INFECTION AND IMMUNITY, Feb. 2009, p. 904–913 Vol. 77, No. 20019-9567/09/$08.00�0 doi:10.1128/IAI.01184-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of a Novel Type III SecretionSystem in trh-Positive Vibrio parahaemolyticus Strain TH3996

Reveal Genetic Lineage and Diversity of PathogenicMachinery beyond the Species Level�†

Natsumi Okada,1 Tetsuya Iida,2* Kwon-Sam Park,3 Naohisa Goto,4 Teruo Yasunaga,4Hirotaka Hiyoshi,1,2 Shigeaki Matsuda,1,2 Toshio Kodama,1 and Takeshi Honda1

Department of Bacterial Infections,1 Laboratory of Genomic Research on Pathogenic Bacteria,2 and Genome Information Research Center,4

Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan, and Department ofFood Science and Technology, College of Ocean Science and Technology, Kunsan National University, Kunsan, Korea3

Received 24 September 2008/Returned for modification 3 November 2008/Accepted 1 December 2008

Vibrio parahaemolyticus is a bacterial pathogen causative of food-borne gastroenteritis. Whole-genome se-quencing of V. parahaemolyticus strain RIMD2210633, which exhibits Kanagawa phenomenon (KP), revealedthe presence of two sets of the genes for the type III secretion system (T3SS) on chromosomes 1 and 2, T3SS1and T3SS2, respectively. Although T3SS2 of the RIMD2210633 strain is thought to be involved in humanpathogenicity, i.e., enterotoxicity, the genes for T3SS2 have not been found in trh-positive (KP-negative) V.parahaemolyticus strains, which are also pathogenic for humans. In the study described here, the DNA regionof approximately 100 kb that surrounds the trh gene of a trh-positive V. parahaemolyticus strain, TH3996, wassequenced and its genetic organization determined. This revealed the presence of the genes for a novel T3SSin this region. Animal experiments using the deletion mutant strains of a gene (vscC2) for the novel T3SSapparatus indicated that the T3SS is essential for the enterotoxicity of the TH3996 strain. PCR analysis showedthat all the trh-positive V. parahaemolyticus strains tested possess the novel T3SS-related genes. Phylogeneticanalysis demonstrated that although the novel T3SS is closely related to T3SS2 of KP-positive V. parahaemo-lyticus, it belongs to a distinctly different lineage. Furthermore, the two types of T3SS2 lineage are also foundamong pathogenic Vibrio cholerae non-O1/non-O139 strains. Our findings demonstrate that these two distincttypes are distributed not only within a species but also beyond the species level and provide a new insight intothe pathogenicity and evolution of Vibrio species.

Vibrio parahaemolyticus is a gram-negative halophilic marineand estuarine bacterium which is an important pathogen caus-ative of food-borne gastroenteritis and traveler’s diarrhea (1).Although most V. parahaemolyticus strains are nonpathogenicfor humans, a limited population of these organisms causeshuman diseases. Almost all clinical V. parahaemolyticus isolatesproduce the thermostable direct hemolysin (TDH) and/or theTDH-related hemolysin (TRH), which are encoded by the tdhand trh genes, respectively (5, 21). The Kanagawa phenome-non (KP), a beta-type hemolysis on a special blood agar (Wa-gatsuma agar) (28), is known as a good marker of pathogenicstrains (5, 21). V. parahaemolyticus strains which exhibit KPpossess the two tdh genes tdhA (tdh2) and tdhS (tdh1) but notthe trh gene (6, 19, 21). In contrast, KP-negative clinical V.parahaemolyticus strains possess the trh gene only or both thetrh and tdh genes, while the majority of the nonpathogenicstrains possess neither tdh nor trh.

TDH and TRH, which have several biological activities in

common (5, 20, 30, 33), are considered to be the major viru-lence factors in clinical V. parahaemolyticus strains (5, 30).However, several studies have demonstrated that although theenterotoxicity was reduced in tdh- or trh-deleted mutant strainsfrom that in the parent strains, the enterotoxic activity of thesemutant strains partially remained (24, 25, 34). These resultssuggest that in addition to TDH and TRH, extra virulencefactors are likely to exist in the organisms.

Whole-genome sequencing of a KP-positive V. parahaemo-lyticus strain, RIMD2210633, has disclosed the presence of twosets of the genes for the type III secretion system (T3SS) onchromosomes 1 and 2, T3SS1 and T3SS2, respectively (16).T3SS is possessed by gram-negative bacteria, especially in an-imal and plant pathogens, and is thought to contribute to thevirulence of these pathogens. T3SS delivers bacterial virulenceeffectors directly into the host cells, which means that thissystem contributes to virulence against the host.

Our previous studies demonstrated that the T3SSs of V.parahaemolyticus RIMD2210633 are important for virulenceof the organism (13, 23, 26). The genes for T3SS1 arepresent in all V. parahaemolyticus strains examined (10, 16,26). T3SS1 of the strain RIMD2210633 is involved in itscytotoxicity (23, 26). In contrast, deletion of the genes forT3SS2 of the RIMD2210633 strain partially eliminated fluid-accumulating activity in rabbit ileal loops, indicating thatT3SS2 is involved in enterotoxicity of this strain (26). So far,

* Corresponding author. Mailing address: Laboratory of GenomicResearch on Pathogenic Bacteria, International Research Center for In-fectious Diseases, Research Institute for Microbial Diseases, Osaka Uni-versity, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-4257. Fax: 81-6-6879-4258. E-mail: iida@biken.osaka-u.ac.jp.

† Supplemental material for this article may be found at http://iai.asm.org/.

� Published ahead of print on 15 December 2008.

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the genes for T3SS2 have been found only in KP-positivestrains (10, 16, 26).

The KP-positive strain-specific T3SS2 genes are present on apathogenicity island (Vp-PAI) consisting of a ca. 80-kb DNAregion on its chromosome 2, and this region was found tocontain the genes for TDH as well (16, 31). Pathogenicityislands (PAIs) are large genomic regions (ca. 10 to 200 kb)which are acquired by horizontal gene transfer. PAIs oftenpossess mobile genetic elements and the genes involved invirulence (3, 22).

It has not been made clear whether trh-positive clinical V.parahaemolyticus strains include any PAIs. Several reportshave suggested, however, that a PAI may be present in theregion surrounding the trh gene in trh-positive V. parahaemo-lyticus strains (8, 31, 32).

To examine whether a PAI is present in trh-positive V. para-haemolyticus strains, in this study we sequenced the regionsurrounding the trh gene on chromosome 2 in V. parahaemo-lyticus TH3996 (a trh-positive strain). This disclosed the pres-ence of an approximately 100-kb DNA region which is consid-ered to be a PAI. Our findings also demonstrated the presenceof a set of T3SS genes, which are related to but of a distinctlydifferent lineage from the T3SS2 genes in RIMD2210633.

MATERIALS AND METHODS

Bacterial strains and growth conditions. Table 1 shows the bacterial strainsand plasmids used in this study. All of the V. parahaemolyticus strains wereobtained from the Laboratory for Culture Collection, Research Institute forMicrobial Diseases, Osaka University. Clinical V. parahaemolyticus strains, in-cluding TH3996, were isolated at the Osaka and Kansai International Airportquarantine stations from patients with traveler’s diarrhea. The bacteria werecultured at 37°C with shaking in Luria-Bertani (LB) broth (tryptone, 1%; yeastextract, 0.5%) with 3% NaCl. The Escherichia coli DH5� and SM10�pir (17)strains were used for general manipulation of plasmids and their mobilizationinto V. parahaemolyticus, respectively. The E. coli strains were grown in LB brothor on LB agar. Thiosulfate-citrate-bile-sucrose agar (Nissui, Tokyo, Japan) wasused for the screening of mutant strains, and LB agar with 3% NaCl was used forcolony hybridization. Antibiotics were used at the following concentrations:ampicillin, 100 �g/ml; kanamycin, 50 �g/ml; and chloramphenicol, 5 �g/ml.

DNA manipulation. Chromosomal DNA from V. parahaemolyticus strains wasextracted from overnight culture of the organism in LB broth with 3% NaCl bymeans of the QIAamp DNA mini kit (Qiagen, Valencia, CA) according to themanufacturer’s protocol. DNA used for subcloning or nucleotide sequence anal-ysis was extracted from E. coli by using the QIAprep Spin miniprep kit (Qiagen)according to the manufacturer’s instructions. Cloning, restriction endonuclease

procedures, DNA ligation, and transformation of E. coli by plasmids were carriedout with previously described standard protocols (29). All of the restrictionenzymes and DNA ligation kits were purchased from Takara Shuzo (Otsu,Japan).

Construction of a fosmid library and screening for clones containing Vp-PAITH3996. A fosmid library was constructed using the CopyControl FosmidLibrary Production Kit (Epicentre Biotechnologies, Madison, WI) according tothe manufacturer’s instructions. Purified genomic DNA of V. parahaemolyticusTH3996 was digested with sonication, and the DNA fragments were ligated intothe Cloning-Ready CopyControl pCC1Fos vector (Epicentre). To screen forclones containing a portion of the target region among the 768 fosmid clones,colony hybridization at 40°C was performed as described previously (7). Fourprobes for the colony hybridization analysis, probe-left, -right, -trh, and -vopC,were prepared by PCR using oligonucleotide primers (see the supplementalmaterial) that were synthesized based on the sequence of two genes of Vp-PAIRIMD2210633 and the trh and vopC genes in strain TH3996, with genomicDNA of strain TH3996 as the template. Each probe was labeled with the PCRDIG probe synthesis kit (Boehringer Mannheim, Mannheim, Germany) withspecific primers.

Construction of deletion mutant strains. PCR-amplified DNA fragments usedfor constructing the in-frame deletion mutation of vscC2 were generated bymeans of overlap PCR as described previously (26) with the PCR primers vscC-1,vscC-2, vscC-3, and vscC-4 (see the supplemental material). Two DNA fragmentswere amplified by PCR with V. parahaemolyticus TH3996 chromosomal DNA asthe template and with the primer pair vscC-1 and vscC-2 and primer pair vscC-3and vscC-4, respectively. The primer vscC-2 included a complementary 15-bpsequence at its 3� end and vscC-3 at its 5� end. The two fragments were then usedas templates for a second PCR with the primers vscC-1 and vscC-4, resulting inthe construction of a fragment with a deletion in the vscC2 gene. The fragmentcontaining the deletion was purified and cloned into the pT7Blue T-vector(Novagen, Inc., Madison, WI). This fragment was then removed from thepT7Blue T-vector by digestion with BamHI and PstI and cloned into a suicidevector, pYAK1, which contains the sacB gene, conferring sensitivity to sucrose.This plasmid was introduced into E. coli SM10�pir and then mated with V.parahaemolyticus strain TH3996. Thiosulfate-citrate-bile-sucrose agar containingchloramphenicol at a concentration of 5 �g/ml was used to screen vscC deletionmutants, then the mutants were selected on LB plates supplemented with 10%sucrose. We compared the growth rates of the parent and mutant strains in LBmedium with 3% NaCl, but we could not detect any significant difference ingrowth rates between the parent and the mutants.

Complementation of the T3SS deletion mutant strain. The vscC2 complemen-tation study was performed as described previously (14, 23, 26). The vscC2 genewas amplified by PCR using the V. parahaemolyticus strain TH3996 chromosomalDNA as the template and the primers comple-F and comple-R (see the supple-mental material). The amplicon was cloned downstream from the tdhA promoterin pSA-tdhP (14) by insertion into the BamHI and SalI sites. The plasmid wasintroduced into the vscC2 mutant strain by electroporation.

DNA sequencing and informatic analysis. To sequence the Vp-PAITH3996

region (approximately 100 kb), we digested the insert fragments of fosmid clonescontaining Vp-PAITH3996. The fosmid clones were cut into smaller fragments byusing the appropriate restriction enzymes and then ligated into the pUC119

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Description Reference or source

V. parahaemolyticusTH3996 Clinical isolate, trh� ure� 34TH3996 �trh TH3996, trh disrupted 34TH3996 �vscC2 TH3996, vscC2 disrupted This studyTH3996 �trh �vscC2 TH3996, trh and vscC2 disrupted This study

E. coliDH5� F� �80dlacZ�M15 �(lacZYA-argF)U169 deoR recA1 endA1 hsdR17

phoA supE44 �� thi-1 gyrA96 relA1Laboratory collection

SM10�pir thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu �pir R6K 17

PlasmidspUC119 Cloning vector, Apr 29pT7Blue T-vector Multicopy (ColE1 ori) TA clonig vector, Apr Novagen, Inc.pYAK1 Suicide vector, R6Kori, sacB, Cmr 12pSA-tdhP pSA19CP-MCS containing tdhA promoter in EcoRI-SmaI site 14

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vector. Gap closure was achieved with PCR direct sequencing, with primers thatwere designed to anneal to each end of neighboring contigs. Nucleotide sequenc-ing was performed with the ABI PRISM 3100 genetic analyzer (Applied Biosys-tems, Foster City, CA) and the BigDye v3.1 cycle sequencing kit (AppliedBiosystems). The Genetyx sequence analysis program (Software Development,Tokyo, Japan) was used for computer analysis of DNA sequences. Homologysearches against deposit sequences were performed via the National Center forBiotechnology Information using the BLAST network service (http://www.ncbi.nlm.nih.gov) and the BLAST service at the Genome Information ResearchCenter (http://genome.naist.jp/bacteria/vpara/). Sequence information was ob-tained from the NCBI. The computer program CLUSTAL W was used for theamino acid sequence alignment and phylogenetic analysis.

Analysis of secreted proteins. Secreted proteins were prepared as describedpreviously (26). Secreted proteins from the parent and mutant strains wereisolated from the supernatants of bacterial cell cultures grown for 6 h at 37°C inLB medium. Secreted proteins were precipitated by the addition of trichloro-acetic acid to a final concentration of 10% (vol/vol). The proteins were collectedby centrifugation at 17,500 g for 30 min at 4°C, and the resultant pellets werewashed in ice-cold 100% acetone and suspended in sodium dodecyl sulfatesample buffer.

Western blot analysis. The secreted proteins used for Western blot analysiswere separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresiswith 12% polyacrylamide. The transferred membrane was first probed withanti-VopD2 polyclonal antibody (14) and then with horseradish peroxidase-conjugated goat antirabbit antibody (Zymed Laboratories, South San Francisco,CA). The blots were developed by using the ECL Western blotting kit (Amer-sham, Piscataway, NJ) according to the manufacturer’s instructions.

Rabbit ileal loop test. The enterotoxic activities of the wild-type and mutantstrains were assessed with a rabbit ileal loop test (26). The two strains werecultured at 37°C with shaking in LB broth (3% NaCl), diluted 100 times with LBbroth (0.5% NaCl), and cultured overnight at 37°C. Next, the cultures werediluted 100 times with LB broth (0.5% NaCl) and cultured at 37°C for 6 h. Theorganisms were harvested by centrifugation at 3,000 g for 10 min and sus-pended in LB broth (0.5% NaCl). The rabbit ileal loop test used 1.5-kg femaleNew Zealand White rabbits, whose small intestine was used to make 5 or 10ligated loops per rabbit. Bacterial cells (109, 108, or 107 CFU) of the wild-type ormutant strains and negative control were injected into the loops as describedpreviously (26), followed by measurement of the fluid accumulation in each loop16 h after challenge. We performed 6 (109 CFU) or 10 (108 and 107 CFU)experiments for each sample using different rabbits.

Oligonucleotide primers and PCR conditions. The supplemental materialshows the oligonucleotide primers used in this study. PCR conditions for theconstruction of mutant strains and probes were as follows: after 2 min of dena-

turation at 94°C, a cycle of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min wasrepeated 30 times. To detect the presence of the T3SS genes, PCR was per-formed using the EX-PCR kit (Takara Shuzo, Kyoto, Japan). The PCR condi-tions were as follows: after initial denaturation at 94°C for 3 min, a cycle of 94°Cfor 30 s, 55°C for 30 s, and 72°C for 1 min or 2 min was repeated 30 times. PCRscanning of Vp-PAITH3996 was performed using genomic DNA as a template anda long accurate PCR kit (Takara Shuzo). The PCR conditions were as follows:after initial denaturation at 94°C for 3 min, a cycle of 94°C for 30 s and 65°C for10 min was repeated 30 times. Custom-synthesized oligonucleotides for the PCRwere purchased from Gene Design (Osaka, Japan).

Statistical analysis. Statistical significance was determined using the t test. AP value of 0.05 was considered statistically significant.

Nucleotide sequence accession number. The nucleotide sequence data re-ported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotidesequence database under accession number AB455531.

RESULTS

Cloning and sequencing of the ca. 100-kb DNA region whichflanks the trh gene. For the cloning of the DNA region flankingthe trh gene on the small chromosome of V. parahaemolyticusstrain TH3996, genomic DNA of the strain was isolated andsonicated to yield approximately 40-kb fragments. The DNAfragments obtained were inserted into the Cloning-ReadyCopyControl pCC1Fos vector (Epicentre Biotechnologies,Madison, WI) to construct a fosmid library. To screen theclones containing the trh gene and surrounding regions fromamong a total of 768 fosmid clones, colony hybridization wascarried out using three probes (probe-left, -trh, and -right)(Fig. 1a) (see the supplemental material). Two probes, probe-left and -right, were generated by amplifying genomic DNAfrom strain TH3996 with primers (see the supplemental mate-rial) based on sequences from the KP-positive strainRIMD2210633. This yielded three clones, which hybridizedwith one each of the probes (Fig. 1a, clones 1 to 3) and weredigested with several restriction enzymes and subcloned intothe corresponding sites of the pUC119 vector. By sequencingthe subclones of clone 3, we identified the vopC gene at the 5�

FIG. 1. DNA region flanking the trh gene on the small chromosome of V. parahaemolyticus TH3996. (a) The four black squares indicate theposition of the four probes (probe-left, -trh, -right, and -vopC) that were prepared and used for colony hybridization. The fosmid clones 1 to 4 wereobtained from the 768 fosmid clones of V. parahaemolyticus TH3996 genomic DNA with four probes, probe-left, -trh, -right, and -vopC,respectively. (b) The two rectangles represent the two contigs obtained in this study. The broken line indicates the region that was not identifiedand corresponds to the inside of VPA1357 of Vp-PAIRIMD2210633. The colored blocks show the 100 ORFs of this region. The outline ofVp-PAITH3996 is indicated, consisting of the trh gene (yellow block), the urease gene cluster (green blocks), T3SS-related genes (red or blue blocks),and transposases (orange blocks), while the other ORFs of this region are marked with gray blocks. The ORFs that flanked Vp-PAITH3996 areindicated with white blocks, and the line marks the small chromosome of TH3996. (c) The 10 lines with arrowheads at both ends, representingPCR-A1 to -A6 and -B1 to -B4, designate the regions that were amplified for PCR scanning.

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end of this clone. To screen new clones containing the vopCgene, which encodes a homologue of the cytotoxic necrotizingfactor (13), from the fosmid library, we prepared a new probe(Fig. 1a, probe-vopC) and performed colony hybridization us-ing this probe. The clone thus obtained (Fig. 1a, clone 4) wassubcloned and sequenced as were the other three. By sequenc-ing these subclones, we could get two contigs, contigs A (ca.55.1 kb) and B (ca. 36.8 kb), which almost cover the approxi-mately 100-kb region surrounding the trh gene (Fig. 1b).

The sequences of the 3� end of contig A (approximately 570 bp)and the 5� end of contig B (approximately 560 bp) showed highhomology with the 5� and 3� ends, respectively, of an open readingframe (ORF), VPA1357, of the RIMD2210633 strain. Compari-son with the nucleotide sequences of the RIMD2210633 genomesuggested that the region between contigs A and B corresponds toVPA1357 of RIMD2210633 (Fig. 1b) (16). VPA1357, 4,869 bp insize, encodes a hypothetical protein which has numerous re-peated sequences, spanning nearly two-thirds of the sequence ofthe gene (16). It was difficult to determine the sequence of thisregion accurately due to the numerous repeats. Instead of com-pleting the sequencing of the region, we therefore estimated itssize by both PCR amplification of the region and construction ofrestriction maps of the fosmid clone 4. We estimated the gapregion between contigs A and B to be approximately 6.1 kb (datanot shown) (Fig. 1b). On the basis of this estimate, we speculatedthat an approximately 7.2-kb ORF which is homologous toVPA1357 of strain RIMD2210633 exists in this region of thesmall chromosome in the TH3996 strain. The estimated size ofthis ORF is obviously larger than that of VPA1357 of theRIMD2210633 strain (4,869 bp).

In order to confirm that the four fosmid clones cover thewhole region flanking the trh gene, PCR scanning with 10 PCRprimer pairs (see the supplemental material) was used for thegenomic DNA of TH3996 (Fig. 1c, PCR-A1 to -A6 and -B1 to-B4). The expected sizes of the amplicons were obtained for allthe primer pairs, suggesting that the fosmid clones cover thewhole target region (data not shown).

The G�C content of the ca. 100-kb region we sequencedaccounted for approximately 39.8%, which is notably lower

than the average G�C content of the small chromosome ofRIMD2210633, which is 45.4% (16).

Annotation of the DNA region revealed that the region pos-sesses a total of 100 ORFs: contigs A and B possess 53 and 46ORFs, respectively, and 1 unsequenced ORF was homologous toVPA1357; and among these ORFs, we identified a set of genesfor the T3SS. Although most of the ORFs encode hypotheticalproteins, this 100-kb region contained known possible virulencefactor genes, including the trh gene and the urease gene cluster(24), in addition to T3SS-related genes, as well as mobile ele-ments, such as transposases (Fig. 1b). Furthermore, as mentionedabove, its G�C content was significantly lower than the genomeaverage. From this series of findings, we hypothesized that the ca.100-kb region on the small chromosome of the TH3996 strain isa PAI, newly identified for this strain, and tentatively named itVp-PAITH3996. The PAI of RIMD2210633, which was referred toas Vp-PAI in previous studies (10, 31), was tentatively namedVp-PAIRIMD2210633 for this report.

At least 14 ORFs showed significant homology with T3SS-related genes that have been reported to date (9, 14, 15, 26).These genes were predicted to encode the apparatus proteinsof T3SS (vscCJQRSTU and vcrD), an ATPase (vscN), translo-cons (vopBD), and effectors (vopCLP) (vopP is also known asvopA) (Fig. 2) (9, 14, 15, 26). A recent study of ours found thatthe TH3996 strain possesses the genes for T3SS1 on its ge-nome, as do other V. parahaemolyticus strains (10). The novelT3SS-related genes found in Vp-PAITH3996 were obviouslydifferent from the T3SS1 genes of the RIMD2210633 strain.The genetic organization of the former was similar to that ofT3SS2 of the RIMD2210633 strain but not identical. TheT3SS-related genes found in Vp-PAITH3996 had 34.5% to89.5% homology with the corresponding T3SS2 genes inVp-PAIRIMD2210633. These results demonstrated that the novelT3SS-related gene set present on the small chromosome of thetrh-positive strain TH3996 can be considered to be T3SS2-related T3SS.

Construction from strain TH3996 of a mutant strain with adeletion in the T3SS-related gene and protein secretion by themutant strain. In Vp-PAITH3996, at least 14 T3SS-related

FIG. 2. Comparison of the gene organization of T3SS-related genes in V. parahaemolyticus and V. cholerae. The genetic organization of theT3SS in Vp-PAITH3996 was compared with those in RIMD2210633 (T3SS2), V. cholerae 1587, and V. cholerae AM-19226. Genes are indicated byarrows. Red arrows indicate the genes encoding putative apparatus proteins of T3SS and blue arrows the genes encoding putative regulatory andeffector proteins of T3SS, and gray arrows indicate the genes encoding hypothetical proteins. The colors of the arrows are identical to thosepreviously used (4).

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genes were found, as mentioned above, and all of these geneswere conserved in T3SS2 on Vp-PAIRIMD2210633 as well. Wetherefore hypothesized that the T3SS genes found in Vp-PAITH3996 may express a functional secretion system likeT3SS2 in Vp-PAIRIMD2210633. To confirm the expression of asecretion system of T3SS in Vp-PAITH3996, T3SS-dependentprotein secretion was analyzed. A T3SS-deficient mutant wasconstructed by disruption of the homologue of the vscC2 gene,which encodes an outer membrane protein of T3SS2 (26), inthe TH3996 strain (resulting in TH3996 �vscC2). The secre-tion of VopD2, a translocon protein of T3SS2, by the parentand mutant strains was examined by means of Western blotanalysis using the anti-VopD2 antibody of strain RIMD2210633 (14). As shown in Fig. 3, VopD2 was detected in thesupernatant of the parent strain but not in the vscC2 deletionmutant strain. The secretion of VopD2 was restored bycomplementation with the vscC2 gene (Fig. 3). These resultssuggested that the genes for T3SS in Vp-PAITH3996 express afunctional secretion system.

Enterotoxicity assay of mutant strains. Previous studieshave demonstrated that T3SS2 of V. parahaemolyticus RIMD2210633 contributes to the enterotoxicity of the organism (26).To determine the possible contribution of the T3SS-relatedgenes encoded in Vp-PAITH3996 to enterotoxicity of the strain,we examined the enterotoxic activity of the wild-type and mu-tant strains in the rabbit ileal loop test. For this test, we usedTH3996 �vscC2 and a mutant strain with a deletion in the trhgene (TH3996 �trh), the contribution of which to the entero-toxicity of the strain was previously reported (34). Further-more, we constructed a double deletion mutant strain withdeletions in both the trh and vscC2 genes (TH3996 �trh�vscC2). The wild-type strain or the mutants (109 CFU [each])or LB broth as a negative control was injected into the ligatedileal loop of rabbits. After 16 h, the small intestines of therabbits were removed and the fluid accumulation in the ligatedileal loops was measured (Fig. 4a). The vscC2 deletion mutantstrain was associated with little fluid accumulation, at levelssimilar to that with the negative control, LB. There was nosignificant difference in fluid accumulation levels betweenresults for the trh gene-disrupted mutant strain and thewild-type strain (Fig. 4a). Results obtained under these exper-imental conditions indicated that the T3SS encoded in Vp-PAITH3996 is an important factor in the enterotoxicity of theorganism. These results also suggested that the T3SS is func-tionally expressed under in vivo conditions.

A previously reported significant reduction of enterotoxicityof trh-deleted mutant strains (34) was not observed during theexperiment (Fig. 4a). However, the dose of the bacteria (109

CFU) injected into ligated ileal loops of the rabbits in our

study differed from the one used in a previous study (107 or 108

CFU) (34). To confirm the effect of the bacterial inoculationdose, we performed additional rabbit ileal loop tests (Fig. 4b).The wild-type strain and the trh deletion mutant were injectedinto the ligated ileal loop of rabbits at doses of 107 CFU and108 CFU for each, and the fluid accumulation in each loop wasmeasured 16 h after challenge. At both doses, fluid accumula-tion with the trh deletion mutant strain was significantly lowerthan that with the wild-type strain (Fig. 4b), thus confirmingthe findings of Xu et al. (34). We can therefore conclude thatthe discrepancy was due to the difference in the dose of thebacterial inoculum.

Distribution of the T3SS-related genes among V. parahae-molyticus strains. To analyze the distribution of the T3SS-relatedgenes found in Vp-PAITH3996 in other V. parahaemolyticusstrains, a PCR assay was performed using oligonucleotide primerpairs (see the supplemental material) targeting the genes presentin Vp-PAITH3996, i.e., trh, ureC, vscC2N2R2S2T2U2, vcrD2,vopB2D2, or vopCLP, for 33 V. parahaemolyticus strains. ThePCR primer pairs were designed based on the sequences of genesin strain TH3996 (see the supplemental material). Since they did

FIG. 3. Western blot analysis of the novel T3SS-dependent se-creted protein in Vp-PAITH3996. Western blot analysis of VopD2 insupernatants of wild-type and mutant strains. Lane 1, strain TH3996;lane 2, vscC2 deletion mutant (TH3996 �vscC2); lane 3, vscC2 comple-mentation in TH3996 �vscC2; lane 4, TH3996 �vscC2 harboring pSA-tdhP. Blots were probed with anti-VopD2 antibody.

FIG. 4. Fluid accumulation in the rabbit ileal loop test. Fluid ac-cumulation is the amount of accumulated fluid (in milliliters) perlength (in centimeters) in ligated rabbit small intestine. (a) WT, strainTH3996; �vscC2, vscC2 deletion mutant (TH3996 �vscC2); �trh, trhdeletion mutant (TH3996 �trh); �trh�vscC2, trh and vscC2 doubledeletion mutant (TH3996 �trh �vscC2); LB, LB broth with 0.5% NaCl(negative control). Means and standard deviations are shown. Aster-isks indicate significant differences from the results obtained with theparent strain (P 0.01). (b) WT, TH3996 strain; �trh, trh deletionmutant (TH3996 �trh); LB, LB broth with 0.5% NaCl (negative con-trol). Bacterial doses in CFU used for the challenges are indicated atthe bottom. Means and standard deviations are shown. Double aster-isks indicate significant differences from the results obtained with theparent strain (P 0.05).

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not amplify the T3SS2 genes of the RIMD2210633 strain (datanot shown), these primer pairs were specific to the genesof TH3996. The 33 strains of V. parahaemolyticus included strainsof various serotypes and had been isolated for years (Table 2). Ofthese strains, 27 were trh positive, 3 KP-positive (tdh positive andtrh negative), and 3 tdh and trh negative.

Most of the genes were amplified by PCR in the 27 trh-positive strains tested (Table 2); however, none of the genestested in any of the KP-positive strains could be amplified.Furthermore, no amplicons were obtained in the tdh- andtrh-negative strains. Although the amplicons for vscT2, vopB2,or vopP were not obtained in a few of the trh-positive strains(Table 2), most of the T3SS-related genes seemed to be con-served in these strains. Although the trh genes can be dividedinto two groups, trh1 and trh2, based on the sequences (11),

there was no difference in the presence of the T3SS-relatedgenes between trh1-positive and trh2-positive strains (Table 2).These findings suggest that all the trh-positive V. parahaemo-lyticus strains tested possess the novel T3SS-related genes,which were found in Vp-PAITH3996. Furthermore, these geneswere not detected in KP-positive V. parahaemolyticus strains orin tdh- and trh-negative V. parahaemolyticus strains.

Phylogenetic analysis of T3SS genes in Vp-PAITH3996. Theresults presented here suggest that trh-positive V. parahaemolyti-cus strains possess T3SS-related genes which are closely related tothe ones present in Vp-PAITH3996. Although genetic organizationof the T3SS-related genes in Vp-PAITH3996 is similar to that ofT3SS2 in Vp-PAIRIMD2210633, there are clear distinctions, includ-ing the different locations of vopP (Fig. 2). Furthermore, PCRanalysis findings suggest that the sequences of the T3SS-related

TABLE 2. Distribution of T3SS-related genes in V. parahaemolyticus strains

KPassessment

Hemolysingene content Strain Serotype Yr Sourceb

Presence of gene

vscC2 vscR2 vscS2 vscT2 vscU2 vcrD2 vscN2 vopB2 vopD2 vopC vopL vopP trh ureC

KP� tdh negative,trh1positive

TH3996 O4:K11 1983 P � � � � � � � � � � � � � �

RIMD2212735 O1:KUTa 2001 P � � � � � � � � � � � � � �RIMD2210536 O3:K6 1985 P � � � � � � � � � � � � � �

tdh negative,trh2positive

RIMD2210001 O1:K1 1950 P � � � � � � � � � � � � � �

RIMD2212673 O1:K56 2000 P � � � � � � � � � � � � � �RIMD22122508 O1:KUT 2005 P � � � � � � � � � � � � � �RIMD2212624 O5:K17 2000 P � � � � � � � � � � � � � �RIMD22121464 O1:K33 2002 P � � � � � � � � � � � � � �RIMD22122382 OUTa:KUT 2005 P � � � � � � � � � � � � � �RIMD22122410 O11:KUT 2005 P � � � � � � � � � � � � � �RIMD22122377 O3:K7 2005 P � � � � � � � � � � � � � �RIMD22121386 O1:K56 2002 P � � � � � � � � � � � � � �RIMD22122421 O3:KUT 2005 P � � � � � � � � � � � � � �RIMD2212888 O1:KUT 2001 P � � � � � � � � � � � � � �RIMD22122489 OUT:KUT 2005 P � � � � � � � � � � � � � �

tdh positive,trh1positive

RIMD2210856 O10:KUT 1991 P � � � � � � � � � � � � � �

RIMD2212746 O8:KUT 2001 P � � � � � � � � � � � � � �RIMD22121037 O10:K52 2001 P � � � � � � � � � � � � � �RIMD22121271 O1:KUT 2002 P � � � � � � � � � � � � � �RIMD2212707 O3:KUT 2001 P � � � � � � � � � � � � � �RIMD2212940 O1:KUT 2001 P � � � � � � � � � � � � � �RIMD2212953 O1:KUT 2001 P � � � � � � � � � � � � � �

tdh positive,trh2positive

RIMD22122886 O8:K56 2005 P � � � � � � � � � � � � � �

RIMD22122682 O1:K20 2005 P � � � � � � � � � � � � � �RIMD22121158 O3:KUT 2000 P � � � � � � � � � � � � � �RIMD22121266 O1:K20 2002 P � � � � � � � � � � � � � �RIMD22122395 O3:K75 2005 P � � � � � � � � � � � � � �

KP� tdh positive,trhnegative

RIMD2210633 O3:K6 1996 P � � � � � � � � � � � � � �

RIMD2210086 O4:K12 1968 P � � � � � � � � � � � � � �RIMD2211499 O2:K3 1994 P � � � � � � � � � � � � � �

KP� tdh negative,trhnegative

RIMD2212201 O3:K20 1999 S � � � � � � � � � � � � � �

RIMD2210384 O4:KUT 1976 F � � � � � � � � � � � � � �RIMD2210470 O5:KUT 2001 S � � � � � � � � � � � � � �

a UT, untypeable.b P, patient; S, seawater; F, food.

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genes in Vp-PAITH3996 are rather dissimilar to those of the T3SS2genes in Vp-PAIRIMD2210633 (Table 2), suggesting that thesetwo gene sets belong to different lineages.

Previous studies reported that the V. cholerae non-O1/non-O139 serogroup strains AM-19226, 1587, and 623-39, which donot have the cholera toxin gene, possess a set of T3SS genes (4,18). In their report, Dziejman et al. pointed out that the geneorganization of the T3SS gene cluster of V. cholerae AM-19226and that of the T3SS2 of V. parahaemolyticus RIMD2210633are similar (Fig. 2) (4). We therefore performed a phylogeneticanalysis of the T3SS genes from the V. parahaemolyticus and V.cholerae strains reported to date.

For the phylogenetic analysis, we used amino acid sequencesof five T3SS-related genes (i.e., vscCNRT and vcrD) of sixstrains of Vibrio species, V. parahaemolyticus TH3996, RIMD2210633 (T3SS1 and T3SS2), and AQ3810 (tdh positive and trhnegative), and V. cholerae strains AM-19226, 1587, and 623-39.In addition, six pathogenic species that are known to possessthe T3SS genes, namely, those from the genera Yersinia,Shigella, Salmonella, Pseudomonas, and Escherichia, wereincluded in the analysis. By using the neighbor-joining method,we constructed phylogenetic trees for each gene.

The analysis clearly demonstrated that the T3SS-relatedgenes in Vp-PAITH3996 are only distantly related to the genesof T3SS1 of RIMD2210633 and are more likely to belong tothe cluster containing T3SS2 of RIMD2210633 and T3SSs ofV. cholerae (here referred to as the T3SS2 family) (Fig. 5).

Unexpectedly, the T3SS-related genes in Vp-PAITH3996

were found to be more closely related to the T3SS genes of V.cholerae strains 1587 and 623-39 than were the T3SS2 genes inVp-PAIRIMD2210633. Furthermore, the T3SS genes of anotherV. cholerae strain, AM-19226, were more closely related to theT3SS2 genes in Vp-PAIRIMD2210633 than were those of V. chol-erae strain 1587 and strain 623-39 (Fig. 5). These resultsprompted us to classify the T3SSs of V. parahaemolyticus andV. cholerae into two phylogroups, one comprising T3SS2 inVp-PAIRIMD2210633 and the T3SS of V. cholerae strain AM-19226 and the other comprising the T3SS in Vp-PAITH3996 andthe T3SSs of V. cholerae strains 1587 and 623-39. We tenta-tively designated the former phylogroup T3SS2� and the latterT3SS2�.

A recent study reported that clinical V. cholerae non-O1/non-O139 serogroup strains V51 and NRT36S also possess theT3SS-related genes in a pathogenicity island (VPI-2) on thosechromosomes and that the genes for T3SS in strains V51,NRT36S, and AM-19226 have only �90% homology withthose of 1587 and 623-39 (18). On the basis of this report andour findings, we suggest that the T3SS-related genes in V51and NRT36S belong to T3SS2�.

DISCUSSION

Vibrio parahaemolyticus is an important human pathogen.Strains isolated from diarrheal patients produce TDH or TRHor both, but the strains isolated from the environment do nothave these properties. TDH, which is produced by KP-positiveV. parahaemolyticus strains, is known as a major virulencedeterminant of these strains. However, whole-genome sequenc-ing of a KP-positive strain, RIMD2210633, revealed the presenceof two sets of the T3SS genes, T3SS1 and T3SS2, on its chromo-

somes 1 and 2, respectively (16). Although the genes for T3SS1are present in all V. parahaemolyticus strains examined (10, 16,24), those for T3SS2 are found only in KP-positive strains (10).The genes for T3SS2 of the RIMD2210633 strain are located ona pathogenicity island (Vp-PAIRIMD2210633) (16).

In our study, the DNA region of approximately 100 kb thatsurrounds the trh gene of trh-positive V. parahaemolyticusTH3996 was sequenced and its gene organization identified.We detected a PAI-like structure (Vp-PAITH3996) in the ge-nome of the strain and demonstrated the presence of the genesfor novel T3SS in the Vp-PAITH3996 region (Fig. 1b and 2).Gene organization and phylogenetic analysis indicated that thenewly discovered T3SS genes in TH3996 are more closely re-lated to the T3SS2 than the T3SS1 genes of RIMD2210633(Fig. 5).

In the KP-positive V. parahaemolyticus strain RIMD2210633,the presence of a number of T3SS2-related genes that encode thestructural components or effectors has been reported (9, 13, 15,23, 24). Construction of the mutant strains of those T3SS2-relatedgenes indicated that T3SS2 is important for enteropathogenicityof the KP-positive V. parahaemolyticus strain (13, 23, 24). Sincethe presence of the novel T3SS-related genes in trh-positive V.parahaemolyticus strains was proven, we investigated whether theT3SS is also involved in the enteropathogenicity of the TH3996strain. Animal experiments using the deletion mutant strains of agene (vscC2) for the T3SS apparatus indicated that the T3SS isessential for the enterotoxicity of the TH3996 strain. A previousreport showed that the deletion of the trh gene in a trh-positive V.parahaemolyticus strain, TH3996, significantly decreased the fluidaccumulation in rabbit ileal loop tests, but the mutant partiallyretained its enterotoxic activity (34). This suggests that the viru-lence factor(s) of the TH3996 strain is not limited to TRH alone,but to date no other virulence factor has been reported in trh-positive strains. In our study, we could demonstrate that theT3SS-related genes present in Vp-PAITH3996 are involved in en-terotoxicity of the trh-positive strains, making them a strong can-didate for this previously unidentified virulence factor.

Although the genetic organization of the T3SS2-relatedgene cluster of Vp-PAITH3996 was similar to that of Vp-PAIRIMD2210633, the homologies of individual genes of TH3996with those of RIMD2210633 varied widely, from 34.5% to89.5%. The PCR with several primer pairs that can amplify thegenes for the T3SS of trh-positive V. parahaemolyticus strainscould not amplify the genes of KP-positive V. parahaemolyticusstrains. This seems to indicate, therefore, that the nucleotidesequences of the T3SS-related genes are different for KP-positive and trh-positive strains. Our findings are supported bythose of a study using comparative genomic hybridization anal-ysis of five trh-positive strains, which could not detect thepresence of the T3SS2-related genes (10). From these results,we conclude that the trh-positive strain-specific T3SS-relatedgene cluster does not occur in KP-positive strains. This indi-cates that a distinct lineage of T3SS2-related genes (T3SS2�and T3SS2�) must exist in KP-positive and trh-positive V. para-haemolyticus strains.

The phylogenetic analysis showed that the two types ofT3SS2, T3SS2� and T3SS2�, are also distributed amongpathogenic V. cholerae non-O1/non-O139 serogroup strains(Fig. 5). However, the gene compositions of VPI-2 of V. chol-erae strains (18), except for the T3SS gene cluster, were not

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similar to those of Vp-PAITH3996 and Vp-PAIRIMD2210633.Thus, the gene compositions of the PAI cassettes in V. para-haemoloyticus and V. cholerae, except for the genes for T3SS,were clearly different, implying that the evolutionary history ofthe PAIs of the two species is also different.

The genetic organization of Vp-PAITH3996 was found tohave features in common with that of Vp-PAIRIMD2210633, withboth PAIs containing the T3SS and hemolysin genes, i.e.,tdh or trh (Fig. 6). Recently the three ORFs on Vp-PAIRIMD2210633, VPA1394, VPA1395, and VPA1396, wereidentified as genes for the Tn7 superfamily of transposons (31).Tn7 is a bacterial transposon that is widespread in diverse

species and is involved in the formation of genomic islands(27). In the aforementioned study by Sugiyama et al., it wasfound that on chromosome 2 of the KP-positive strainRIMD2210633, Vp-PAIRIMD2210633 was flanked by 5-bp directrepeats (DRs) and was inserted between VPA1309 andVPA1397 (31). It was thus speculated by the authors that thisTn7 superfamily-like genetic element is involved in the forma-tion of Vp-PAIRIMD2210633 on chromosome 2 of strainRIMD2210633. In the trh-positive strain TH3996, we detectedthe presence of three ORFs in Vp-PAITH3996 that showed highhomology with VPA1394, VPA1395, and VPA1396, which, asmentioned above, are components of the Tn7 superfamily (Fig.

FIG. 5. Phylogenetic analysis of the T3SS-related genes. Phylogenetic trees of the five T3SS-related genes (vscCNRT and vcrD), constructedusing the neighbor-joining method. Abbreviations of the 12 strains that were used in the analysis are as follows: VpTH3996-T3SS2, V. parahaemo-lyticus strain TH3996; VpRIMD2210633-T3SS2, V. parahaemolyticus strain RIMD2210633 (T3SS2); VpRIMD2210633-T3SS1, V. parahaemolyticusstrain RIMD2210633 (T3SS1); VpAQ3810, V. parahaemolyticus strain AQ3810; VcAM19226, V. cholerae strain AM-19226; Vc1587, V. choleraestrain 1587; Vc623-39, V. cholerae strain 623-39; Yp, Yersinia pestis strain CO92 (pCD1); Ye, Y. enterocolitica strain 8081 (pYVe8081); Sf, Shigellaflexneri strain M90T (pWR100); SPI1, Salmonella enterica serovar Typhimurium strain LT2(SPI1); SPI2, S. enterica serovar Typhimurium strainLT2(SPI2); Pa, Pseudomonas aeruginosa strain PAO1; EPEC, enteropathogenic Escherichia coli strain E2348/69; EHEC, enterohemorrhagic E. coliO157:H7 strain Sakai. Sequence information was obtained from the NCBI. The computer program CLUSTAL W was used for the amino acidsequence alignment and phylogenetic analysis.

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6). In addition, we found that Vp-PAITH3996 was flanked by5-bp DRs (Fig. 6). Since these structural features of Vp-PAITH3996 were similar to those of Vp-PAIRIMD2210633, it wasspeculated that the Tn7 superfamily transposons found in Vp-PAITH3996 also might be involved in the initial formation of thePAI cassettes. However, since the entire set of Tn7 transposongenes was not conserved in Vp-PAITH3996, as was reported inthe case of Vp-PAIRIMD2210633, the Tn7 superfamily trans-posons in Vp-PAITH3996 and Vp-PAIRIMD2210633 might nolonger be functional as transposable elements.

Vp-PAITH3996 and Vp-PAIRIMD2210633, large gene clustersof more than 80 kb, were acquired as a result of horizontalgene transfer in V. parahaemolyticus. However, it is unclearhow V. parahaemolyticus integrated the PAI cassette into itschromosome after acquisition of the foreign PAI cassette intoits cytoplasm. As mentioned above, the Tn7 superfamily in Vp-PAIRIMD2210633 and Vp-PAITH3996 may no longer be functional,and there are no reports of such a large DNA region beingtransferred horizontally by the Tn7 superfamily, which might thusnot be involved in insertion of the PAI cassette in the V. parah-

FIG. 6. Structure of pathogenicity island of V. parahaemolyticus strains. Schematic representation of the structure of Vp-PAITH3996 in thetrh-positive V. parahaemolyticus strain TH3996 and of Vp-PAIRIMD2210633 in the KP-positive strain RIMD2210633. The region, flanked by 5-bpDRs, shows Vp-PAITH3996 and Vp-PAIRIMD2210633, which are inserted into the region between the two white arrows, which indicate the two ORFs(one encoding the hypothetical protein and the other the Acyl-CoA thioester hydrolase-related protein). The outlines of the region, consisting ofthe trh gene and tdh genes, are indicated by yellow arrows. The T3SS2-related gene cluster is represented by the blue blocks and the genes of theTn7 superfamily by the orange arrows, and other ORFs of the region are represented by black bold lines, which indicate chromosome 2 of strainsTH3996 and RIMD2210633.

FIG. 7. Schematic representation of the hypothetical evolutionary acquisition of a T3SS-related gene cluster in V. parahaemolyticus and V.cholerae. Lineage is based on the presence of each of the determinants, for example, tdh, trh, CTX, and T3SS2. The shaded ellipses show theT3SS-related gene clusters. Bold lines represent the evolutionary process. Circles indicate the strains of V. parahaemolyticus and V. cholerae.Among them, shaded circles indicate that the strains possess T3SS� or T3SS�. The broken lines indicate that the T3SS-related gene clusters orCTX has been acquired by horizontal gene transfer while the organisms were evolving.

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aemolyticus chromosome. It is considered likely that the exchangeof O-antigen-encoding cassettes, which are large DNA fragments(more than 32 kb in size), in V. cholerae is mediated by homolo-gous recombination, because the regions flanking the cassettesshow high homology (2). Similarly, the sequences flanking Vp-PAITH3996 and Vp-PAIRIMD2210633 in V. parahaemolyticus werehighly conserved. This implies that the large PAI cassettes may beintegrated into the chromosome by means of homologous recom-bination in V. parahaemolyticus.

It is interesting that the distribution of T3SS2� and T3SS2�is not limited to being found within a species but goes beyondthe species level (Fig. 7). The presence of common secretionsystem genes in organisms beyond the species level suggeststhat the possession of such secretion systems may confer somecommon beneficial effect(s) on the organisms. Although thenature of such benefit(s) is as yet unknown, attempts to iden-tify the role of those secretion systems in aquatic environmentsmay lead to a better understanding of the life cycles of humanpathogens in nature. This points to the significance of theresults of our phylogenetic analysis of the T3SS genes, becausewe believe they provide a new insight into the pathogenicityand evolution of Vibrio species.

ACKNOWLEDGMENTS

This work was supported by Grants-in-Aid for Scientific Researchon Priority Areas and for Scientific Research from the Ministry ofEducation, Culture, Sports, Science and Technology of Japan.

We thank the staff of the Kansai International Airport QuarantineStation for supplying the V. parahaemolyticus strains.

REFERENCES

1. Blake, P. A., R. E. Weaver, and D. G. Hollis. 1980. Diseases of humans (otherthan cholera) caused by vibrios. Annu. Rev. Microbiol. 34:341–367.

2. Blokesch, M., and G. K. Schoolnik. 2007. Serogroup conversion of Vibriocholerae in aquatic reservoirs. PLoS Pathog. 3:e81.

3. Dobrindt, U., B. Hochhut, U. Hentschel, and J. Hacker. 2004. Genomicislands in pathogenic and environmental microorganisms. Nat. Rev. Micro-biol. 2:414–424.

4. Dziejman, M., D. Serruto, V. C. Tam, D. Sturtevant, P. Diraphat, S. M.Faruque, M. H. Rahman, J. F. Heidelberg, J. Decker, L. Li, K. T. Montgom-ery, G. Grills, R. Kucherlapati, and J. J. Mekalanos. 2005. Genomic char-acterization of non-O1, non-O139 Vibrio cholerae reveals genes for a type IIIsecretion system. Proc. Natl. Acad. Sci. USA 102:3465–3470.

5. Honda, T., and T. Iida. 1993. The pathogenicity of Vibrio parahaemolyticusand the role of the thermostable direct haemolysin and related haemolysin.Rev. Med. Microbiol. 4:106–113.

6. Iida, T., and K. Yamamoto. 1990. Cloning and expression of two genesencoding highly homologous hemolysins from a Kanagawa phenomenon-positive Vibrio parahaemolyticus T4750 strain. Gene 93:9–15.

7. Iida, T., O. Suthienkul, K. S. Park, G. Q. Tang, R. K. Yamamoto, M.Ishibashi, K. Yamamoto, and T. Honda. 1997. Evidence for genetic linkagebetween the ure and trh genes in Vibrio parahaemolyticus. J. Med. Microbiol.46:639–645.

8. Iida, T., K. S. Park, O. Suthienkul, J. Kozawa, Y. Yamaichi, K. Yamamoto,and T. Honda. 1998. Close proximity of the tdh, trh and ure genes on thechromosome of Vibrio parahaemolyticus. Microbiology 144:2517–2523.

9. Iida, T., K. S. Park, and T. Honda. 2006. Vibrio parahaemolyticus, p. 340–348.In F. L. Thompson, B. Austin, and J. Swings. (ed.), The biology of vibrios.ASM Press, Washington, DC.

10. Izutsu, K., K. Kurokawa, K. Tashiro, S. Kuhara, T. Hayashi, T. Honda, andT. Iida. 2008. Comparative genomic analysis using microarray demonstratesa strong correlation between the presence of the 80-kilobase pathogenicityisland and pathogenicity in Kanagawa phenomenon-positive Vibrio para-haemolyticus strains. Infect. Immun. 76:1016–1023.

11. Kishishita, M., N. Matsuoka, K. Kumagai, S. Yamasaki, Y. Takeda, and M.Nishibuchi. 1992. Sequence variation in the thermostable direct hemolysin-

related hemolysin (trh) gene of Vibrio parahaemolyticus. Appl. Environ. Mi-crobiol. 58:2449–2457.

12. Kodama, T., Y. Akeda, G. Kono, A. Takahashi, K. Imura, T. Iida, and T.Honda. 2002. The EspB protein of enterohaemorrhagic Escherichia coliinteracts directly with alpha-catenin. Cell Microbiol. 4:213–222.

13. Kodama, T., M. Rokuda, K. S. Park, V. V. Cantarelli, S. Matsuda, T. Iida,and T. Honda. 2007. Identification and characterization of VopT, a novelADP-ribosyltransferase effector protein secreted via the Vibrio parahaemo-lyticus type III secretion system 2. Cell Microbiol. 9:2598–2609.

14. Kodama, T., H. Hiyoshi, K. Gotoh, Y. Akeda, S. Matsuda, K. S. Park, V. V.Cantarelli, T. Iida, and T. Honda. 2008. Identification of two transloconproteins of Vibrio parahaemolyticus type III secretion system 2. Infect. Im-mun. 76:4282–4289.

15. Livermans, A. D., H. C. Cheng, J. E. Trosky, D. W. Leung, M. L. Yarbrough,D. L. Burdette, M. K. Rosen, and K. Orth. 2007. Arp2/3-independent as-sembly of actin by Vibrio type III effector VopL. Proc. Natl. Acad. Sci. USA104:17117–17122.

16. Makino, K., K. Oshima, K. Kurokawa, K. Yokoyama, T. Uda, K. Tagomori,Y. Iijima, M. Najima, M. Nakano, A. Yamashita, Y. Kubota, S. Kimura, T.Yasunaga, T. Honda, H. Shinagawa, M. Hattori, and T. Iida. 2003. Genomesequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct fromthat of V cholerae. Lancet 361:743–749.

17. Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector and its usein construction of insertion mutations: osmoregulation of outer membraneproteins and virulence determinants in Vibrio cholerae requires toxR. J.Bacteriol. 170:2575–2583.

18. Murphy, R. A., and E. F. Boyd. 2008. Three pathogenicity islands of Vibriocholerae can excise from the chromosome and form circular intermediates. J.Bacteriol. 190:636–647.

19. Nishibuchi, M., and J. B. Kaper. 1990. Duplication and variation of thethermostable direct haemolysin (tdh) gene in Vibrio parahaemolyticus. Mol.Microbiol. 4:87–99.

20. Nishibuchi, M., A. Fasano, R. G. Russell, and J. B. Kaper. 1992. Entero-toxigenicity of Vibrio parahaemolyticus with and without genes encodingthermostable direct hemolysin. Infect. Immun. 60:3539–3545.

21. Nishibuchi, M., and J. B. Kaper. 1995. Thermostable direct hemolysin geneof Vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium.Infect. Immun. 63:2093–2099.

22. Oelschlaeger, T. A., and J. Hacker. 2004. Impact of pathogenicity islands inbacterial diagnostics. APMIS 112:930–936.

23. Ono, T., K. S. Park, M. Ueta, T. Iida, and T. Honda. 2006. Identification ofproteins secreted via Vibrio parahaemolyticus type III secretion system 1.Infect. Immun. 74:1032–1042.

24. Park, K. S., T. Iida, Y. Yamaichi, T. Oyagi, K. Yamamoto, and T. Honda.2000. Genetic characterization of DNA region containing the trh and uregenes of Vibrio parahaemolyticus. Infect. Immun. 68:5742–5748.

25. Park, K. S., T. Ono, M. Rokuda, M. H. Jang, T. Iida, and T. Honda. 2004.Cytotoxicity and enterotoxicity of the thermostable direct hemolysin-dele-tion mutants of Vibrio parahaemolyticus. Microbiol. Immunol. 48:313–318.

26. Park, K. S., T. Ono, M. Rokuda, M. H. Jang, K. Okada, T. Iida, and T.Honda. 2004. Functional characterization of two type III secretion systems ofVibrio parahaemolyticus. Infect. Immun. 72:6659–6665.

27. Parks, A. R., and J. E. Peters. 2007. Transposon Tn7 is widespread in diversebacteria and forms genomic islands. J. Bacteriol. 189:2170–2173.

28. Sakazaki, R., K. Tamura, T. Kato, Y. Obara, and S. Yamai. 1968. Studies onthe enteropathogenic, facultatively halophilic bacterium, Vibrio parahaemo-lyticus. 3. Enteropathogenicity. Jpn. J. Med. Sci. Biol. 21:325–331.

29. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, NY.

30. Shirai, H., H. Ito, T. Hirayama, Y. Nakamoto, N. Nakabayashi, K. Kumagai,Y. Takeda, and M. Nishibuchi. 1990. Molecular epidemiologic evidence forassociation of thermostable direct hemolysin (TDH) and TDH-relatedhemolysin of Vibrio parahaemolyticus with gastroenteritis. Infect. Immun.58:3568–3573.

31. Sugiyama, T., T. Iida, K. Izutsu, K. S. Park, and T. Honda. 2008. Preciseregion and the character of the pathogenicity island in clinical Vibrio para-haemolyticus. J. Bacteriol. 190:1835–1837.

32. Tagomori, K., T. Iida, and T. Honda. 2002. Comparison of genome struc-tures of vibrios, bacteria possessing two chromosomes. J. Bacteriol. 184:4351–4358.

33. Takeda, Y. 1982. Thermostable direct hemolysin of Vibrio parahaemolyticus.Pharmacol. Ther. 19:123–146.

34. Xu, M., K. Yamamoto, and T. Honda. 1994. Construction and characteriza-tion of an isogenic mutant of Vibrio parahaemolyticus having a deletion in thethermostable direct hemolysin-related hemolysin gene (trh). J. Bacteriol.176:4757–4760.

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