A Clostridioides difficile bacteriophage genome encodes functional binary toxin-associated genes

Highlights

• First instance of a C. difficile toxin locus being encoded by an associated bacteriophage.

• Putative mobility of toxins in C. difficile.

• Expression of genes associated with the toxins.

Abstract

Pathogenic clostridia typically produce toxins as virulence factors which cause severe diseases in both humans and animals. Whereas many clostridia like e.g., Clostridium perfringens, Clostridium botulinum or Clostridium tetani were shown to contain toxin-encoding plasmids, only toxin genes located on the chromosome were detected in Clostridioides difficile so far. In this study, we determined, annotated, and analyzed the complete genome of the bacteriophage phiSemix9P1 using single-molecule real-time sequencing technology (SMRT). To our knowledge, this represents the first C. difficile-associated bacteriophage genome that carries a complete functional binary toxin locus in its genome.

Introduction

Spore-forming pathogenic clostridia typically produce toxins as virulence factors which can cause severe diseases of both humans and animals. These toxins are often located on the chromosome, but in some cases they were also found to be encoded on plasmids, e.g., in Clostridium perfringens (Adams et al., 2014, Gurjar et al., 2010, Li et al., 2007, Li et al., 2013, Miyamoto et al., 2006, Miyamoto et al., 2008, Miyamoto et al., 2011, Popoff and Bouvet, 2009, Rood, 1998, Rood, 2004, Sayeed et al., 2007, Sayeed et al., 2010), Clostridium botulinum (Adams et al., 2014, Hill et al., 2007, Hill and Smith, 2013), Clostridium tetani (Adams et al., 2014, Brüggemann et al., 2003, Finn et al., 1984, Popp et al., 2012) or Paeniclostridium sordellii (formerly Clostridium sordellii) (Couchman et al., 2015, Sasi Jyothsna et al., 2016).

In contrast, only little is known about the presence and functions of extrachromosomal elements in Clostridioides difficile (formerly Clostridium difficile) (Lawson et al., 2016). Plasmids of sizes between 4.5 and 75 kb were identified in a few isolates of C. difficile (Muldrow et al., 1982). The model strain 630 harbors a cryptic plasmid pCD630 which encodes eleven open reading frames associated with phage genes (Sebaihia et al., 2006, Monot et al., 2011). This plasmid got subsequently lost in this strain (Riedel et al., 2015a, Dannheim et al., 2017). The plasmid pCD630 was not reported to occur in the erythromycin-sensitive derivative C. difficile strain 630Δerm (Hussain et al., 2005, Van Eijk et al., 2015), but was detected during a recent resequencing study of the same strain 630Δerm (Hussain et al., 2005, Dannheim et al., 2017). Another well-characterized C. difficile plasmid is the 6.8 kb plasmid pCD6 isolated from C. difficile strain CD6. In contrast to pCD630 (Sebaihia et al., 2006, Dannheim et al., 2017) the plasmid pCD6 encodes a repA plasmid replication gene (Purdy et al., 2002). In addition to plasmids, very few studies reported extrachromosomal bacteriophage sequences in C. difficile: Strain DSM 1296^T was found to encode two extrachromosomal elements associated with bacteriophages (Riedel et al., 2015b, Wittmann et al., 2015), and in strain BI1 two putative extrachromosomal bacteriophage-associated elements were detected (He et al., 2010).

In comparison to other clostridia no extrachromosomal element encoding toxin genes was found in C. difficile so far. In this study, we determined, annotated, and analyzed the complete genome of the C. difficile bacteriophage phiSemix9P1 using single-molecule real-time (SMRT) sequencing technology. To our knowledge, this represents the first C. difficile associated bacteriophage genome which encodes a complete functional toxin locus (CdtLoc) suggesting a putative mobile character of C. difficile toxins.