Global Transcriptional Response of Clostridium difficile Carrying the ϕCD38-2 Prophage

Sekulović, Ognjen; Fortier, Louis‐Charles

doi:10.1128/aem.03656-14

Cited by 58 publications

(71 citation statements)

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“…Inactivation of recV prevents recombination of the epigenetic switch controlling transcription of cwpV (Emerson et al ., ), thereby allowing the isolation of clones in which the switch is locked in the ON or OFF configuration. The absence of cwpV expression in R20291 OFF was confirmed by RT‐qPCR, Western blot and immunofluorescence (Sekulovic and Fortier, ).…”

Section: Resultsmentioning

confidence: 90%

“…In a previous transcriptomic study with C. difficile R20291, we isolated a lysogen carrying the episomal ϕCD38‐2 prophage in which the cwpV gene (CDR20291_0440) was upregulated about 20 fold (Sekulovic and Fortier, ). This observation was intriguing, and although it is still unknown how ϕCD38‐2 interferes with cwpV expression, it raised interesting questions.…”

Section: Resultsmentioning

confidence: 99%

“…We used the R20291 strain, a ribotype 027 epidemic isolate that is susceptible to infection by three related temperate siphophages from our collection: ϕCD38-2, ϕCD111 and ϕCD146 (Sekulovic et al, 2011;2014). We also used the R20291 OFF strain, a R20291 mutant in which cwpV is not expressed (Sekulovic and Fortier, 2015) (Table 1). This mutant was created by inactivating the recV gene encoding the site-specific recombinase RecV, using the ClosTron system (Heap et al, 2007;Reynolds et al, 2011).…”

Section: Cwpv Protects Against Phage Infectionmentioning

confidence: 99%

“…Inactivation of the bacterial recombinase RecV from strain R20291 (CDR20291_1004) using the ClosTron system has been described previously (Reynolds et al, 2011;Sekulovic and Fortier, 2015). Colony PCR on putative recV mutants was performed in order to identify clones for which the genetic switch controlling the expression of the cwpV gene (Emerson et al, 2009) was locked either in the 'ON' (primer pair LCF 801 + LCF 714) or 'OFF' (primer pair LCF 796 + LCF 797) configuration (Table S1).…”

Section: Isolation Of R20291off and R20291on Clonesmentioning

confidence: 99%

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The Clostridium difficile cell wall protein CwpV confers phase‐variable phage resistance

Sekulović

Bedoya

Fivian‐Hughes

et al. 2015

Molecular Microbiology

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SummaryBacteriophages are present in virtually all ecosystems, and bacteria have developed multiple antiphage strategies to counter their attacks. C lostridium difficile is an important pathogen causing severe intestinal infections in humans and animals. Here we show that the conserved cell‐surface protein CwpV provides antiphage protection in C . difficile. This protein, for which the expression is phase‐variable, is classified into five types, each differing in their repeat‐containing C‐terminal domain. When expressed constitutively from a plasmid or the chromosome of locked ‘ON’ cells of C . difficile R20291, CwpV conferred antiphage protection. Differences in the level of phage protection were observed depending on the phage morphological group, siphophages being the most sensitive with efficiency of plaquing (EOP) values of < 5 × 10−7 for phages ϕCD38‐2, ϕCD111 and ϕCD146. Protection against the myophages ϕMMP01 and ϕCD52 was weaker, with EOP values between 9.0 × 10−3 and 1.1 × 10−1. The C‐terminal domain of CwpV carries the antiphage activity and its deletion, or part of it, significantly reduced the antiphage protection. CwpV does not affect phage adsorption, but phage DNA replication is prevented, suggesting a mechanism reminiscent of superinfection exclusion systems normally encoded on prophages. CwpV thus represents a novel ubiquitous host‐encoded and phase‐variable antiphage system in C . difficile.

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Section: Resultsmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Cwpv Protects Against Phage Infectionmentioning

confidence: 99%

Section: Isolation Of R20291off and R20291on Clonesmentioning

confidence: 99%

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The Clostridium difficile cell wall protein CwpV confers phase‐variable phage resistance

Sekulović

Bedoya

Fivian‐Hughes

et al. 2015

Molecular Microbiology

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“…These are in part derived from technical spike-in controls (phiX174) [48]: 21/62 of the ST1/1*, 0/4 of the ST11/11* and 1/6 of the ST37/37* ECEs correspond to this phage. Clostridium phage phiCD38-2 [49] was also common: 20/62 ST1/1*, 1/4 ST11/11* and 4/6 ST37/37* ECEs correspond to this phage. Finally, in ST11, Clostridium phage phiCD6356 [50] was identified once.…”

Section: Resultsmentioning

confidence: 99%

Anin silicosurvey ofClostridioides difficileextrachromosomal elements

Hornung

Kuijper

Smits

2019

Preprint

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19The gram-positive enteropathogen Clostridioides difficile is the major cause of healthcare 20 associated diarrhoea and is also an important cause of community-acquired infectious 21 diarrhoea. Considering the burden of the disease, many studies have employed whole 22 genome sequencing to identify factors that contribute to virulence and pathogenesis. Though 23 extrachromosomal elements such as plasmids are important for these processes in other 24 bacteria, the few characterized plasmids of C. difficile have no relevant functions assigned 25 and no systematic identification of plasmids has been carried out to date. Here, we perform 26 an in silico analysis of publicly available sequence data, to show that ~13% of all C. difficile 27 strains contain extrachromosomal elements, with 1-6 elements per strain. Our approach 28 identifies known plasmids (e.g. pCD6, pCD630 and cloning plasmids) and 6 novel putative 29 plasmid families. Our study shows that plasmids are abundant and may encode functions 30 that are relevant for C. difficile physiology. The newly identified plasmids may also form the 31 basis for the construction of novel cloning plasmids for C. difficile that are compatible with 32 existing tools. 33 35 36Clostridioides difficile (Clostridium difficile) [1] is a gram-positive, endospore forming, 37 anaerobic bacterium. It is an opportunistic pathogen in humans, and is the causative agent 38 of most cases of antibiotic associated diarrhoea [2]. In recent years, the bacterium is also 39 increasingly found in cases of infectious diarrhoea that cannot be linked to healthcare 40 exposure [2]. C. difficile infections can be refractory to antimicrobial therapy and even when 41 initial cure is observed, relapses are frequent [2]. Typing methods for C. difficile include 42 (capillary) PCR ribotyping, multilocus sequence typing (MLST) and single nucleotide 43 polymorphism (SNP) typing after whole genome sequencing [3]. Since the beginning of the 44 21 th century, an increase in C. difficile infections due to epidemic types such as PCR 45 ribotype 027 and 078 has been noted [4, 5]. Although the molecular mechanisms underlying 46 the epidemicity are poorly understood and remain under debate [6], robust toxin production 47 and sporulation [7], altered surface properties [8], resistance to antimicrobials [5, 9] and an 48 increased ability to metabolize certain sugars [10] have been implicated. 49In other organisms, the contribution of plasmid-encoded functions to virulence and 50 pathogenesis is well-documented [11][12][13]. By contrast, only a limited number of plasmids has 51 been identified in C. difficile and all of these are cryptic, i.e. no traits have been associated 52 with plasmid carriage. Commonly used cloning vectors for C. difficile make use of a replicon 53 derived from the 6.8 kb plasmid pCD6 [14]. The reference strain 630 contains a single 54 plasmid, pCD630, that is part of a larger family of 7.8-11.8 kb plasmids [15, 16]. And recently 55 several large (>42 kb) plasmids were described [17]. Neverthele...

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Characterization of Functional Prophages in Clostridium difficile

Sekulović

Fortier

2016

Methods in Molecular Biology

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Bacteriophages (phages) are present in almost, if not all ecosystems. Some of these bacterial viruses are present as latent "prophages," either integrated within the chromosome of their host, or as episomal DNAs. Since prophages are ubiquitous throughout the bacterial world, there has been a sustained interest in trying to understand their contribution to the biology of their host. Clostridium difficile is no exception to that rule and with the recent release of hundreds of bacterial genome sequences, there has been a growing interest in trying to identify and classify these prophages. Besides their identification in bacterial genomes, there is also growing interest in determining the functionality of C. difficile prophages, i.e., their capacity to escape their host and reinfect a different strain, thereby promoting genomic evolution and horizontal transfer of genes through transduction, for example of antibiotic resistance genes. There is also some interest in using therapeutic phages to fight C. difficile infections.The objective of this chapter is to share with the broader C. difficile research community the expertise we developed in the study of C. difficile temperate phages. In this chapter, we describe a general "pipeline" comprising a series of experiments that we use in our lab to identify, induce, isolate, propagate, and characterize prophages. Our aim is to provide readers with the necessary basic tools to start studying C. difficile phages.

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Global Transcriptional Response of Clostridium difficile Carrying the ϕCD38-2 Prophage

Cited by 58 publications

References 68 publications

The Clostridium difficile cell wall protein CwpV confers phase‐variable phage resistance

The Clostridium difficile cell wall protein CwpV confers phase‐variable phage resistance

Anin silicosurvey ofClostridioides difficileextrachromosomal elements

Characterization of Functional Prophages in Clostridium difficile

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