Spo0A Suppresses sin Locus Expression in Clostridioides difficile

Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

among their respective parent strains. As previously reported, the levels of sinR and sinI transcripts were increased several-fold ( Fig. 1A) in all three spo0A mutants compared to their parent strains. An approximately 2-fold reduction in the sinR and sinI transcripts could be observed in the complemented strains. To further confirm this result, we performed Western blot analysis using SinR-specific antibodies. We grew the mutants and the respective parent strains in TY (tryptose and yeast extract) medium for 10 h and observed the levels of SinR in their cytosol. We found that spo0A mutants of all three strains produced larger amounts of SinR compared to their respective parents (Fig. 1B). However, in our complementation of JIR8094::spo0A and UK1::spo0A, we observed a partial reduction of SinR levels (Fig. 1B). Reduction in SinR level was not obvious in the R20291::spo0A complemented strain. Failures to complement an spo0A mutation had been previously observed in C. difficile. Two independent studies showed incomplete restoration of the sporulation phenotype in R20291::spo0A (28,30). However, when Deakin et al. tested the spo0A mutants of 630Δerm and R20291 strains, they found in vitro levels of sporulation to be restored to wild-type levels in their complemented derivative (31). When they tested the R20291 strains for toxin production, however, the complemented strain still produced increased toxin levels compared to the wild type (31). These observations suggest that introducing spo0A using a multicopy plasmid may not be a suitable method for complementation considering the Spo0A regulatory networks' complex nature. In a recent study, Dembek et al. successfully placed Ptet regulatory elements upstream of the spo0A gene, generating 630erm::PtetSpo0A. This strain can be artificially induced to sporulate by adding anhydrotetracycline (ATc) (32). We obtained this strain and performed SinR Western blotting upon induction of Spo0A. A reduction in sinR and sinI transcription levels could be seen as Spo0A production increases ( Fig. 2A). This observation was further confirmed by Western blot analysis of the Spo0A-induced cultures with SinR-specific antibodies. These results together suggest Spo0A as a negative regulator of the sin locus (Fig. 2B).
Spo0A represses sin locus expression. Spo0A is a transcriptional regulator and is a DNA binding protein. Spo0A binds to specific DNA sequences in the promoter region of its target gene to regulate their expression. To determine if the elevated levels of SinR observed in spo0A mutants are due to the repressor activity of Spo0A, we performed reporter fusion assays. We fused 600 bp of the sin locus upstream DNA with the gusA reporter gene coding for ␤-glucuronidase, and the construct was introduced into the R2091::spo0A mutant and its parent strain. The plasmid carrying a promoterless gusA gene was used as a negative control. We also cloned the promoter region of spoIIAB, known to be regulated by Spo0A, with the gusA gene and used this construct as a positive control. The spoIIAB promoter is positively regulated by Spo0A and was found to be active only in the parent strain and not in the spo0A mutant (Fig. 3A). We observed significantly higher ␤-glucuronidase activity when it was expressed from the  sin locus promoter in the R20291::spo0A mutant strain compared to the parent strain, where very minimal reporter activity was recorded. This observation is consistent with our Western blot results, where we detected elevated levels of SinR in spo0A mutant strains. Taken together, these results suggest that Spo0A represses the transcription of sinR either directly or indirectly. To narrow down the Spo0A-controlled region in the sin locus promoter, we cloned 475 and 340 bp of the upstream DNA with the gusA gene and performed the reporter fusion assays. The levels of reporter gene activity were similar in the cultures carrying the 600-and 340-bp upstream fusions (Fig. 3B). This result indicates that both the sin locus promoter and the Spo0A-regulated regions are present within this 340-bp region.
Spo0A binds to the sin locus upstream region. The results in Fig. 3A and B show that expression of Psin-gusA was lower in the R20291 background, while the expression of the reporter gene was at higher levels in the R20291::spo0A background. To determine whether the repression of sinR by Spo0A is due to Spo0A binding specifically to the promoter region of sinR, we carried out a DNA binding experiment. Considering that Spo0A needs to be phosphorylated to bind to the target DNA, we did not attempt the in vitro electrophoretic gel shift assay. Instead, we used a biotin-labeled DNA pulldown assay to determine the DNA binding ability of Spo0A under native conditions. The DNA segment representing the promoter region of sinR was biotinylated and was coupled to immobilized monomeric avidin resin. This bead-DNA complex was incubated with the cell lysate from the parent R20291 strain. The bound proteins were eluted, run in SDS-PAGE, and immunoblotted with the Spo0A antibody. We first standardized the binding experiment by using the spo0IIAB promoter region as a positive control. Spo0A protein could be detected in the eluates when the spo0IIAB upstream DNA was used as the bait. The biotinylated gluD upstream DNA and the beads alone were also processed similarly and served as negative controls. We applied the same protocol using the biotinylated 340-bp sin upstream DNA as bait. The results showed that it could pull down Spo0A, suggesting that Spo0A binds specifically to the promoter region of the sin locus ( Fig. 4A and B). Next, to narrow down the Spo0A binding site within that 340 bp, we created three biotin-labeled fragments covering the first 118 bp (340 to 222 bp upstream), the last 140 bp, and the overlapping 135-bp midregion (237 to 102 bp upstream) ( Fig. 4A and C) and used them as bait in the pulldown experiment. Spo0A was detected when the 140-bp midregion and the first 118 bp were used as baits (Fig. 4C). Since the biotin-labeled DNA pulldown assay is semiquantitative, this assumption needs further validation. Spo0A could not be recovered from the eluate from the binding of the gluD upstream region or with beads alone (not shown), suggesting the specificity of the Spo0A binding with the sin locus promoter.
Mutational analysis of the sinR upstream region. In B. subtilis, Spo0AϳP is known to bind to the 7-bp DNA element 5=-TGNCGAA-3=, commonly known as the Spo0A box (33). However, there are certain exceptions in which Spo0A binds to degenerated Spo0A boxes with mismatches in the upstream region of some targets (34,35). The DNA binding domain of C. difficile Spo0A is highly homologous to B. subtilis Spo0A, and the key residues of Spo0A known to mediate the interaction with the bases of the 0A box are highly conserved in Bacillus and Clostridium species (24,36). In C. difficile, Spo0A is known to bind to spo0A upstream and sigH upstream. Both of these genes have the TGTCGAA consensus Spo0A box sequence (23,37). C. difficile Spo0A also binds upstream of an spoIIAA-spoIIE-spoIIGA operon with low affinity, where the binding sequence is a degenerated Spo0A box with TACGACA sequence (23). We scanned the upstream region of sinR for potential Spo0A binding consensus sequence. We could predict that Spo0A binds to sequences within the 340 to 102 bp upstream of the sin locus from the biotin pulldown experiment. A classical Spo0A binding box (TTCTACA, complementary to TGTAGAA [marked as R1]) could be identified 274 bp upstream of the start codon. A potential degenerated Spo0A box (TTCGTTT [marked as R2]) was located 230 bp upstream. Two repeats with TATTGTAG sequences could also be seen in this region. We included all four regions for further analysis. We mutated the C and G residues in these chosen regions with T, and these mutated sin upstream regions were used to create reporter fusions. Mutations in the repeat sequences didn't affect the expression of the reported genes. However, mutations in the predicted Spo0A boxes (R1 and R2) affected the expression of the reporter fusion (Fig. 4D). These mutations relieved the repression observed in the parent strain, and the reporter activity was similar to the one observed in the spo0A mutant. To further confirm this result, we performed a biotin pulldown assay with the sin locus upstream DNA carrying both the R1 and R2 mutations. Spo0A couldn't be pulled down when this mutated DNA fragment was used as a bait (Fig. 4B). These results demonstrate that Spo0A binds to the R1 and R2 regions in the sin locus upstream to repress its expression.

DISCUSSION
Sporulation in a cell is an intense response to stress and is particularly expensive in both time and materials (38). The exact conditions and timing for sporulation are likely to be under strong selective pressure as both premature spore production and belated production can have disastrous effects on cell growth and survival. In B. subtilis, the sin (sporulation inhibition) operon is central to the timing and early dynamics of this network (39)(40)(41), and its regulation is controlled by the sporulation master regulator Spo0A itself. In this study, we have demonstrated that similar to B. subtilis, the sin locus in C. difficile is also regulated by Spo0A.
Like in many Gram-positive bacteria, Spo0A is the master regulator of sporulation in C. difficile (24,30,31). When the post-exponential phase begins, Spo0A activates the expression of the genes involved in the sporulation initiation process and positively regulates the sigma factor cascade required for sporulation (29). In many other pathogenic spore-forming bacteria, the gene regulatory networks that influence sporulation and virulence are closely linked with each other (42)(43)(44)(45)(46). In C. difficile, the mutation in spo0A affected many pathogenic traits, including toxin production, flagellum expression, and biofilm formation (5,28,30,31,47). Mackin et al. observed a clear increase in the production of toxins A and B upon disruption of spo0A in the ribotype 027 isolates R20291 and M7404 (30). In a similar study, Deakin et al. found that an R20291 spo0A mutant caused more severe disease in a murine model than the wild-type strain and associated this increase in severity with an increase in the amount of toxins A and B produced by the mutant in vitro (31). Dawson et al. showed that Spo0A in the 630Δerm strain promotes a sporulation cascade and biofilm formation and negatively regulates expression of virulence factors (toxins and flagella) (28). We found the UK1::spo0A strain to produce higher levels of toxins than its parent strain, while no significant difference was observed between the JIR8094 parent and JIR8094::spo0A mutant (see Fig. S3A in the supplemental material). This observation was consistent with the previous report, where a mutation in spo0A influenced the toxin production only in the 027 ribotype, which includes the UK1 and R20291 strains, but not in the 630Δerm strain, which belongs to the 012 ribotype like the JIR8094 strain. Reduced biofilm formation was also found only in R20291::spo0A and UK1::spo0A ( Fig. S3B and C). The mechanism of Spo0A regulation over these pathways remains to be answered. In the C. difficile genome, Ͼ100 open reading frames have potential 0A boxes within 500 bp of their start codons, indicating direct regulation by Spo0A (24). However, tcdA and tcdB, encoding toxins A and B, respectively, are not among them, indicating the indirect influence of Spo0A on toxin production (24). Spo0A could indirectly control motility and biofilm formation since many candidate regulators are encoded by the genes putatively under the direct control of Spo0A in C. difficile (24,31). Our current finding of Spo0A-mediated sin locus regulation can partly explain many of the phenotypes displayed by spo0A mutants, especially in the ribotype 027 strains (28,30). In our initial characterization of the sin locus, we showed decreased toxin production and motility in the absence of SinR and SinI (15). The expression of sinR alone was sufficient to complement these phenotypes and suggested SinR as a positive regulator of these pathways (15). We have further shown that SinR controls toxin production by regulating sigD, a sigma factor that positively regulates tcdR, which is needed for the transcription of toxin genes (15,48,49). SigD is also needed for the transcription of the flagellar operon in C. difficile (48,49). This study has shown increased SinR production in the absence of Spo0A (Fig. 1A and B). qRT-PCR results showed increased expression of sigD, tcdR, and tcdB in the R20291:: spo0A and UK1::spo0A strains compared to their respective parent strains (Fig. S3C). Increased sigD expression can lead to increased flagellar and toxin production and reduced biofilm formation in the spo0A mutant (22, 28) (Fig. S3).
In this study, we have shown that Spo0A binds to the C. difficile sin locus promoter and suppresses the expression of both sinR and sinI. We had previously shown that disruption of sinR by insertion mutagenesis affects transcription of both sinR and sinI (15), suggesting that sinRI is transcribed as a bicistronic message. Our qRT-PCR analysis detected lower levels of sinI transcripts than sinR transcripts. Since the reduction is observed in both the parent strain and spo0A mutants, we can conclude that this effect is independent of Spo0A.
In summary, we have demonstrated that Spo0A, the master regulator of sporulation, regulates sin locus expression. We have further shown that Spo0A can bind to the upstream region of the sin locus and have successfully mapped the region to which it binds. This finding adds a new detail to C. difficile's virulence gene regulatory network.
General DNA techniques. Chromosomal DNA was extracted from C. difficile cultures with the DNeasy blood and tissue kit (Qiagen). PCRs were carried out using gene-specific primers (see Table S2 in the supplemental material). PCR products were extracted from the gel with the Geneclean kit (mpbio). Plasmid DNA was extracted using the QIAprep spin miniprep kit (Qiagen). Standard procedures were used to perform routine cloning.
Construction and complementation of C. difficile spo0A mutant strains. The spo0A mutants of the JIR8094 and UK1 strains were created using the ClosTron gene knockout system as described previously (24,25,28,31). Briefly, for spo0A disruption, the group II intron insertion site between nucleotides 178 and 179 in the spo0A gene in the antisense orientation was selected using a web-based design tool called the Perutka algorithm. The designed retargeted intron was cloned into pMTL007-CE5, as described previously (54). The resulting plasmid, pMTL007-CE5::spo0A-178-179a, was transferred into C. difficile UK1 and JIR8094 cells by conjugation. The potential Ll.ltrB insertions within the target genes were conferred by the selection of lincomycin-resistant transconjugants in 20-g/ml lincomycin plates. PCR using gene-specific primers (Table S2) in combination with the EBS(U) universal primer was performed to identify putative C. difficile mutants. C. difficile spo0A mutants were complemented by introducing pRG312, which contains the spo0A gene with the 300-bp upstream region, through conjugation. Complementation was confirmed by PCR and Western blot analysis.
Western blot analysis. C. difficile cultures for Western blot analysis were harvested and washed in 1ϫ phosphate-buffered saline (PBS) solution. The pellets were resuspended in sample buffer (80 mM Tris, 2% SDS, 10% glycerol) and lysed by sonication. The whole-cell extracts were then centrifuged at 17,000 ϫ g at 4°C for 1 min. The lysate was heated at 100°C for 7 min, and the proteins were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membrane. The blots were then probed with specific primary and secondary antibodies at a dilution of 1:10,000. Immunodetection of proteins was done using the ECL enhanced chemiluminescence kit (Millipore) following the manufacturer's recommendations and were developed using the G-Box iChemi XR scanner. Blot images were overlapped with the original images of the membrane to visualize the prestained marker.
Construction of reporter plasmids and ␤-glucuronidase assay. The sin locus upstream DNA regions of various lengths were amplified by PCR using specific primers with KpnI and SacI (Table S2) recognition sequences. R20291 strain chromosomal DNA was used as a template for this amplification. Plasmid pRPF185 carries a gusA gene for ␤-glucuronidase under a tetracycline-inducible (tet) promoter. The tet promoter was removed using KpnI and SacI digestion and was replaced with sin locus upstream regions of various lengths to create plasmids pBA009, pBA029, pBA037, pBA038, and pBA039 (Table S1). The control plasmid pBA040 with a promoterless gusA gene was created by digestion with KpnI and SacI to remove the tet promoter and then self-ligated after creating blunt ends. Plasmids were introduced into the R20291 and R20291::spo0A strains through conjugation as described previously (15,27). The transconjugants were grown in TY medium in the presence of thiamphenicol (15 g/ml) overnight. These overnight cultures were then used as an inoculum at a 1:100 dilution to start a new culture. Bacterial cultures were harvested at 10 h of growth, and the amount of ␤-glucuronidase activity was assessed as described elsewhere (55,56).
Mutagenesis of sin locus promoter region. A Quick Change Lightning site-directed mutagenesis kit (Agilent Technologies) was used to carry out site-directed mutagenesis whereby G and C residues of the potential Spo0A binding 0A boxes were substituted for with A residues. The mutagenic oligonucleotide primers used are listed in Table S2. Synthetic DNA fragments with R1 and R2 mutations (Fig. 4A) were delivered cloned into pUC57 by Genewiz, which were later used to create reporter fusions and for the biotin pulldown assays.
Biotin pulldown assays. Biotin pulldown assays were carried out as described elsewhere (57). Briefly, the PsinR DNA fragment was biotin labeled and was coupled to immobilized monomeric avidin resin (G Biosciences) in B/W buffer (57). PgluD (upstream of gluD coding for glutamate dehydrogenase) and bead-alone negative controls were treated alongside test samples. The DNA and the beads were incubated at room temperature for 30 min in a rotor. The bead-DNA complex was washed with TE buffer to remove any unbound DNA. To prepare cell lysates, C. difficile R20291 strain was grown to the late exponential phase (16 h) in 500 ml TY medium at pH 7.4. After washing with 1ϫ PBS, the cells were resuspended in BS/THES buffer (57) and lysed using a French press. The whole lysate was centrifuged at 20,000 ϫ g for 30 min at 4°C. The supernatant, along with salmon sperm DNA as a nonspecific competitor, was incubated with the bead-DNA complex and allowed to rotate at 4°C overnight. The bead-DNA-protein complex was washed with BS/THES buffer (5 times). Elution was carried out with 50, 100, and 200 mM NaCl in Tris-HCl at pH 7.4. The eluates were analyzed by SDS-PAGE and Western blotting using Spo0A-specific antibody.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. TEXT S1, DOCX file, 0.02 MB.