INFECTION AND IMMUNITY, July 2010, p. 3280–3287 Vol. 78, No. 70019-9567/10/$12.00 doi:10.1128/IAI.00050-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Effect on Human Cells of Environmental Vibrio parahaemolyticusStrains Carrying Type III Secretion System 2�

Greta Caburlotto,1 Maria M. Lleo,1* Tamara Hilton,2 Anwar Huq,3Rita R. Colwell,3 and James B. Kaper2

Dipartimento di Patologia, Sezione di Microbiologia, Universita di Verona, Strada Le Grazie 8, 37134 Verona, Verona, Italy1;Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore,

Maryland 212012; and Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland 207423

Received 15 January 2010/Returned for modification 19 February 2010/Accepted 4 May 2010

Vibrio parahaemolyticus is an inhabitant of estuarine and marine environments that causes seafood-bornegastroenteritis worldwide. Recently, a type 3 secretion system (T3SS2) able to secrete and translocate virulencefactors into the eukaryotic cell has been identified in a pathogenicity island (VP-PAI) located on the smallerchromosome. These virulence-related genes have previously been detected only in clinical strains. Classicalvirulence genes for this species (tdh, trh) are rarely detected in environmental strains, which are usuallyconsidered to lack virulence potential. However, during screening of a collection of environmental V. parah-aemolyticus isolates obtained in the North Adriatic Sea in Italy, a number of marine strains carrying virulence-related genes, including genes involved in the T3SS2, were detected. In this study, we investigated thepathogenic potential of these marine V. parahaemolyticus strains by studying their adherence ability, theircytotoxicity, their effect on zonula occludin protein 1 (ZO-1) of the tight junctions, and their effect ontransepithelial resistance (TER) in infected Caco-2 cells. By performing a reverse transcription-PCR, we alsotested the expression of the T3SS2 genes vopT and vopB2, encoding an effector and a translocon protein,respectively. Our results indicate that, similarly to clinical strains, marine V. parahaemolyticus strains carryingvopT and vopB2 and that other genes included in the VP-PAI are capable of adhering to human cells and ofcausing cytoskeletal disruption and loss of membrane integrity in infected cells. On the basis of data presentedhere, environmental V. parahaemolyticus strains should be included in coastal water surveillance plans, as theymay represent a risk for human health.

Vibrio parahaemolyticus is an halophilic inhabitant of estua-rine and marine environments and a leading cause of seafood-borne gastroenteritis worldwide, frequently due to ingestion ofuncooked shellfish.

The pathogenicity of V. parahaemolyticus classically has beencorrelated with production of haemolytic toxins TDH andTRH, the first toxin being responsible for the Kanagawa phe-nomenon (6, 8, 14, 17, 27). TDH causes a number of cytotoxiceffects, including erythrocyte lysis, disruption of the microtu-bule cytoskeleton, ion influx into cultured cells, cell rounding,and disruption of epithelial barrier function (4, 7, 22). Less isknown about the targets of TRH, although studies with thepurified protein have shown that the toxin induces lysis oferythrocytes and fluid accumulation in the rabbit ileal loopmodel (6, 23). Kanagawa-positive V. parahaemolyticus strainscarrying the tdh gene show a very high capability of adhering tohuman intestinal cells (5) and compromising the integrity ofthe epithelial barrier. The loss of membrane integrity, whichcan be monitored by measuring the transepithelial resistance(TER), may contribute to the diarrhea associated with V. para-haemolyticus infections, similar to the effects caused by otherenterovirulent bacteria.

Some studies examining the contribution of TDH and TRH

to V. parahaemolyticus virulence have focused on effects in-duced by the two individual purified toxins that cause drasticdisruption of the epithelial barrier function (4, 16, 22, 23).However, Lynch et al. (11) reported that destruction of theepithelial barrier can occur independently of toxin production,as shown from studies conducted on strains lacking the tdh andtrh genes. In addition, some strains lacking classical virulencefactors caused a decrease in TER values in eukaryotic cells,accompanied by an increase in paracellular permeability asso-ciated with dramatic disruption of the actin cytoskeleton. Inthe same report (11), it was demonstrated that purified TDHprotein was not able to alter the epithelial barrier. Lynch andcollaborators concluded that previous reports indicating a rolefor TDH in cytoskeleton disruption resulted from use of ele-vated toxin concentrations, seemingly higher than those en-countered in the bacterial culture supernatant (11). A role forTDH and/or TRH was not excluded, but it was suggestedthat these toxins may exert their effects at later stages ofinfection. These data, together with other data obtained inrecent years (12, 19, 28), indicated that TDH or TRH is notthe only bacterial factor determining V. parahaemolyticusvirulence and that other factors might contribute to thepathogenic potential directed to human hosts. A number ofthese factors have been subsequently detected and wellcharacterized (10, 12, 20, 28).

Genome sequencing of the V. parahaemolyticus clinicalstrain RIMD2210633 revealed that this bacterial species pos-sesses two sets of type 3 secretion system (T3SS) genes: T3SS1,located on chromosome 1, and T3SS2, located on chromosome

* Corresponding author. Mailing address: Dipartimento di Patolo-gia, Sezione di Microbiologia, Universita di Verona, Strada Le Grazie8, 37134 Verona, Italy. Phone: 39 045 8027194. Fax: 39 045 8027101.E-mail: maria.lleo@univr.it.

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2 (12). T3SS1 genes display high similarity with those fromYersinia T3SS, while T3SS2 genes are only partially similar toother bacterial secretion systems reported to date (20). Fol-lowing this discovery, a number of studies have been conductedto elucidate the role of these genes in the pathogenicity process(1, 9, 10, 20). Using in vitro human cell lines and ileal loop tests,it was shown, using clinical strains, that T3SS1 is responsiblefor the cytotoxicity of V. parahaemolyticus for eukaryotic cells(20) while T3SS2 is involved in cytotoxicity and enterotoxicity(9, 20).

A good model for in vitro studies on the intestinal barrier isrepresented by the human intestinal Caco-2 cell line, which hasbeen used to test the cytotoxicity caused by V. parahaemolyti-cus. Although the cytotoxicity of this bacterial species to hu-man cells has been clearly proven (22), the virulence factorsinvolved in this phenomenon have not been fully described. AV. parahaemolyticus mutant strain with both copies of tdh de-leted, which completely abolished the hemolytic activity, stillshowed fluid accumulation in the rabbit intestine, suggestingthat an unknown virulence factor(s) may be involved in thetoxicity caused by V. parahaemolyticus (21).

Until recently, no data were available on secretion systemgenes in environmental V. parahaemolyticus and those strainswere assumed to lack the T3SS2 system (12). For this reason,the recently reported finding that some environmental strainscan carry a T3SS2 system (3) represents an interesting discov-ery regarding the potential virulence of marine bacterialstrains. In the current study, we investigated the pathogenicpotential of environmental V. parahaemolyticus strains isolatedfrom the Venice Lagoon area in the Northern Adriatic Sea(Italy) by analyzing their ability to adhere to eukaryotic cells,the damage they might cause to the cell structures, and theircytotoxic effect on these cells.

MATERIALS AND METHODS

Bacterial strains and growth conditions. A collection of 59 environmental V.parahaemolyticus strains isolated from the North Adriatic Sea (Italy) was ana-lyzed in this study concerning the pathogenicity potential.

The entire collection was screened for adherence and cytotoxicity (Table 1). Asubset of six strains (see Table 1 and below), representing the different classes ofenvironmental strains and carrying specific virulence-related genes, were se-lected and used to further evaluate the pathogenic potential of the marine strains

(tests on transepithelial resistance and cell structural damage). Two of thesestrains possessed the tdh and orf8 genes that have been considered markers of thepandemic O3:K6 V. parahaemolyticus strain (15). These two strains also con-tained a sequence variation in the toxRS gene known as toxRS/new that is alsoconsidered to be a marker for pandemic strains (18). The six strains subjected tothe more extensive characterization were as follows.

(i) Two strains (VPeVEpan and VPeVEpan2) carrying pandemic markers tdhand orf8 (13, 15) and the toxRS/new gene (18) and isolated from water andplankton, respectively, at the site Caleri in the Venetian Lagoon (2).

(ii) Two strains (VPe23 and VPe28, both tdh negative and trh negative)representing strains containing T3SS2 genes such as vopB2 and vscC2, encodingtranslocation proteins (10, 26), vopT and vopP, encoding effector proteins (9, 26),and other genes located on the pathogenicity island (VP-PAI), such as vopC,encoding a gene homologous to the Escherichia coli cytotoxic necrotizing factor,and VPA1376, encoding a protein homologous to Vibrio cholerae VPI protein(12).

(iii) Two strains (VPe93 and VPe122, negative for tdh, trh, and T3SS2 genes)representative of strains not carrying the considered virulence-related genes.

Bacteria were grown in tryptic soy broth (TSB) (Difco Laboratories, Detroit,MI) supplemented with 1% NaCl at 37°C, unless otherwise indicated. Specialgrowth conditions for the different types of experiments are described in thefollowing protocols. Cell growth was monitored with an LKB spectrophotometerat a 640-nm wavelength (optical density of 640 nm [OD640]).

Detection of mRNA by RT-PCR. Experiments were conducted with bacterialstrains grown in two different media, Luria-Bertani (LB) broth supplementedwith 3% NaCl and Dulbecco’s modified Eagle medium (DMEM) supplementedwith 5% fetal bovine serum. For RNA extraction and DNase treatment, theRNase minikit (Qiagen) was used. A quantity of extracted RNA, ranging be-tween 2.5 and 5 �g, was reverse transcribed using Superscript III First-Strandsynthesis system (Invitrogen, according to the manufacturer’s instructions). Foreach reverse transcription (RT) reaction, an RT-negative (RT�) control includ-ing all components except the SuperScriptIII RT enzyme was prepared. Thefirst-strand cDNA obtained was amplified directly by PCR, using the Easy-Ahigh-fidelity PCR cloning enzyme (5 U/�l).

Adherence test. Bacterial cells were grown in brain heart infusion broth con-taining 0.5% mannitol and 3% NaCl (BHIM3) and incubated at 37°C withoutshaking until an optical density of ca. 0.8 to 0.9 (A620) was obtained. For theseexperiments, HeLa cells were grown in monolayer in Dulbecco’s modified Eaglemedium supplemented with 10% fetal bovine serum and 1% mannose (DMEM/F12) and used at passage level 22 to 25. After the HeLa cells were washed withphosphate-buffered saline (PBS), bacterial cells were added to the medium at amultiplicity of infection (MOI) of 10:1, mixed, and incubated at 37°C for twodifferent periods of time, 30 and 60 min. After incubation, cells were washedthree times with PBS, fixed in 70% methanol for 10 min, and Giemsa stained for30 min. Cells were washed three times with PBS and dried. Finally, the coverslipswere removed from the wells, assembled on the microscope slides, and examinedmicroscopically to enumerate adherent bacteria.

For each bacterial strain tested, 100 HeLa cells were observed, on average, atthe microscope and the number of bacteria adhering to each of the eukaryoticcells was counted. At least two separate experiments were conducted in dupli-

TABLE 1. Bacterial strains analyzed in this study grouped according to the specific genetic virulence profile observed

Bacterial strainsd Virulence genes detected Adherence capabilitya

(no. of strains)Cytotoxicityb

(no. of strains)

QM97097 (O3:K6 reference strain) tdh toxRS/new orf8 VP0394,c T3SS2 genes Very good Very highVPeVEpan, VPeVEpan2 tdh toxRS/new orf8 VP0394c vopP vcsC2

vopB2 vopT vopC VPA1376cVery good (2) High (2)

VPe23, VPe28, VPe6, VPe19, VPe167, VPe8,VPe3, VPe25, VPe2, VPe26, VPe27

vopP vcsC2 vopB2 vopT vopC VPA1376c Very good-good (7) Medium (10)Medium (4) Low (1)

VPe17, VPe24, VPe15, VPe10, VPe1, VPe11,VPe31, VPe35

trh ure Very good-good (3) High (1)Medium (5) Low (7)

VPe27, VPe4, VPe19, VPe32, VPe20, VPe130 VP0394c Good (3) Medium (1)Medium (3) Low (5)

VPe93, VPe122, and 30 other strains None of the considered virulence genes Good (11) Medium (3)Medium (16) Low (29)Poor/no adherence (5)

a Adherence phenotypes: very good, 11 to 15 bacteria/cell; good, 7 to 10 bacteria/cell; medium, 3 to 6 bacteria/cell; and poor/no adherence, 0 to 2 bacteria/cell.b Cytotoxicity phenotypes (in percent cytotoxicity): very high, 80 to 90%; high, 60 to 80%; medium, 45 to 55%; and low, 25%.c VP0394 encodes a DNA methyltransferase while VPA1376 codes for a homologue of a V. cholerae pathogenicity island gene.d Strains used in the tests regarding transepithelial resistance and cell structural damage are shown in bold.

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cate, and three different examiners read the slides. After the averaged number ofadhered bacteria/cell was calculated for each one of the strains, the adherenceindex was determined by referring to one of the four defined ranges: 11 to 15bacteria/cell, 7 to 10 bacteria/cell, 3 to 6 bacteria/cell, and 0 to 2 bacteria/cell.

Cytotoxicity test. For cytotoxicity assays, three separate cultures of bacterialcells were grown in TSB-1% NaCl, pH 8.2, at 37°C overnight with shaking.Caco-2 cells were seeded at 2 � 104 cells/well in 96-well plates and cultured for48 h to confluence. Cells were used between passages 18 and 24. Three differentCaco-2 cell monolayers were cocultured with PBS-washed bacteria at a multi-plicity of infection (MOI) of 10:1 for 5 h. The release of lactate dehydrogenase(LDH) into the medium was quantified using a Cytotox96 nonradioactive cyto-toxicity kit (Promega), following the manufacturer’s instructions. The resultsrepresent the means of six independent determinations, with bars showing onestandard deviation.

Because different types of target cells release different amounts of LDH, apreliminary experiment was performed comparing LDH released from Caco-2cells and other cell lines (T84 intestinal cells, HCT-8 cells, HeLa cell line), usingdifferent concentrations of human cells to determine the most appropriate con-ditions for in vitro study.

Measurement of transepithelial resistance (TER). Transwell (12-well polycar-bonate transwell filter support, 0.4-�m pore size, and 12-mm diameter; CorningInc., NY) filters were treated with sterile type I collagen (10 �l of collagen, 0.5mg/ml on each coverslip) and sterilized under UV light overnight before Caco-2cells were added to the apical compartment of each well. Caco-2 cells wereseeded at an initial density of 8 � 104 cells/cm2 at passage level 18 to 24 inDMEM supplemented with 10% fetal bovine serum, 100 U ml�1 penicillin, 100mg ml�1 streptomycin, 1� nonessential amino acids (NEAA), and 1 mM sodiumpyruvate. The bacterial cells were added gently drop by drop (final volume, 500�l), and 1.5 ml of the same medium was added to the basolateral compartment.The cells were incubated at 37°C in a 5% CO2 atmosphere until polarizedmonolayers were formed, approximately 21 days, and the medium was changedevery second day. After 10 days, the TER was tested using an EVOM-G ohm-meter (World Precision Instruments).

The TER was successively tested after 21 days in the first experiment, 22 daysin the second experiment, and 25 days in the third experiment. Only wellsdisplaying baseline resistance readings greater than 350 �/cm2 were used forexperiments. Before the experiments were started, the instrument was preparedso that the resistance range was approximately 2,000 � in order to have readingsof 100 �, 200 �, etc. In every experiment a blank (Transwell without cells) wasused and measured hourly. A volume of 2 �l of V. parahaemolyticus overnightculture was added to the upper chamber of each well, and TER measurementswere obtained hourly. Also, the TER of two transwells containing uninfectedcells was measured hourly. For this experiment, strains were grown overnight inLB broth supplemented with 3% NaCl at 37°C with shaking.

Evaluation of cell structural damage by immunofluorescence microscopy.Caco-2 cells were seeded at an initial density of 8 � 104 cells/cm2 onto type Icollagen-coated (0.5 mg/ml) coverslips and incubated at 37°C in 24 wells in thepresence of 5% CO2 for 5 days in high-glucose medium (DMEM supplementedwith 5% fetal bovine serum, 1� NEAA, and 1 mM sodium pyruvate). Type Icollagen-coated coverslips were previously sterilized by overnight incubationunder UV. The medium was removed from the wells, and each well was gentlywashed with sterile phosphate-buffered saline (PBS) three times and filled with500 �l of medium. Cells were infected with 2 �l of an overnight culture ofbacteria grown in LB broth supplemented with 3% NaCl at 37°C with shaking.Cells were incubated at 37°C for two different periods of time, 3 h and 6 h.Following infection, coverslips were washed three times with PBS for 5 min andfixed in 2.5% paraformaldehyde for 10 min at 37°C, after which the cells wererinsed with PBS supplemented with 0.5% Triton X-100 three times before beingblocked for 30 min with 1% normal goat serum in 500 �l PBS/well, supplementedwith 0.1% Triton X-100. The cells were incubated for 45 min with rabbit anti-zonula occludin protein 1 (ZO-1) (N-term) (diluted 1:80; Zymed, San Francisco,CA) in PBS supplemented with 1% normal goat serum and 0.2% bovine serumalbumin. The cells were washed three times with PBS and labeled with Alexa-Fluor 568 goat anti-rabbit antibody (diluted 1:80; Molecular Probes) in PBSsupplemented with 1% normal goat serum and 0.2% bovine serum albumin foran incubation period of 30 min. Cells were washed three times with PBS andlabeled to detect actin protein with Alexa-Fluor 488 phalloidin (diluted 1:200;Molecular Probes) in PBS for an incubation period of 20 min. The coverslipswere washed with PBS three times for 5 min, dried, coated with ProLongantifade kit, and dried overnight covered by foil. Finally, samples were visualizedusing an inverted microscope (Nikon Eclipse TE 2000-E).

Statistical analysis. The results were statistically analyzed with analysis ofvariance (ANOVA) at the P � 0.05 significance level with the SPSS software.

Data corresponding to adherence and cytotoxicity assays were also comparedusing the Student’s t test with a significance level of P � 0.01.

RESULTS

Expression of type 3 secretion system genes carried by V.parahaemolyticus environmental strains. In a recent study, wedemonstrated the presence of T3SS2 and other virulence-re-lated genes in a number of environmental V. parahaemolyticusstrains isolated in the North Adriatic Sea (Italy) in the area ofthe Venetian Lagoon (3). In addition to T3SS2 genes (vopB2,vscC2, vopT, and vopP) and genes vopC and VPA1376 encodingproteins homologous to E. coli and V. cholerae virulence fac-tors, the virulence genes included in the environmental strainscreening were trh and ure encoding TRH and urease, respec-tively, and the virulence-related open reading frame (ORF)gene VP0394, carried on a 22-kb pathogenicity island-like ele-ment located on chromosome I and whose predicted proteinproduct has homology to a DNA methyltransferase protein(28). Since the presence of a gene in a bacterial genome doesnot ensure that this gene is functional, it is necessary to dem-onstrate gene transcription by detecting the correspondingmRNA using reverse transcriptase PCR or other methods.

In a previous report (3), the transcription of the genes vopCand vcsC2 was tested. In the present study, we focused on theexpression of two other T3SS2 genes, vopT and vopB2, whichencode proteins that function as effector and translocon, re-spectively, as recently described (10). These two genes havebeen detected in the two marine pandemic strains and 12environmental strains carrying only the secretion system. Thetranscription of these two genes was evaluated in a V. para-haemolyticus strain carrying the genetic pandemic markers tdh,orf8, and toxRS/new and some T3SS2 genes (VPeVEpan), inthe strain VPe23 carrying only the T3SS2 genes, and in thereference pandemic strain QM97097 (see Table 1). The genegyrB was included in the study as a constitutively expressedhousekeeping gene.

As demonstrated previously (3) for genes vopC and vcsC2,the results obtained indicate that vopT and vopB2 are tran-scribed in the three V. parahaemolyticus strains, under ourexperimental conditions, as shown by RT-PCR analysis andresulting in production of amplified corresponding cDNA(Fig. 1).

Adherence capability of the V. parahaemolyticus environmen-tal strains. An initial step in the pathogenic process for manybacteria is adherence to the host cell before delivering toxinsor causing damage to the cell structure. Earlier studies re-ported the capability of Kanagawa phenomenon (KP)-positivestrains to adhere to intestinal cells (5). Therefore, we investi-gated whether environmental strains from our study were ca-pable of adhering to human cells.

Preliminary experiments were performed to identify the op-timal infection period that allowed bacterial adherence to hu-man cells. An infection time of 60 min was found to be optimal,since a longer period of incubation resulted in nonspecificattachment to the coverslips by most of the strains, making itdifficult to evaluate the specific capability to adhere to humancells.

Evaluation of adherence capability of the strains was basedon an arbitrary designed adhesion index, considering that max-

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imum adherence (very good adherence, 11 to 15 bacteria/cell)corresponded to the capability to adhere to eukaryotic cellsof the reference strain QM97097 (KP-positive clinicalstrain). Other adhesion ranges were defined as good adhe-sion (7 to 10 bacteria/cell), medium adhesion capability (3 to6 bacteria/cell), and poor or no adhesion capability (0 to 2bacteria/cell).

Of the 59 environmental strains tested for adherence toHeLa cells, 13% showed very strong adherence, 30% goodadherence, 48% medium adherence, and 8% poor or no ad-herence. Among strains showing very strong or good adher-ence, 54% carried virulence-related genes, while among strainsshowing medium or poor/no adherence, only 37% carried vir-ulence-related genes (Table 1).

Results for strains representative of the entire screened col-lection are shown in Fig. 2. As seen in this figure, the strain notcarrying virulence-related genes (VP393) showed poor adher-ence to HeLa cells, compared to the strain carrying the T3SS2genes (VPe23), which showed good capability to adhere tohuman cells. This effect was more evident in the VPeVEpanstrain, which showed an adherence capability similar to that ofthe reference clinical strain QM97097. Adherence phenotypes

shown by the other strains are presented in Table 1. It shouldbe noted, however, that some strains lacking virulence genesshowed good adherence (Table 1), indicating that adhesion isa necessary but not sufficient condition for bacteria to succeedin the infection process. The results indicate that there is not astrict correlation between presence or absence of the genestested in this study and adherence.

Cytotoxic effect of environmental V. parahaemolyticus strainson Caco-2 cells. The cytotoxicity analysis was performed bymeasuring release of lactate dehydrogenase (LDH) by Caco-2cells infected with the different environmental strains (LDH isreleased upon cell lysis).

In order to use a consistent MOI (multiplicity of infection)index of 10 bacteria:1 cell in each experiment, standard growthcurves were prepared.

Results shown in Fig. 3 indicate that, compared to the ref-erence strain, the highest cytotoxic activity was detected in thestrain VPeVEpan containing tdh and T3SS2 genes, a lower butstill relevant activity in strains carrying T3SS2 but not tdh(VPe23), and very low cytotoxic activity in strains not carryingvirulence-associated genes (VPe93). The results represent themeans of six independent determinations, with bars showingone standard deviation. As shown in Table 1, the same exper-iment conducted with strains belonging to the different groupsgave similar results, without statistically significant differences(Student’s t test), compared with the corresponding represen-tative strains presented in the figure.

Effect on cell permeability (TER) of epithelial cells infectedwith bacterial strains possessing or lacking virulence genes.The effects of infection by environmental V. parahaemolyticusstrains on epithelial barrier integrity were examined by mea-suring TER (transepithelial resistance) across a Caco-2 cellmonolayer.

As shown in Fig. 4, at time zero (T0), cells infected with anyof the strains tested gave epithelial resistance values of ca. 400�. During the first hour of infection (T1), the values decreaseddramatically (70 to 80 �) in all cases except uninfected cells(negative control) and cells infected with strains lacking viru-lence-related genes, for which the values slightly decreased(only by a few ohms). The TER values of uninfected cellsremained fairly constant, showing a slight decrease of about100 � over 7 h (Fig. 4), and cells infected with strains lackingvirulence-related genes decreased slightly more, about 150 �over 7 h. Strains VPeVEpan and Vpe23 (both containingT3SS2) showed a gradual decrease after the first hour, whichbecame more marked in the VpeVEpan strain by the thirdhour of infection (T3) and then remained roughly constantduring the rest of the experiment. The KP-positive referencestrain QM97097 induced a gradual decrease in resistance valueuntil T6 (about 4 h of infection), and then resistance droppeddramatically for the remaining 3 h.

Alterations induced on eukaryotic cell tight junctions by V.parahaemolyticus strains possessing or lacking virulence genes.Alterations in TER are often accompanied by changes in lo-calization of the tight junction proteins. The effect of infectingCaco-2 cells with environmental V. parahaemolyticus strainscarrying virulence-related genes on localization of ZO-1 (pe-ripheral protein) and filamentous actin was examined usingimmunofluorescence microscopy.

In uninfected cells, the protein ZO-1 and filamentous actin

FIG. 1. Amplification products obtained by reverse transcription ofgyrB (housekeeping gene) (A), vopT (encoding effector T3SS2) (B),and vopB2 (encoding part of the translocon T3SS2) (C). Each panelcontains a molecular marker in the left lane with six sample lanes asfollows: lanes 1 and 2, VPeVEpan; lanes 3 and 4, VPe23; lanes 5 and6, AN2416 (reference KP-positive strain, given by R. Y. C. Kong).Lanes 1, 3, and 5 are RT�; lanes 2, 4, and 6 are RT�.

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were located at the periphery of the apical side of the cellmonolayer; the location of both proteins revealed the integrityof the cells showing intact and relaxed borders (Fig. 5A). Thesame localization of ZO-1 and filamentous actin was observedin cells infected with strains lacking virulence-related genes(Fig. 5B). No disruption of the cell monolayer was observed,confirming the absence of cytotoxicity in cells infected withthese strains. The only detectable effect on the cells was inborder shape, which assumed a curly profile. In contrast, whencells were infected with the KP-positive pandemic strain usedas a positive reference strain, the cell monolayer was disrupted

and presented a discontinuity in the peripheral borders of thecells, and a complete rearrangement of the cytoskeleton wasseen (Fig. 5D). This result was expected, since the capability ofstrains producing both TDH and TRH toxins to disrupt the cellbarrier is well established.

Infection of cell monolayers with environmental strains car-rying T3SS2 genes (VPe23 and VPe28) resulted in a cleardisruption of ZO-1 and filamentous actin (Fig. 5C). The dis-ruption was even stronger when the infection was induced bythe VPeVEpan strain carrying both T3SS2 and tdh, comparedto that induced by strains carrying only T3SS2; the cytoskele-

FIG. 2. Adherence test micrographs showing HeLa cells infected with representative environmental V. parahaemolyticus strains. (A) KP-positive clinical strain (QM 97097); (B) VPeVEpan (tdh� T3SS2�); (C) strain not carrying virulence-related genes (VPe93); (D) strain carryingonly T3SS2 genes (VPe23); (E) trh-positive environmental V. parahaemolyticus strain (VPe10).

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ton completely lost integrity, and drastic rearrangement ofactin was observed (Fig. 5E and F). Moreover, in experimentsperformed with strains carrying virulence-related genes, mostof the cells detached from the coverslip, indicating cytotoxicitycaused by the infecting bacterial cells and the capability ofthese bacteria to produce virulence proteins.

DISCUSSION

The pathogenicity of V. parahaemolyticus classically hasbeen correlated with production of hemolytic toxins TDHand TRH, the first toxin being responsible for the Kanagawaphenomenon.

A number of data obtained in recent years (12, 19, 28)indicated that TDH or TRH is not the only bacterial factordetermining V. parahaemolyticus virulence and that other fac-

tors might contribute to the pathogenic potential directed tohuman hosts. Several of these factors have been successivelydetected and well characterized (10, 12, 20, 28), as is the roleof secretion systems in mediating the delivery of bacterial vir-ulence proteins to eukaryotic cells. In V. parahaemolyticus ithas been demonstrated that at least two sets of T3SS genesexist: T3SS1, located on chromosome 1 and responsible for thecytotoxicity of the bacterial species for eukaryotic cells (20),and T3SS2, shown to be involved in cytotoxicity and entero-toxicity (9, 10, 20). Other genes have been proposed as beingassociated with the V. parahaemolyticus pathogenic potential,e.g., urease and methyltransferase (3).

To evaluate if a direct correlation exists between the pres-ence of virulence genes, such as tdh, trh, the T3SS2 genes, andother virulence-associated genes, and the potential capabilityof environmental strains to cause infection in humans, theeffective pathogenic potential of some marine V. parahaemo-lyticus strains isolated from the Italian coastline was analyzedby studying the in vitro interaction with human cells, includingadherence capability, cytotoxicity, and the ability to inducestructural alterations in infected cells.

An initial step in this study, the transcription of T3SS2, wasconfirmed via RT-PCR, showing that two T3SS2 genes, vopT,encoding a homolog of the Pseudomonas exoenzyme T andcontributing to cytotoxicity, and vopB2, encoding a transloca-tor involved in the contact-dependent activity of pore forma-tion in infected cells, are expressed under standard growthconditions. Transcription of other T3SS2 genes carried bystrains from the same environmental V. parahaemolyticus col-lection was previously reported (3).

Our data on the adherence phenotype of more than 50environmental strains indicate that there is not an unequiv-ocal direct correlation between the presence of tdh, trh,T3SS2, or other virulence genes and the ability of the envi-ronmental strains to adhere to human cells. Hence, adher-ence, demonstrated under conditions employed in thisstudy, does not seem to be a pathogenic factor specific for V.parahaemolyticus isolates carrying virulence genes. This re-sult is consistent with other data in the literature (25) and

FIG. 3. Percent cytotoxic activity, measured as LDH concentrations released from Caco-2 cells infected with environmental V. parahaemolyticusstrains. From left to right: strain VPe93 without virulence genes (tdh and T3SS2 negative); strain VPe23 (tdh negative, T3SS2 positive) carryingonly the genes belonging to the secretion system; the pandemic strain VPeVEpan (tdh and T3SS2 positive) carrying the secretion system genes andthe tdh gene, and, in black, the KP-positive clinical strain (QM 97097) as a reference. The results represent the means of six independentdeterminations, with bars showing one standard deviation. The differences in cytotoxicity values between strain VPe23, strain VPe93, and theclinical strain QM 97097 are statistically significant (Student’s t test, P � 0.01).

FIG. 4. Transepithelial resistance values of Caco-2 cell monolay-ers infected with V. parahaemolyticus environmental strains. Bacte-rial strains used in this experiment were the pandemic strain VPe-pan, carrying the tdh and T3SS2 genes, the strain VPe23, carryingonly the T3SS2 genes, and the strain VPe93, as a representative ofstrains without virulence genes. A reference strain (QM97097) wasalso included. The same experiment conducted with strains VPe-pan2, VPe28, and VPe122 gave similar results without statisticallysignificant differences (Student’s t test) compared with the onepresented in the figure. Three separate experiments were conductedin duplicate, and results were averaged.

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indicates that adherence cannot be used as a discriminatoryelement to distinguish pathogenic and nonpathogenic V.parahaemolyticus strains.

To investigate further the pathogenic potential of envi-ronmental V. parahaemolyticus strains, the cytotoxicity ofstrains carrying and not carrying T3SS2 genes was evaluated.The cytotoxic effect of the environmental V. parahaemolyti-cus strains seems to be associated more with the TDH pro-tein, as shown by results obtained with strains VPeVEpanand VPeVEpan2 (a high level of cytotoxicity, similar to thatcaused by the reference pandemic strain QM97097), in com-parison to the strains carrying only the T3SS2 genes anddemonstrating a cytotoxicity half that of the referencestrain. Although a previous study on tdh-deprived mutantsexcluded a role for TDH in cytotoxicity (21), other reports(22, 24) have provided support for the cytotoxic effect ofTDH on human cells. Our results are in accordance with thelatter and, moreover, suggest an additive effect of T3SS2and TDH on the cytotoxic damage caused by the environ-mental strain VPeVEpan.

On the other hand, the cytotoxicity of strains not carryingvirulence genes was detectable, measured as release of LDH,although at a much lower level (25%). The possibility thatother genes not analyzed in this study are in some way involvedin the toxicity to eukaryotic cells cannot be excluded.

In the experiments performed using Caco-2 cell monolayers,two representative strains among those carrying T3SS2 (VPe23and VPe28) induced a decrease in TER values which wasassociated with a profound reorganization of the cytoskeleton:infection led to the redistribution and aggregation of actin andZO-1 within Caco-2 cells in accordance with previous reports(11) for strains lacking the tdh and trh genes. Because theability to cause disruption of tight junctions and a conse-quent decrease of TER values is very similar for strainscarrying only T3SS2 and those with the tdh gene, it appearsthat these effects are mainly associated with the secretionsystem, as reported for strains lacking TDH prior to theidentification of T3SS2 as a virulence factor (11). The effectson the cytoskeleton and tight junctions could be assumed tocomprise a phenotype distinguishing environmental V. para-

FIG. 5. Immunofluorescence micrographs of Caco-2 cells double labeled with Alexa488 phalloidin for filamentous actin (green) andAlexa568 for rabbit antibodies against ZO-1 (red). (A) Uninfected cells; (B) cells infected with a strain not carrying virulence-related genes(VPe93); (C) cells infected with a strain carrying T3SS2 genes (VPe23); (D) cells infected with KP-positive reference strain QM 97097; (Eand F) cells infected with the VPeVEpan strain (T3SS2 and tdh positive). Photographs are representative of the results obtained in twoseparate experiments.

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haemolyticus with pathogenic potential from nonpathogenicV. parahaemolyticus.

On the basis of data obtained from the experimentsdescribed here employing representative V. parahaemolyticusstrains, it can be concluded that the environmental strainsanalyzed, including those lacking the classical pathogenic fac-tors tdh and trh but carrying other virulence-associated genessuch as those belonging to T3SS2, showed at least some of thevirulence characteristics typical of clinical strains: they adhereefficiently to human cells and once in contact with humanintestinal cells cause disruption of the membrane tight junc-tions, compromising the intestinal barrier. We hypothesize arole for this secretion system in the pathogenicity of the marinestrains, but further studies are needed to provide direct evi-dence to support this hypothesis. The results reported in thispaper support the fact that environmental V. parahaemolyticusstrains might constitute a public health concern and a risk tohuman health and should be taken into consideration in waterquality surveillance plans.

ACKNOWLEDGMENTS

The V. parahaemolyticus environmental strains analyzed in this studywere isolated within the framework of the international researchproject VibrioSea, cofunded by the Centre National d’Etudes Spatiales(CNES), the Institut Pasteur, France, and the Universities of Veronaand Genoa, Italy. This project has been developed in the context of adoctorate fellowship, Cooperint2007, funded by the University of Ve-rona, Italy, and assigned to G. Caburlotto. This work was supported byNIH grant R01 AI 19716 to J. B. Kaper and partially by grantNBCH2070002 to R. R. Colwell received from the Department ofHomeland Security.

We thank Patricia B. Lodato and Jane Michalski (Department ofMicrobiology and Immunology, University of Maryland School ofMedicine, Baltimore, Maryland) for their suggestions and technicalsupport in the part concerning the expression of virulence-relatedgenes. We thank Alessio Fasano (University of Maryland School ofMedicine, Baltimore, Maryland) for the technical support in immuno-fluorescence microscopy.

REFERENCES

1. Akeda, Y., K. Okayama, T. Kimura, R. Dryselius, T. Kodama, K. Oishi, T.Iida, and T. Honda. 2009. Identification and characterization of a type IIIsecretion-associated chaperone in the type III secretion system 1 of Vibrioparahaemolyticus. FEMS Microbiol. Lett. 296:18–25.

2. Caburlotto, G., V. Ghidini, M. Gennari, M. C. Tafi, and M. M. Lleo. 2008.Isolation of a Vibrio parahaemolyticus pandemic strain from a marinewater sample obtained in the northern Adriatic. Eurosurveillance 13(11):pii�8068.

3. Caburlotto, G., M. Gennari, V. Ghidini, M. C. Tafi, and M. M. Lleo. 2009.Presence of virulence-related genes in tdh-negative Vibrio parahaemolyticusenvironmental strains isolated from marine samples in the area of the Ve-netian Lagoon, Italy. FEMS Microbiol. Ecol. 70:506–514.

4. Fabbri, A., L. Falzano, C. Frank, G. Donelli, P. Matarrese, F. Raimondi, A.Fasano, and C. Fiorentini. 1999. Vibrio parahaemolyticus thermostable directhemolysin modulates cytoskeletal organization and calcium homeostasis inintestinal cultured cells. Infect. Immun. 67:1139–1148.

5. Hackney, C. R., E. G. Kleeman, B. Ray, and M. L. Speck. 1980. Adherenceas a method for differentiating virulent and avirulent strains of Vibrio para-haemolyticus. Appl. Environ. Microbiol. 40:652–658.

6. Honda, T., Y. Ni, and T. Miwatani. 1988. Purification and characterization ofa hemolysin produced by a clinical isolate of Kanagawa phenomenon-nega-tive Vibrio parahaemolyticus and related to the thermostable direct hemoly-sin. Infect. Immun. 56:961–965.

7. Honda, T., Y. Ni, T. Miwatani, T. Adachi, and J. Kim. 1992. The thermo-stable direct hemolysin of Vibrio parahaemolyticus is a pore-forming toxin.Can. J. Microbiol. 38:1175–1180.

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

9. 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 parahae-molyticus type III secretion system 2. Cell. Microbiol. 9:2598–2609.

10. 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.

11. Lynch, T., S. Livingstone, E. Buenaventura, E. Lutter, J. Fedwick, A. G.Buret, D. Graham, and R. DeVinney. 2005. Vibrio parahaemolyticus disrup-tion of epithelial cell tight junctions occurs independently of toxin produc-tion. Infect. Immun. 73:1275–1283.

12. Makino, K., O. Kenshiro, 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.

13. Meador, C. E., M. M. Parsons, C. A. Bopp, P. Gerner-Smidt, J. A. Painter,and G. J. Vora. 2007. Virulence gene- and pandemic group-specific markerprofiling of clinical Vibrio parahaemolyticus isolates. J. Clin. Microbiol. 45:1133–1139.

14. Miyamoto, Y., T. Kato, S. Obra, S. Akiyama, K. Takizawa, and S. Yamai.1969. In vitro characteristic of Vibrio parahaemolyticus: its close correlationwith human pathogenicity. J. Bacteriol. 100:1147–1149.

15. Myers, M. L., G. Panicker, and A. K. Bej. 2003. PCR detection of a newlyemerged pandemic Vibrio parahaemolyticus O3:K6 pathogen in pure culturesand seeded waters from the Gulf of Mexico. Appl. Environ. Microbiol.69:2194–2200.

16. 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.

17. 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.

18. Okura, M., R. Osawa, A. Iguchi, E. Arakawa, J. Terajima, and H. Watanabe.2003. Genotypic analyses of Vibrio parahaemolyticus and development of apandemic group-specific multiplex PCR assay. J. Clin. Microbiol. 41:4676–4682.

19. 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.

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

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

22. Raimondi, F., J. P. Y. Kao, C. Fiorentini, A. Fabbri, G. Donelli, N. Gaspa-rini, A. Rubino, and A. Fasano. 2000. Enterotoxicity and cytotoxicity ofVibrio parahaemolyticus thermostable direct hemolysin in in vitro systems.Infect. Immun. 68:3180–3185.

23. Takahashi, A., N. Kenjyo, K. Imura, Y. Myonsun, and T. Honda. 2000. Cl�

secretion in colonic epithelial cells induced by the Vibrio parahaemolyticushemolytic toxin related to thermostable direct hemolysin. Infect. Immun.68:5435–5438.

24. Tang, G. Q., T. Iida, K. Yamamoto, and T. Honda. 1995. Ca(2�)-indepen-dent cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin(TDH) on Intestine 407, a cell line derived from human embryonic intestine.FEMS Microbiol. Lett. 134:233–238.

25. Vongxay, K., S. Wang, X. Zhang, B. Wu, H. Hu, Z. Pan, S. Chen, and W.Fang. 2008. Pathogenetic characterization of Vibrio parahaemolyticus iso-lates from clinical and seafood sources. Int. J. Food Microbiol. 126:71–75.

26. Vora, G. J., C. E. Meador, M. M. Bird, C. A. Bopp, J. D. Andreadis, andD. A. Stenger. 2005. Microarray-based detection of genetic heterogeneity,antimicrobial resistance, and the viable but nonculturable state in humanpathogenic Vibrio spp. Proc. Natl. Acad. Sci. U. S. A. 102:19109–19114.

27. Wagatsuma, S. 1968. A medium for the test of the haemolytic activity ofVibrio parahaemolyticus. Media Circle 13:159.

28. Wang, H. Z., M. M. Wong, D. O’Toole, M. M. Mak, R. S. Wu, and R. Y.Kong. 2006. Identification of a DNA methyltransferase gene carried on apathogenicity island-like element (VPAI) in Vibrio parahaemolyticus and itsprevalence among clinical and environmental isolates. Appl. Environ. Mi-crobiol. 72:4455–4460.

Editor: S. M. Payne

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