Spores of Clostridioides difficile are toxin delivery vehicles

Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells.

Deletion and mutational analysis of the tcdR regulatory region.For deletion analysis of the tcdR promoter a fragment of 183 bp (fragment PtcdR-D) was fused to SNAP Cd in pFT47, according to the general strategy outlined above for the full-length fusion (Fig. S3).The fragment was PCR-amplified using the primer pair PtcdR-A-SNAP-EcoRI-Fw/ PtcdR-SNAP-XhoI-Rev and inserted between the EcoRI/XhoI sites of pFT47, yielding pCC20.The plasmid was introduced into E. coli HB101 (RP4) yielding strain AHEC237 and transferred to C. difficile 630Δerm by conjugation, producing strain AHCD795.
To introduce point mutations at the -10 region of the σ D promoter located upstream of tcdR (Fig. S3) overlap extension PCR was performed, initially using the primer pairs PtcdR-SNAP-EcoRI-Fw/ PsigDmut-Rev and PsigDmut-Fw/ PtcdR-SNAP-XhoI-Rev that originated fragments of 741 and 195 bp, and then, after mixing the two fragments, with PtcdR-SNAP-EcoRI-Fw/ PtcdR-SNAP-XhoI-Rev.The final PCR product was cloned between EcoRI/XhoI sites of pFT47 to give pCC27.The plasmid was transformed into E. coli HB101 (RP4) yielding strain AHEC274 and then transferred to C. difficile 630Δerm by conjugation, producing strain AHCD821.
Construction of a ΔtcdR mutant through allele coupled exchange.Allele coupled exchange using a pyrE strain 2 was used to obtain in frame deletions of the tcdR gene in C. difficile 630Δerm and R20291.pyrE negative strains previously constructed 2 were used in order to allow for a positive/negative selection.An allele exchange cassette was obtained by overlap extension PCR using the primer pair tcdR-AscI-Fw/tcdR-SOE-Rev for the left-hand homology arm (LHA) originating a 631 bp fragment and tcdR-SOE-Fw/tcdR-SbfI-Rev for the right-hand homology arm (RHA) originating a 522 bp fragment.The final PCR was performed with the primer pair tcdR-AscI-Fw/ tcdR-SbfI-Rev and the resulting fragments inserted between the SbfI and AscI sites of pMTL-YN3, to give pSR3 (for the construction of the ΔtcdR mutant) in 630Δerm, and pMTL-YN4, to give pSR2 (for the construction of the ΔtcdR mutant in R20291).pSR3 and pSR2 were transformed into E. coli HB101 (RP4) to give AHEC239 and AHEC227, respectively, and then moved from these strains, by conjugation, into C. difficile 630ΔermΔpyrE and R20291ΔpyrE 2 .C. difficile transconjugants were selected by sub-culturing on BHI agar containing thiamphenicol (15 µg/mL) and cefoxitin (25 µg/mL) and re-streaked twice in this medium for 630Δerm and four times for R20291.Colonies were screened by colony PCR with DNA obtained using 5% chelex resin (Sigma-Aldrich) to identify singlecrossover mutants.For the tcdR gene the primer pairs YN3-vef-Fw/ tcdR-vef-Fw and YN3-vef-Rev/ tcdR-vef-Rev for 630Δerm or YN4-vef-Fw/ tcdR-vef-Fw and YN4vef-Rev/ tcdR-vef-Rev for R20291.The single-crossover mutants identified were streaked onto CDMM 3 with 1% agar supplemented with 5-Fluoroorotic acid (2 mg/mL) and uracil (5 µg/mL) in order to select for plasmid excision.The isolated FOA-resistant colonies were screened by colony PCR as above using the primer pair tcdR-vef-Fw/ tcdR-vef-Rev.The primers used flank the upstream and downstream homology regions.The double-crossover mutants that were thiamphenicol sensitive (AHCD811 in 630Δerm and AHCD1093 in R20291) were reverted to a PyrE+ phenotype through conjugation with plasmid pMTL-YN1 for 630Δerm and pMTL-YN2 for R20291.The resulting colonies were streaked onto non-supplemented CDMM agar and the restoration of the pyrE allele was confirmed by colony PCR using the primer pair pyrE-vef-Fw/pyrE-vef-Rev.Isolates with the expected structure were identified and named AHCD820 (ΔtcdR in 630Δerm) and AHCD1102 (ΔtcdR in R20291).Plasmids pMS470 and pMS464 (above; Table S3) were then introduced into the 630Δerm ΔtcdR mutant (AHCD820) by conjugation with E. coli HB101 (RP4) derivatives, which resulted in strains AHCD840 and AHCD841 for ΔtcdR, respectively.

Construction of ΔtcdA and ΔtcdB mutants and of a ΔtcdAΔtcdB double
mutant through allele coupled exchange.Allelic exchange 2 was also used to construct ΔtcdA and ΔtcdB mutants and the double mutant ΔtcdAΔtcdB.Plasmids pSR7 and pSR8 were used to delete tcdA and tcdB, respectively.For the construction of pSR7 the allele exchange cassettes were obtained by overlap extension PCR using the primer pairs tcdA-AscI-Fw/tcdA-SOE-Rev for the LHA originating a 626 bp fragment and tcdA-SOE-Fw/ tcdA-SbfI-Rev for the RHA originating a 544 bp fragment.The final PCR was obtained with the primer pair tcdA-AscI-Fw/ tcdA-SbfI-Rev.For the construction of pSR8, the allele exchange cassettes were obtained by overlap extension PCR using the primer pairs tcdB-AscI-Fw/ tcdB-SOE-Rev for the LHA originating a 635 bp fragment and tcdB-SOE-Fw/ tcdB-SbfI-Rev for the RHA originating a 497 bp fragment.The final PCR was obtained with the primer pair tcdB-AscI-Fw/ tcdB-SbfI-Rev.All plamids were verified by DNA sequencing.The plasmids obtained were transformed into E. coli HB101 (RP4) yielding strains AHEC498 (for ΔtcdA) and AHEC499 (for ΔtcdB).The E. coli strains for the construction of the ΔtcdA and ΔtcdB mutants were subsequently mated with C. difficile 630ΔermΔpyrE and transconjugants were selected by subculturing on BHI agar containing thiamphenicol (15 µg/mL) and cefoxitin (25 µg/mL) and re-streaked twice in this medium.Colonies were screened by colony PCR with DNA prepared using 5% chelex resin (Sigma-Aldrich) to identify single-crossover mutants.For the tcdA gene the primer pairs YN3-vef-Fw/ tcdA-vef-Fw and YN3-vef-Rev/ tcdA-vef-Rev were used, while for tcdB the primer pairs YN3-vef-Fw/ tcdB-vef-Fw and YN3-vef-Rev/ tcdB-vef-Rev were used.The single-crossover mutants identified were streaked onto CDMM 3 with 1% agar supplemented with 5-Fluoroorotic acid (2 mg/mL) and uracil (5 µg/mL) in order to select for plasmid excision.The isolated FOA-resistant colonies were screened by colony PCR using the primer pair tcdA-vef-Fw/tcdA-vef-Rev for tcdA and tcdB-vef-Fw/tcdB-vef-Rev for tcdB, which flank the upstream and downstream homology regions.
The double-crossover mutants that were thiamphenicol sensitive (AHCD999 for tcdA and AHCD1415 for tcdB) were reverted to a PyrE+ phenotype through conjugation with the plasmid pMTL-YN1.The resulting colonies were streaked onto non-supplemented CDMM agar and the restoration of the pyrE allele was confirmed by colony PCR using the primer pair pyrE-vef-Fw/pyrE-vef-Rev.This produced strains AHCD1002 (ΔtcdA) and AHCD1439 (ΔtcdB).The double-crossover ΔtcdA mutant that was thiamphenicol sensitive (pyrE-) was used to mate with E. coli AHEC499 to construct the double ΔtcdA/ΔtcdB mutant AHCD1046, using the same methodology outlined above for the single mutant.This strain was reverted to a PyrE+ phenotype through conjugation with pMTL-YN1.The resulting colonies were streaked onto non-supplemented CDMM agar and the presence of a restored pyrE allele was confirmed by colony PCR using the primer pair pyrE-vef-Fw/ pyrE-vef-Rev.This screen led to the isolation of strain AHCD1066.
Complementation through allelic exchange at the pyrE locus.In order to complement the ΔtcdR mutation, the coding sequence of the tcdR gene and its promoter region, a total of 1460 bp, was PCR amplified from 630Δerm or R20291 genomic DNA, using the primer pair tcdR-comp-BamHI-Fw/tcdR-comp-HindIII-Rev and the fragment inserted between the BamHI and HindIII sites of either pMTL-YN1C, yielding pSR5 (for 630Δerm) and pMTL-YN2C, yielding pSR34 (for R20291).pSR5 and pSR34 were transformed into E. coli HB101 (RP4) yielding strains AHEC285 and AHEC661, respectively, and subsequently mated with C. difficile 630ΔermΔpyrEΔtcdR and R20291ΔpyrEΔtcdR.The transconjugants obtained were streaked onto CDMM and tested by colony PCR using primer pair tcdR-vef-Fw/ tcdR-vef-Rev to test for both tcdR complementation and with primer pair pyrE-vef-Fw/pyrE-vef-Rev for pyrE reversion; clones with the expected changes were identified and named AHCD828, for 630Δerm, and AHCD1161, for R20291.Plasmids pMS470 and pMS464 (above; Table S3) were introduced into the complementation strain through matting from E. coli HB101 (RP4) derivatives, resulting in strains AHCD845 and AHCD846, respectively.
Placing tcdR under the control of forespore-or mother cell-specific promoters.The sspA or spoIIIAA promoters were fused to the tcdR gene using overlap extension PCR.The tcdR gene was PCR amplified from C. difficile 630Δerm genomic DNA with an optimized RBS using the primer pair TcdR-RBSopt-Fw/ TcdR-comp-HindIII-Rev, producing a 723bp.The PCR fragment was inserted between the XhoI/HindIII sites of pMTL-YN1C, to produce pCC44.The sspA and spoIIIAA promoter regions were PCR amplified using primer pairs PsspA-BamHI-Fw/ PsspA-SNAP-XhoI-Rev and PspoIIIAA-BamHI-Fw/ PspoIIIAA-SNAP-XhoI-Rev, producing fragments of 482 and 494 bp which were inserted between the BamHI/XhoI sites of pCC44 to give pCC45 (PsspA-tcdR) and pCC46 (PspoIIIA-tcdR).
The plasmids were transformed into E. coli HB101 (RP4) yielding strains AHEC391 and AHEC389, respectively, and subsequently transferred to C. difficile 630ΔermΔpyrEΔtcdR by conjugation.
The transconjugants obtained were streaked onto CDMM and tested by colony PCR using the primer pair PsspA-BamHI-Fw/ TcdR-comp-XhoI-Rev and PspoIIIAA-BamHI-Fw/ TcdR-comp-XhoI-Rev to test for integration and with the primer pair pyrE-vef-Fw/pyrE-vef-Rev to test for pyrE reversion, yielding strains Construction of a ΔspoVT in-frame deletion mutant using CRISPR-Cas9.A CRISPR-Cas9-based approach 4 was used for the in-frame deletion of the spoVT gene in the C. difficile 630Δerm sigG mutant previously constructed using the ClosTron system 1 .The sgRNA, used to target the Cas9 nuclease to the spoVT gene, was obtained by a primer dimer PCR reaction.The primers for amplification were designed by adding the 20 nucleotides SEED region identified with the Benchling software (https://benchling.com/crispr) to the universal forward primer (SpoVT_sgRNA_Fw) together with the universal reverse primer sgRNA_Rev.The allele exchange cassette composed of a left-hand homology arm (LHA) and a righthand homology arm (RHA) was obtained by overlap extension PCR using the primer pair spoVT-AscI-Fw/spoVT-SOE-Rev for the LHA originating a 701 bp fragment and spoVT-SOE-Fw/spoVT-AsiSI-Rev for the RHA originating a 709bp fragment.The final PCR was done with the primer pair spoVT-AscI-Fw/spoVT-AsiSI-Rev originating a 1410 bp fragment that was inserted between the AscI/AsiSI sites of pMTL-Cas9 4 together with the sgRNA fragment that was cloned between the SalI/AsiSI sites, to yield pCC86, the sequence of which was verified.pCC86 was introduced into E. coli HB101 (RP4) to give AHEC855 and then introduced into C. difficile 630Δerm sigG by conjugation.Transconjugants were selected by subculturing on BHI agar containing thiamphenicol (15 µg/mL) and cefoxitin (25 µg/mL) and then re-streaked in this medium.The selected transconjugants were screened by colony PCR using the primer pair spoVT-vef-Fw/spoVT-vef-Rev.A strain with the expected structure was isolated and named AHCD1418.

-Plasmids used in this study.
ermB, erythromycin resistance determinant.12. El Meouche, I. et al.Characterization of the SigD Regulon of Clostridium difficile and Its Positive Control of Toxin Production through the Regulation of tcdR.PLoS One 8, (2013).13.Kurka, H. et al.Sequence Similarity of Clostridium difficile Strains by Analysis of Conserved Genes and Genome Content Is Reflected by Their Ribotype Affiliation.PLOS ONE 9, e86535 (2014). *