BEC, a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks

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Shinya Yonogi, Shigeaki Matsuda, Takao Kawai, Tomoko Yoda, Tetsuya Harada, Yuko Kumeda, Kazuyoshi Gotoh, Hirotaka Hiyoshi, Shota Nakamura, Toshio Kodama and Tetsuya Iida Infect. Immun. 2014, 82(6):2390. DOI: 10.1128/IAI.01759-14. Published Ahead of Print 24 March 2014.

BEC, a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks Shinya Yonogi,a Shigeaki Matsuda,b Takao Kawai,a Tomoko Yoda,a Tetsuya Harada,a Yuko Kumeda,a Kazuyoshi Gotoh,c Hirotaka Hiyoshi,b Shota Nakamura,c,d Toshio Kodama,e Tetsuya Iidab,c,e

Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.

C

lostridium perfringens, a spore-forming anaerobic rod, is a member of the normal intestinal flora in humans and animals and a component of soil and sewage microbiota (1–4). C. perfringens is the causative agent of various human diseases, including gas gangrene and food-borne gastroenteritis (5–10). The pathogenicity of C. perfringens is attributed to various toxins produced by the organism, including alpha, beta, epsilon, and iota toxins that classify C. perfringens isolates into five toxin types (A to E), theta toxin, NetB, and C. perfringens enterotoxin (CPE) (10–13). CPE, which is mainly produced by type A C. perfringens, is associated with human gastrointestinal (GI) illnesses, such as food-borne gastroenteritis, antibiotic-associated diarrhea, and sporadic diarrhea (14–16). CPE is a 35-kDa protein, and the cpe gene is located in the chromosome or on a plasmid (17–20). After orally ingested CPE-positive C. perfringens reaches the GI tract, sporulating C. perfringens produces CPE, and the toxin causes clinical symptoms, such as diarrhea and abdominal cramping. In the clinical diagnosis of gastroenteritis due to C. perfringens, it is important to detect CPE in patient fecal specimens and examine CPE production of C. perfringens isolates by reversed passive latex agglutination (RPLA) or cpe-specific PCR. Recently, we experienced two outbreaks of food-borne acute gastroenteritis in which C. perfringens was strongly suspected based on the epidemiological information and fingerprinting of the isolates. However, the C. perfringens isolates of these outbreaks were negative for the cpe gene by PCR and for CPE by RPLA. This prompted us to explore the possibility that the isolates produce a novel enterotoxin distinct from CPE.

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In this study, we demonstrated enterotoxic activity in culture supernatants of C. perfringens isolates from the outbreaks and identified a novel binary enterotoxin, BEC, which is composed of BECa and BECb. The becAB genes were located on a 54.5-kb pCP13-like plasmid. The recombinant BECb (rBECb) alone showed fluid-accumulating activity in the suckling mouse assay. Although the recombinant BECa (rBECa) alone did not show activity, the enterotoxicity of rBECb was enhanced by the addition of rBECa. We constructed a becB mutant by intron insertion from one of the C. perfringens isolates from the outbreaks. The fluid-accumulating activity in the culture supernatants of the mutant was dramatically decreased compared to that of the parental strain, indicating the involvement of BEC in enterotoxicity. These results suggest that BEC is a novel enterotoxin produced by C. perfringens and that BEC-producing C. perfringens can be a causative agent of acute gastroenteritis in humans.

Received 13 March 2014 Returned for modification 15 March 2014 Accepted 19 March 2014 Published ahead of print 24 March 2014 Editor: S. R. Blanke Address correspondence to Tetsuya Iida, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.01759-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.01759-14

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Division of Bacteriology, Department of Infectious Disease, Osaka Prefectural Institute of Public Health, Osaka, Osaka, Japana; Laboratory of Genomic Research on Pathogenic Bacteriab and Pathogenic Microbes Repository Unit,e International Research Center for Infectious Diseases, and Department of Infection Metagenomicsc and Department of Genome Informatics,d Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan

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MATERIALS AND METHODS

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The desalted solution was applied to DEAE Sepharose FF (GE Healthcare) anion-exchange chromatography in a linear gradient of 20 mM TrisHCl (pH 8.0) plus 60 to 140 mM NaCl. The DEAE fractions in which enterotoxic activity was detected were applied to SP Sepharose HP (GE Healthcare) cation-exchange chromatography in a linear gradient of sodium acetate buffer (pH 4.8) plus 100 to 200 mM NaCl. The SP fractions with enterotoxic activity were subjected to a CHT-II hydroxyapatite chromatography column (Bio-Rad) equilibrated with 1 mM phosphate buffer (pH 7.2) and run through a linear gradient of 100 to 200 mM phosphate buffer. At each purification step, each fraction was tested for fluid accumulation activity with the suckling mouse assay and subjected to SDS-PAGE. A SuperSep Ace 5 to 20% gel (Wako) was used for SDS-PAGE. The concentration of the protein was measured by the Bradford method with a Coomassie protein assay kit (Thermo Scientific). The final purified protein was applied to SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The sequence of the Nterminal amino acid residues then was determined with a protein sequencer (Procise 492cLC; Life Technologies). Sequencing of genomic DNA from OS1 and TS1 strains and bioinformatic analysis. The genomic DNA of C. perfringens TS1 and OS1 was extracted with a DNeasy blood and tissue kit (Qiagen). Each genomic DNA sample was subjected to high-throughput DNA sequencing with the 454 GS Junior platform (Roche). The obtained sequences were assembled using a Newbler program (Roche). The amino acid sequence determined by protein N-terminal sequencing was searched in the obtained contigs with a tblastn program. The becAB sequences obtained by 454 sequencing were proofread using Sanger sequencing with an ABI Prism 3100 (Life Technologies). Toxins similar to the determined toxin sequences were searched using a blastp program. A multiple-sequence alignment was executed using ClustalW (28). The phylogenetic tree of BECb and related toxins was constructed with ClustalW using the neighbor-joining method. The tree was visualized using GENETYX-Tree (GENETYX). The signal peptide prediction was performed using the SignalP4.1 server (http: //www.cbs.dtu.dk/services/SignalP/). Expression and purification of recombinant BECa and BECb. The becAB genes of C. perfringens OS1 were PCR amplified using two sets of primers. The primer pairs for becA (rbecA F and rbecA R; see Table S2 in the supplemental material) and becB (rbecB F and rbecB R; see Table S2) were designed from the sequences obtained by high-throughput sequencing of the genomic DNA. PCR was performed in a 25-␮l reaction mixture containing 1⫻ PrimeSTAR GXL buffer (TaKaRa), 0.2 mM deoxynucleoside triphosphate (dNTP) mixture, 0.6 U of PrimeSTAR GXL DNA polymerase (TaKaRa), 0.4 ␮M primers, and 1.5 ␮l of template solution. The following PCR conditions were used: denaturation at 94°C for 1 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 45°C to becA or 50°C to becB for 15 s, extension at 68°C for 3 min, and a final extension step at 68°C for 10 min. The PCR products were analyzed by electrophoresis on 1.5% agarose gels. The 1.2-kb and 2.4-kb fragments were purified by agarose gel electrophoresis. After the addition of 3= A overhangs, the PCR products were ligated with pCR2.1-TOPO TA vector (Life Technologies). The ligated vector was transformed into One Shot TOP10 chemically competent E. coli (Life Technologies). The transformants were inoculated onto LB plates containing 50 ␮g/ml kanamycin (Km) and cultured at 37°C. Km-resistant colonies were picked and cultured in Terrific broth for plasmid purification. Each plasmid DNA was purified with a PureLink HiPure plasmid DNA purification miniprep kit (Invitrogen). The purified plasmids were digested with restriction enzymes SmaI and XhoI and ligated into a pGEX-4T-2 (GE Healthcare) vector. Both rBECa and rBECb of C. perfringens strain OS1 were expressed as glutathione S-transferase (GST) fusion proteins in E. coli BL21. Both recombinant proteins were expressed in LB broth containing 100 ␮g/ml ampicillin with shaking. rBECa was expressed at 25°C with 0.1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG), and rBECb was expressed at 30°C with 1 mM IPTG. The recombinant toxins were purified by GSTrap affinity chromatography

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Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are summarized in Table S1 in the supplemental material. C. perfringens strains OS1, TS1, Iz1, Ta1, NCTC8239, NCTC8798, and Y1– Y34 were isolated from human feces. C. perfringens strain NCTC8239 was used as a CPE-positive control in the rabbit ileal loop assay. All of the C. perfringens strains were cultured in modified Duncan-Strong medium at 37°C (21). The heat-stable enterotoxin-producing and the heat-labile enterotoxin-nonproducing (ST⫹, LT⫺) Escherichia coli strain 18H278 (O128:H12) was used as a positive control in the suckling mouse assay (22). Rabbit ileal loop assay. The bacteria were grown in modified DuncanStrong medium (21) for 24 h at 37°C. The bacterial cultures were centrifuged at 5,000 ⫻ g for 10 min. The supernatants were passed through a 0.22-␮m membrane filter. The supernatants were then concentrated by membrane ultrafiltration with a molecular weight cutoff of 10,000 (Vivaspin; GE Healthcare) and dialyzed with phosphate-buffered saline (PBS). The protein concentrations of each supernatant sample were determined with a Coomassie protein assay kit (Thermo Scientific). The supernatant samples were then diluted with PBS to a final concentration of 1.0 mg/ml. Rabbit ileal loop tests were performed as previously described (23–25). Briefly, 1 ml of diluted supernatant samples was injected into ligated ileal loops of rabbits, and fluid accumulation in each loop was measured 15 h after inoculation. The FA ratio is the amount of accumulated fluid (ml) per length (cm) of ligated rabbit small intestine. Suckling mouse assay. The suckling mouse assay was conducted to examine the fluid-accumulating activity of the culture supernatant, the fraction at each purification step, and the recombinant toxins. The bacteria were grown in modified Duncan-Strong medium (21) for 24 h at 37°C. The culture supernatants were concentrated by membrane ultrafiltration with a molecular weight cutoff of 10,000 (Vivaspin, GE Healthcare) and dialyzed with PBS. The samples were then diluted with PBS to a final concentration of 0.5 mg/ml. The protein concentrations of each sample were determined with a Coomassie protein assay kit (Thermo Scientific). Each mouse was intragastrically inoculated with 100 ␮l of sample. Four hours after inoculation at 26°C, the fluid accumulation in the intestine was observed. The ratio of the intestinal weight to the remaining body weight (FA ratio) was measured as previously described (22). PFGE. Pulsed-field gel electrophoresis (PFGE) with SmaI-digested DNA was performed by a previously reported method (26), with some modifications. Briefly, isolates of C. perfringens were grown anaerobically in BHI broth at 37°C. PFGE was performed on a 1.0% SeaKem gold agarose gel (Cambrex Bio Science). The cells embedded in the agarose gel were treated with lysis buffer (6 mM Tris, 1 M NaCl, 100 mM EDTA, 0.5% Brij58, 0.2% sodium deoxycholate, 0.5% N-lauroyl sarcosine) containing RNase (20 ␮g/ml), lysozyme (1 mg/ml), and mutanolysin (20 U/ml) at 37°C overnight. The plugs were restricted with 30 U of SmaI (Roche) at 25°C overnight. Electrophoresis was performed on a CHEF-DRIII apparatus (Bio-Rad Laboratories) in 0.5⫻ Tris-borate-EDTA buffer at 14°C and 6 V/cm. A linearly ramped switching time of 0.5 to 40 s was applied for 20 h. The gels were stained with ethidium bromide, destained, and photographed under UV light. The PFGE patterns were analyzed by following the established criteria for bacterial strain typing by PFGE methodology (27). Purification of BECb and N-terminal amino acid sequencing. C. perfringens strain TS1 was cultured in modified Duncan-Strong medium for 16 h at 37°C under anaerobic conditions. The cultured medium was centrifuged at 15,000 ⫻ g for 30 min at 4°C. After centrifugation, the supernatant was filtered through a 0.22-␮m membrane. The filtrate was precipitated by ammonium sulfate saturation up to 70%. The saturated solution was then centrifuged at 14,000 ⫻ g for 45 min at 4°C, and the pellet was collected and resuspended with 20 mM Tris-HCl buffer (pH 8.0). The suspension was desalted by a PD-10 column (GE Healthcare) against 20 mM Tris-HCl buffer (pH 8.0) not containing ammonium sulfate.

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polymerase (TaKaRa), 0.8 ␮M primers, and 1 ␮l of template solution. The following PCR conditions were used: denaturation at 94°C for 1 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, extension at 72°C for 90 s, and a final extension step at 72°C for 10 min. If the intron was not inserted, an 821-bp fragment was observed. If the intron was successfully inserted, a 1.7-kb fragment was detected. Four primers (becB 1285a-IBS, becB 1285a-EBS1d, bec_S3, and becB F; see Table S2) were used for sequencing. The constructed mutant was cured of pJIR750becB and applied to the suckling mouse assay. Detection of becAB genes in C. perfringens isolates from human feces. The prevalence of the becAB genes in C. perfringens from humans was investigated by PCR. Single colonies of each strain were picked and suspended in distilled water. The suspension was boiled for 10 min and then centrifuged at 15,000 ⫻ g for 5 min. The supernatants were used as the template for PCR. Two primer pairs for the becA gene (becA F and becA R; see Table S2 in the supplemental material) and for the becB gene (becB F and becB R; see Table S2) were used. PCR was performed in a 25-␮l reaction mixture containing 1⫻ Ex Taq buffer (TaKaRa), 0.2 mM dNTP mixture, 0.5 U of Ex Taq DNA polymerase (TaKaRa), 0.8 ␮M primers, and 1 ␮l of template solution. The following PCR conditions were used: denaturation at 94°C for 1 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and a final extension step at 72°C for 10 min. The PCR products were analyzed by electrophoresis on 1.5% agarose gels. Possession of each gene was confirmed by the presence of PCR products (499 bp for becA and 416 bp for becB). Sequence determination of the genetic region around becAB. The genetic regions around the becAB genes were determined by assembling the 454 contigs with gap closure using PCR amplification and Sanger sequencing. The genomic DNA of OS1 and TS1 was further subjected to sequencing with a MiSeq platform (Illumina). Sequences of 235,805,331 bp and 189,325,261 bp were obtained for TS1 and OS1, respectively. MiSeq reads were mapped to the above-described draft sequence using CLC Genomics Workbench (CLC Bio) to correct sequence errors. Open reading frames were predicted and annotated using Glimmer3, GenomeGambler 1.5, and the NCBI BLAST network service. Ethics statement. Animal studies were conducted in strict accordance with the guidelines for the Care and Use of Laboratory Animals at the Research Institute for Microbial Diseases, Osaka University, and the guidelines for animal care and use of the Osaka Prefectural Institute of Public Health, which are based on the Fundamental Guidelines for the Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan, 2006. All rabbit experiments were performed by following an experimental protocol approved by the Animal Care and Use Committee of the Research Institute for Microbial Diseases, Osaka University (permit numbers Biken-APH21-04-0 and Biken-AP-H25-01-0). All mouse experiments were approved by the Animal Care and Use Committee of the Osaka Prefectural Institute of Public Health (permit numbers H24-2 and H25-6). All C. perfringens isolates from human feces were collected in routine microbiological investigations based on the Japanese Food Sanitation Act at the Tochigi Prefectural Institute of Public Health and the Osaka Prefectural Institute of Public Health, Japan, except NCTC8239 and NCTC8798, which were in the culture collection of the Osaka Prefectural Institute for Public Health. All samples were deidentified. According to the Ethical Guidelines for Epidemiological Studies of the Ministry of Health, Labor and Welfare, Japan, an informed consent from patients involved is not required for isolation of bacterial strains from human fecal specimens for diagnostic purposes and use of the strains in research. Statistical analysis. The statistical analysis was performed using a oneway analysis of variance (ANOVA), followed by Dunnett’s multiple-comparison test. Nucleotide sequence accession numbers. The nucleotide sequences determined in the course of this work have been deposited at DDBJ/

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(GE Healthcare). The purified recombinant toxins were diluted to 200 ␮g/ml and then incubated with thrombin (1.6 NIH U/ml) to cleave the GST domain for 6 h. To remove the GST tag, the reaction mixtures were reapplied to a GSTrap affinity chromatography column (GE Healthcare), and the flowthrough fractions were collected. The purified recombinant proteins were applied to suckling mouse assays. The concentration of the proteins was measured by the Bradford method with a Coomassie protein assay kit (Thermo Scientific). ADP ribosylation assay. The ADP ribosylation assay was performed as described previously (29, 30). Briefly, two ␮g of non-muscle actin (Cytoskeleton, Inc.) was incubated with 300 ng of each recombinant toxin component in the presence of 10 ␮M biotinylated NAD⫹ (Trevigen) or nonlabeled NAD⫹ in the reaction buffer (20 mM Tris-HCl at pH 7.5, 1 ␮M dithiothreitol, 40 ␮M ATP, 40 ␮M CaCl2, and 5 ␮M MgCl2) at 37°C for 60 min, followed by the addition of 3⫻ SDS sample buffer to terminate reactions. The reaction mixture was boiled at 95°C for 5 min and then subjected to SDS-PAGE and silver staining. For detection of biotinylated ADP-ribosylated actin, proteins of gels were transferred to PVDF membrane and detected with horseradish peroxidase-conjugated streptavidin (Thermo Scientific). Microscope analysis. Vero cells were seeded on collagen-coated coverslips at 2 ⫻ 104 cells/well in 24-well plates and grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Just prior to assay, the purified recombinant toxins were treated with trypsin from bovine pancreas (Sigma) at 37°C for 30 min, followed by the addition of trypsin inhibitor from Glycine max (Sigma) to terminate the reaction. Forty-eight h postseeding, the culture medium was replaced with DMEM containing 1% FBS, and then cells were exposed to toxins for 3 h. The cells were fixed with 4% paraformaldehyde for 30 min, washed with Dulbecco’s PBS, and incubated with 5 ␮g/ml Alexa 594conjugated wheat germ agglutinin (Invitrogen) to label plasma membranes. Cells then were permeabilized with 0.1% Triton X-100 and incubated with Alexa 488-conjugated phalloidin (Invitrogen) and Hoechst 33258 (Life Technologies). The coverslips were analyzed by fluorescence microscopy using a Biozero BZ-8000 (Keyence). For Giemsa staining, cells treated with toxins were fixed with methanol, followed by staining. Construction of the becB suicide plasmid. The TargeTron gene knockout system (Sigma) was used to disrupt the becB gene. The identified nucleotide sequence of the becB gene was submitted to the Sigma TargeTron design site, and primer information (IBS, EBS2, and EBS1/␦) to retarget the group II intron was obtained. The position 1284/1285 from the ATG start codon in becB was selected for TargeTron modification. PCR was carried out with BECb1285a-IBS, BECb1285a-EBS2, BECb1285a-EBS1/␦, and EBS universal primers (see Table S2 in the supplemental material) according to the instructions of the TargeTron gene knockout system (Sigma). After ethanol precipitation and resolution of the obtained 350-bp PCR products, the products and pJIR750ai (Sigma) were digested with HindIII and BsrGI. The digested PCR products and pJIR750ai were ligated with a DNA ligation kit, version 2.1 (TaKaRa). The ligation mixtures were transformed into E. coli XL1-Blue cells. The transformants were inoculated on LB plates containing 10 ␮g/ml chloramphenicol (Cm) and cultured at 37°C. Cm-resistant colonies were picked and cultured in LB broth with 10 ␮g/ml Cm for plasmid purification. DNA from the plasmid (named pJIR750becB) was purified with a PureLink HiPure plasmid DNA purification Midiprep kit (Invitrogen). Construction of the becB mutant. The purified suicide plasmid was electroporated into C. perfringens strain OS1. To induce gene disruption, the transformants were cultured in TGY broth with 1 mM IPTG at 37°C for 4 h. The culture medium was spread onto GAM/2 plates containing 10 ␮g/ml Cm and cultured overnight at 37°C under anaerobic conditions. The resultant mutants were isolated and screened for gene disruption by PCR and sequencing. The primer pair bec_S3 and becB F (see Table S2 in the supplemental material) was used for PCR. PCR was performed in a 25-␮l reaction mixture containing 1⫻ Ex Taq buffer (TaKaRa), 0.2 mM deoxynucleoside triphosphate (dNTP) mixture, 0.5 U of Ex Taq DNA

Novel Enterotoxin of C. perfringens

EMBL/GenBank under accession numbers AP013033 (pCP-OS1) and AP013034 (pCP-TS1).

RESULTS

Non-CPE-producing C. perfringens from outbreaks of foodborne gastroenteritis. Recently, two distinct outbreaks of foodborne gastroenteritis occurred in Japan. Case 1. On 15 August 2009, 420 individuals had a buffet-style lunch at a hotel in Osaka, Japan. Among these individuals, 84 presented with GI symptoms, primarily diarrhea and abdominal pain. Among the seven patients examined, C. perfringens was isolated from the feces of five patients. Case 2. In January 2010, 171 individuals had a buffet-style dinner at a hotel in Tochigi, Japan. Seventy-nine individuals presented with diarrhea and abdominal pains. Among the 37 patients examined, C. perfringens was isolated from the feces of 27 patients and a food sample. The C. perfringens isolates from the two outbreaks were type A, i.e., positive for the alpha toxin gene and negative for the beta, beta 2, iota, and epsilon toxin genes by PCR (31, 32), but they were negative for CPE by RPLA and negative for the cpe gene by PCR; thus, they were non-CPE producers. In both cases, other possible pathogens of acute gastroenteritis were not detected. The isolates were subjected to fingerprinting by pulsed-field gel electrophoresis (PFGE) analysis. The PFGE patterns of the five isolates from Osaka were indistinguishable from each other (Fig. 1A). The PFGE patterns of the six isolates from the five fecal samples and the food sample (roast beef) in the Tochigi case were also indistinguishable from each other (Fig. 1B). Therefore, C. perfringens isolates were strongly suspected to be the cause of each outbreak. C. perfringens isolates from the two outbreaks, however, showed clearly distinct PFGE patterns (Fig. 1B), indicating that the genetic backgrounds of the C. perfringens isolates from the two outbreaks were not the same.

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FIG 1 PFGE patterns of SmaI-digested genomic DNA of C. perfringens strains. (A) PFGE patterns of SmaI-digested genomic DNA from C. perfringens strains isolated from the Osaka outbreak. Lanes: M, Salmonella enterica serovar Braenderup H9812 (a size marker); 1 to 5, C. perfringens isolates from diarrheal patient feces. (B) PFGE patterns of SmaI-digested genomic DNA from C. perfringens strains isolated from the Tochigi outbreak. Lanes: M, S. Braenderup H9812 (a size marker); 1 to 5, C. perfringens isolates from diarrheal patient feces in the Tochigi outbreak; 6, cpe-positive C. perfringens isolate from healthy food handler feces from the Tochigi outbreak; 7, C. perfringens isolate from a roast beef sample from the Tochigi outbreak; 8, C. perfringens isolate from the Osaka outbreak.

Fluid-accumulating activity in supernatants of C. perfringens strains TS1 and OS1 in rabbit ileal loop and suckling mouse assays. To examine the enterotoxic activity of the C. perfringens isolates, we selected one isolate from each outbreak and designated the Tochigi outbreak as TS1 and the Osaka outbreak as OS1. The fluid-accumulating activity of TS1 and OS1 in the cultured supernatants was assessed by rabbit ileal loop and suckling mouse assays. In the rabbit ileal loop assay, 1 ml of concentrated C. perfringens culture supernatant (adjusted to 1.0 mg/ml of protein) was inoculated. The supernatants of the TS1 and OS1 strains and CPEproducing C. perfringens strain NCTC8239 demonstrated significant fluid accumulation in the rabbit small intestine, while the non-CPE-producing negative-control strains Ta1 and Iz1 did not (Fig. 2A and B). In the suckling mouse assay, concentrated culture supernatants (adjusted to 0.5 mg/ml; 0.1 ml for each) of C. perfringens strains TS1 and OS1 showed significant fluid accumulation. In contrast, concentrated culture supernatants of Ta1, Iz1, and the CPE-producing NCTC8239 strain did not show significant fluid accumulation (Fig. 2C). These results indicate that both TS1 and OS1 C. perfringens strains produce an enterotoxic substance that is distinct from CPE. Purification of the enterotoxin produced by C. perfringens strain TS1. Because both the rabbit ileal loop and suckling mouse assays demonstrated enterotoxic activity in the culture supernatants of TS1 and OS1, we attempted to purify the enterotoxic substance. The culture supernatants of TS1 were subjected to a series of purification steps, including ammonium sulfate precipitation, DEAE Sepharose, SP Sepharose, and hydroxyapatite column chromatography, using the suckling mouse assay for the detection of enterotoxicity. This resulted in the purification of an enterotoxic protein showing a single band on SDS-PAGE with a molecular mass of approximately 80 kDa (Fig. 3A). The band was cut out and subjected to N-terminal amino acid sequence analysis. The N-terminal amino acid sequence was determined to be MINNTFFMGYYF. Identification of novel enterotoxin genes. To identify the gene encoding the novel enterotoxin, we carried out highthroughput DNA sequencing of the genomic DNA from C. perfringens strain TS1 using the 454 GS Junior platform (Roche). A single run produced 105,983 reads and yielded a total of 39,004,133 bases. The obtained sequences were assembled using a Newbler program, resulting in 498 contigs, and the total number of contig bases was 3,356,939 bp. We searched the 12 residues of the amino acid sequence described above in the contigs with the tblastn program and found a perfectly matched sequence in an open reading frame (ORF). The region containing the ORF was proofread and confirmed by Sanger sequencing. The ORF encoded a protein composed of 799 amino acid residues. The 12 amino acid residues revealed by the N-terminal amino acid sequencing were located at the N terminus of the deduced protein. The sequence of deduced protein was predicted not to possess a signal peptide sequence by the SignalP4.1 server, which is consistent with the result of N-terminal sequencing. The theoretical pI of the ORF was predicted to be 5.03 by ProtParam (33). The protein encoded by this ORF had significant similarity to binding components of ADP-ribosylating binary toxins, such as C. perfringens iota toxin Ib (YP_004670323.1) (43% amino acid identity), C. spiroforme Sb component (CAA66612.1) (40% identity), C. difficile CdtB (AEC11585.1) (43% identity), C. botulinum

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adjusted to a protein concentration of 1.0 mg/ml, and PBS, a negative control, were inoculated into ligated ileal loops of rabbits. After 15 h, fluid accumulation in each loop was measured. Shown are the macroscopic appearance of rabbit ileal loops (A) and the enterotoxicity of each sample (B). The fluid accumulation (FA) ratio was the amount of accumulated fluid (ml) per length (cm) of ligated rabbit small intestine. The strains tested were the following: OS1, isolate from the Osaka outbreak; TS1, isolate from the Tochigi outbreak; Ta1 and Iz1, CPE-negative C. perfringens isolates from human feces; NCTC8239, CPE-positive C. perfringens NCTC8239. The values are averages from six assays for each sample, and the error bars represent the standard deviations. Dunnett’s multiplecomparison test of C. perfringens strains OS1, TS1, and NCTC8239 against PBS showed a significant difference (*, P ⬍ 0.01). (C) Suckling mouse assay. Concentrated culture supernatants (0.1 ml; adjusted to 0.5 mg/ml) of each strain and PBS were intragastrically inoculated into suckling mice. The culture supernatant of the heat-stable enterotoxin (ST⫹, LT⫺)-producing E. coli. Strain 18H278 (O128:H12) was used without concentration. After 4 h, fluid accumulation in the intestines was measured. The FA ratio is gut weight (mg) per remaining body weight (mg). The remaining body weight (mg) was calculated as the total body weight minus the weight of the intestines (mg). The strains tested were OS1, TS1, Ta1, Iz1, NCTC8239 (CPE positive), and the heat-stable enterotoxin (ST⫹, LT⫺)-producing E. coli 18H278 (O128:H12) strain. The values are averages from quintuple assays for each sample, and the error bars represent the standard deviations. Dunnett’s multiple-comparison test of C. perfringens strains OS1 and TS1 and E. coli strain 18H278 against PBS showed a significant difference (*, P ⬍ 0.01).

C2 toxin II (BAH29486.1) (41% identity), Brevibacillus laterosporus Isp1a protein (CAI40767.1) (34% identity), and Bacillus thuringiensis Vip1 (AEI87572.1) (36% identity) (see Table S3B and Fig. S1 in the supplemental material). Therefore, we designated this protein BECb (binary enterotoxin of Clostridium perfringens component b). BECb had limited similarity to other ADP-ribosylating binary toxins. For example, although BECb showed the highest similarity to the C. perfringens iota toxin Ib, it shared only

FIG 3 SDS-PAGE of purified native BECb, rBECa, and rBECb. Purified native BECb, rBECa, and rBECb were separated by 5 to 20% SDS-PAGE. Native BECb was purified with DEAE Sepharose, SP Sepharose, and hydroxyapatite chromatography CHT-II columns. rBECa and rBECb were purified with affinity chromatography GSTrap columns. After cleaving the GST domain with thrombin, purified rBECa and rBECb were reapplied to a GSTrap column to remove the GST tag. Prestained protein marker, broad range (7 to 175 kDa; New England BioLabs) was used as a size marker. The SDS-PAGE gels were stained with Bio-safe Coomassie (Bio-Rad). (A) Native BECb; (B) rBECa; (C) rBECb.

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43% identity at the amino acid level. A phylogenetic tree including BECb and related toxins was constructed (Fig. 4). BECb made an independent branch, indicating that BECb was not simply a variant of other ADP-ribosylating binary toxins but represented a novel toxin. An ORF corresponding to the enzymatic component of ADPribosylating binary toxins was identified upstream of the BECb gene (becB). The protein encoded by the ORF (becA) was designated BECa (binary enterotoxin of Clostridium perfringens component a). BECa was composed of 419 amino acid residues with a molecular mass of 47 kDa, and the predicted pI was 5.46. We also searched the sequence of BECa for the presence of the signal peptide using the SignalP4.0 server, and the results suggested that BECa also did not possess the signal sequence. The protein had similarities to C. perfringens iota toxin Ia (YP_004670324.1) (44% identity), C. difficile CdtA (AEC11575.1) (43% identity), C. spiroforme Sa (CAA66611.1) (42% identity), B. laterosporus Isp2b (CAI43279.1) (34% identity), B. thuringiensis Vip2 (AER12133.1) (28% identity), and C. botulinum C2 I (YP_004385832.1) (29% identity) (see Table S3A in the supplemental material). BECa possessed the three highly conserved regions of ADP-ribosylating family toxins, which play an important role in enzymatic activity (see Fig. S2) (34). We carried out similar high-throughput DNA sequencing of the genomic DNA from the C. perfringens OS1 strain. A single sequencing run with the 454 GS Junior platform produced 104,218 reads and yielded a total of 37,577,144 bases. Newbler assembly of the sequences resulted in 253 contigs, and the total number of bases of contigs was 3,278,000 bp. In one of the contigs, we identified both the becA and becB genes. The sequences were proofread by Sanger sequencing, revealing that they were completely identical to those of becA and becB in the TS1 strain. These results demonstrate that the TS1 and OS1 strains share identical

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FIG 2 Rabbit ileal loop and suckling mouse assay of culture supernatants. (A and B) Rabbit ileal loop assay. One milliliter of culture supernatant from each strain,

Novel Enterotoxin of C. perfringens

genes for the novel binary toxin, despite having distinct genetic backgrounds (Fig. 1). rBEC functions as a binary actin ADP-ribosylating toxin in vitro. To examine the biological activities of BECa and BECb, the recombinant proteins encoded in the identified ORFs (becA and becB) were expressed in Escherichia coli with the pGEX vector system, purified by a GSTrap affinity chromatography column (GE Healthcare), and treated with thrombin to remove the GST tag (Fig. 3B and C). Because BEC belongs to the binary toxin family and the highly conserved regions in the enzymatic components of ADP-ribosylating toxins were also conserved in the sequence of BECa, it is possible that BECa can also catalyze ADP ribosylation of actin. To examine whether BEC shows an ADP-ribosylating activity, purified actin was incubated with rBECa or rBECb in the presence of the biotinylated NAD⫹. As expected, the incorporation of biotinylated ADP into purified actin was observed only when BECa was challenged (see Fig. S3 in the supplemental material), indicating that BECa ADP ribosylates actin in the presence of NAD⫹. We also examined the effects of BECa and BECb on cell morphology. Vero cells were incubated with rBECa and/or rBECb, followed by actin staining or Giemsa staining. In the cells treated with rBECa and rBECb simultaneously, cell rounding occurred at 3 h, while neither rBECa nor rBECb alone affected cell morphology (see Fig. S4 in the supplemental material). Although we have not directly evaluated whether or not BECb delivers BECa into cells yet, these results indicate that BEC performs as an actin

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ADP-ribosyltransferase at least in vitro, which is similar to the behavior of typical binary toxins. rBECb alone shows fluid-accumulating activity in the suckling mouse assay. We next examined the enterotoxic activity of BECa and BECb using the recombinant proteins in a suckling mouse assay (Fig. 5). When 1 ␮g or more of rBECb was inoculated, the FA ratio significantly increased compared to that of the PBS negative control. The increase was dose dependent (Fig. 5A). In contrast, rBECa alone did not show apparent fluid-accumulating activity in the assay (Fig. 5A). These results suggest that BECb alone has fluid-accumulating activity in the suckling mouse assay, while BECa alone does not. Enterotoxic activity of rBECb is enhanced by the addition of rBECa. In the above-described assay, rBECa alone did not show clear fluid-accumulating activity. However, because BECa and BECb had significant similarities to the previously reported binary toxins, we examined whether there was a synergetic effect between rBECa and rBECb in the suckling mouse assay (Fig. 5B). As previously demonstrated, rBECa did not show any enterotoxic activity in the absence of rBECb (Fig. 5B, white bars). However, 500 ng and 5 ␮g of rBECa significantly enhanced the enterotoxic activity of 500 ng of rBECb (Fig. 5B, black bars). Even 100 ng of rBECb, a level that did not exert enterotoxicity alone (Fig. 5A), showed fluid-accumulating activity in the suckling mouse assay upon simultaneous addition of 1 ␮g or larger amounts of rBECa

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FIG 4 Phylogenetic tree of BECb and the binding components of binary toxins. A phylogenetic tree was constructed using ClustalW with neighbor-joining bootstrapping (1,000 interactions). The tree was visualized using GENETYX-Tree. The toxins and proteins that used were the following: C. perfringens Ib (GenBank accession number ZP_02632016.1), B. thuringiensis Ib (ZP_04111675.1), B. cereus Ib (ZP_03115351.1), C. spiroforme Sb (CAA66612.1), C. botulinum C2II (ADD91306.1, BAA32537.1, BAH29486.1, YP_002650775.1, YP_003034266.1, and YP_004385833.1), C. difficile CdtB (AEC11576.1, AEC11570.1, AEC11585.1, AEC11561.1, AEC11582.1, AAF81761.1, AEC11564.1, and AEC11585.1), C. difficile ADP-ribosyltransferase binding component (AAB67305.1), B. laterosporus Isp1a (CAI40767.1), B. laterosporus Isp1b (CAI43278.1), B. cereus Vip (AEH05932.1), B. thuringiensis Vip1 and Vip1A (AEI87572.1 and ABR68093.1), B. cereus PA (YP_002752995.1 and YP_003786968.1), B. anthracis PA (AAT98414.1), and B. anthracis Pag (CAC93935.1).

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Individual effects of rBECa and rBECb. rBECa or rBECb was intragastrically inoculated into suckling mice. After 4 h, fluid accumulation in the intestine was measured. White bars indicate rBECa, and black bars indicate rBECb. (B) Synergetic effects between rBECa and rBECb. Mixtures of rBECa and rBECb were intragastrically inoculated into suckling mice. After 4 h, fluid accumulation in the intestine was measured. White bars indicate the absence of rBECb. Gray and black bars indicate 100- and 500-ng additions of rBECb, respectively. The FA ratio was calculated as gut weight (mg) per remaining body weight (mg). The remaining body weight (mg) was the total weight minus the weight of the intestines (mg). The values are averages of triplicate assays for each sample, and the error bars represent standard deviations. Significant differences (*, P ⬍ 0.01; **, P ⬍ 0.05) between each sample and the PBS control by Dunnett’s multiple-comparison test are indicated.

(Fig. 5B, gray bars). These results suggest that although BECa itself is not enterotoxic, BECa can enhance the enterotoxicity of BECb. The becB mutant loses fluid-accumulating activity in the suckling mouse assay. To evaluate the contribution of BECb to the virulence of the C. perfringens strains, an isogenic becB mutant of C. perfringens OS1 was constructed by an intron insertion in the becB gene using the TargeTron gene knockout system. The enterotoxic activity in the culture supernatants of the obtained mutant (OS1⌬becB) was compared to that of the parental OS1 strain (Fig. 6). The FA ratio of OS1⌬becB was 0.0478 ⫾ 0.0045, while that of strain OS1 was 0.0864 ⫾ 0.0079. Thus, the enterotoxicity of the becB mutant was significantly decreased compared to that of the parental strain (P ⬍ 0.01). The FA ratio of the becB mutant was indistinguishable from that of PBS (Fig. 6). These results indicate that BECb is essential for the enterotoxic activity in the supernatants of the C. perfringens OS1 strain, suggesting that BEC is responsible for the virulence of the strain. becAB are encoded on a large plasmid. To analyze the genetic region flanking the becAB genes in strain OS1, we assembled the contigs produced by 454 sequencing using gap closure with PCR amplification and Sanger sequencing. The assembling resulted in

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an approximately 54.5-kb circular contig containing the becAB genes. This suggested that the becAB genes of C. perfringens strain OS1 were located on a large circular plasmid. By adding Illumina sequencing, as described in Materials and Methods, we proofread the circular contig and finally obtained a complete sequence of the plasmid. The plasmid, designated pCP-OS1, was composed of a 54,535-bp nucleotide sequence. The G⫹C content of pCP-OS1 was 25.1%, which was lower than that of the previously sequenced chromosomes of C. perfringens strains (strain 13, 28.6%; ATCC 13124, 28.4%). We identified 55 putative ORFs that covered 84.6% of the plasmid with an average length of 839 bp. Although 39 ORFs were classified as hypothetical with unknown functions, 16 ORFs were function assigned. We found a virulence-associated gene, the collagen adhesin gene (cna), in addition to the becAB genes. Notably, 38 ORFs on pCP-OS1 had high similarities with genes on the plasmid pCP13 from C. perfringens strain 13 (35), including the plasmid partitioning-associated genes (soj and parB), resolvase gene (resP), type I topoisomerase gene (topA), and cna gene. A comparison of the pCPOS1 and pCP13 sequences showed high similarities (92 to 99% identity at the nucleotide level) across ⬃38 kb, which covers 69% of the plasmid pCP-OS1 (Fig. 7). Around the becAB genes, six unique ORFs were present between the PCP25 and PCP33 homologues. Two of these ORFs were function assigned: one encoded a transposon resolvase, and the other encoded a putative RNA polymerase sigma factor. We also analyzed the genomic regions around becAB of strain TS1. The becAB genes were also located on a large circular plasmid, termed pCP-TS1, in strain TS1 (Fig. 7). The size of pCP-TS1 was 54,478 kb, and its G⫹C content was 25.0%. We predicted 55 ORFs in pCP-TS1 that covered 83.8% of pCP-TS1 with an average

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FIG 5 Fluid-accumulating activity of rBECa and rBECb in suckling mice. (A)

FIG 6 Fluid-accumulating activity of C. perfringens OS1 and isogenic mutant OS1⌬becB in suckling mice. Concentrations (0.1 ml; adjusted to 0.5 mg/ml) of culture supernatants of C. perfringens, unconcentrated culture supernatants of ST-producing E. coli 18H278, and PBS were intragastrically inoculated into suckling mice. After 4 h, fluid accumulation in the intestine was measured. The FA ratio was calculated as gut weight (mg) per remaining body weight (mg). The remaining body weight (mg) was calculated by the total body weight minus the weight of the intestines (mg). The strains tested were OS1, OS1 isogenic mutant OS1⌬becB, and the heat-stable enterotoxin (ST⫹, LT-)-producing E. coli 18H278 (O128:H12) strain. The values are averages from quintuple assays for each sample with error bars representing the standard deviations. Significant differences (*, P ⬍ 0.01) between each sample and the PBS control by Dunnett’s multiple-comparison test are indicated.

Novel Enterotoxin of C. perfringens

perfringens strain 13 (GenBank accession number NC_003042) (35) are demonstrated. The red arrows indicate virulence-associated genes including adhesins. Plasmid partitioning-associated genes are shown as green arrows. DNA replication, DNA repair, and transcription-related genes are indicated by blue arrows. Other genes are represented by colored arrows: resolvase genes, orange; genes for cell wall-binding proteins, brown; ABC transporter genes, violet; type II restriction enzyme genes, yellow. Pink shading indicates the regions of similarity (⬎90% identity at the nucleotide level).

length of 830 bp. Interestingly, the overall structure of pCP-TS1 was almost identical to that of pCP-OS1 (99% identity at the nucleotide level). Taken together, our results indicate that the OS1 and TS1 strains harbor a closely related large plasmid carrying the becAB genes, despite their distinct genetic backgrounds (Fig. 1). DISCUSSION

In this study, we identified a novel binary toxin, BEC, composed of BECa and BECb, that was produced by C. perfringens isolates from outbreaks of human acute gastroenteritis. Purified rBECb alone had enterotoxic activity (Fig. 5A), and disruption of becB diminished the enterotoxicity of the C. perfringens isolate (Fig. 6). rBECa itself did not show apparent enterotoxicity but did enhance the enterotoxic activity of rBECb when simultaneously challenged in an animal model (Fig. 5B). Thus, this study showed that BEC is a novel enterotoxin produced by C. perfringens and suggested that BEC-producing C. perfringens strains are causative agents of acute gastroenteritis in humans. Toxins belonging to the binary toxin family are composed of an enzymatic component and a binding component (36). These include C. perfringens iota toxin (34), C. spiroforme iota-like toxin (37), C. difficile Cdt (38), and C. botulinum C2 toxin (39, 40). Enzymatic components of clostridial binary toxins destroy filamentous actin via mono-ADP ribosylation of globular actin, while binding components bind to receptors on targeted cells and translocate the enzymatic components into the cytosol of the cells (34, 41). BECa and BECb have significant similarities to the enzymatic and binding components, respectively, of previously reported binary toxins (Fig. 4; also see Fig. S1 and S2 in the supplemental material). Our in vitro ADP-ribosylation assay showed that BECa has an ADP-ribosylating activity (see Fig. S3). Moreover, BECa induced the morphological changes in cultured cells only in the presence of BECb (see Fig. S4), a result consistent with the nature of the binary toxin. Although we have not directly evaluated whether or not BECb delivers BECa into cells in this study, these results suggest that BECb translocates BECa into the cytosol of targeted cells. Taken together, these findings demonstrate that BEC is a novel binary toxin. It is known that the components of the iota-like toxins are interchangeable among the toxins (41). The BEC toxin identified in this study is similar to the binary toxin family toxins; the three highly conserved regions of the toxin family are conserved in BECa, and BECb is predicted to consist of four domains, like the binding component of other related toxins (see Fig. S1 and S2 in the supplemental material). Therefore, it is possible that the com-

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ponents of BEC can also be interchangeable with the components of the related binary toxins. Although we have not directly evaluated whether the components of BEC and other binary toxins are functionally interchangeable in this study, this issue will be addressed in our next study. Binary toxins are thought to have an effect on target cells only when both components act together; the components alone do not exert biological activities (36, 42–44). However, our suckling mouse assay demonstrated that BECb alone exerts the enterotoxic activity even without BECa (Fig. 5A). Recently, there were several reports showing that C. perfringens Ib, the binding component of iota toxin, showed biological activities even in the absence of the enzymatic component Ia (45, 46). Richard et al. (45) showed that Ib alone decreases the transepithelial resistance of Caco-2 cell monolayers without affecting the actin cytoskeleton. Nagahama et al. (46) reported that Ib shows cytotoxic activity to limited types of cell lines, such as A431 and A549. Both activities were thought to be related to pore formation by Ib, raising the possibility that binding components alone can exert biological activities. The present study demonstrated that BEC represents another example of binary toxins that have biological activity even without the enzymatic component. Although it is unknown how BECb causes fluid accumulation in the intestines, it is possible that BECb induces the enterotoxicity due to its pore-forming activity. The mode of action of BECb on target cells remains to be explored. The fluid-accumulating activity of rBECb was enhanced by rBECa (Fig. 5B). Thus, BECa is considered to have synergistic activity on the unique effect of BECb. BECa possesses ADP-ribosylating activity of actin (see Fig. S3 in the supplemental material), and the activity affects the morphology of target cells (see Fig. S4). This might be involved in the perturbation of the intestinal epithelial cell monolayer, leading to the enhancement of enterotoxicity. This possibility remains to be further elucidated. Synergy between the enzymatic and binding components of binary ADP-ribosylating toxins are drastic. When C. perfringens Ia and Ib were combined in equimolar amounts, there was at least a 64-fold increase in biological activity over individual components in the guinea pig dermonecrotic assay (47). On the contrary, enhancement of the enterotoxic activity of BECb by BECa is comparatively small. BECb alone showed fluid accumulation with 1 ␮g or higher, while in the presence of BECa, 100 ng or higher BECb caused fluid accumulation (Fig. 5); thus, there was 10-fold enhancement at most. These results also suggest that BECb is the primary factor responsible for the enterotoxicity, and BECa has a marginal (or just additional) role in it.

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FIG 7 Genetic organization of becAB-carrying plasmids. Genetic features of plasmids pCP-OS1 and pCP-TS1 compared with plasmid pCP13 derived from C.

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ACKNOWLEDGMENTS We thank M. Nagahama, Tokushima Bunri University, for providing anti-C. perfringens beta toxin serum, N. Goto, Osaka University, for setting up GenomeGambler, members of Tochigi Prefectural Institute of Public Health and Environmental Science for providing the C. perfringens isolates from food and feces of diarrheal patients and a healthy food handler, and H. Nariya, Kagawa University, for helpful suggestions. We also thank K. Miyano, R. Kawahara, M. Kanki, and K. Seto, Osaka Prefectural Institute of Public Health, for technical support. This study was supported by Grants-in-Aid for Scientific Research for Challenging Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (24659310), the Program of Funding Research Centers for Emerging and Reemerging Infectious Diseases by MEXT, the project for the International Research Center for Infectious Diseases, Research Institute for Microbial Diseases,

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Osaka University, by MEXT, and a grant from the Project for New Bioscience by the Osaka prefectural government.

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Preliminary screening of the distribution of the becAB genes in C. perfringens isolates from human feces (10 CPE-positive strains from diarrheal patients, 9 CPE-negative strains from diarrheal patients, and 1 CPE-positive and 16 CPE-negative strains from healthy individuals) (see Table S1 in the supplemental material) detected only one becAB-positive strain from a healthy individual (CPE negative). This suggests that the prevalence of BEC-producing C. perfringens among human feces isolates is rare, at least so far. However, only CPE-producing C. perfringens strains have been examined in clinical settings as a causative agent of acute gastroenteritis. More extensive investigations on the prevalence of becAB among C. perfringens isolates from human feces should clarify the relevance of BEC and BEC-producing C. perfringens to human GI illness. In the Tochigi outbreak, the BEC-producing C. perfringens was isolated from roast beef served at the dinner. In the Osaka outbreak, although BEC-producing C. perfringens was not isolated, roast beef was suspected as the cause of the acute gastroenteritis by epidemiological investigation. Thus, it is possible that beef is contaminated with the bacteria. Because C. perfringens exists in the intestines of cattle and other livestock, it is necessary to survey the prevalence of BEC-producing C. perfringens in edible meat, meat products, and feces of cattle and other livestock. PFGE analysis clearly demonstrated that the genetic backgrounds of the Tochigi and Osaka strains were distinct (Fig. 1). Nevertheless, the strains shared nearly identical plasmids carrying becAB (Fig. 7). This indicates that the strains acquired the becAB genes through horizontal gene transfer mediated by plasmids. This suggests that the becAB gene can be transferred to a wider range of C. perfringens populations, and that the spread of BECproducing C. perfringens could occur. The behavior of this emerging pathogen should be monitored. In conclusion, we identified BEC as a novel enterotoxin of C. perfringens and suggest that BEC-producing C. perfringens can be the cause of acute gastroenteritis in humans. Several aspects remain to be explored regarding BEC and BEC-producing C. perfringens, such as the mode of interaction between BECa and BECb, the mechanism of enterotoxic activity, and the prevalence of becAB-possessing C. perfringens in human and animal intestines and environments. Investigations from various aspects, including basic research and in public health, are needed for this novel pathogen. The present study represents the first step in elucidating the potential threat of BEC-producing C. perfringens to human health.

Novel Enterotoxin of C. perfringens

June 2014 Volume 82 Number 6

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