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Microbial Pathogenesis 51 (2011) 378e383

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Microbial Pathogenesis

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Short Communication

A MinD mutant of enterohemorrhagic E. coli O157:H7 has reduced adherenceto human epithelial cells

Rajinder P. Parti a, Debabrata Biswas a,1, Monica Wang a, Mingmin Liao a, Jo-Anne R. Dillon a,b,*

aVaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK, Canada S7N 5E3bDepartment of Biology, University of Saskatchewan, W.P. Thompson Building, 112 Science Place, Saskatoon, SK, Canada S7N 5E2

a r t i c l e i n f o

Article history:Received 22 July 2010Received in revised form29 June 2011Accepted 6 July 2011Available online 20 July 2011

Keywords:Enterohemorrhagic E. coli O157:H7AdherenceMicrocolonyminD

Abbreviations: EHEC, Enterohemorrhagic E. coreceptor; A/E, Attaching and effacing; Hela, Human ecells; Caco-2, Human colonic adenocarcinoma cells.* Corresponding author. Department of Biology, Uni

Thompson Building, 112 Science Place, Saskatoon, SK,966 1535; fax: þ1 306 966 7478.

E-mail address: j.dillon@usask.ca (J.-A.R. Dillon).1 Present address: Department of Animal and A

Maryland, College Park, MD, USA.

0882-4010/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.micpath.2011.07.003

a b s t r a c t

Adherence to epithelial cells is a prerequisite for intestinal colonization by the bacterial pathogen,enterohemorrhagic Escherichia coli (EHEC). The deletion of minD, a cell division gene, in EHEC causedreduced adherence to human epithelioid cervical carcinoma (HeLa) and human colonic adenocarcinoma(Caco-2) cells as compared to wild-type. The minD mutant formed minicells and filaments owing toaberrant cytokinesis. Moreover, its ability to form microcolonies as typically seen in the co-cultures ofwild-type with Caco-2 cells, was abolished. In conclusion, the present study highlights the importance ofminD in regards to EHEC adherence to human epithelial cells.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 isa food-borne pathogen that causes severe human diseasesincluding non-bloody diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome. It primarily colonizes the epithelium of the largeintestine by forming distinct histopathological attaching andeffacing (A/E) lesions. These lesions are characterized by bacteria-engulfing actin-rich pedestals formed by epithelial cells, degener-ation of brush border microvilli and intimate bacterial attachment[1,2].

Bacterial colonization, a multi-step process begins with theinitial adherence of EHEC to epithelial cells, which in turn inducesthe formation of a host cell-bacteria bridge (translocon) involvingbacterial-encoded EspA, EspB and EspD proteins [3]. The trans-located intimin receptor (Tir) and other bacterial effector proteinsare injected through the translocon into the epithelial cell. In the

li; Tir, translocated intiminpithelioid cervical carcinoma

versity of Saskatchewan, W.P.Canada S7N 5E2. Tel.: þ1 306

vian Science, University of

All rights reserved.

last step, the bacteria intimately attach to and develop micro-colonies on the epithelial cells, inducing condensation of F-actinunderneath them [4]. The intimate attachment is mediated byintimin, an outer membrane protein of EHEC which recognizes Tiror recognizes the host cell integrin or nucleolin [4]. EHEC fimbrialadhesions such as long polar (LP) fimbriae [5], E. coli laminin-binding fimbrae (ELF) [6], the E. coli pilus (ECP) [7], curli [8] andthe F9 pilus [9] also contribute to adherence. Several other viru-lence factors such as shiga toxin and TccP (Tir cytoskeleton-coupling protein) have also been associated with colonization[4,10,11].

In EHEC, microcolony formation is associated with increasedadherence to human epidermoid cancer cells as a consequence ofmultiple pch (perC homologue) genes [12]. In Pseudomonas aeru-ginosa, microcolony formation correlated with more binding tobuccal epithelial cells [13]. Although themechanism ofmicrocolonyformation in EHEC is not very clear, a bacterial effector such asEspL2 has recently been implicated in the process via interactionwith Annexin A2 [11]. The polar type 4 pili of EHEC, also known ashemorrhagic coli pili, are involved in adherence and biofilmformation [14]. In enteropathogenic E. coli, bundle-forming pilimediated the initial binding to epithelial cells, the formation ofmicrocolonies, and interbacterial interactions [15].

The arrangement of cells within a microcolony is a function ofthe cell’s shape, size, mode of cell wall growth, bacterial polymerproduction, and cell division events. Dependence on cell shape was

R.P. Parti et al. / Microbial Pathogenesis 51 (2011) 378e383 379

exemplified by defective biofilms of aberrantly-shaped E. coli withsuccessive deletions of penicillin binding proteins [16,17]. Changesin cell shape are frequently brought about by regulating celldivision in bacteria such as E. coli and Mycobacterium tuberculosisthrough control of FtsZ oligomerization [18,19]. Cell division eventsalso control bacterial aggregation as seen in the V-shaped daughterpairs of Corynebacterium spp. resulting from snapping cytokinesis[20].

In gram-negative rod-shaped bacteria such as E. coli, cell divi-sion involves MinC, MinD, and MinE proteins that participate in theidentification of the midcell site for division septum placement andsite-specific inhibition of septation at the cell poles [21]. MinCprevents cell division by inhibiting the formation of FtsZ rings thatare critical in initating septum formation [22,23]. MinD, an ATPaserecruits MinC to the membrane, thereafter activating its inhibitoryaction [24,25]. Moreover, it sensitizes MinC to suppression by MinEthat causes MinC and MinD to dissociate from the membrane andco-oscillate from one pole to the other [24,26,27]. This movementproduces a concentration gradient leaving the midcell free of MinCand MinD, thereby allowing FtsZ to form a ring and promptingdivisome formation. In the absence ofminC orminD gene products,septation frequently occurs adjacent to the cell pole instead of atmidcell, leading to the formation of either small spherical minicellsthat lack chromosomal DNA or long filamentous cells [28]. Similarcell division phenotypes in E. coli involving Min proteins fromNeisseria gonorrhoeae [25,29] have also been observed.

Regulation of bacterial cell division events in response to hostdefense can be manifested as altered virulence [19,30]. The aim ofthe present study was to determine the influence of an EHEC celldivision defect on its virulence. More specifically, a minD deletionmutant of EHEC EDL933 was constructed and its adherence tohuman epithelioid cervical carcinoma (HeLa) and human colonicadenocarcinoma (Caco-2) cells was compared to the wild-type. Wedemonstrate that EHEC minD deletion strain produces filamentsand minicells with defective microcolonies and reduced adherenceto human epithelial cells.

2. Results

2.1. EHEC minD deletion mutant, MEDL1, displays aberrant cellmorphology

As observed by Differential Interference Contrast microscopy,EHEC MEDL1 exhibited a mixed phenotype of filaments and mini-cells signifying aberrant cell division, whilewild-type EHEC EDL933cells displayed typical rod-shaped morphology (Fig. 1A and B).

Fig. 1. Effect of minD deletion on cell morphology of EHEC. Differential interference contras(B). Both panels are of the same magnification, and the scale bar in panel B represents 4 mm.arrows) and minicells (short arrows).

Moreover, EHEC MEDL1 formed larger colonies on LB agar platesrelative to those of wild-type EHEC (data not shown).

2.2. minD mutation in EHEC causes decrease in adherence tohuman epithelial cells with no effect on shiga toxin production

To compare the binding efficiency of EHEC EDL933 and MEDL1strains, HeLa and Caco-2 cell monolayers were infected with eachstrain for 1, 2 and 3 h. The adherence of EHEC MEDL1 to HeLa cellswas significantly lower than that of EHEC EDL933 at all 3 timepoints (P< 0.008, 0.0108 and 0.0498, respectively) (Fig. 2A). Simi-larly, as shown in Fig. 2B, EHEC MEDL1 exhibited four- to fivefoldlower levels of cell binding thanwild-type EDL933 after 1, 2 and 3 hof Caco-2 cells infection (P< 0.0062, 0.0056 and 0.001, respec-tively). Therefore, this adherence assay clearly reveals the signifi-cance of minD in the association of EHEC with human epithelialcells. The results for these adherence assays were not influenced bybacterial growth rates as both strains grew at similar rates in tissueculture medium for the first 6 h under ex vivo assay conditions(data not shown).

RT-PCR using primers specific for slt (shiga-like toxin)-II [31]revealed that slt mRNA in EHEC EDL933 is expressed at levelssimilar to that in theminDmutant strain (Fig. 2C). The production ofshiga toxin in EHEC is not affected by minD disruption.

2.3. minD mutant of EHEC is defective in microcolony formation

Scanning electron microscopic analysis showed that the typicalrod-shaped cells of EHEC EDL933 attach onto the surface of theCaco-2 cell monolayer as microcolonies (Fig. 3A). Moreover, within1 h of infection, wild-type EDL933 appeared very tightly opposed tothe plasma membrane (Fig. 3A and C).

In contrast, EHEC MEDL1, occurring in filamentous and minicellmorphologies, adhered to Caco-2 cells in a diffuse pattern 1 h post-infection (Fig. 3B and D). At 6 h post-infection, lysis of epithelialcells was observed in the EHEC EDL933-infected monolayer incontrast to the intact Caco-2 cells incubated with EHEC MEDL1(Fig. 3E and F). This cell death may be the result of wild-typebacteria-host cell interactions mediated by tight adherence whilelow and diffused association of MEDL1 with Caco-2 cells explainsthe maintenance of epithelial cell integrity.

3. Discussion

The minD mutant of EHEC forms minicells and filamentsbecause of the frequent placement of the septum at the cell polesinstead of the midcell. These aberrantly shaped mutant bacteria

t micrographs of wild-type EHEC EDL933 (A) and EHEC minD deletion mutant, MEDL1EHEC MEDL1 exhibited abnormal cell morphologies (B) including long filaments (long

Fig. 2. Decreased adherence of minD mutant, EHEC MEDL1 to Hela (A) and Caco-2 (B) cells as compared to the wild-type EHEC EDL933. Percentage adherence was determinedfollowing infection with EHEC EDL933 and EHEC MEDL1 for 1, 2 and 3 h. Results expressed as the percentage of original inocula are the average� SD of three independentexperiments. *P value of <0.05 compared to the wild-type strain EDL933. (C) RT-PCR of slt mRNA from EDL933 and MEDL1 strains of EHEC grown under optimal conditions (late logphase, 37 �C, 200 rpm shaking). Lanes 1e2, slt-II-specific 254 bp amplicons from EDL933 and MEDL1, respectively; lanes 3e4, no RT controls; lanes 5e6, negative (no RNA) controls;lanes 7e8, positive mRNA controls in EDL933 and MEDL1 respectively. GeneRuler 1 kb Plus DNA Ladder was used as a molecular weight marker.

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showed reduced adherence to cultured Hela and Caco-2 cells. Infact, the percentage adherence levels of the mutant bacteria toepithelial cells were so low that even a gradual increase in incu-bation time with cultured cells to 3 h did not make a significantdifference to the binding values. This decreased adhesion mightpossibly be either due to changes in cell surface characteristics suchas hydrophobicity or shape. In a recent study, an aberrantly shapeddip1281 mutant strain of Corynebacterium diphtheria displayedaltered cell surface protein patterns and a simultaneous decrease inadherence and internalization in epithelial cells [32]. Bacterialshape does contribute to virulence as confirmed by the correlationof pathogenesis with cell division protein-mediated shape changesin uropathogenic E. coli and M. tuberculosis [19,30].

EHEC MEDL1, a minD mutant of EHEC, did not aggregate intomicrocolonies which is so very essential to epithelial cell adherenceas shown earlier [12]. This may again be explained by the abnormalshapes of the mutant cells, although the effects of a minD deletionon microcolony-promoting factors such as EspL2 cannot be ruledout. Based on our results, we can conclude thatminD is essential foradherence via its role in bacterial shape maintenance and micro-colony formation. Future studies on the effects of a minD deletionon known EHEC adhesins such as intiminwould further unravel themechanisms of adherence to epithelial cells. In a recent publication,we showed a clear involvement of min genes in the adherence ofN. gonorrhoeae to epithelial cells [33]. Studies done in Francisellatularensis have also attributed MinD a role in virulence, thatincluded the facilitation of bacterial survival in macrophages[34,35].

EHEC is bound to the epithelial cell surface in microcolonies,beneath which F-actin is assembled into focussed pedestals [4,10].In our investigation, wild-type EHEC EDL933 attached intimately inmicrocolonies to Caco-2 cells 1 h post-infection, thereby triggeringthe host cell defense machinery. This interaction culminated aslysed cells 6 h post-infection, possibly owing to bacterial- or self-induced cell death. In the co-culture of Caco-2 with EHEC MEDL1,

the lack of microcolony formation explains reduced interactionwith epithelial cells and consequently, the cell monolayer wasintact. In future studies on EHEC MEDL1-infected epithelial cells, itwould be interesting to study actin pedestal formation. However,using confocal microscopy on Caco-2 cells infected for 6 h, weobserved both the wild-type and mutant adherent bacteria dis-playing associated actin fluorescence (data not shown).

Finally, the present study reveals the significance of the EHECcell division gene, minD, in adherence to human epithelial cells.Moreover, its relevance to EHEC microcolony formation and inter-actions with Caco-2 cells is also demonstrated.

4. Materials and methods

4.1. Bacterial strains and culture conditions

Wild-type EHEC EDL933 [36] was used to construct the isogenicminD (MEDL1) mutant. E. coli SY327lpir (D(lac pro) argE(Am) rifnalA recA56 (lpirR6K); [37]) was used as a host for the amplificationof E. coli suicide plasmids, and E. coli Sm10pir (thi thr leu tonA lacYsupE recA::RP4-2Tc::Mu (lpirR6 K), Kmr; [38]) as a host for har-bouring conjugative plasmids and as a donor for subsequentconjugation experiments. All E. coli strains were grown at 37 �C onLB medium (Difco) supplemented with appropriate antibiotics asindicated.

4.2. Construction of minD deletion mutant of EHEC

An EHEC minD deletion mutant was created in EHEC EDL933using the allelic exchange deletion method as described previously[39]. E. coli suicide plasmid pRE112 [39]was used as a cloning vectorto construct a minD deletion plasmid. Initially, the flanking region(1.2 kb) immediately upstream of theminDwas PCR-amplified fromEHEC EDL933 using primers Up1 (50-gcggtctagagaaaaaatacacttgcctcaatactg-30) and Up2 (50-gcgcgagctcaaattccttgttaaaaagggatc-30).

Fig. 3. Scanning electron and confocal microscopy of Caco-2 cells infected with EHEC EDL933 and EHEC MEDL1. Microcolonies of EHEC EDL933 attached intimately to epithelial cells1 h post-infection (A, C), and lysed eukaryotic cell membranes 6 h post-infection (E). Diffuse adherence of EHEC MEDL1 was observed 1 h post-infection to Caco-2 cells (B, D), whilecultured cells are intact even after 6 h of infection (F). Confocal images of DAPI-stained bacteria (blue; shownwith arrows) display compact (C) or sparse arrangement (D) on Caco-2monolayer stained for actin (red; Alexa Fluor� 546 phalloidin). Arrowheads are used to show cell nuclei stained blue by DAPI. Scale bars for SEM and confocal are 1 and 5 mm,respectively (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

R.P. Parti et al. / Microbial Pathogenesis 51 (2011) 378e383 381

Primers contained restriction endonuclease digestion sites XbaI andSacI, respectively. The amplicon was cloned into the XbaI/SacI sitesof pRE112, generating pRE112MinDup. Subsequently, a down-stream flanking region (1 kb) of minD was PCR-amplified usingprimers Dn1 (50-atatgagctcccagaggatcaatcagtattgc-30) and Dn2 (50-gtgtcccgggatggcatggaatcgaaatcagt-30); the primers contained SacI/SmaI endonuclease restriction enzyme sites. The amplicon wassubsequently cloned into the SacI/SmaI sites of pRE112 MIinDup,generating plasmid pRE112MinD. pRE112MinD contained sequ-ences upstreamanddownstreamofminDbut lackedminD. TheE. colisuicide plasmids (pRe112, pRE112MinDup and pRE112MinD) wereamplified in E. coli SY327lpir. DNA sequence fidelity of plasmidpRE112MinD was confirmed by DNA sequencing analysis of theinserted amplicons (PBI-NRC, Saskatoon, SK, Canada).

Plasmid pRE112MinD was transformed into E. coli Sm10pir togenerate a donor strain for mating with EHEC EDL933. The trans-formants were grown on LB agar containing chloromphenical (Cm,30 mg/ml). In conjugation assays, mid-log phase cells of the donor

strain [E. coli Sm10pir (pRE112MinD)] and the recipient strain(EHEC EDL933) were co-incubated (1:1) at 35 �C for 2 h. Cells werecultured on LB plates containing Nal to select single cross-overmutants of EHEC EDL933, generating a mutant of EHEC EDL933with a minD deletion (EHEC MEDL1). EHEC MEDL1 cells wereidentified by sucrose selection [39]. The absence of minD in EHECMEDL1 was confirmed by lack of PCR amplification using primerscorresponding to minD sequences and by Western Blot analysisusing the anti-MinD (E. coli) polyclonal antibody [25,40].

4.3. Reverse-transcriptase polymerase chain reaction

EHEC EDL933 and MEDL1 cultures were grown by inoculatinga loopful of bacteria into LB broth and incubating at 37 �C withshaking (200 rpm) till late log phase. Genomic DNA-free RNA wasextracted from cultures using the Ribopure-Bacteria Kit (Ambion,USA). For amplification by RT-PCR (Qiagen OneStep RT-PCR Kit,Cat# 210210), 0.25 mg of bacterial RNA/reaction was used. A primer

R.P. Parti et al. / Microbial Pathogenesis 51 (2011) 378e383382

pair [31] targeting the specific sequence of the slt-II toxin gene wasused to compare transcription of Shiga toxin in the two EHECstrains. Positive mRNA controls were evaluated using primers fortufA, an elongation factor gene constitutively expressed in livingbacterial cells [31]. Negative controls lacked template RNA in orderto detect possible contamination of reaction components. To detectDNA contamination, control (no RT) reactions were kept on ice andplaced in the thermal cycler only after it had reached 95 �C for theHotStarTaq DNA Polymerase activation step.

4.4. Epithelial cell culture and adherence assay

HeLa and Caco-2 cells were grown in minimal essential mediumwith 10% foetal bovine serum at 37 �C in a humidified atmosphereof 5% CO2. Medium used for Caco-2 cells was supplemented with L-glutamine (2 mM), 100 mMnonessential amino acid solution (GibcoBRL), and 1 mM sodium pyruvate. Confluent cultures werepassaged by trypsinization (0.25% trypsin, 0.1% EDTA), and resee-ded at a density of 105 cells/ml in 75 cm2

flasks.The adherence assay was performed as described by Samadder

et al. [6]. HeLa or Caco-2 cells were seeded in 24-well tissue cultureplates (Falcon, USA) at a density of 105 cells/ml/well. After over-night growth, cell monolayers reached semi-confluence and werethen infected with bacteria at a multiplicity of infection of 1:100(100 bacteria per epithelial cell). Cell monolayers were washed fivetimes with antibiotic-free medium 1 h, 2 h and 3 h post-infectionfollowed by lysis with 1% saponin for 15 min and serial dilutionsof the lysate were plated on LB agar to determine the number ofviable bacteria.

4.5. Light microscopy

Cultures grown overnight of EHEC EDL933 and MEDL1 werediluted 1:100 with fresh LB medium and further incubated for 3 hat 37 �C, followed by fixation as previously described [25]. Differ-ential interference contrast microscopy was done on fixed bacterialcells using an Olympus BX61 microscope fitted with a 100� oilimmersion objective and a Photometrics CoolSnap ES camera.Image processing, de-convolution and cell count were facilitatedwith ImagePro 6.0 software.

4.6. Scanning electron and confocal microscopy

Caco-2 cells were grown on MatTek plates or plastic cover-slips(Sumitomo, Japan) for confocal and scanning electron microscopy(SEM), respectively. Cells were then infected as described above forthe adherence assay. For SEM, infected cells were rinsed withphosphate buffered saline and fixed overnight at 4 �C with 2%glutaraldehyde (Sigma, USA). After fixation, cells werewashed withphosphate buffered saline and dehydrated using a series ofincreasing concentrations of ethanol (50, 80, 90, 95 and 100%), for5 min at each concentration, with the final concentration repeatedtwice. Cells were dried in a critical point-drier, mounted on a stabculture and ion-coated with gold 200�A using an ion coater. Finally,cells were observed using a S-8600 scanning electron microscope(Hitachi, Japan) at the Electron Microscope Unit of the University ofSaskatchewan.

As described previously [15], confocal microscopy was per-formed on infected caco-2 cells by fixing (15 min) them in 3.7%formaldehyde followed by permeabilization (10 min) with 0.1%Triton X-100 (in PBS). Then, samples were washed three times withPBS, stained with DAPI (Sigma) to stain bacteria and/or phalloidin(Molecular Probes) to stain cytoskeletal actin. Caco-2 cell prepa-rations were examined using a Leica TCS SP5 Spectral ConfocalMicroscope, equipped with an Argon (458, 477, 488, 514, 488 nm)

and two Helium/Neon (543, 633 nm) lasers and 10�/20�/40�/63�PL APO objectives.

4.7. Statistical analysis

Statistical analysis was performed using Graphpad Prism 5.0. Foradherence assays, a non-parametric analysis (KruskaleWallis test)was done on bacterial colony counts. P< 0.05 was defined assignificant.

Acknowledgements

We thank Daryoush Hajinezhad, WCVM, University of Sas-katchewan, Saskatoon, Canada, for assistance in confocal micros-copy. We thank Dr. Lawrence Rothfield (Department of Molecular,Microbial and Structural Biology, University of Connecticut HealthCenter, Farmington, CT, USA) for kindly providing anti-MinD (E. coli)antibody. This work was supported by the Canadian Institutes ofHealth Research and the Saskatchewan Health Research Founda-tion Regional Partnership Program (CIHR-RPP; Grant No.G00005398), the SHRF New Investigator Establishment Grant(Grant No. 1866-2007), and the Natural Sciences and EngineeringResearch Council of Canada (Grant No. 203651-2006 RGPIN).

Appendix. Supplementary material

Supplementary data associated with this article can be found, inthe on-line version, at doi:10.1016/j.micpath.2011.07.003.

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