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Microbial Pathogenesis 45 (2008) 377–385

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Microbial Pathogenesis

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Escherichia albertii and Hafnia alvei are candidate enteric pathogenswith divergent effects on intercellular tight junctionsq

Kevin A. Donato a,c, Mehri Zareie a, Agatha N. Jassem a, Narveen Jandu a, Nelab Alingary a,Soo Chan Carusone a, Kathene C. Johnson-Henry a, Philip M. Sherman a,b,c,*

a Cell Biology Program, Research Institute, Hospital for Sick Children, University of Toronto, Room 8409, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8b Department of Paediatrics, University of Toronto, Toronto, Canadac Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

a r t i c l e i n f o

Article history:Received 24 January 2008Received in revised form 27 August 2008Accepted 12 September 2008Available online 1 October 2008

Keywords:Tight junctionEpithelial barrier functionBacterial infectionIntestine

q Research performed at: Hospital for Sick ChildRoom 7142, Toronto, Ontario, Canada M5G 1X8. Supptutes of Health Research (grant FRN 9317 to P.M.S.).

* Corresponding author. Cell Biology Program, ReSick Children, University of Toronto, Room 8409, 555Ontario, Canada M5G 1X8. Tel.: þ1 416 813 7734; fax

E-mail address: philip.sherman@sickkids.ca (P.M. S

0882-4010/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.micpath.2008.09.004

a b s t r a c t

Attaching-effacing lesion-inducing Escherichia albertii and the related, but non-attaching–effacingorganism, Hafnia alvei, are both implicated as enteric pathogens in humans. However, effects of thesebacteria on epithelial cells are not well-characterized. Related enteropathogens, including enter-ohemorrhagic Escherichia coli O157:H7, decrease epithelial barrier function by disrupting intercellulartight junctions in polarized epithelia. Therefore, this study assessed epithelial barrier function and tightjunction protein distribution in polarized epithelia following bacterial infections. Polarized epithelial(MDCK-I and T84) cells grown on filter supports were infected apically with E. coli O157:H7, E. albertii,and H. alvei for 16 h at 37 �C. All strains decreased transepithelial electrical resistance and increasedpermeability to a dextran probe in a host cell-dependent manner. Immunofluorescence microscopyshowed that both E. coli O157:H7 and E. albertii, but not H. alvei, caused a redistribution of the tightjunction protein zona occludens-1. In contrast to E. coli O157:H7, E. albertii and H. alvei did not redis-tribute claudin-1. Western blotting of whole cell protein extracts demonstrated that each bacteriumcaused differential changes in tight junction protein expression, dependent on the host cell. Thesefindings demonstrate that E. albertii and H. alvei are candidate enteric pathogens that have both strain-specific and host epithelial cell-dependent effects.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Escherichia albertii and Hafnia alvei are members of the familyEnterobacteriaceae and have been associated with cases of diarrheain humans, but whether these bacteria can be regarded as enter-opathogens remains the subject of controversy [1–3]. Formerly,these bacteria were classified exclusively as H. alvei, including twopathotypes distinguished by their geographical origin, genotypeand interaction with epithelial cells. The first pathotype includedbacterial isolates from Bangladesh (e.g. strain 19982), whichcontain the Escherichia coli attaching–effacing (eae) gene encodinga 94 kDa outer membrane protein, intimin, that imparts the abilityto induce attaching-and-effacing (A/E) lesions [4]. A/E lesions arecharacterized by cytoskeletal rearrangements in epithelial cells asa result of intimate bacterial attachment [5]. Following apical

ren, 555 University Avenue,orted by the Canadian Insti-

search Institute, Hospital forUniversity Avenue, Toronto,

: þ1 416 813 6531.herman).

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attachment to epithelial cells, A/E lesion-inducing pathogensincluding enterohemorrhagic E. coli utilize a type-III secretionsystem which acts as a molecular syringe to inject effectors,including the translocating intimin receptor (Tir), that cause therecruitment of F-actin and crosslinking molecules, including a-actinin, to form attachment pedestals at the host cell plasmamembrane underneath adherent organisms [4,5]. The second H.alvei pathotype includes strains of Canadian origin (H. alvei strainsH1 through H9, D46 and D67). We have previously shown thatthese strains do not contain the eae gene and do not induce A/Elesions in response to infection of epithelial cells [4].

Following discovery of eaeþ H. alvei, further characterization ofthese bacteria revealed differences in metabolism, conditions foroptimal growth and nucleic acid sequences between eaeþ and eae�

H. alvei [6,7]. As a result, eaeþ strains originally classified as H. alveiwere subsequently reclassified under the genus Escherichia, and arenow referred to as E. albertii [8,9].

Diarrheal illness can result from dismantling the tight junctionbarrier apparatus between polarized epithelial cells [10]. The type-III secretion is also required for the disruption of tight junctions inA/E E. coli [11]. We hypothesized that epithelial cell responses to E.albertii and H. alvei infections could be distinct. However, the effects

K.A. Donato et al. / Microbial Pathogenesis 45 (2008) 377–385378

of E. albertii and H. alvei on epithelial barrier function have not beenexplored previously. Therefore, the aim of this study was to definethe effects of these bacteria on epithelial permeability and tightjunction integrity of polarized epithelial cell monolayers.

2. Results

2.1. E. albertii and H. alvei infections decrease transepithelialelectrical resistance of polarized epithelial monolayers

To assess epithelial barrier function with respect to passive ionflow, the transepithelial electrical resistance (TER) of polarizedMDCK-I and T84 cells was measured before and after infection, andcompared to uninfected monolayers, which were used as negativecontrols. As shown in Fig. 1A, after 16 h of infection, EHEC O157:H7,

Fig. 1. E. albertii and H. alvei infections decrease epithelial barrier function in a hostcell-specific manner. (A) Post-infection transepithelial electrical resistance (TER) inMDCK-I (open bars) and T84 (solid bars) cells was recorded as a percentage of pre-infection resistance and normalized to uninfected monolayers (expressed as 100%).n¼ 7–16 Separate trials for MDCK-I cells, n¼ 11–19 separate trials for T84 cells(duplicate monolayers were used for each trial). Uninfected MDCK-I and T84 mono-layers developed mean resistances of 3835� 386 U cm2 and 2385� 519 U cm2 after16 h, respectively. (B) Alexa 647-conjugated dextran (10 kDa) was added to the apicalaspect of infected MDCK-I (open bars) and T84 (solid bars) monolayers and allowed todiffuse into the basolateral medium for an additional 5 h. Basolateral medium sampleswere collected and scanned in an Odyssey� infrared imager with 169 mm resolutionusing the 700 nm channel. Sample intensities were normalized to those taken fromuninfected monolayers (intensity¼ 1.0). n¼ 7 Separate trials for MDCK-I cells, n¼ 4separate trials for T84 cells (duplicate monolayers were used for each trial). ANOVA*P< 0.05, **P< 0.01 compared to uninfected monolayers of the same cell line.

strain CL56, significantly decreased TER in both MDCK-I and T84monolayers (28.1�5.9% and 35.8� 8.3% of uninfected epithelia,respectively; ANOVA P< 0.001, n¼ 16 for MDCK-I cells, n¼ 11 forT84 cells), a finding supported by earlier studies [12]. Incubation ofMDCK-I cells with either E. albertii, H. alvei strain H2, or H. alveistrain D46 also resulted in a significant decrease in resistance(41.6� 5.3%, 53.6� 6.3%, and 45.9� 5.3%, respectively; P< 0.001,n¼ 12–16). E. albertii and H. alvei H2 also decreased the electricalresistance of T84 cells, but to a lesser degree compared with MDCK-I monolayers (88.4� 3.5% and 80.1�3.9%, respectively; P< 0.05,n¼ 11–19). By contrast, H. alvei, strain D46 did not induce a drop inthe resistance of T84 cells (96.9� 3.1%; P> 0.05, n¼ 11).

Treatment of MDCK-I cells with formaldehyde-fixed or heat-killed bacteria, or the addition of chloramphenicol to the co-culturemedium abolished the bacterial-induced decrease in TER (Table 1),indicating that live and metabolically active bacteria with thecapacity to synthesize new proteins were required for pathogen-esis. Conditioned medium and bacterial culture supernatants didnot decrease TER in MDCK-I epithelia (Table 1) indicating thatsecreted factors present in the culture medium were not respon-sible for the disruption of epithelial barrier integrity, and that directcontact of live bacteria binding to the host cell surface was requiredto induce the observed effects. Similar results were obtained usingT84 cells (data not shown).

Table 1Post-treatment transepithelial electrical resistance and dextran permeability ofMDCK-I cells.

Treatmenta TERb n Basal mediumfluorescence intensityc

n

Uninfected cells 100 16 1.00 7

Live, untreated bacteriaEHEC O157:H7 28.1� 5.9** 16 8.62� 2.1** 7E. albertii 41.6� 5.3** 16 5.90� 0.9** 7H. alvei H2 53.6� 6.3** 16 1.33� 0.1 7H. alvei D46 45.9� 5.3** 12 3.44� 0.7* 7

Chloramphenicol (20 mg/ml) in apical mediumEHEC O157:H7 100 3 0.89� 0.08 3E. albertii 100 3 0.88� 0.07 3H. alvei H2 99.0� 1.0 3 0.80� 0.08 3H. alvei D46 97.3� 1.8 3 0.79� 0.07 3

Formalin-fixed bacteria (10%, 24 h at 4 �C)EHEC O157:H7 100 2 0.95 2E. albertii 98.5 2 0.86 2H. alvei H2 98.5 2 0.84 2H. alvei D46 97.5 2 0.82 2

Heat-killed bacteria (boil> 45 min)E. albertii 98.2� 1.0 7 1.10� 0.11 3H. alvei H2 94.0 1 0.83 1H. alvei D46 100 2 0.84 2

Conditioned mediumd

EHEC O157:H7 100 2 0.90 2E. albertii 100 2 0.94 2H. alvei H2 100 2 0.74 2H. alvei D46 100 2 0.92 2

Bacterial culture supernatantd

EHEC O157:H7 100 1 0.95 1E. albertii 100 1 0.87 1H. alvei H2 100 1 1.92 1H. alvei D46 100 1 1.15 1

ANOVA *P< 0.05, **P< 0.01.a Multiplicity of infection 100:1, 16 h, 37 �C.b Values (mean� SEM for n� 3) represented as percentage of uninfected

monolayers.c Values (mean� SEM for n� 3) represented as integrated intensity relative to

uninfected monolayers.d Filtered and mixed (1:1) with fresh antibiotic-free medium and added to apical

aspect of fresh monolayers for 16 h at 37 �C.

K.A. Donato et al. / Microbial Pathogenesis 45 (2008) 377–385 379

2.2. E. albertii and H. alvei D46 increase the permeability ofpolarized monolayers to macromolecules

Complimentary to measuring TER, epithelial barrier functionwas then assessed by measuring the diffusion of a larger macro-molecule, dextran (10-kDa) conjugated to an infrared-sensitivefluorophore, from the apical to the basolateral aspect of bothinfected and uninfected epithelial monolayers. As shown in Fig. 1B,tissue culture medium samples taken from the basolateralcompartment of E. coli O157:H7-infected MDCK-I monolayers hadgreater fluorescent intensity relative to uninfected monolayers(relative fluorescent intensity [RI] of basolateral medium samplesfrom infected cells¼ 8.62� 2.1; P< 0.01, n¼ 7; RI of samples fromuninfected cells¼ 1.0).

Samples from the basolateral compartments of E. albertii and H.alvei D46-infected MDCK-I monolayers also demonstratedenhanced permeability to the probe, compared to uninfectedcontrols (RI¼ 5.90� 0.9 and 3.44� 0.7, respectively; P< 0.05,n¼ 7). By contrast, samples collected from H. alvei H2-infectedMDCK-I monolayers were not significantly different from unin-fected cells (RI¼ 1.33� 0.1; P> 0.05, n¼ 7). As in TER studies, theeffects observed were dependent on the type of host cell infected,since neither E. albertii (RI¼ 0.94� 0.25) nor the two strains of H.alvei (strain H2: RI¼ 1.66� 0.43; strain D46: RI¼ 1.09� 0.27),increased the dextran permeability of T84 monolayers (P> 0.05,n¼ 4). Formalin-fixation, heat-killing or chloramphenicol treat-ment abolished the observed increases in macromolecularpermeability in MDCK-I cells. Conditioned medium and bacterialculture supernatants did not increase epithelial permeability to thedextran probe, compared to uninfected cells (Table 1).

2.3. Bacterial specific effects on the tight junction proteins ZO-1and claudin-1

Confocal immunofluorescence micrographs of ZO-1, a tightjunction anchorage protein [13] in uninfected MDCK-I (Fig. 2A) andT84 (Fig. 2B) monolayers when visualized ‘en-face’ had well-cir-cumscribed, intact junctional belts. By contrast, ZO-1 distribution ofE. coli O157:H7-infected monolayers displayed disturbances in thejunctional belt formations, including strand breaks and segments ofpunctate-like discontinuity (Fig. 2C and D), consistent withprevious observations [12,14]. A loss of polarity in the EHEC-infected epithelial monolayers was visualized longitudinally in thexz-plane, compared to uninfected cells which had tight junctionslocated in a normal orientation along the apical aspect of polarizedcells. E. albertii infection also caused the ZO-1 assembly to fragmentin both MDCK-I (Fig. 2E) and T84 cells (Fig. 2F), but polarization wasmaintained. By contrast, H. alvei H2 and D46 infections did notresult in redistribution of ZO-1 in either epithelial cell type (Fig. 2Gthrough J).

As shown in Fig. 3B, EHEC-infected MDCK-I monolayers hada claudin-1 staining pattern that was more diffuse, poorly cir-cumscribed and inadequately polarized, compared with uninfectedmonolayers which had intact junctional belts (Fig. 3A). Although E.albertii redistributed ZO-1, infected monolayers retained normalclaudin-1 distribution and polarization (Fig. 3C). Neither of the twostrains of H. alvei had a discernible effect on the distribution ofclaudin-1 (Fig. 3D and E).

2.4. Tight junction protein expression profiles followingbacterial infection

To complement confocal imaging findings, tight junction proteinexpression in whole cell extracts was quantified by using immu-noblotting. Only EHEC O157:H7 infection caused a significantdecrease in the expression of ZO-1 in MDCK-I cells (relative band

intensity to uninfected control and normalized to b-actin[RI]¼ 0.27� 0.05% of uninfected cells; P< 0.01, n¼ 3; Fig. 4A). Bycontrast, in T84 cells, only H. alvei D46 caused a decrease in theexpression of ZO-1 (RI¼ 0.4� 0.1; P< 0.05, n¼ 4; Fig. 4B).

As shown in Fig. 4C, compared to uninfected MDCK-I cells, EHECO157:H7 infection resulted in a shift in the occludin band pattern tothe lower molecular weight (that is, hypophosphorylated) isoform(ratio of band intensity of hyperphosphorylated isoform to totaloccludin band intensities¼ 0.71�0.04 relative to uninfected cellswith their ratio normalized to 1.0). Similar results were notobserved following E. albertii and H. alvei infections(RI¼ 0.96� 0.02 for E. albertii, 0.94� 0.03 for H. alvei H2, and0.92� 0.03 for H. alvei D46; P> 0.05, n¼ 4). H. alvei H2 was the onlyorganism to cause a marginal decrease in T84 cell occludin phos-phorylation (RI¼ 0.84� 0.09; P< 0.05, n¼ 4; Fig. 4D).

There was no decrease in the levels of either claudin-1 orclaudin-4 in any of the infected monolayers, compared withuninfected controls (Fig. 4E–H). On the other hand, claudin-5expression was marginally reduced in T84 cells infected by each ofthe bacterial strains (Fig. 4I; RI¼ 0.76� 0.14 for EHEC, 0.57� 0.10for E. albertii, 0.62� 0.21 for H. alvei H2, and 0.72� 0.12 for H. alveiD46; P¼ 0.06, n¼ 4). As reported previously by other investigators[15] MDCK-I cells did not express detectable levels of claudin-5(data not shown).

2.5. Host cell specific bacterial adherence of E. albertii and H. alvei

To determine whether differential host cell responses followinginfection could have related to binding properties of the infectingorganism, the amount of bacterial adhesion to polarized mono-layers was then quantitated. As shown in Table 2, after 16 h ofinfection (MOI 100:1, 37 �C), each of the four bacterial strains testedadhered in comparable amounts (that is, less than one log unitdifference between the numbers of adherent bacteria) to the samecell type. However, the bacterial strains adhered approximately 10times less (one log unit) to T84 cells, compared with MDCK-I cells.

2.6. LEE genes (Tir/EspE and EspF) in EHEC O157:H7 are present inE. albertii, but not H. alvei

The presence of selected EHEC O157:H7 secreted effector geneswas tested by polymerase chain reaction with primers designedbased on known gene sequences in EHEC O157:H7, strain EDL933and E. albertii, strain TW0617. As shown in Fig. 5, primers specificfor EHEC espE yielded a product of the predicted size (357 bp),corresponding to the primer pair design solely in the sampleprepared from EHEC O157:H7, strain CL56 cDNA. Primers specificfor E. albertii espE yielded a product (793 bp) only in E. albertii,strain 19982 extracts. Primers for EHEC O157:H7 and E. albertii espFyielded species-specific single gene products of predicted sizes of254 bp and 222 bp, respectively.

3. Discussion

In this study we demonstrate, for the first time, that responses ofpolarized epithelia to E. albertii strain 19982, formerly classifiedunder the genus Hafnia [8], and H. alvei strains H2 and D46 differwith respect to effects on epithelial barrier function and theintegrity of intercellular tight junctions. Similar to EHEC O157:H7, E.albertii and H. alvei strains cause significant decreases in trans-epithelial electrical resistance (TER) of MDCK-I monolayers. Thesefindings show that Hafnia strains have the ability to disruptepithelial barrier function, albeit to a weaker extent than therelated E. albertii and EHEC O157:H7, even though the organismlacks the locus of enterocyte effacement (LEE) pathogenicity islandwhich contains the necessary factors for the bacterium to induce A/

Fig. 2. Strain dependent effects on ZO-1 distribution in epithelial monolayers. Confocal laser scanning micrographs (xy-plane or ‘en-face’) of monolayers immunofluorescentlystained using a specific polyclonal primary antibody for ZO-1 and Cy2-conjugated secondary antibody (green). An xz-plane image is placed below the original to visualize monolayerpolarity. Nuclei are stained with DAPI (blue). (A and B) Uninfected control monolayers had intact ZO-1 junctional belts. (C and D) EHEC O157:H7 infection for 16 h, caused ZO-1redistribution in MDCK-I and T84 monolayers (arrows) with a loss of epithelial polarity. (E and F) E. albertii infection caused ZO-1 redistribution (arrows), but polarity is largelymaintained. (G through J) H. alvei H2 and H. alvei D46 infections did not redistribute ZO-1. Images were collected at 1 mm intervals. Scale bars (yellow) equal 10 mm. En-face imageswere created by scanning with 630� optical magnification and digitally magnified, by two, using the manufacturer’s operating software and deleting signal from the DAPI-detectingchannel, for greater detail. Images are representative of at least three separate trials.

K.A. Donato et al. / Microbial Pathogenesis 45 (2008) 377–385380

E lesions. Although there are differences between observations inTER and permeability assays to an inert probe, such as dextran, theobserved findings are not entirely surprising, since tight junctionshave been demonstrated previously to form distinct barrierstoward ions and macromolecules and, therefore, need to beconsidered separately [16,17].

Microorganisms employ various modes of action to disruptapical junction complexes, including direct attachment to host cellsand the secretion of mediators into the external environment,which then interact with epithelial surfaces [10]. In this study, wedemonstrate that E. albertii and H. alvei strains must be live,metabolically active and have the capacity to synthesize newproteins in order to induce damage to polarized epithelia. Inaddition, bacterial factors secreted into the host-bacterial co-culture medium do not appear to be involved in mediating injury totight junctions, indicating that bacterial attachment to the cell

monolayer is likely to be required. Such a contact-dependent modeof action is similar to previous observations described using EHECO157:H7 [14].

Whether E. albertii and H. alvei can be regarded as intestinalpathogens remains a topic of controversy. Five E. albertii strains,originally isolated at the International Center for Diarrhoeal DiseaseResearch Branch in Bangladesh, were associated with infantilediarrhea [6]. H. alvei, on the other hand, is more frequently isolated,from patients with either intestinal or extra-intestinal illnesses inNorth America [2,18,19] and Europe [20–22]. H. alvei can also beisolated from environmental reservoirs including mammals, fish,birds, foodstuffs and water [3,23]. H. alvei is considered an oppor-tunistic pathogen that is often nosocomially acquired in immunecompromised patients through wounds, invasive procedures (e.g.catheterization) or in subjects with underlying chronic illnesses[24]. To determine whether these bacteria have the capacity to

Fig. 3. EHEC O157:H7, but not E. albertii or H. alvei, causes claudin-1 redistribution in MDCK-I monolayers. Confocal micrographs (xy-plane, ‘en-face’) of epithelial cells stained forclaudin-1 (green). An xz-plane image is placed below the original to visualize monolayer polarity. Nuclei are stained with DAPI (blue). (A) Uninfected control monolayers displayedwell-circumscribed claudin-1 junctional belts. (B) EHEC O157:H7 infection for 16 h caused claudin-1 distribution to distort (arrows) and the monolayers to lose their polarity. (Cthrough E) E. albertii, H. alvei H2 and H. alvei D46 did not redistribute claudin-1. Images were collected at 1 mm intervals. Scale bars (yellow) equal 10 mm. En-face images werecreated by scanning with 630� optical magnification and digitally magnified, by two, using the manufacturer’s operating software and deleting signal from the DAPI-detectingchannel, for greater detail. Images are representative of at least three separate experiments.

K.A. Donato et al. / Microbial Pathogenesis 45 (2008) 377–385 381

cause disease, we employed a reductionist epithelial barrier modelinfected with either of E. albertii or H. alvei, compared to EHECO157:H7 infection, which induces well-characterized pathogeniceffects on barrier function [12,14,25,26].

Epithelial barrier function is disrupted when apical junctioncomplex proteins are either dismantled from the apical juxta-membrane regions of cells or when they are reduced in levels ofexpression. Pathogenic Enterobacteriaceae, including enteropatho-genic E. coli strain E2348/69 [27], enterohemorrhagic E. coliO157:H7 [12,14,25,26] and the related mouse enteric pathogen,Citrobacter rodentium [28], each disrupt epithelial tight junctionstructure and decrease junction protein expression resulting indecreases in TER and increased permeability to macromolecules. Inthe current study, we show that E. albertii and H. alvei also decreaseresistance of polarized epithelia.

Given that epithelial barrier function was disrupted by bacterialinfections, we then delineated the nature of the host response toinfection by focusing on intercellular tight junction morphologyand protein expression. Tight junctions are part of an apicallyapposed intercellular adhesion complex, serving to seal the para-cellular space and polarize epithelia [29]. Transmembrane proteinsthat comprise tight junctions include claudins, occludin and junc-tion adhesion molecules. Cytoplasmic zona occludens (ZO) proteinsmediate attachment of transmembrane tight junction proteins tothe F-actin cytoskeleton to form a scaffold, which recruits cellsignaling molecules and forms a contractile ring regulating thefunctional integrity of tight junctions [30,31].

The barrier defects caused by E. albertii and H. alvei were moreevident in MDCK-I cells, compared to T84 cells. The secretorynature of T84 human colonic crypt-like epithelial cells couldprovide a protective barrier that inhibits binding of bacteria [32,33].MDCK-I cells, derived from the canine renal collecting duct are

absorptive in nature and express different glycosphingolipidsurface content than T84 cells [34,35], which could account for theobserved increase in binding of bacteria. Microbial adherence tohost cells also influences bacterial pathogenicity. For instance, thereare differences in the amino acid sequences of outer membraneproteins, such as intimin, across various attaching–effacingbacteria, which influence host cell responses to infection [36].Furthermore, the EHEC O157:H7 genome encodes additionaltranslocated effectors, including EspF, that directly mediatedisruption of intercellular tight junctions [25]. We have shown thatE. albertii, but not H. alvei, also expresses homologs of both thetranslocated intimin receptor (Tir/EspE) and EspF, which couldexplain why only E. albertii disrupts ZO-1 distribution in tightjunctions.

With the use of confocal immunofluorescence microscopy toassess tight junction morphology, we demonstrate that E. albertiitargets intercellular tight junctions, but does so in a slightlydifferent manner than E. coli O157:H7. Although ZO-1 is displaced,as is seen in EHEC O157:H7 infection [12,14], host cell polarity wasstill maintained. Following E. albertii infection, claudin-1 is unaf-fected, both in its spatial distribution and in the amount of totalprotein expression, whereas EHEC O157:H7 dismantles this proteinfrom tight junctions [12].

To further define the nature of abnormalities observed in tightjunctions, we characterized the expression profiles of tightjunction proteins in infected polarized epithelia, because thepermeability properties of tight junctions are dependent on boththe quantity and type of junction protein expressed [37,38]. Forexample, occludin dephosphorylation causes its withdrawal fromthe tight junction apparatus and a resulting decrease in barrierfunction [11,25,27]. In this study, we show that only EHECO157:H7 caused the dephosphorylation of occludin in MDCK-I

Fig. 4. Divergent tight junction protein expression profiles are observed following E. coli O157:H7, E. albertii and H. alvei infections. Representative western blots for tight junctionproteins using MDCK-I and T84 whole cell extracts. (A and B) ZO-1 blot in MDCK-I and T84 cells, respectively. (C and D) Occludin blot in MDCK-I and T84 cells, respectively, anddisplaying high molecular weight (HMW, hyperphosphorylated) and low molecular weight (LMW, hypophosphorylated) isoforms. (E and F) Claudin-1 blot in MDCK-I and T84 cellextracts, respectively. (G and H) Claudin-4 blot in MDCK-I and T84 cell extracts, respectively. (I) Claudin-5 blot in T84 cell protein extracts.

K.A. Donato et al. / Microbial Pathogenesis 45 (2008) 377–385382

cells, while H. alvei H2 caused a marginal decrease in occludinphosphorylation.

The expression of claudin-5 in EHEC O157:H7, E. albertii and H.alvei-infected T84 monolayers has not been explored previously.Claudin-5 confers increased epithelial barrier function to sodiumions and high transepithelial electrical resistance [15,39]. In addi-tion, levels of claudin-5 are reduced in inflamed intestinal tissue ofactive Crohn’s disease and in in vitro models of Helicobacter pyloriinfection [40,41]. All the bacterial strains in this study causeda moderate decrease in the level of expression of claudin-5 in T84

Table 2Bacterial adherence to polarized epithelial monolayers following 16 h of infection.

Bacterium, strain Adherent bacteria per epithelial cell (log10)

MDCK-I T84

EHEC O157:H7, CL56 2.01� 0.12 1.35� 0.03E. albertii, 19982 2.32� 0.12 1.62� 0.06H. alvei, H2 2.99� 0.04 2.09� 0.09H. alvei, D46 2.59� 0.17 1.69� 0.15

Initial inoculum 4.5�107 bacteria for MDCK-I and 3.0�108 bacteria for T84 cells;multiplicity of infection¼ 100:1 for 16 h at 37 �C. Values are reported as mean-s� SEM, n¼ 3 for both MDCK-I and T84 cells.

cells, even though levels of claudin-1 and claudin-4 were notperturbed.

Conversely, claudin-2 is known to increase the permeability ofintercellular tight junctions and is expressed in very low amountsin high resistance T84 monolayers, and not at all in MDCK-Imonolayers [42,43]. Infections with EHEC, E. albertii or H. alvei didnot induce expression of claudin-2 in either T84 or MDCK-I cells,indicating that the observed decreases in barrier function were notdue to an increased proportion of conductive proteins present inthe tight junction. Taken together, the results of this studydemonstrate that decreased epithelial barrier function in responseto bacterial infection is due to abnormalities in both spatial distri-bution and quantitative expression of selected intercellular tightjunction proteins.

4. Experimental/materials and methods

4.1. Epithelial cell cultures

Madin Darby Canine Kidney type-I (MDCK-I) cells (kindlyprovided by Dr. R. Worrell, Vontz Center for Molecular Studies,University of Cincinnati, Cincinnati, OH) were grown in Dulbecco’s

Fig. 5. E. albertii, but not H. alvei, express the LEE encoded effectors EspE and EspF. PCR products of genes encoding E. coli secreted protein (Esp) E (also referred to as Tir) and EspFfrom EHEC O157:H7, strain CL56; E. albertii, strain 19982; and H. alvei strains H2 and D46. The glyceraldehyde-3-phosphate dehydrogenase gene, gapA, was used as a positive control(predicted product size of 627 bp). RNA- and cDNA-template negative samples were used as negative controls.

K.A. Donato et al. / Microbial Pathogenesis 45 (2008) 377–385 383

modified Eagle Medium (DMEM; Gibco, Grand Island, NY) sup-plemented with 10% (v/v) fetal bovine serum (Gibco) and 2% (v/v)penicillin–streptomycin (Gibco). T84 cells (American Type CultureCollection, Manassas, VA) were grown in a 1:1 mixture of DMEMand Ham’s F12 medium (Gibco) supplemented with 10% (v/v) fetalbovine serum, 2% (v/v) penicillin–streptomycin, 2% (v/v) sodiumbicarbonate (Gibco), and 0.5% (v/v) L-glutamine (Gibco). Cells wereincubated in 5% CO2 at 37 �C.

4.2. Bacterial strains and infection conditions

E. albertii, strain 19982 (International Center for DiarrhoealDisease Research, Dhaka, Bangladesh), H. alvei, strain H2 (Hospitalfor Sick Children, Toronto, Ontario, Canada), H. alvei, strain D46,isolated from an outbreak of diarrheal illness in a NewfoundlandHospital [2], and E. coli strain CL56 (serotype O157:H7) (Hospital forSick Children) were plated onto Columbia agar with 5% sheep blood(BBL, Sparks, MD). Prior to infection, bacteria were re-grown in10 ml of static, non-aerated Luria–Bertani (LB) broth at 37 �C toa concentration of w109 CFU/ml. Cell monolayers were apicallyinfected with one of the bacterial strains (MOI 100:1) and incubatedfor 16 h at 37 �C and 5% CO2.

Prior to some experiments, apical wells were filled withmedium containing 20 mg/ml chloramphenicol to inhibit bacterialprotein synthesis [44]. Chloramphenicol treatment did not causethe death of bacteria, since post-infection co-culture media swab-bed onto blood agar plates and incubated overnight yielded viablecolonies. Other experiments utilized 10% formalin-fixed (24 h) or-boiled (100 �C for >45 min) bacteria. Samples of formalin-fixedand heat-killed bacteria were also swabbed onto agar plates toconfirm bacterial death.

To determine if either bacterial or host cell derived secretedfactors played a role in the disruption of barrier function, filteredtissue culture medium obtained from monolayers infected for 16 h,or bacterial culture supernatant from bacteria grown in antibiotic-free eukaryotic culture medium for 16 h, were mixed (1:1 ratio)

with fresh medium and added to untreated monolayers for 16 h at37 �C.

4.3. Measurement of transepithelial electrical resistance (TER)

Confluent flasks of epithelial cells were trypsinized and platedonto 12 mm diameter Transwells (1.5–2.0�105 MDCK-I cells/cm2

or 106 T84 cells/cm2; pore size¼ 0.4 mm, polystyrene membranes;Costar, Corning, NY). Monolayers were grown to allow the devel-opment of intercellular tight junctions and TER to reach>1000 U cm2. Prior to infection with bacteria, cell culture mediumwas changed to antibiotic-free medium. TER was measured witha Millicell-ERS voltmeter and chopstick electrodes (Millipore,Bedford, MA), in units of U cm2, before and 16 h after infection. Dataare reported as the percentage of pre-infection values normalizedagainst uninfected control monolayers.

4.4. Dextran permeability assay

Infected and non-infected tissue culture cells grown on12 mm diameter Transwell supports were rinsed four times inDulbecco’s PBS (Gibco) to remove non-adherent bacteria. Alexa647-conjugated dextran (10 kDa, 100 mg/ml, Molecular Probes,Eugene, OR; diluted in DMEM) was added (0.2 ml) to the apicalaspect of each filter support and DMEM added (0.4 ml) to thebasolateral compartment. Monolayers were incubated for 5 h at37 �C and samples then taken from the basolateral compartmentof each well. After dilution (1:20) in DMEM, samples were loadedinto 96-well microtiter plates (Corning) and read using aninfrared imaging system (Odyssey�; LI-COR Biosciences, Lincoln,NE) in a single scan with 169 mm resolution using the 700 nmchannel. Quantification of signal intensity was performed withcommercial analysis software provided by LI-COR Biosciences.Data recorded from the raw integrated intensity values wereexpressed relative to the intensity of samples collected fromuninfected monolayers.

K.A. Donato et al. / Microbial Pathogenesis 45 (2008) 377–385384

4.5. Confocal immunofluorescence microscopy

MDCK-I and T84 cells were seeded into 6.5 mm Transwells andinfected under the same experimental conditions used for TERexperiments. MDCK-I cell monolayers were rinsed in PBS followedby fixation and permeabilization in 100% methanol (�20 �C)(Caledon Laboratories, Georgetown, Ontario, Canada) for 5 min. T84cells were fixed in 3.7% formaldehyde for 10 min followed by per-meabilization in 0.1% Triton X-100 for 10 min. Cells were incubatedin 10% (v/v) normal goat serum (Jackson Immunoresearch, WestGrove, PA) in PBS for 1 h at room temperature and then incubatedwith primary rabbit anti-ZO-1 or rabbit anti-claudin-1 (Zymed, SanFrancisco, CA) for 1 h at 37 �C. T84 cells were incubated withprimary antibody mixture supplemented with 0.01% Triton X-100(to increase permeability). After rinsing in PBS to remove unboundprimary antibody, cells were incubated in secondary Cy2-conju-gated goat anti-rabbit IgG (1:200 dilution; Zymed) for 1 h at roomtemperature. An aliquot of 300 nM 40, 6-diamidino-2-phenylindole(DAPI) dilactate was added for 5 min to stain nuclei. After rinsing,monolayers on their mesh supports were excised from the wells,mounted onto glass slides and examined using two photon confocallaser scanning microscopy (Zeiss LSM510, Frankfurt, Germany).

4.6. Western blotting for tight junction proteins

Whole cell protein extracts were prepared from epithelial cellmonolayers grown in Transwells. Cells were rinsed, scraped andpelleted in cold PBS. Pellets were re-suspended in protein extrac-tion–cell lysis buffer, as described previously [12]. Samples wereadded (1:2) to a volume of sodium dodecyl sulfate loading bufferwith 1:20 (v/v) dithiothreitol, boiled for 5 min and subjected toelectrophoresis through Tris–HCl gels (Ready Gel�; Bio-Rad Labo-ratories, Hercules, CA) and transferred onto nitrocellulosemembranes (BioTrace NT; Pall Corporation; Ann Arbor, MI) asdescribed previously [12]. Membranes were then blocked for 1 hwith Odyssey blocking buffer (LI-COR Biosciences) and probedovernight at 4 �C with either rabbit anti-claudin-1, -2, -4, and -5,occludin or ZO-1 (1:1000–1:2500; Zymed) and with mouse anti-b-actin (1:5000; Sigma, St. Louis, MO) diluted in Odyssey blockingbuffer supplemented with 0.1% Tween-20. Membranes werewashed in PBS with 0.1% Tween-20 (PBS-T) and then simulta-neously probed with IRDye 800 goat anti-rabbit IgG (1:10,000;Rockland Immunochemicals, Gilbertsville, PA) and Alexa Fluor�

680 goat anti-mouse IgG (1:20,000; Molecular Probes) diluted inOdyssey blocking buffer with 0.1% Tween-20 for 1 h at roomtemperature. Membranes were rinsed before imaging with aninfrared-based detection system (Odyssey�) in both 700 nm and800 nm channels in a single scan at 169 mm resolution. Quantifi-cation was performed with accompanying analysis software.Western blot intensity measurements were recorded as the ratio ofthe integrated intensities of the tight junction protein band ofinterest relative to the integrated intensity of the corresponding b-actin band from the same sample. For occludin, the ratio of the highmolecular weight band intensity (hyperphosphorylated,membrane localized) to the total occludin intensity (including thedephosphorylated band, cytoplasm localized) was determined [11].

4.7. Bacterial adhesion assay

Epithelial cells were seeded in 12 mm diameter Transwell platesat a density of 1.5�105 MDCK-I cells and 106 T84 cells/cm2 andthen grown to confluency. Triplicate monolayers were infectedwith one of the four strains of bacteria (MOI 100:1) for 16 h at 37 �Cin 5% CO2, rinsed two times in Dulbecco’s PBS to remove non-adherent bacteria, lysed in 0.5 ml sterile 1% Triton X-100 in distilledwater and scraped from wells to release adherent bacteria.

Triplicate test samples were pooled before spreading serial 10-folddilutions onto blood agar plates, which were then incubatedovernight at 37 �C with subsequent counting of bacterial colony-forming units (CFU). Each bacterial adhesion assay was repeated onthree separate occasions.

4.7.1. RNA extractionRNA extracts of EHEC O157:H7, strain CL56; E. albertii, strain

19982; H. alvei, strain H2; and H. alvei, strain D46 were obtainedusing the Qiagen RNeasy min-prep kit (Qiagen, Mississauga, ON,Canada).

4.7.2. cDNA synthesisFor PCR analysis, cDNA synthesis was performed by using the

Invitrogen Thermoscript kit (Invitrogen, Carlsbad, CA). Briefly, 1 mgof RNA-template was reverse transcribed using random hexamerprimers in buffered conditions (1� synthesis buffer; 0.1 M DTT,40 U/ml RNaseOUT�; 15 U/ml Thermoscript� RT). The reaction wasincubated for 50 min at 60 �C and terminated at 85 �C for 5 min. Atthe end of the cycle, 1 ml RNase H (Invitrogen) was added to eachreaction tube and samples incubated at 37 �C for 20 min and thenstored at�20 �C. An RNA-template free sample (water) was used asa negative control.

4.7.3. Primers and PCR reactionsPrimers were designed (PrimerQuest, Integrated DNA Technolo-

gies, Coralville, IA) specific for locus of enterocyte effacement path-ogenicity island encoded E. coli O157:H7 gene espE (variouslyreferred to as the translocating intimin receptor, Tir) and espF inEHEC O157:H7, strain EDL933 (GenBank accession no. AE005174) andE. albertii, strain TW07627 (GenBank accession no.NZ_ABKX01000002): espE EHEC EDL933: forward 50-ACG CGC ATAAGT GCT TTG AAT CCC-30 and reverse 50-ACA AGC GCA CGT ACG GTAGAG AAT-30; espE E. albertii TW07627: forward 50-TGC TTG CGC CTGAGC ATT ACT TTC-30 and reverse 50-TCC GCT ACG TAT TGC TGC ATCTGA-30; espF EHEC EDL933: forward 50-TAA TGC CTG TGC AAT GGGCGG TAA-30 and reverse 50-ATG TGA GCA GCC AGG TGA CTT CAT-30;and espF E. albertii TW07627: forward 50-AAA GAC TGG GAT GAA CCAGAT GCC-30 and reverse 50-TGG CAC TTC AAC CCT TAA ACC AGC-30.Primers for the housekeeping gene encoding D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), gapA were used as a control,synthesized as follows from EHEC O157:H7, strain EDL933: forward50-GAT TAC ATG GCA TAC ATG CTG-30 and reverse 50-CAG ACG AACGGT CAG GTC AA-30. The cDNA-templates were amplified using 1 mgof each specific primer, 0.2 mM dNTP, 25 mM MgCl2, and 1.25 U Taqpolymerase (Fermentas, Burlington, ON, Canada) in supplied buffer(10 mM Tris–HCl, pH 8.8; 50 mM KCl, 0.08% NP40). A cDNA-templatefree sample (water) was used as a negative control.

The reaction was incubated at 95 �C for 10 min, followed by 45cycles at 95 �C for 30 s, 55 �C for 60 s, and 72 �C for 30 s. PCRproducts were resolved on a 2% agarose gel in 1� TAE buffer andthen stained with ethidium bromide (50 mg/ml) for visualization ofbands under UV light illumination.

4.8. Data analyses

Data are reported as means� SEM. To test for significanceamong multiple groups, one-way analysis of variance was used fornon-Gaussian distributions, and a P-value of <0.05 consideredsignificant.

Acknowledgments

This research was supported by an operating grant from theCanadian Institutes of Health Research (CIHR). K.A. Donato was therecipient of a Canadian Association of Gastroenterology Summer

K.A. Donato et al. / Microbial Pathogenesis 45 (2008) 377–385 385

Studentship Award and a CIHR Canada Graduate ScholarshipAward. P.M. Sherman is the recipient of a Canada Research Chair inGastrointestinal Disease.

Portions of the work described in this manuscript were pre-sented at the annual general meeting of the American Society forMicrobiology 2007 in Toronto, Ontario, Canada.

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