title: Prophage-stimulated toxin production in Clostridium difficile NAP1/027 lysogens.
abstract: TcdA and TcdB exotoxins are the main virulence factors of Clostridium difficile, one of the most deadly nosocomial pathogens. Recent data suggest that prophages can influence the regulation of toxin expression. Here we present the characterization of ϕCD38-2, a pac-type temperate Siphoviridae phage that stimulates toxin expression when introduced as a prophage into C. difficile. Host range analysis showed that ϕCD38-2 was able to infect 99/207 isolates of C. difficile representing 11 different PCR ribotypes. Of 89 isolates corresponding to the NAP1/027 hypervirulent strain, which recently caused several outbreaks in North America and Europe, 79 (89%) were sensitive to ϕCD38-2. The complete double-stranded DNA (dsDNA) genome was determined, and a putative function could be assigned to 24 of the 55 open reading frames. No toxins or virulence factors could be identified based on bioinformatics analyses. Our data also suggest that ϕCD38-2 replicates as a circular plasmid in C. difficile lysogens. Upon introduction of ϕCD38-2 into a NAP1/027 representative isolate, up to 1.6- and 2.1-fold more TcdA and TcdB, respectively, were detected by immunodot blotting in culture supernatants of the lysogen than in the wild-type strain. In addition, real-time quantitative reverse transcriptase PCR (qRT-PCR) analyses showed that the mRNA levels of all five pathogenicity locus (PaLoc) genes were higher in the CD274 lysogen. Our study provides the first genomic sequence of a new pac-type Siphoviridae phage family member infecting C. difficile and brings further evidence supporting the role of prophages in toxin production in this important nosocomial pathogen.
authors: Département de Microbiologie et d'Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001 12e Ave. Nord, Sherbrooke, Québec J1H 5N4, Canada.
authors: Ognjen
authors: O
authors: Sekulovic
authors: Mathieu
authors: M
authors: Meessen-Pinard
authors: Louis-Charles
authors: LC
authors: Fortier
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis*; D001427:Bacterial Toxins / Q000096:biosynthesis*; D016360:Clostridioides difficile / Q000302:isolation & purification / Q000472:pathogenicity* / Q000821:virology*; D003015:Clostridium Infections / Q000382:microbiology; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004768:Enterotoxins / Q000096:biosynthesis*; D005060:Europe; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D016679:Genome, Viral; D058507:Host Specificity; D015151:Immunoblotting; D008242:Lysogeny; D008969:Molecular Sequence Data; D009656:North America; D016366:Open Reading Frames; D039002:Prophages / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017906:Siphoviridae / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004279:DNA, Viral; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile