Microbial Pathogenesis 1996; 21: 111–123

Vibrio cholerae hemagglutinin/protease (HA/protease)causes morphological changes in cultured epithelialcells and perturbs their paracellular barrier function

Zhengyang Wu,1 Debra Milton,2 Pia Nybom,1 Anita Sjo1 and

Karl-Eric Magnusson1,∗

1Department of Medical Microbiology, Linkoping University, S-581 85, Linkoping,Sweden, 2Department of Applied Cell and Molecular Biology, University of Umea,S-901 87, Umea, Sweden

(Received January 15, 1996; accepted in revised form March 22, 1996)

Wu, Z. (Department of Medical Microbiology, Linkoping University, S-581 85, Linkoping,Sweden) D. Milton, P. Nybom, A. Sjo and K.-E. Magnusson. Vibrio cholerae hemagglutinin/protease (HA/protease) causes morphological changes in cultured epithelial cells andperturbs their paracellular barrier function. Microbial Pathogenesis 1996; 21: 111–123.

In this report, we describe the cytotoxic activity of the cholera hemagglutinin/protease(HA/protease). A concentrated protein sample from the 37°C overnight culture supernatantof CVD110, a DctxA, Dzot, DAce and hlyA::(ctxB mer) mutant of El Tor biotype Ogawaserotype strain E7946 caused morphological changes in cultured MDCK-I epithelial cells andaltered their arrangement of filamentous actin (F-actin) and Zonula occludens-associatedprotein ZO-1. The drastic morphological changes can be inhibited by Zincov, a specificbacterial metalloprotease inhibitor. The cytotoxic fractions of the sample after FPLC gel-filtration fractionation showed two visible protein bands with molecular weights of ap-proximately 34- and 32 kDa. Microsequencing of these two proteins revealed that theywere the cholera HA/protease. 1996 Academic Press Limited

Key words: Vibrio cholerae; MDCK-I; F-actin; ZO-1; electrical resistance; hemagglutinin;protease.

Introduction

Vibrio cholerae produces several hemagglutinins, and one of them is the solublehemagglutinin/protease (HA/protease).1,2 It is a member of a large family ofmetalloproteases widely distributed in pathogenic bacteria.3 The V. cholerae HA/protease cleaves several physiologically important substrates, including mucin,fibronectin and lactoferrin,4 and activates the A subunit of CT by nicking it to A1and A2 fragments.5 However, there is no clear evidence showing that it is directlyinvolved in the virulence of V. cholerae. One study suggests an indirect involvementby aiding the detachment of V. cholerae from intestinal epithelium, thus allowingfurther transmission of the bacteria.6

In this work, we demonstrate that the cholera HA/protease causes drasticmorphological changes in Madin Darby canine kidney cell line I (MDCK-I) cells,and rearranges their filamentous actin (F-actin) and Zonula occludens-associated

∗Author for correspondence.

0882–4010/96/080111+13 $18.00/0 1996 Academic Press Limited

Z. Wu et al.112

Fig. 1. Morphological changes of the MDCK-I cells. The pictures were taken at the same time, c.40 min after addition of the samples. Bar: 100 lm. Panel A shows MDCK-I cells treated with 10% (v/v)PBS (pH 7.3). Similar normal morphology was also observed in MDCK-I cells treated with 10% (v/v)concentrated protein samples from 37°C overnight culture supernatants of the E. coli strain DH-1, theV. cholerae strains CVD101 and 395 (data not shown). Panel B shows MDCK-I cells treated with 10%(v/v) concentrated 30°C overnight culture supernatant of CVD110. Panel C shows MDCK-I cells treatedwith 10% (v/v) concentrated 37°C overnight culture supernatant of CVD110. Panel D shows MDCK-Icells treated with 10% (v/v) concentrated 37°C overnight culture supernatant of E7946.

protein ZO-1. The cytotoxicity of the cholera HA/protease indicates that it mightparticipate in the pathogenesis of V. cholerae.

Results

Culture supernatant of CVD110, a DctxA, Dzot, DAce and hlyA::(ctxB mer) mutant

(phenotype CTA−, ZOT−, Ace− and hemolysin−) of El Tor biotype Ogawa serotype

strain E7946 8,9 caused morphological changes of the MDCK-I cells

Cultured epithelial cells have been used widely as cell models for virulence studiesof enteropathogenic bacteria. In this work, Madin Darby canine kidney cell line I(MDCK-I) was used. This cell line forms a well-polarized epithelium when culturedon porous or other solid culture supports,10,11,12 making it suitable for use as anepithelial model.

When the concentrated culture supernatant of CVD110 grown at 37°C was addedto MDCK-I cells, remarkable morphological changes were observed [Fig. 1(C)].These morphological changes could be divided into two stages: elongation anddetachment. Elongation of the cells occurred 20–40 min after addition of theconcentrated supernatant, and after a further 2–4 h incubation, the cells detachedfrom the culture plates, remarkably as a whole sheet rather than as individualcells or cell clusters. Occasional visible destruction of the cell monolayer could

V. cholerae HA/protease perturbs normal barrier function of epithelial cells 113

be observed. We also found that subconfluent cells were more sensitive to theculture supernatant of CVD110 than confluent cells, and new-confluent cells weremore sensitive than the cells that had been confluent for several days.

Concentrated culture supernatant of the wildtype parent strain of CVD110, theV. cholerae El Tor Ogawa strain E7946 caused a similar elongation of the cells,and the effect was roughly equivalent to that of CVD110. However, more severedestruction of the cell-monolayer was also observed [Fig. 1(D)]. Culturesupernatants from the V. cholerae classical Ogawa strain 395 and its DctxA mutantCVD101, and the negative control, culture supernatant from an E. coli strain DH-1 caused no or slight and irregular morphological changes in a portion of theMDCK-I cells. Moreover, neither elongation nor complete detachment of the cellswas observed after overnight incubation (data not shown). This result indicatesthat none of the cholera toxin (CT), the Zonula occludens toxin (ZOT)12,13 and theAcessory cholera enterotoxin (Ace)14 produced by the 395, E7946 and/or CVD101was involved. If once treated with the 37°C culture supernatant of CVD110, thesecells developed the same drastic morphological changes. Whereas, when theMDCK-I cells were treated with a culture supernatant of CVD110 grown at 30°C,little or no change of the morphology occurred [Fig. 1(B)].

Morphological changes were also observed using HT29 and Caco-2 cells, twohuman adenocarcinoma cell lines which form polarised cell monolayersresembling normal intestinal epithelium.15,16 However, these cells seemed tocontract instead of being elongated, and they detached rapidly as whole sheetswithout visible destruction (data not shown).

These morphological changes were dose-dependent. When the concentrated37°C culture supernatant of CVD110 was diluted 10 times, it still causedmorphological changes in MDCK-I cells, though the time required increased from20 min to >2 h (data not shown). When the overnight culture supernatants of thebacteria were used without being concentrated, no morphological changes of thecells could be observed within 6 h, however detachment occurred after anovernight incubation.

When the cell culture medium containing the culture supernatant of CVD110was replaced with fresh normal medium, the detachment of the MDCK-I cellsceased but the previously detached cells were unable to re-adhere within 5 h.After overnight incubation, however, the detached cells adhered again to thesurface of the culture plates and grew. This indicates that the effect of the virulenceactivity is reversible. The reversibility depended on the time that the MDCK-I cellshad been exposed to the CVD110 supernatant. Longer exposure times led to alower capability for the cells to re-adhere and grow.

Changes in the transepithelial electrical resistance (TER) of the MDCK-I cell

monolayers

To clarify if the barrier property of the MDCK-I cell monolayer was affected, westudied its TER. When the cells were challenged with the concentrated 37°Cculture supernatant of CVD110, the TER increased initially and then decreased toa very low level (Fig. 2). This effect was dose-dependent. Diluted supernatantcaused similar but slower TER changes. The time required for TER changes wasconsistent with the time for the morphological changes observed above. Cellstreated with the concentrated 37°C culture supernatants of V. cholerae strains 395,CVD101 or the E. coli strain DH-1 showed slight or irregular alterations in TER(data not shown). Caco-2 cells showed similar TER-changes when they were

Z. Wu et al.114

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Fig. 2. A representative experiment showing changes of the transepithelial electrical resistance(TER) of MDCK-I cells. The rate of the TER changes was dependent on the batch of the bacterialsupernatant samples. In this experiment, the cells were treated with 1% (-Φ-), 2% (-Ε-), 4% (-Β-), and8% (-X-) (v/v) of the concentrated 37°C overnight culture supernatant of CVD110. The negative controlwas the cells treated with 10% (v/v) PBS (pH 7.3) (-Α-). Cells treated with the culture supernatants ofthe E. coli strain DH-1, the V. cholerae strains 395 and CVD101 showed slight and irregular TER changes(data not shown).

treated with the CVD110 37°C culture supernatant (data not shown). This resultindicates that the barrier function of the cell monolayers was affected.

Distribution and rearrangement of F-actin and ZO-1 in MDCK-I cells

Because cell shape is determined by the cytoskeleton, we studied the distributionand arrangement of one of the major cytoskeletal protein fibres, filamentous-actin (F-actin) in the cells. In cells treated with phosphate buffered saline (PBS)(17) pH 7.3, F-actin was normally distributed [Fig. 3(A)]. There was diffuse F-actindistribution in the cytosol and clearer F-actin arrangement as bundles at theapical, lateral and basal domains. However, when the cells were challenged withthe 37°C culture supernatant of CVD110 or the cytotoxic fractions of the supernatantafter fast performance liquid chromatography (FPLC) size-fractionation, asignificant redistribution of F-actin from cytosol to the lateral domain was observed[Fig. 3(B)]. After prolonged incubation, a drastic increase of F-actin amount aroundthe apical domain and a significant decrease of F-actin at the basal domain couldbe seen [Fig. 3(C)&(D)]. The clear F-actin distribution that distinguished betweenthe cells was also altered.

Tight junctions join the epithelial cells together and form a barrier which controlsthe paracellular permeability and thereby the barrier function of the epithelium.18

To clarify whether permeability and barrier function of the MDCK-I cell monolayerwas affected at the tight junctions, we studied the distribution and arrangementof ZO-1, an important tight junction-associated protein19 in both confluent andsubconfluent MDCK-I cells. In cells treated with PBS (pH 7.3), ZO-1 was distributedas narrow ribbons around cell-cell borders [Fig. 4(A)]. However, in cells treatedwith the concentrated 37°C culture supernatant of CVD110, no ZO-1 around cell-cell borders could be observed [Fig. 4(C)]. Degraded ZO-1 arrangement could beobserved in the subconfluent cells challenged with 30°C culture supernatant ofCVD110 [Fig. 4(B)].

V. cholerae HA/protease perturbs normal barrier function of epithelial cells 115

Fig. 3. Distribution of F-actin in confluent MDCK-I cells. The bright areas/ribbons represent labelledF-actin. The cells were treated with either PBS (pH 7.3) (Panel A), or the cytotoxic FPLC fraction 53 ofthe concentrated supernatant proteins from CVD110 grown at 37°C for 45 min (Panel B), 90 min (PanelC), and 180 min (Panel D). Arrows indicate the F-actin bundles around the basal domain of the cells.They are not visible in the panel C and D. a: apical; b: basal.

Characterization and identification of the cytotoxic factor

To study if the cytotoxic factor is exclusively secreted into the medium, thecytotoxicity of the CVD110 cell extract was examined. The cell extract could notcause morphological changes in MDCK-I cells even after overnight incubation(data not shown), indicating that the functional form of the cytotoxic factor wasexclusively exported into the culture supernatant.

Because the classical strain 395 and its DctxA mutant CVD101 could not changethe morphology of MDCK-I cells as the El Tor strains E7946 and CVD110 did, andthe El Tor hemolysin is the only known cholera toxic factor which exists commonlyin El Tor strains but seldom in classical strains,20 we studied the haemolytic activityof CVD110. On sheep blood-agar plates, the wild-type strain E7946 showedcomplete haemolytic activity. As negative controls, the classical strain 395 and itsDctxA mutant CVD101 showed slight haemolytic activity. As anticipated, CVD110

Z. Wu et al.116

Fig. 4. Distribution of ZO-1 in subconfluent MDCK-I cells 20 min after addition of samples. The brightribbons represent labelled ZO-1. The cells were treated with PBS (pH 7.3) (Panel A), the concentrated30°C overnight culture supernatant of CVD110 (Panel B) and the concentrated 37°C overnight culturesupernatant of CVD110 (Panel C). Confluent cells showed similar but slower disappearance of ZO-1when they were challenged with the 37°C culture supernatant of CVD110, and the 30°C culturesupernatant did not degrade their ZO-1 in 20 min.

showed only slight haemolytic activity on mercury-containing sheep blood-agarplates. This result indicates that the El Tor hemolysin was not produced by CVD110and thereby can not be responsible for the morphological changes of the MDCK-I cells.

To obtain some idea on the size of the cytotoxic factor, the concentrated 37°Cculture supernatant of CVD110 was size-fractionated by fast performance liquidchromotography (FPLC). Nine consecutive fractions of total 95 fractions (fractions49 to 57) altered the morphology of MDCK-I cells after overnight incubation. Thefractions 53 and 54 showed strongest activity and altered the morphology of thecells after 2 h. The time required for the morphological changes to occur waslonger than that of the unfractionated concentrated supernatant. This could bedue to the dilution effect of the FPLC gel filtration, possible instability of this

V. cholerae HA/protease perturbs normal barrier function of epithelial cells 117

34

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Fig. 5. SDS-PAGE analysis of some FPLC fractions of the concentrated supernatant proteins fromCVD110 grown at 37°C. Lanes:- 1: fraction 37; 2: fraction 41; 3: fraction 45; 4: fraction 47; 5: fraction49; 6: fraction 51; 7: fraction 53; 8: fraction 55; 9: fraction 57; 10: fraction 59; 11: fraction 61; 12: fraction64; 13: fraction 68. Fractions 49 to 57 harbored the cytotoxic activity.

cytotoxic factor at room temperature, and/or shortage of a co-factor. To analysethe size of the proteins in the cytotoxic fractions sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) was run. Only two protein bands,approximately 32- and 34 kDa were visible (Fig. 5). These two proteins were notdetectable on the gel in the inactive fractions which exhibited protein bands ofother sizes. Thus, it is likely that one or both of these two proteins are responsiblefor the cytotoxic activity.

Proteolytic analysis with SDS-gelatin-polyacrylamide gels showed a proteaseactivity of approximately 32 kDa, predominantly in the cytotoxic FPLC-fractions[Fig. 6(A)]. This size is similar to that of the predominant proteolytic form of theHA/protease found in non-O1 V. cholerae.21 A protease acitivity of the same sizewas also found in the culture supernatants of CVD110 and E7946 [Fig. 6(B)]. Incontrast, concentrated culture supernatants of DH-1, CVD101 and 395 did notshow any protease activity on the gels (data of DH-1 and CVD101 not shown).The culture supernatant of CVD110 grown at 30°C showed non or a much weakerprotease activity. Besides the 32 kDa protease activity, a minor protease activityof approximately 22 kDa was also present in the culture supernatants of CVD110and E7946. However, the FPLC-fractions containing this protease activity did notshow any cytotoxic activity in the MDCK-I cell morphology assay, indicating thatit is not responsible for the cytotoxicity of the CVD110 culture supernatant. Thepresence of this 32 kDa protease activity in the samples is consistent with theircytotoxicity, thus we concluded that this protease is responsible for the cytotoxicity.

Sequences of these two proteins obtained by trypsin digestion and peptidesequencing showed 100% match to the sequence of the cholera HA/protease22

(data not shown). Thus, they are most likely the 34 kDa and 32 kDa forms of theHA/protease found in O1- and/or non-O1 V. cholerae.21,22

To further verify that the cholera HA/protease is directly involved in thecytotoxicity of the CVD110 culture supernatant, a Zincov inhibition test wasdone. Zincov is a specially designed bacterial metalloprotease inhibitor23 whichspecifically and effectively inhibits the cholera HA/protease.24 When the MDCK-I

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Fig. 6. Proteolytic analysis with SDS-gelatin-polyacrylamide gels. The clearing zones represent theposition of protease(s) which degraded the gelatin. Panel A shows FPLC fractions of the concentratedsupernatant proteins from CVD110 grown at 37°C. Lanes:- 1: 37°C culture supernatant of CVD110; 2:fraction 47; 3: fraction 49; 4: fraction 51; 5: fraction 52; 6: fraction 53; 7: fraction 54; 8: fraction 57; 9:fraction 59; 10: fraction 61; 11: fraction 64. Panel B shows concentrated protein samples from thebacterial overnight culture supernatants. The bacteria were grown at 37°C if not stated otherwise.Lanes:- 1: V. cholerae classical strain 395; 2: V. cholerae El Tor strain CVD110; 3: E7946, the wild-typeparent strain of CVD110; 4: CVD110 grown at 30°C.

cells were treated with the culture supernatant of CVD110 together with 100 lm

Zincov, the time required for the morphological changes increased from 20 minto >5 h. If 500 lm Zincov was used, the cells did not show any remarkablemorphological changes after overnight incubation. This result confirmed that thecholera HA/protease is responsible for the cytotoxicity.

V. cholerae HA/protease perturbs normal barrier function of epithelial cells 119

Discussion and conclusions

One of the V. cholerae hemagglutinins, the soluble hemagglutinin/protease1,2 is amember of the Zn-containing bacterial metalloprotease family.3 It cleaves severalphysiologically important substances, e.g. mucin, fibronectin, and lactoferrin.4 Itis regarded as a kind of ‘detachase’ which degrades protein structures requiredfor V. cholerae to attach to the intestinal epithelium and thus releases them forfurther transmission.6 However, since non-pathogenic V. cholerae strains alsoproduce the HA/protease, it is not regarded as a primary virulence determinant.

In this work, we initially observed that the classical strain 395 and its DctxAmutant CVD101 could not change the morphology of MDCK-I cells as the El Torstrain E7946 and its derivative CVD110 did. This phenomenon led us to suspectthat the El Tor hemolysin might be responsible, as active hemolysin is commonlyassociated with the El Tor biotype but not the classical biotype of V. cholerae.20

However, several lines of evidence showed that the observed cytotoxic activitycould not be caused by the El Tor hemolysin. First, the hlyA-gene of CVD110 wasinactivated by deletion of a 400-bp fragment plus insertion of a mercury resistancegene, and CVD110 did not exhibit strong hemolytic activity on mercury sheepblood agar plates as its wildtype parent strain E7946 did. Second, the size of thiscytotoxic factor, i.e. around 32- to 34 kDa, is much smaller than that of the El Torhemolysin, which is 65.3 kDa after posttranslational modification.25,26 Our findingsdemonstrate instead that it is the HA/protease.

The HA/protease apparently affects protein structures required for the MDCK-Icells to keep their normal morphology and to adhere to the culture support, viathe rearrangement of filamentous-actin (F-actin) and the tight junction-associatedprotein ZO-1. That subconfluent MDCK-I cells were more sensitive than confluentcells suggests that the HA/protease may be especially effective at the basolateraldomain of the cells, or the sensitivities of the MDCK-I cells to the HA/proteasemay depend on the cell growth stages. Caco-2 cells detached more rapidly thanMDCK-I cells did, indicating higher sensibility or response to the HA/protease. Itcould also be due to that the HA/protease diffused more easily across the Caco-2 cell monolayer, which usually has much lower barrier capability.9,16

Many bacterial toxic factors modify the F-actin arrangement in the host cells.27

They include several ADP-ribosylating toxins of Clostridium,28 the YopE of Yersinia,29

and the E. coli cytotoxic necrotizing factor type 1.30 In this work, we demonstratethat the HA/protease also modifies the F-actin structure. More and more evidencethus suggests that modification of host F-actin filament system, i.e. thecytoskeleton is one major event caused by many pathogenic bacteria.

In the challenged MDCK-I cells, F-actin became first redistributed from thecytosol to the lateral domain. After a longer incubation time, a drastic relativeincrease of F-actin amount at the apical domain and a significant decrease of F-actin amount at the basal domain were seen, and the earlier accumulation of F-actin around the lateral domain became less distinct. The initial redistribution ofF-actin to the lateral domain, perhaps together with the later accumulation of F-actin at the apical domain may explain the strengthened barrier function of thecell monolayer, i.e. the initial increase of TER, while the decrease of F-actin amountat the basal domain may have directly caused the detachment of the cells andthereby the later rapid decrease of TER. Since the cholera HA/protease is a solubleprotease and has not been shown to be able to penetrate the cell membrane, westill do not know the mechanism behind this F-actin rearrangement. The HA/protease may degrade special membrane protein structures, thereby triggeringsignals for rearrangement of F-actin in the cells.

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Tight junctions form the major intestinal barrier for water and ion transport,and decreased tight junctions lead directly to an increased paracellularpermeability. The effect of HA/protease on the tight junction structures viadegradation of the tight junction-associated protein ZO-1 might be involvedindirectly in the mild diarrhea caused by CVD110,31 though no direct evidence hasbeen found yet to support that the HA/protease is a primary virulence factor.

The supernatants of classical strain 395 and its DctxA mutant CVD101 did notshow HA/protease activity when they were tested with SDS-gelatin-polyacrylamidegels. Nevertheless, it does not necessarily mean that they can not produce theHA/protease. Hanne and Finkelstein2 showed that the production of HA/proteaseby V. cholerae is greatly affected by the culture conditions and growth stages ofthe bacteria. Culture supernatant of CVD110 grown at 30°C showed no or low HA/protease activity. However, this experiment indicated at the same time that noneof the CT, ZOT and Ace produced by CVD101 and/or 395 could induce this specificmorphological changes and TER reduction in MDCK-I cells.

Zn-metalloproteases are widely distributed among pathogenic bacteria.3 In arecent study, all Helicobater pylori strains investigated harbored a protease whosegene showed 99% homology to the cholera hap gene.32 In another study, a proteasepurified from V. cholerae O1 culture supernatant enhanced the enterotoxicity ofboth V. cholerae O1 and non-O1.33 There is accumulated evidence suggesting thatbacterial proteases may play an important role in the virulence of the bacteria.34

The Pseudomonas aeruginosa elastase, a homologue to the V. cholerae HA/protease35 also perturbs the paracellular barrier function of epithelial monolayers.36

However, though our studies in this work demonstrate that the HA/protease ishighly cytotoxic to the cultured MDCK-I cells, more evidence is needed to provethat it is involved in the virulence of V. cholerae.

To date, it is not fully understood about what happens to epithelial cells uponinteraction to V. cholerae. The discovery of this cytotoxic activity of V. choleraeHA/protease, together with further investigations on the virulence mechanismsof pathogenic V. cholerae will probably give more detailed information on V.cholerae pathogenesis. It might also aid to the development of safer and moreeffective live oral vaccines against cholera and a better understanding of regulatoryfunctions of the intestinal mucosal barrier.

Materials and methods

Chemicals. These were purchased from Sigma Chem. Co. (St. Louis, MO) if not mentionedotherwise.

Bacterial strains and culture conditions. The wildtype pathogenic V. cholerae O1 strains,the El Tor biotype Ogawa serotype strain E7946 and the classical biotype Ogawa serotypestrain 395 were kindly provided by Dr. G. Jonson, Dept. of Medical Microbiology andImmunology, University of Goteborg, Sweden. The CVD110 strain, a DctxA, Dzot, Dace andhlyA::(ctxB mer) mutant of E7946,10,11 and the CVD101 strain, a DctxA mutant of pathogenicV. cholerae classical strain 395,37 were kindly provided by Dr. J. Kaper, Dept. of Medicine,University of Maryland, USA. The E. coli strain DH-1 (recA1, endA1, thi-1, hsdR17, supE44,gyr96, relA1)38 was also used. Culture medium for the bacteria was Luria Bertani medium17

pH 7.0. For cultivation of CVD110 mercury chloride was always added to 20 lm/ml. Thebacteria were grown overnight at 37°C if not mentioned otherwise.

To examine the haemolytic activity of CVD110, the bacteria were spread onto 5% sheepblood-agar plates containing 20 lm/ml mercury chloride and incubated at 37°C overnight.The parent wild-type strain of CVD110, El Tor Ogawa strain E7946 was used as a positive

V. cholerae HA/protease perturbs normal barrier function of epithelial cells 121

control, and the classical Ogawa strains 395 and 101 were used as negative controls. Thecontrol bacteria were spread onto the same type plates but without mercury.

Epithelial cell (MDCK-I) culture conditions. The MDCK-I cells (kindly provided by Dr. KaiSimons, EMBL, Heidelberg) were grown in Dulbecco’s modified Eagle medium (DMEM)supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (0.1 mg/ml) and glutamine (2 mm). Cell-cultures were maintained in 80 cm2 flasks at 37°C in ahumidified 5% CO2 incubator (Kebo-ASSAB, Sundbyberg, Sweden). The medium wasreplaced every 2–3 days. The cells were harvested every 5–7 days using 0.25% trypsin and0.02% EDTA and replated (1:5) for further experiments.

Cell culture media and other related materials were obtained from NordCell AB(Stockholm, Sweden) except the fetal bovine serum which was from GIBCO-BRL LifeTechnologies, Inc. (Grand Island, NY).

Preparation of protein samples. For all culture supernatant protein preparations, thebacterial overnight cultures were centrifuged and the supernatants filter-sterilized through0.45 lm Minisart N sterile filters (Sartorius AG, Gottingen, Germany). Then, ammoniumsulphate was added to 100% saturation. After being stirred at 4°C for 2 h, the solutionswere centrifuged at 5000×g at 4°C for 1 h. The pellets were dissolved in PBS (pH 7.3) anddialysed at 4°C against 200 volumes of PBS (pH 7.3) with three buffer changes with 2-hintervals and one buffer change with an overnight interval. Then, concentration dialysisagainst polyethylene glycol 8000 (USB Co., Cleveland, OH) was done. The final concentrationof the supernatant proteins were approximately 100–200 fold of the original culturesupernatants. For all dialysis steps, Slide-A-Lyzer dialysis cassettes (Pierce, Beijerland, TheNetherlands) with a molecular weight cut-off of 10 kDa were used. The concentrated proteinsamples were aliquoted and kept at −20°C until use. LB-medium treated in the same waywas used as a negative control.

Protein preparations from the whole CVD110 cells were prepared as follows: CVD110cells from an overnight culture grown at 37°C were pelleted, washed once with PBS (pH7.3), resuspended in 1/200 volume of PBS (pH 7.3), and sonicated. After sonication, thelysate was centrifuged at 14 000×g for 30 min at 4°C to remove insoluble substances. Theclear cell extract was used for test of the cytotoxicity as described above.

Assay for the cytotoxic factor by studying morphological changes of the MDCK-I cells.MDCK-I cells were grown on glass coverslips (Novakemi AB, Stockholm, Sweden) inmultiwell culture dishes until confluent. Then, the concentrated protein samples from thebacterial culture supernatants were added to 10% v/v unless otherwise stated, and thecells were returned to the cell culture incubator. An Olympus Nomarski light microscopewas used for the observation of the cells at various time points. This assay was used forscreening of the cytotoxic activity in various samples and Zincov-inhibition test.

Transepithelial electrical resistance (TER) measurement. MDCK-I cells were grown onCostar Transwell polycarbonate culture inserts of 6.5 mm diameter and 3 lm pore-size(Corning Costar Co., Cambridge, MA) until their TER became at least 1 kX per filter. Variousamount of the concentrated bacterial culture supernatants were added to the apical sideof the cell monolayers, and the cells were then incubated at 37°C. A Millicell ERS apparatus(Millipore, MA) was used to measure TER at various time points.

Analysis of F-actin and ZO-1 arrangement in MDCK-I cells using confocal laser scanningmicroscopy. MDCK-I cells were grown on glass coverslips or Costar Transwell cultureinserts until confluent unless otherwise stated, and the concentrated protein samples fromthe bacterial culture supernatants were added to 10% (v/v) or FPLC-fractions to 50% (v/v).After incubation at 37°C for various time periods, the cells were fixed with ice-cold 2.5%paraformadehyde for 30 min and permeabilized with −20°C 100% methanol for 4 min. F-actin was stained with rhodamine-labelled phalloidin (Molecular Probes Inc., Eugene, OR),while ZO-1 was indirectly labelled first with rat anti-ZO-1 antibodies (Chemicon Int. Inc.,Temecula, CA) then with rhodaminated rabbit anti-rat antibodies (Jackson Immunores.Labs., West Grove, PA). The arrangement of the labelled F-actin and ZO-1 was studiedusing a confocal laser scanning microscope (Phoibos 2000, Molecular Dynamics, Sunnyvale,CA). The 514nm-line of the Argon laser was used to excite fluorescence.

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Identification of the cytotoxic factor. The supernatant proteins of CVD110 were size-fractionated by FPLC using a Pharmacia Biotech (Uppsala, Sweden) FPLC system. Thecolumn was packed with AcA44 Ultrogel (LKB, Bromma, Sweden) with a separation rangefrom 10 kDa to 150 kDa. Proteins in a 500 ml culture supernatant of CVD110 grown at 37°Cwere concentrated 500-fold as described above. The sample was centrifuged at 14 000×gfor 30 min to remove insoluble substances before loading. PBS (pH 7.3) was used as theelution buffer. The FPLC was run at room temperature, and the time required was around10 h. The fractions (1.5 ml each) were collected and their cytotoxic activity was tested oncultured MDCK-I cells as described above. The size of the proteins in the fractions weredetermined by SDS-PAGE analysis.39

Proteolytic activity of the concentrated protein samples from the bacterial culturesupernatants and the FPLC fractions was detected with SDS-gelatin-polyacrylamide gelsas described.40 Briefly, the proteins were separated with usual SDS-PAGE except that 0.2%gelatin was co-polymerized in the gels. FPLC fractions (20 ll of each), or 2 ll of the 100- to200-fold concentrated bacterial culture supernatants was applied into each lane. Afterelectrophoresis the gels were first soaked at 37°C for 1 h in 2.5% Triton X-100 at roomtemperature, then incubated at 37°C for 3.5 h in 0.1 m glycine (pH 8.3). After that the gelswere fixed and stained with 0.25% Coomassie brilliant blue R-250 (LKB, Bromma, Sweden)in acetic acid-methanol-water (10:30:60), and then destained with acetic acid-methanol-water (10:30:60). Protease activity was detected by measuring the clearing zones in thegels.

Microsequencing of the 34- and the 32 kDa proteins present in the active FPLC fractionswas carried out by Dr. B. Ek at Department of Cell Research, Swedish University ofAgriculture Sciences, Uppsala, Sweden. The sequences were compared to the publishedHA/protease protein sequence.22

To further confirm that the cholera HA/protease is responsible for this cytotoxicity, 100-to 500 lm Zincov (Calbiochem, La Jolla, CA) was included in the Zincov-inhibition test,using the MDCK-I cell morphology assay as described above.

This research was supported by the Swedish Medical Research Council (project No. 6251),King Gustav V:th 80-year Foundation, the Professor Nanna Svartz Foundation, the Magn.Bergvall Foundation, and the Swedish Research Council for Engineering Sciences. Specialthanks to Tommy Sundqvist and Tony Forslund for helpful discussions. We are grateful toDr. J. Kaper for providing us the CVD110 and CVD101 strains and to Dr. G. Jonson for theEl Tor strain E7946 and the classical strain 395.

References

1. Finkelstein RA, Hanne LF. Purification and characterisation of the soluble hemagglutinin (choleralectin) produced by Vibrio cholerae. Infect Immun 1982; 36: 1199–208.

2. Hanne LF, Finkelstein RA. Characterization and distribution of the hemagglutinins produced byVibrio cholerae. Infect Immun 1982; 36: 209–14.

3. Hase CC, Finkelstein RA. Bacterial extracellular Zinc-containing metalloproteases. Microbiol Rev1993; 57: 823–37.

4. Finkelstein RA, Boesman-Finkelstein M, Holt P. Vibrio cholerae hemagglutinin/lectin/proteasehydrolyzes fibronectin and ovomucin: F. M. Burnet revisited. Proc Natl Acad Sci USA 1983; 80:1092–5.

5. Booth BA, Boesman-Finkelstein M, Finkelstein RA. Vibrio cholerae soluble hemagglutinin/proteasenicks cholera enterotoxin. Infect Immun 1984; 45: 558–60.

6. Finkelstein RA, Boesman-Finkelstein M, Chang Y, Hase CC. Vibrio cholerae hemagglutinin/protease,colonial variation, virulence, and detachment. Infect Immun 1992; 60: 472–78.

7. Misfeldt DS, Mammannoka ST, Pitelka DR. Transepithelial transport in cell culture. Proc Natl AcadSci USA 1976; 73: 1212–15.

8. Cereijido M, Robbins ES, Dolan WJ, Rotonno CA, Sabatini DD. Polarized monolayers formed byepithelial cells on a permeable and translucent support. J Cell Biol 1978; 77: 853–80.

9. Richardson JC, Scalera V, Simmons NL. Identification of two strains of MDCK cells which resembleseparate nephron tubule segments. Biochim Biophys Acta 1981; 673: 26–36.

10. Ketley JM, Michalski J, Galen J, Levine MM, Kaper JB. Construction of genetically marked Vibriocholerae O1 vaccine strains. FEMS Microbiol Lett 1983; 111: 15–22.

11. Michalski J, Galen JE, Fasano A, Kaper JB. CVD110, an attenuated Vibrio cholerae O1 El Tor liveoral vaccine strain. Infect Immun 1993; 61: 4462–68.

V. cholerae HA/protease perturbs normal barrier function of epithelial cells 123

12. Fasano A, Baudry B, Pumplin DW et al. Vibrio cholerae produces a second enterotoxin, whichaffects intestinal tight junctions. Proc Natl Acad Sci USA 1991; 88: 5242–46.

13. Baudry B, Fasano A, Ketley J, Kaper JB. Cloning of a gene (zot) encoding a new toxin producedby Vibrio cholerae. Infect Immun 1992; 60: 428–34.

14. Trucksis M, Galen JE, Michalski J, Fasano A, Kaper JB. Accessory cholera enterotoxin (Ace), thethird toxin of a Vibrio cholerae virulence cassette. Proc Natl Acad Sci USA 1993; 90: 5267–71.

15. Pinto M, Appay MD, Simon-Assmann P et al. Enterocytic differentiation of cultured human cancercells by replacement of glucose by galactose in the medium. Biol Cell 1982; 44: 193–96.

16. Pinto M, Robine-Leon S, Appay MD et al. Enterocyte-like differentiation and polarization of thehuman colon carcinoma cell line Caco-2 cells in culture. Biol Cell 1983; 47: 323–30.

17. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual. 2nd edn. Cold SpringHarbor, NY: Cold Spring Harbor Laboratory Press, 1989; B12 and A1.

18. Schneeberger EE, Lynch RD. Structure, function and regulation of cellular tight junctions. Am JPhysiol 1992; 262: L647–61.

19. Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA. Identification of ZO-1, a high molecularweight polypeptide associated with the tight junctions (Zonula occludens) in a variety of epithelia.J Cell Biol 1986; 103: 755–66.

20. Liu PV. Studies on the hemolysin of Vibrio cholerae. J Infect Dis 1959; 104: 238–59.21. Naka A, Yamamoto K, Miwatani T, Honda T. Characterization of two forms of hemagglutinin/

protease produced by Vibrio cholerae non-O1. FEMS Microbiol Lett 1992; 77: 197–200.22. Hase CC, Finkelstein RA. Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/

protease (HA/protease) gene and construction of an HA/protease-negative strain. J Bacteriol 1991;173: 3311–17.

23. Nishino N, Powers JC. Design of potent reversible inhibitors for thermolysin. Peptides containingZinc coordinating ligands and their use in affinity chromatography. Biochem 1979; 18: 4340–7.

24. Booth BA, Boesman-Finkelstein M, Finkelstein RA. Vibrio cholerae soluble hemagglutinin/proteaseis a metalloprotease. Infect Immun 1983; 42: 639–44.

25. Goldberg SL, Murphy JR. Cloning and characterization of the hemolysin determinants from Vibriocholerae RV79(Hly+), RV79(Hly−), and 569B. J Bacteriol 1985; 162: 35–41.

26. Hall RH, Dvasar BS. Vibrio cholerae HlyA hemolysin is processed by proteolysis. Infect Immun1990; 58: 3375–79.

27. Donelli G, Fiorentini C. Bacterial protein toxins acting on the cell cytoskeleton. Microbiologica1994, 17: 345–62.

28. Aktories K. Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins. Mol CellBiochem 1994; 138: 167–76.

29. Rosqvist R, Forsberg A, Wolf-Watz H. Intracellular targeting of the Yersinia YopE cytotoxin inmammalian cells induces actin filament disruption. Infect Immun 1991; 59: 4562–69.

30. Falzano L, Fiorentini C, Boquet P, Donelli G. Interaction of Escherichia coli cytotoxic necrotizingfactor type 1 (CNF1) with cultured cells. Cytotechnol 1993, 11: S56–58.

31. Tacket CO, Losonsky G, Nataro JP, Cryz SJ, Edelman R, Fasano A, Michalski J, Kaper JB, LevineMM. Safety and immunogenicity of live oral cholera vaccine candidate CVD110, a DctxA, Dzot,Dace derivative of El Tor Ogawa serotype Vibrio cholerae. J Infect Dis 1993; 168: 1536–40.

32. Smith AW, Chahal B, French GL. The human gastric pathogen Helicobacter pylori has a geneencoding an enzyme first classified as a mucinase in Vibrio cholerae. Mol Microbiol 1994; 13:153–60.

33. Ichinose Y, Ehara M, Honda T, Miwatani T. The effect on enterotoxicity of protease purified fromVibrio cholerae O1. FEMS Microbiol Lett 1994; 115: 265–71.

34. Travis J, Potempa J, Maeda H. Are bacterial proteases pathogenic factors? Trends in Microbiol1995; 3: 405–7.

35. Hase CC, Finkelstein RA. Comparison of the Vibrio cholerae hemagglutinin/protease and thePseudomonas aeruginosa elastase. Infect Immun 1990; 58: 4011–15.

36. Azghani AO, Gray LD, Johnson AR. A bacterial protease perturbs the paracellular barrier functionof transporting epithelial monolayers in culture. Infect Immun 1993; 61: 2681–86.

37. Kaper J, Lockman H, Baldini MM, Levine MM. A recombinant live oral cholera vaccine. Biotechnol1984; 2: 345–49.

38. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol 1983; 166:557–80.

39. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophageT4. Nature 1970; 227: 680–85.

40. Milton DL, Norqvist A, Wolf-Watz H. Cloning of a metalloprotease gene involved in the virulencemechanism of Vibrio anguillarum. J Bacteriol 1992; 174: 7235–44.