Materials and Methods

Function of the CRISPR-Cas System of the Human PathogenClostridium difficile

MATERIALS AND METHODSPlasmid and bacterial strain construction and growth conditions. C. difficile and E. coli strains and plasmids used in this study together with detailed descriptions of plasmid and bacterial strain construction are presented in Table S4 in the supplemental material. C. difficile strains were grown anaerobically (5% H2, 5% CO2, and 90% N2) in TY (53) or brain heart infusion (BHI) (Difco) medium in an anaerobic chamber (Jacomex). When necessary, cefoxitin (Cfx) (25 µg·ml−1) and thiamphenicol (Tm) (15 µg·ml−1) were added to C. difficile cultures. E. coli strains were grown in LB broth (54), and when needed, ampicillin (100 µg·ml−1), chloramphenicol (15 µg·ml−1), tetracycline (15 µg·ml−1), kanamycin (50 µg·ml−1), or streptomycin (50 µg·ml−1) was added to the culture medium. All primers used in this study are listed in Table S6.RNA-seq analysis. Total RNA was isolated from late-exponential-growth-phase cultures (grown for 6 h) of C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 grown in TY medium as previously described (55), and mRNA was enriched from total RNA using MicrobExpress kit (Ambion). Nonoriented RNA-seq library construction was performed with the TruSeq RNA sample prep kit from Illumina as previously described (56) and subjected to Illumina HiSeq 2000 sequencing.In silico analysis of CRISPR array spacer content and cas locus. CRISPRdb tools (11), the CRISPRTarget program (28), or BLASTN (27) were used for spacer homology search in the available sequences (April 2015). Several potential CRISPR arrays predicted by CRISPRdb within the tcdA coding region were excluded from further analysis for the following reasons: (i) their location within the toxin-encoding genes in all analyzed C. difficile strains as a part of the cell wall-binding repeat regions within the TcdA amino acid sequence; (ii) the prediction of the corresponding arrays as “questionable sequences” by the CRISPRdb program for several analyzed C. difficile strains; (iii) the absence of a characteristic RNA-seq profile for CRISPR arrays within the tcdA coding region; (iv) the absence of potential targeting of corresponding spacers for the known sequences; (v) the differences in the length and sequence of associated direct repeats with those of active CRISPR arrays. Thus, in such particular cases, the CRISPRdb predictions within repeated coding regions should be considered with caution and would need experimental confirmation.For general CRISPR spacer homology search, the sequences presented ≤7 single nucleotide polymorphisms (SNPs) (80% match or ≥30/37 nucleotides) were considered positive hits. The raw sequencing read data of published genome sequences from 2,207 C. difficile strains (57,–62) were used to search for the presence of cas loci homologous to the CD2982-CD2975 and CD2455-CD2451 cas operons from strain 630 and the CDR20291_2998-2994 operon from strain {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. For each strain, the sequencing reads were mapped on the sequence of corresponding cas locus using Bowtie (63). Coverage values of ≥80% were considered positive hits for the presence of corresponding cas loci in a given strain. The multilocus sequence typing (MLST) scheme of Lemee et al. (64) has also been inferred from raw sequencing read data.Phage infection assays. Phage host range determination was done as previously described (29) using spot tests with diluted phage lysates and exponentially grown C. difficile cultures.Plasmid conjugation efficiency assays. Derivatives of pMTL84121 and pRPF185 plasmids used to estimate the conjugation efficiency were transformed into the E. coli HB101 (RP4) and subsequently mated with C. difficile 630Δerm mutant strain on BHI agar plates for 24 h at 37°C. The proportion of C. difficile transconjugants was estimated by subculturing the cell conjugation mixture on BHI agar containing Tm (15 µg·ml−1) and Cfx (25 µg·ml−1) and comparing the number of CFU obtained after plating serial dilutions on BHI agar plates with Cfx lacking Tm.Plasmid-based interference assays in E. coli. The transformation efficiency of pT7Blue derivative plasmids (see Table S4 in the supplemental material) was monitored with E. coli strains lacking endogenous cas genes and carrying C. difficile CRISPR “miniarray” within their genome, including a CRISPR spacer targeting pT7Blue constructs and the plasmids pRSF-1b and/or pCDF-1B expressing C. difficile cas genes under the control of T7 RNAP promoter. Cas protein and crRNA production was induced with 0.5 to 1 mM l-arabinose and 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), and electrocompetent cells were prepared under these inducing conditions. Plasmids were then introduced by electroporation, and undiluted and serially diluted aliquots of the transformation mixture were spotted on LB agar plates containing ampicillin and either streptomycin or kanamycin/streptomycin to measure the transformation efficiency.Data access. The complete genome sequences of phiCD24-1, phiCD111, phiCD146, phiCD211, phiCD481-1, phiCD505, phiCD506, phiMMP01, phiMMP03, and phiCD52 were deposited in EMBL-EBI database under accession no. {"type":"entrez-nucleotide","attrs":{"text":"LN681534","term_id":"732169326","term_text":"LN681534"}}LN681534, {"type":"entrez-nucleotide","attrs":{"text":"LN681535","term_id":"732169390","term_text":"LN681535"}}LN681535, {"type":"entrez-nucleotide","attrs":{"text":"LN681536","term_id":"732169446","term_text":"LN681536"}}LN681536, {"type":"entrez-nucleotide","attrs":{"text":"LN681537","term_id":"1217107185","term_text":"LN681537"}}LN681537, {"type":"entrez-nucleotide","attrs":{"text":"LN681538","term_id":"732169690","term_text":"LN681538"}}LN681538, {"type":"entrez-nucleotide","attrs":{"text":"LN681539","term_id":"732169745","term_text":"LN681539"}}LN681539, {"type":"entrez-nucleotide","attrs":{"text":"LN681540","term_id":"732169822","term_text":"LN681540"}}LN681540, {"type":"entrez-nucleotide","attrs":{"text":"LN681541","term_id":"732169876","term_text":"LN681541"}}LN681541, {"type":"entrez-nucleotide","attrs":{"text":"LN681542","term_id":"732169958","term_text":"LN681542"}}LN681542, and PRJEB7856, respectively. RNA-seq coverage visualizations of the CRISPR loci are available for strain 630 through https://mmonot.eu/COV2HTML/visualisation.php?str_id=-17 and for strain {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 through http://mmonot.eu/COV2HTML/visualisation.php?str_id=-18 (65).

Article TitleFunction of the CRISPR-Cas System of the Human PathogenClostridium difficile

Abstract

Clostridium difficileis the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen,C. difficilemust be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays inC. difficilereference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain{"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, an epidemicC. difficilestrain. Through genome sequencing and host range analysis of several newC. difficilephages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in bothC. difficilestrains. We further demonstrate thatC. difficileCas proteins are capable of interference in a heterologous host,Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites.


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