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Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile.

Boudry P, Semenova E, Monot M, Datsenko KA, Lopatina A, Sekulovic O, Ospina-Bedoya M, Fortier LC, Severinov K, Dupuy B, Soutourina O - MBio (2015)

Bottom Line: Clostridium difficile is the major cause of nosocomial infections associated with antibiotic therapy worldwide.We provide experimental evidence for the function of the C. difficile CRISPR system against plasmid DNA and bacteriophages.These data demonstrate the original features of active C. difficile CRISPR system and bring important insights into the interactions of this major enteropathogen with foreign DNA invaders during its infection cycle.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France.

No MeSH data available.


Related in: MedlinePlus

Functionality of C. difficile cas genes for plasmid interference in E. coli. The transformation efficiency was estimated with pT7Blue derivative plasmids carrying the wild-type (wt) protospacer corresponding to the first spacer of the CRISPR 16 array (CR16) (rows 2 and 5) or a mutated protospacer CR16 (rows 3 and 6) compared to the pT7Blue empty vector used as a negative control (rows 1 and 4). The protospacer plasmid used is indicated to the left of the photographs together with schematic representation of E. coli strains carrying engineered CRISPR arrays with the corresponding spacer under the control of T7 RNAP promoter (T7). E. coli KD623 strain (rows 1 to 3) carries C. difficile CRISPR “miniarray” with the first spacer of CRISPR 16 array flanked by repeats, and E. coli KD626 strain (rows 4 to 6) carries reduced “miniarray” with one repeat lacking spacer sequence. The CRISPR “leader” region (LDR) is indicated. Both strains were transformed with pCDF1-b vector derivative, allowing the expression of C. difficile cas gene set lacking cas1 and cas2 (from CD2982 to CD2977). The Cas protein production and crRNA expression were induced by the addition of 1 mM l-arabinose and 1 mM IPTG. The serial dilutions of transformation mixtures deposited on LB plates with ampicillin are indicated (ND, not diluted).
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fig7: Functionality of C. difficile cas genes for plasmid interference in E. coli. The transformation efficiency was estimated with pT7Blue derivative plasmids carrying the wild-type (wt) protospacer corresponding to the first spacer of the CRISPR 16 array (CR16) (rows 2 and 5) or a mutated protospacer CR16 (rows 3 and 6) compared to the pT7Blue empty vector used as a negative control (rows 1 and 4). The protospacer plasmid used is indicated to the left of the photographs together with schematic representation of E. coli strains carrying engineered CRISPR arrays with the corresponding spacer under the control of T7 RNAP promoter (T7). E. coli KD623 strain (rows 1 to 3) carries C. difficile CRISPR “miniarray” with the first spacer of CRISPR 16 array flanked by repeats, and E. coli KD626 strain (rows 4 to 6) carries reduced “miniarray” with one repeat lacking spacer sequence. The CRISPR “leader” region (LDR) is indicated. Both strains were transformed with pCDF1-b vector derivative, allowing the expression of C. difficile cas gene set lacking cas1 and cas2 (from CD2982 to CD2977). The Cas protein production and crRNA expression were induced by the addition of 1 mM l-arabinose and 1 mM IPTG. The serial dilutions of transformation mixtures deposited on LB plates with ampicillin are indicated (ND, not diluted).

Mentions: As a first step to mechanistic studies of the C. difficile CRISPR-Cas system, we established a heterologous system in a surrogate E. coli host that had its own CRISPR-Cas system removed. E. coli was chosen as a host that is easier to manipulate genetically than C. difficile. E. coli plasmids expressing the conserved and complete cas operon from C. difficile strain 630 containing eight cas genes (CD2982-CD2975) were created. The first part of the C. difficile cas operon (from CD2982 to CD2977 encoding the interference components) was cloned into the pCDF-1b expression vector (pDIA6351), and the rest of the operon (cas1 [CD2976] and cas2 [CD2975] genes) was cloned into the pRSF-1b vector (pDIA6349) under the control of T7 RNA polymerase (T7 RNAP) promoter. Next, E. coli host strains containing minimized C. difficile CRISPR arrays were created. Sequences of the highly expressed C. difficile 630 CRISPR 12 or CRISPR 16 arrays (Fig. 2; see Fig. S1 in the supplemental material) were selected for this purpose. The third “miniarray” containing only the leader region with direct repeat but without the spacer sequence was used as a negative control. These CRISPR arrays, flanked by a T7 RNAP promoter and transcriptional terminator sequences, were introduced into the genome of the E. coli BL21-AI_ΔCRISPR strain lacking endogenous cas genes and carrying the T7 RNAP-encoding gene under the control of the arabinose-inducible araBAD promoter (strains KD620, KD623, and KD626 [Table S4]). To monitor the CRISPR interference, strains KD620, KD623, and KD626 harboring the C. difficile cas expression plasmids were transformed with the compatible pT7Blue-based plasmids containing the protospacer-matching spacers within CRISPR “miniarrays.” Each strain was transformed with the pT7Blue derivatives containing the CCA PAM followed by either a protospacer perfectly matching the CRISPR spacer (pDIA6361 or pDIA6363), a protospacer with a single mismatch at the first position (pDIA6362 or pDIA6364), or an empty control pT7Blue vector (Table S4). Upon induction of C. difficile subtype I-B CRISPR-Cas in E. coli in the presence of l-arabinose, we observed a decrease in the transformation efficiency of plasmids containing protospacers fully matching the CRISPR array spacers and no difference in the transformation efficiency with a control strain carrying a CRISPR array without a spacer. Mutation in the first position of the protospacer “seed” region abolished the observed interference leading to the transformation efficiencies similar to those obtained with the empty vector (Fig. 7).


Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile.

Boudry P, Semenova E, Monot M, Datsenko KA, Lopatina A, Sekulovic O, Ospina-Bedoya M, Fortier LC, Severinov K, Dupuy B, Soutourina O - MBio (2015)

Functionality of C. difficile cas genes for plasmid interference in E. coli. The transformation efficiency was estimated with pT7Blue derivative plasmids carrying the wild-type (wt) protospacer corresponding to the first spacer of the CRISPR 16 array (CR16) (rows 2 and 5) or a mutated protospacer CR16 (rows 3 and 6) compared to the pT7Blue empty vector used as a negative control (rows 1 and 4). The protospacer plasmid used is indicated to the left of the photographs together with schematic representation of E. coli strains carrying engineered CRISPR arrays with the corresponding spacer under the control of T7 RNAP promoter (T7). E. coli KD623 strain (rows 1 to 3) carries C. difficile CRISPR “miniarray” with the first spacer of CRISPR 16 array flanked by repeats, and E. coli KD626 strain (rows 4 to 6) carries reduced “miniarray” with one repeat lacking spacer sequence. The CRISPR “leader” region (LDR) is indicated. Both strains were transformed with pCDF1-b vector derivative, allowing the expression of C. difficile cas gene set lacking cas1 and cas2 (from CD2982 to CD2977). The Cas protein production and crRNA expression were induced by the addition of 1 mM l-arabinose and 1 mM IPTG. The serial dilutions of transformation mixtures deposited on LB plates with ampicillin are indicated (ND, not diluted).
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Related In: Results  -  Collection

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fig7: Functionality of C. difficile cas genes for plasmid interference in E. coli. The transformation efficiency was estimated with pT7Blue derivative plasmids carrying the wild-type (wt) protospacer corresponding to the first spacer of the CRISPR 16 array (CR16) (rows 2 and 5) or a mutated protospacer CR16 (rows 3 and 6) compared to the pT7Blue empty vector used as a negative control (rows 1 and 4). The protospacer plasmid used is indicated to the left of the photographs together with schematic representation of E. coli strains carrying engineered CRISPR arrays with the corresponding spacer under the control of T7 RNAP promoter (T7). E. coli KD623 strain (rows 1 to 3) carries C. difficile CRISPR “miniarray” with the first spacer of CRISPR 16 array flanked by repeats, and E. coli KD626 strain (rows 4 to 6) carries reduced “miniarray” with one repeat lacking spacer sequence. The CRISPR “leader” region (LDR) is indicated. Both strains were transformed with pCDF1-b vector derivative, allowing the expression of C. difficile cas gene set lacking cas1 and cas2 (from CD2982 to CD2977). The Cas protein production and crRNA expression were induced by the addition of 1 mM l-arabinose and 1 mM IPTG. The serial dilutions of transformation mixtures deposited on LB plates with ampicillin are indicated (ND, not diluted).
Mentions: As a first step to mechanistic studies of the C. difficile CRISPR-Cas system, we established a heterologous system in a surrogate E. coli host that had its own CRISPR-Cas system removed. E. coli was chosen as a host that is easier to manipulate genetically than C. difficile. E. coli plasmids expressing the conserved and complete cas operon from C. difficile strain 630 containing eight cas genes (CD2982-CD2975) were created. The first part of the C. difficile cas operon (from CD2982 to CD2977 encoding the interference components) was cloned into the pCDF-1b expression vector (pDIA6351), and the rest of the operon (cas1 [CD2976] and cas2 [CD2975] genes) was cloned into the pRSF-1b vector (pDIA6349) under the control of T7 RNA polymerase (T7 RNAP) promoter. Next, E. coli host strains containing minimized C. difficile CRISPR arrays were created. Sequences of the highly expressed C. difficile 630 CRISPR 12 or CRISPR 16 arrays (Fig. 2; see Fig. S1 in the supplemental material) were selected for this purpose. The third “miniarray” containing only the leader region with direct repeat but without the spacer sequence was used as a negative control. These CRISPR arrays, flanked by a T7 RNAP promoter and transcriptional terminator sequences, were introduced into the genome of the E. coli BL21-AI_ΔCRISPR strain lacking endogenous cas genes and carrying the T7 RNAP-encoding gene under the control of the arabinose-inducible araBAD promoter (strains KD620, KD623, and KD626 [Table S4]). To monitor the CRISPR interference, strains KD620, KD623, and KD626 harboring the C. difficile cas expression plasmids were transformed with the compatible pT7Blue-based plasmids containing the protospacer-matching spacers within CRISPR “miniarrays.” Each strain was transformed with the pT7Blue derivatives containing the CCA PAM followed by either a protospacer perfectly matching the CRISPR spacer (pDIA6361 or pDIA6363), a protospacer with a single mismatch at the first position (pDIA6362 or pDIA6364), or an empty control pT7Blue vector (Table S4). Upon induction of C. difficile subtype I-B CRISPR-Cas in E. coli in the presence of l-arabinose, we observed a decrease in the transformation efficiency of plasmids containing protospacers fully matching the CRISPR array spacers and no difference in the transformation efficiency with a control strain carrying a CRISPR array without a spacer. Mutation in the first position of the protospacer “seed” region abolished the observed interference leading to the transformation efficiencies similar to those obtained with the empty vector (Fig. 7).

Bottom Line: Clostridium difficile is the major cause of nosocomial infections associated with antibiotic therapy worldwide.We provide experimental evidence for the function of the C. difficile CRISPR system against plasmid DNA and bacteriophages.These data demonstrate the original features of active C. difficile CRISPR system and bring important insights into the interactions of this major enteropathogen with foreign DNA invaders during its infection cycle.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France.

No MeSH data available.


Related in: MedlinePlus