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Paragraph: C. difficile 630Δerm Spo0A Regulates Sporulation, but Does Not Contribute to Toxin Production, by Direct High-Affinity Binding to Target DNA

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485338/

SHA: f0fb3bbd96dad4c907c7fd456cd5783ed8fa7bd6

Authors: Rosenbusch, Katharina E.; Bakker, Dennis; Kuijper, Ed J.; Smits, Wiep Klaas
Date: 2012-10-31
DOI: 10.1371/journal.pone.0048608
License: cc-by

Abstract: Clostridium difficile is a Gram positive, anaerobic bacterium  that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative) targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds. Spo0A is the key regulator for sporulation [1, 2] . Most of our knowledge about the protein is based on work in Bacilli. Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA [3] [4] [5] . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay [6] . The signaling cascade allows for the integration of environmental signals into the regulation of Spo0A dependent processes, including sporulation. The two functional domains, the N-terminal phosphorylation and dimerization domain (receiver domain), and the C-terminal DNA binding (effector) domain are separated by a hinge region that is relatively poorly conserved [7] . Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization [8, 9] , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation [10] . Extensive characterization of Spo0A targets has revealed a motif that represents a high affinity Spo0A binding site, the 0A box [10, 11] . The crystal structure of the DNA binding domain confirms specific and non-specific contacts between the protein and the consensus sequence [12, 13] . It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis [14] [15] [16] , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis [17] [18] [19] [20] [21] , and solvent production in Clostridium acetobutylicum [22, 23] . C. difficile is a   that is the causative agent of C. difficile infection (CDI) (for recent reviews see [24, 25] ). Though many people are asymptomatically colonized by C. difficile, the bacterium can cause serious health problems, such as pseudomembranous colitis and toxic megacolon, under the influence of risk factors such as age and antibiotic use. As a result, CDI was long regarded a nosocomial infection. Recently, however, an increase in the cases of community acquired CDI can be observed [26] . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 (BI/ NAP1) and 078 [27, 28] . Its main virulence factors are the major clostridial toxins A and B [29, 30] . In addition, certain strains of C. difficile, including ribotypes 027 and 078, additionally encode a binary toxin [31, 32] . C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow [24, 25] . There is limited knowledge about the regulation of sporulation in C. difficile. It has been reported that spo0A, as expected, is required for the formation of spores [33] and the gene is required for persistence and transmission in mice [34] . Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A [2] . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A [35] . Similarly, it was reported that multiple orphan histidine kinases can phosphorylate Spo0A in C. acetobutylicum [36] . Recently, it was reported that spo0A can be transcribed from a SigH-dependent promoter [37] . It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes. Escherichia coli strains were routinely grown in Luria-Bertani broth or plates, supplemented with appropriate antibiotics. Chloramphenicol was used at a final concentration of 20 mg/mL for agar plates and 10 mg/mL for liquid cultures. Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL. Cloning was carried out using E. coli DH5a, overexpression was performed in E. coli Rosetta(DE3) pLysS (Novagen). C. difficile strains were grown in a glucose-free trypton-yeast based medium (TTY; 3% w/v bactotrypton (BD), 2% yeast extract (Fluka), 0.1% w/v thioglycollate (Sigma) pH 7.4), supplemented with 20 mg/mL of lincomycin when appropriate, or on CLO or TSS plates (Biomerieux). All plasmids are listed in Table 1 . Primers (obtained from Sigma Aldrich) are listed in Text S1 and specific cycling conditions are available on request. Unless noted otherwise, PCR reactions were carried out using Pfu polymerase (Fermentas) according to the instructions of the manufacturer. Plasmid pWKS1251, for the overproduction of Spo0A-DBD carrying a C-terminal 66His-tag, was constructed as follows. A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO (Invitrogen), yielding pWKS1247. This plasmid was digested with NdeI and XhoI, separated on a 1% agarose/0.56 TAE (20 mM Tris Acetate, 0.5 mM EDTA) gel, the fragment corresponding to the DNA binding domain was recovered by gel-isolation (using a GeneJET Gel Extraction kit, Fermentas) and cloned into similarly digested pMF14 [10] that had been gel-isolated in the same manner. The construct was verified by PCR, restriction analyses and DNA sequencing using primers oWKS-135 and oWKS-136 (see below). Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO. The inserts, and in the case of the mutated PabrB promoters the presence of the desired point mutations in the consensus 0A box, were verified by DNA sequencing using primers oWKS-24 and oWKS-25 (see below). Sequence grade plasmids were isolated using a Nucleospin Plasmid QuickPure kit (Macherey Nagel) according to the manufacturer's instructions, except that two lysis reactions were combined onto a single filter and eluted with 65uC prewarmed AE buffer. All constructs were sequenced using BigDye Terminator chemistry (Invitrogen) on an ABI3130 sequencer (Perkin Elmer), according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix (Invitrogen) in a final volume of 20 mL. After thermocycling, DNA was precipitated and washed with 65% isopropanol, and dissolved in 12 mL HiDi formamid (Invitrogen) at 96uC for 2 mins and stored in the dark at 4uC until the sequencing run. Sequence analyses were performed in CloneManager Professional Suite 7 (SciEd) and Geneious version 5.6.2 (Biomatters Ltd). Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta(DE3) pLysS (Novagen). Transformants were used to inoculate 25 mL of LB with appropriate antibiotics. After overnight incubation, the cells were 1:100 diluted in 500 mL fresh medium containing appropriate antibiotics. Protein production was induced with 1 mM IPTG at an OD600 of 0.7 and growth was continued for another three hours before harvesting. Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described [10] . In short, cells were disrupted in 4 mL lysis buffer (2 mM PMSF, 10 mM imidazole, 5 mM beta-mercaptoethanol, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9). Cleared cell lysates we incubated with 2 mL pre-equilibrated 50% TALON slurry (Clontech) in a final volume of 15 mL lysis buffer for 1 hr. The resin was allowed to settle on a Poly-Prep column (BioRad) and washed with 2 mL wash buffer (20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9). The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column (identical to wash buffer but with 50, 100, 250 or 500 mM imidazole). The whole procedure was carried out at 4uC. Fractions were assayed for purity and yield and suitable fractions were dialysed against 26 1L dialysis buffer (50 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM DTT) using Slide-A-Lyzer cassettes with a molecular weight cut-off of 3.5 kDa (Pierce). Proteins were stored at 280uC in storage buffer (identical to dialysis buffer but containing 20% glycerol). Protein concentrations were determined using Bradford reagent (BioRad), according to the manufacturer's instructions. DNA fragments for use in EMSA experiment were generated by PCR using GoTaq polymerase (Promega) and chromosomal DNA from B. subtilis JH642 (Bacillus Genetic Stock Center 1A96; http://www.bgsc.org), plasmids listed in Table 1 , or chromosomal DNA from C. difficile 630Derm [38] as a template. Primers and specific cycling conditions for generation of the EMSA probes are listed in Text S1. DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS) and a QIAExII kit (Qiagen), according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase (Invitrogen) according to the instructions of the manufacturer. Specific activity was determined on a LS6000 scintillation counter (Beckman). EMSA conditions were based on previous studies [10] . In short, binding reactions were carried out in binding buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol) in the presence of 200 mg/mL bovine serum albumin (NEB) and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer.

Question: What is Clodstridium difficile?
Gram positive, anaerobic bacterium