-52% of wild-type activity. Taken together, these data suggest that CdtR is activated by phosphorylation of Asp61. The same principles were applied to assess the function of PCR ribotype 078-derived CdtR, which was shown to be non-functional owing to polymorphisms present within its coding gene. Conversely, polymorphisms present within its promoter region, provide significantly enhanced promoter activity compared with its PCR ribotype 027 counterpart. To ensure our data were representative for each ribotype, we determined that the cdtR nucleotide sequence was conserved in a small library of eight PCR ribotype 027 clinical isolates and nineteen PCR ribotype 078 isolates from clinical and animal origin.

Development of Clostridium difficile R20291ΔPaLoc model strains and in vitro methodologies reveals CdtR is required for the production of CDT to cytotoxic levels.

Assessing the regulation of Clostridium difficile transferase (CDT), is complicated by the presence of a Pathogenicity locus (PaLoc) which encodes Toxins A and B. Here we developed R20291ΔPaLoc model strains and cell-based assays to quantify CDT-mediated virulence. Their application demonstrated that the transcriptional regulator, CdtR, was required for CDT-mediated cytotoxicity.