probiotic colonisation of mucus-adherent ht29mtxe12 cells

Probiotic colonisation of mucus-adherent HT29MTXE12 cells 1

attenuates Campylobacter jejuni virulence properties. 2

3

Abofu Alemka 1,2

, Marguerite Clyne 1,2

, Fergus Shanahan 4, Thomas Tompkins

3, 4

Nicolae Corcionivoschi 1,2

, and Billy Bourke1,2

* 5

6

1School of Medicine and Medical Science, Conway Institute, University College Dublin, 7

Belfield, Dublin 4, Ireland. 8

2The Children’s Research Centre, Our Lady’s Childrens’Hospital, Crumlin, Dublin 12, 9

Ireland. 10

3Institut Rosell-Lallemand, 6100 Royalmount, Montreal, QC Canada. 11

4Alimentary Pharmabiotic Centre, University College Cork, Ireland. 12

13

Running title: Probiotics attenuate C. jejuni virulence properties. 14

Keywords: Campylobacter jejuni, mucus, probiotics 15

16

*Corresponding author: Billy Bourke 17

The Children’s Research Centre, Our Lady’s Childrens’ Hospital, 18

Crumlin, Dublin 12, Ireland. Email: [email protected] 19

Tel: 0035314096272 Fax: 0035314555307 20

21

22

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01249-09 IAI Accepts, published online ahead of print on 22 March 2010

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Summary 23

24

The HT29MTXE12 (E12) cell line harbours an adherent mucus layer, providing a novel 25

technique to model mucosal infection in vitro. In this study, we have characterised the 26

interaction of Campylobacter jejuni with E12 cells and exploited its unique mucus layer 27

to examine the potential efficacy of probiotic treatment to attenuate C. jejuni virulence 28

properties. C. jejuni 81-176 colonised and reproduced in E12 mucus. Adhesion to and 29

internalisation of C. jejuni was enhanced in E12 cells harbouring mucus compared to 30

parental cells without mucus. Translocation of C. jejuni occurred at early time points 31

following infection. C. jejuni aligned with tight junctions and colocalised with the tight 32

junction protein occludin, suggesting a paracellular route of translocation. Probiotic 33

strains, Lactobacillus rhamnosus R0011, L. helveticus R0052, L. salivarius AH102, 34

Bifidobacterium longum AH1205, a commercial combination of L. rhamnosus R0011 and 35

L. helveticus R0052 (LacidofilTM), and a cocktail consisting of L. rhamnosus, L. 36

helveticus and L. salivarius (RhHeSa), colonised E12 mucus and bound to underlying 37

cells. Probiotics attenuated C. jejuni association with, internalisation into E12 cells and 38

translocation to the basolateral medium of transwells. Live bacteria and prolonged 39

precolonisation of E12 cells with probiotics were necessary for probiotic action. These 40

results demonstrate the potential for E12 cells as a model of mucosal pathogenesis and 41

provide a rationale for the further investigation of probiotics as prophylaxis against 42

human campylobacteriosis. 43

44

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INTRODUCTION 46

47

The food-borne pathogen C. jejuni is among the commonest bacterial causes of 48

gastroenteritis (43). Serious postinfectious sequelae such as reactive arthritis (35), 49

irritable bowel syndrome (37), and the paralytic neuropathy, Guillain-Barre syndrome 50

(30) also have been associated with antecedent C. jejuni infections. Treatment of 51

Campylobacter infections is usually supportive while antibiotics are reserved for invasive 52

disease (43). Currently there is no vaccine against Campylobacter and preventive 53

measures aimed at reducing environmental contamination are largely ineffective. Thus 54

there is a need for alternative strategies that would complement currently employed 55

methods aimed at reducing C. jejuni induced disease burden in humans. 56

57

Probiotics, ‘live microorganisms which when administered in adequate amounts confer a 58

health benefit on the host’ (44) or ‘commensal microorganisms that can be harnessed for 59

health benefits’ (34), are an attractive alternative intervention strategy both in poultry and 60

in humans for the interruption of the C. jejuni infection cycle and / or treatment of active 61

Campylobacter-related disease. Probiotics have been shown to attenuate the virulence of 62

several enteropathogens in vitro (9, 36). Preliminary data from coculture (7) and animal 63

studies (29, 41, 45) point to the potential effectiveness of probiotics in inhibiting C. jejuni 64

infection. In a recent paper, Wine et al. (46) provide evidence for strain and cell-type 65

specific reduction in C. jejuni invasiveness following coculture with probiotics. However, 66

in general, the effect of probiotics on the pathogenicity of C. jejuni has received little 67

attention. 68


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Campylobacter research has been hampered by the absence of a simple and reliable 69

animal model that mimics human campylobacteriosis (17) and by concern for the 70

development of serious sequelae following experimental human challenge with C. jejuni. 71

For these reasons, in vitro cell culture methods have been extensively used to study the 72

virulence of this organism. However, a key disadvantage in the use of conventional cell 73

culture methods is the lack of an associated mucus gel layer. The HT29MTXE12 (E12) 74

cell line, a goblet cell like subclone of the human colon carcinoma HT29 cell line, 75

uniquely secretes an adherent mucus layer and forms tight junctions when polarised on 76

transwell inserts (3). This novel model provides an opportunity to study in vitro the role 77

of mucus and the relationship of mucus associated factors such as endogenous flora and 78

probiotic organisms on colonisation and pathogenic behaviour of enteric pathogens. 79

80

In this study, we have characterised and exploited this novel model in order to examine 81

the effectiveness of probiotics in protection against C. jejuni. We demonstrate that the 82

presence of an associated mucus layer enhanced C. jejuni pathogenicity and we provide 83

evidence for bacterial translocation through the paracellular route. We show that some 84

probiotic strains attenuate the virulence of C. jejuni. Further, we show that 85

precolonisation of E12 cells with probiotics is necessary for probiotic efficacy. These 86

data provide evidence for the potential use of certain probiotics in intervention strategies 87

against C. jejuni. In particular, these results suggest that the effectiveness of probiotics 88

against human C. jejuni infection may require anticipatory use of this therapy to prevent 89

infection in those at high risk rather than as active treatment in infected individuals. 90

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Materials and methods 92

I: Infection assays 93

Bacterial strains, routine maintenance and culture 94

95

C. jejuni 81-176 is a well characterised and invasive strain that has been used in several 96

studies (14). It was originally isolated in a milk-borne outbreak (20). C. jejuni 81-176 was 97

routinely cultured on Mueller Hinton agar (Oxoid), supplemented with Campylobacter 98

selective supplement (Skirrow) at 37˚C under microaerophilic conditions using 99

Campygen gas packs (Oxoid). Lactobacillus salivarius AH102 (9) and Bifidobacterium 100

longum AH1205 (9) previously have been characterised at the Pharmabiotic Centre, 101

Cork. Lactobacillus helveticus R0052 (16, 36), Lactobacillus rhamnosus R0011 (36) and 102

LacidofilTM (5: 95 mixture of L. helveticus and L. rhamnosus) were obtained as 103

lyophilised products from the Institut Rosell-Lallemand, Montreal, Canada, and stored at 104

4°C. L. salivarius, L. rhamnosus and L. helveticus were routinely grown in de Man, 105

Rogosa, and Sharpe (MRS) broth. RhHeSa was a combination of L. rhamnosus, L. 106

helveticus and L. salivarius prepared by mixing equal volumes and optical densities 107

(OD600 nm) of these bacteria in tissue culture medium. B. longum was grown in MRS 108

broth supplemented with 0.05% cysteine hydrochloride. All probiotic strains were grown 109

at 37°C under microaerophilic conditions. 110

111

112

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Cell lines 114

115

The human colon adenocarcinoma cell line HT29 (22) was purchased from the American 116

Type Culture Collection (ATCC HTB-38). HT29MTXE12 (E12) cells, which are 117

methotrexate (MTX) adapted HT29 cells (HT29MTX) (22) grown on transwell inserts 118

and selected on the basis of the formation of an adherent mucus layer, tight junctions and 119

a confluent monolayer, were a kind gift from Prof. Per Artursson of the University of 120

Uppsala, Sweden (3). 121

122

Cell culture 123

124

E12 cells were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM- 125

Biowhittaker) supplemented with 10% foetal bovine serum (FBS) (Sigma), 2mM L-126

glutamine (Gibco), and 1% non essential amino acids (Biowhittaker). HT29 cells were 127

grown in McCoy’s 5A modified medium (Sigma) supplemented with 10% FBS. All cells 128

were routinely maintained in 75 cm2 tissue culture flasks and incubated at 37˚C in 5% 129

CO2 in a humidified atmosphere. Cells were passaged when the confluency of the flasks 130

was about 80%. Cells were trypsinised and seeded onto 24 well PVDF plates (Corning). 131

In order to polarise cells, they were seeded on transwell inserts (0.4µm pore size, 12mm 132

diameter) (Corning) where they formed a polarised monolayer and tight junctions. Cells 133

were seeded at a density of 1 x 105 cells / well. Cells were grown for 48 hours on 24 well 134

plates prior to use, and up to 21 days on transwell inserts. Penicillin (10 U/ml), 10 ug / ml 135

streptomycin and 0.25 ug/ ml amphotericin B were added to the medium prior to seeding 136


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on transwells. Antibiotics were removed from the cells at least 24 hours before infection 137

assays. 138

139

Culture of C. jejuni for infection assays 140

141

C. jejuni 81-176 was cultured from frozen stocks on Mueller Hinton agar for 24 hours. 142

One loop full of bacteria was then transferred to biphasic medium in 25 cm2 tissue culture 143

flasks (Corning) consisting of Mueller Hinton agar and 6 ml of DMEM prepared as 144

described above but supplemented with 2% FBS, and without antibiotics, for 18 to 21 145

hours. All bacterial incubations were under microaerophilic conditions generated by 146

Campygen gas packs. Bacteria were harvested from the biphasic medium, washed once in 147

DMEM and then diluted to an optical density OD600 ~ 0.2 or the required OD where 148

indicated. 149

150

Total association and gentamycin protection assay 151

152

E12 and HT29 cells grown in 24 well plates (non polarised and no mucus) or polarised 153

HT29 (21 days) and polarised E12 cells (7, 14 and 21 days) seeded on 24 well plates or 154

0.4µm pore size transwells (Corning), respectively, were infected with C. jejuni harvested 155

from Mueller Hinton Agar / DMEM biphasic medium at a multiplicity of infection (MOI) 156

of 500. Infected cells were incubated at 37˚C under microaerophilic conditions using 157

Campygen gas packs (Oxoid) for 3, 24 and 48 hours. Infection of E12 cells for 24 and 48 158

hours with C. jejuni did not affect the viability of cells, as shown by trypan blue 159


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exclusion assays. One set of wells was washed six times with PBS and the cells were 160

lysed with 100 µl of 0.1% Triton-X-100 in PBS for 15 minutes, after which serial 161

dilutions of the cell lysate in PBS were plated on Mueller Hinton agar. To determine the 162

amount of internalised bacteria, a second set of wells was washed with tissue culture 163

medium and 400 µg / ml gentamycin sulphate in DMEM was added on both chambers of 164

the transwells for 2 hours. Cells were washed in PBS, lysed with 100 µl of 0.1% Triton-165

X-100 in PBS and serial dilutions of the lysate plated on Mueller Hinton agar plates. 166

Mueller Hinton agar plates were incubated at 37˚C under microaerophilic conditions for 167

48 to 72 hours after which bacterial colony forming units (CFU) were enumerated. 168

169

To quantify bacteria that bound to underlying cells after penetrating the E12 mucus layer, 170

10 mM N-acetylcysteine (NAC Sigma) in PBS containing 0.2 µM calcium chloride, 0.5 171

mM magnesium chloride and 15 mM glucose, for 1 hour with agitation at 70 rpm, was 172

used to remove the mucus layer after the C. jejuni infection period. Cells were washed 173

four times in PBS to remove NAC, lysed with 100 µl of 0.1% Triton-X-100 in PBS for 174

15 minutes, and serial dilutions of the cell lysates made in PBS. 175

176

Reproduction in E12 mucus 177

178

E12 cells were infected with C. jejuni as described above (3 sets of wells). After 3 hours, 179

all wells were washed and a total association assay was performed on one set of wells. 180

The remaining two sets of wells were further incubated with bacteria free medium for 24 181

and 48 hours after which a total association assay was again performed. All wells were 182


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washed six times in tissue culture medium prior to total association assays. As a control, 183

DMEM alone (supplemented with 2% FBS) was investigated for bacterial replication 184

after 24 hours. C. jejuni reproduction was also investigated directly in mucus harvested 185

from E12 cell culture supernatants. Mucus was pooled from confluent E12 cell culture 186

supernatants by centrifugation at 1500 rpm for 5 mins. Pelleted mucus was coincubated 187

with C. jejuni in DMEM supplemented with 2% FBS (OD600 ~ 0.15) over a 24 hour 188

period. As a control, C. jejuni was incubated in DMEM alone without mucus over a 24 189

hour period. Bacterial CFU was enumerated in mucus and medium alone (controls) at 0, 190

5 and 24 hours as described above. 191

192

Translocation and transepithelial resistance (TEER) measurements 193

194

The amount of bacteria that translocated to the basolateral medium of the transwells after 195

apical infection with C. jejuni was determined by making serial dilutions of the 196

basolateral medium in PBS which was then plated on Mueller Hinton agar and incubated 197

at 37˚C under microaerophilic conditions. The basolateral medium was plated 5 and 24 198

hours postinfection. To investigate the effect of C. jejuni on the barrier properties of E12 199

cells, following the infection of E12 cells with C. jejuni, transepithelial resistance (TEER) 200

was measured at 0, 5, 24 and 48 hours postinfection using an EVOM X meter (World 201

Precision Instruments) coupled to an Endohm chamber (World Precision Instruments) 202

and compared to non infected cells whose TEER was measured at the same time points. 203

The effect on E12 cell TEER of coincubation of probiotics and C. jejuni was also 204

determined. E12 cells were precolonised with L. rhamnosus for 15 hours prior to 205


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infection with C. jejuni for a further 48 hours. TEER of E12 cells was measured at 0, 15, 206

24 and 48 hours postinfection in coincubated cells, in cells infected with L. rhamnosus or 207

C. jejuni alone and in uninfected cells. 208

209

Infection studies with probiotics and C. jejuni 210

211

Probiotics were grown in MRS broth for 6 to 9 hours under microaerophilic conditions. 212

Probiotics were pelleted by centrifugation at 3000 rpm for 5 mins, resuspended in 213

prepared tissue culture medium (DMEM or RPMI) at an OD600 ~ 0.3 to 0.4. 300 µl of 214

probiotics in tissue culture medium was used to infect cells for an initial period of either 4 215

hours or 15 hours. The medium on the cells was then replaced with 300 µl OD600 ~ 0.15 216

C. jejuni harvested from biphasic medium. Infected cells were further incubated for 24 217

hours. To minimise changes in the pH due to the probiotics (probiotics reduce the pH of 218

medium), medium on cells was replaced with fresh medium at the 6th and 12th hours 219

following infection. Medium replenishment was done carefully and did not disturb the 220

mucous layer with its associated colonising organisms. Medium replenishment did not 221

affect probiotic colonisation levels as shown by total association assays. Typically pH 222

levels were maintained above 5.5. Coincubation with probiotics did not affect the 223

viability of E12 cells as shown by trypan blue exclusion assays. After the incubation 224

period, a total association and invasion assay was performed to quantify bound and 225

internalised C. jejuni and probiotics. As a control, heat treated L. rhamnosus (100˚C for 226

10 minutes) was also used to infect E12 cells. Heat treatment resulted in dead but 227

physically intact bacteria as verified by gram staining. Translocation of the bacteria from 228


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the apical to the basolateral aspect of cells was determined by making serial dilutions of 229

the basolateral medium in PBS and plating on Mueller Hinton agar to quantify bacterial 230

CFU in the basolateral medium. Mueller Hinton agar supplemented with Campylobacter 231

Selective Supplement (Skirrows) was used to select for C. jejuni CFU while MRS agar 232

(MRS broth supplemented with 1% agar) was used to select for probiotics. For selection 233

of C. jejuni when coincubated with L. rhamnosus, Campylobacter blood free selective 234

agar base (modified CCDA Oxoid) supplemented with Campylobacter selective 235

supplement was used. 236

237

Statistical analysis 238

239

Experiments were conducted on at least three separate occasions in triplicates. Results are 240

presented as the means ± standard deviations (error bars) of replicate experiments. TEER 241

experiments were conducted with a sample size (n) of at least 5. Graphs were drawn 242

using Microsoft Excel and the unpaired student t test was used to estimate statistical 243

significance. P<0.05 was considered significant. 244

245

246

247

248

249


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II: Microscopy 250

251

Embedding polarised E12 cells in liver 252

253

The HT29MTXE12 cell line is a subclone of the parent HT29 cell line, which secretes an 254

adherent mucus gel when it becomes polarised on transwell inserts for 21 days (3). The 255

mucus layer typically increased in thickness with time. Conventionally, E12 cells were 256

studied and used at 7, 14, and 21 days post seeding on transwells. After cells were grown 257

on transwells for the required time, the transwell inserts were washed with PBS, excised, 258

embedded between two flat pieces of chicken liver tissue to protect the mucus layer (18), 259

frozen in isopentane cooled by liquid nitrogen, and placed in Cryomold Intermediates 260

(Tissue-Tek) containing OCT compound (Santa Cruz). The embedded transwell inserts 261

were further frozen in isopentane cooled by liquid nitrogen, prior to storage at -20˚C. 262

Sections were made using a cryostat and fixed onto poly-L lysine precoated microscope 263

slides (VWR International) using 4% formaldehyde in PBS (formalin). Slides were 264

examined by fluorescence microscopy (see Imaging section below). 265

266

PAS Staining for mucus 267

268

Slides were immersed in periodic acid solution (Sigma) for 5 minutes, Schiff’s reagent 269

(Sigma) for 15 minutes and counterstained with haematoxylin solution (Sigma) for 90 270


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seconds. Slides were rinsed with 6 changes of distilled water between reagents, left to dry 271

and mounted in DPX (BDH) prior to fluorescence microscopy. 272

273

TAMRA staining of C. jejuni and probiotics 274

275

Bacteria were fluorescently labelled with carboxytetramethylrhodamine (TAMRA, 276

Molecular Probes) (6, 28) and used in infection assays. TAMRA stains the cell cytoplasm 277

by reacting with amines to form stable amide bonds, and is conventionally used in our 278

laboratory for vital staining (6). Bacteria were incubated in 10 µg / ml of TAMRA in PBS 279

for 30 minutes at 37˚C in the dark, followed by 4 washes in PBS (6, 28). 280

281

Antibody detection of C. jejuni colonisation of E12 mucus 282

283

E12 cells were infected with C. jejuni, and the transwells containing cells were embedded 284

in chicken liver as described above. Slides were fixed for 15 minutes with 4% 285

formaldehyde in PBS, blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour, 286

then incubated with 1/200 of rabbit C. jejuni antiserum (28) in 1% BSA for 1 hour. Cells 287

were incubated with a 1/500 dilution of goat anti-rabbit-IgG conjugated to Alexafluor488 288

(Invitrogen) for 1 hour followed by a 1/5000 dilution of 4’, 6-Diamidino-2-phenylindole 289

dihydrochroride (DAPI, Sigma) in PBS for 30 minutes in the dark, to stain cell nuclei. 290

Slides were washed in PBS, air dried and then mounted in Dako fluorescent mounting 291

medium. All washes between treatments were carried out six times in PBS. All antibody 292


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dilutions were in 1% BSA in PBS. Slides were visualised by immunofluorescence 293

microscopy. 294

295

Colocalisation of C. jejuni with occludin 296

297

E12 cells were cultured for 14 days and infected with C. jejuni for 3 hours or 24 hours. 298

For some experiments, TAMRA stained bacteria were used to infect cells. Cells were 299

washed six times in PBS and the mucolytic agent NAC was incubated with cells for 2-3 300

hours to remove the mucus layer. Following the removal of mucus, transwells were 301

washed in PBS and fixed with 4% formalin for 1 hour. In the case where TAMRA (red) 302

stained bacteria were used to infect E12 cells, the following procedure was used to stain 303

the tight junction protein occludin after fixing in formalin: Cells were blocked with 20% 304

rabbit serum (Dako) in 1% BSA for 1 hour, incubated for 1 hour with a 1: 100 dilution of 305

goat anti-occludin antibody (Santa Cruz) followed by a 1: 500 dilution of rabbit anti-goat 306

IgG conjugated to Alexafluor488 (Invitrogen). Thus C. jejuni was stained red and 307

occludin stained green. All washes between treatments were six times with PBS. 308

Transwells were allowed to dry, the inserts were excised from the transwell and mounted 309

onto a microscope slide using Dako fluorescent mountant. Slides were visualised by both 310

immunofluorescence and confocal microscopy (see Imaging section below). 311

312

313

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Differential staining 315

316

This protocol was used to discriminate between adherent and internalised bacteria (6). 317

E12 cells were cultured for 14 days and infected with TAMRA stained C. jejuni for 24 318

hours or 48 hours, the mucus layer removed and the cells fixed as described above. Cells 319

were blocked with 20% goat serum in 1% BSA in PBS. To stain adherent bacteria, cells 320

were incubated with a 1:200 dilution of rabbit anti-C. jejuni followed by a 1: 500 dilution 321

of goat anti-rabbit IgG conjugated to Alexafluor 488. Adherent bacteria stained yellow 322

(green and red) while internalised bacteria stained red (TAMRA only). All washes 323

between treatments were carried out six times in PBS. Transwells were allowed to dry, 324

inserts excised and mounted onto a microscope slide using Dako fluorescent mountant. 325

This procedure was repeated after C. jejuni was used to infect E12 cells for 48 hours to 326

reveal bacteria that remained adherent and internalised 48 hours postinfection. 327

328

Differential staining of probiotics and C. jejuni 329

330

L. rhamnosus was stained with TAMRA as described above for C. jejuni. TAMRA (red) 331

stained L. rhamnosus was used to infect E12 cells for an initial period of 4 hours, after 332

which the medium on the cells was replaced with 300 µl of OD600 ~ 0.15 C. jejuni. 333

Probiotics and C. jejuni were then further incubated for 24 hours after which the filter 334

was excised from the transwell, embedded in chicken liver, sections made in a cryostat 335

and fixed on to a microscope slide as decribed above. Medium on the infected cells was 336

replaced with fresh medium at the 6th and 12th hours postinfection to minimise changes in 337


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pH. Sections were then stained with a 1:200 dilution of rabbit raised anti-C. jejuni 338

antibody and revealed with a 1:400 dilution of donkey anti-rabbit IgG conjugated to 339

Alexafluor488 (Molecular Probes). All antibodies were diluted in 1% BSA in PBS and 340

incubations were for 1 hour at room temperature. DAPI (1:4000) was added to the 341

secondary antibody solution to detect cell nuclei. Red stained L. rhamnosus and green 342

stained C. jejuni could be observed in mucus and bound to cells by both 343

immunofluorescence and laser scanning confocal microscopy. 344

345

To detect both C. jejuni and L. rhamnosus bound to cells, 10 mM NAC was used to 346

remove the mucus layer as described above, immediately after the incubation period. 347

Cells were then fixed in 4% formaldehyde in PBS for 30 mins after which rabbit raised 348

anti C. jejuni and donkey raised anti rabbit IgG conjugated to Alexafluor488 were used to 349

detect C. jejuni bound to cells as described above. Red stained probiotics and green 350

stained C. jejuni bound to cells were observed by both immunofluorescnce and confocal 351

microscopy. 352

353

Imaging 354

355

Immunofluorescence microscopy was carried out using an Olympus BX 51 light 356

microscope and a JFX U-RFL-T fluorescent unit. Confocal microscopy was carried out 357

using an LSM 510 laser scanning confocal microscope. 358

359

360


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Scanning electron microscopy (SEM) 361

362

Scanning electron microscopy (SEM) was carried out according to a protocol described 363

by Sherman et al (2005). E12 cells were cultured for 14 days and infected with C. jejuni 364

for 24 hours as described above. Uninfected cells were used as controls. Cells were 365

washed in PBS and fixed with 2.5% glutaraldehyde in Sorenson’s phosphate buffer pH 366

7.4 overnight. The fixing solution was replaced with Sorenson’s phosphate buffer for 10 367

mins after which cells were incubated in 1% osmium tetroxide for 1 hour at room 368

temperature. Cells were then dehydrated in increasing grades of ethanol (from 70% to 369

100%). Cells were critically point dried, sputter coated with gold and visualised using a 370

scanning electron microscope (JSM 5410 LV). 371

372

RESULTS 373

374

I: The interaction of C. jejuni with mucus-adherent E12 cells 375

376

Growth, polarization and C. jejuni colonization of E12 mucus 377

378

The thickness of the mucus layer and the transepithelial resistance of E12 cells typically 379

increased from 7, through 14 to 21 days postseeding (Fig 1A(i),(ii)). Using 380

immunofluorescence microscopy, C. jejuni could be seen abundantly colonising E12 381

mucus by 24 hours postinfection (Fig 1B (ii)). The organisms penetrated the mucus layer 382


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and could be found attached to and beneath the monolayer by 24 hours postinfection (Fig 383

1B (iii)). In addition, there was a modest increase in C. jejuni numbers due to 384

reproduction in the monolayer between 3 hours (6.9 x 106 cfu / well) and 24 hours (1.9 x 385

107 cfu / well) and a further increase at 48 hours following infection (3.6 x 107 cfu / well) 386

(Fig 1C). Furthermore, direct incubation of C. jejuni in mucus harvested from E12 cell 387

culture supernatants led to a 10 fold increase in the number of recovered bacteria after 24 388

hours coincubation, compared with bacteria recovered from tissue culture medium alone 389

(data not shown). This indicates that E12 cells can sustain C. jejuni viability and E12 390

mucus encourages reproduction of the organism. 391

392

C. jejuni colonised mucus, bound to and invaded E12 cells 393

394

C. jejuni adhesion and internalisation into underlying E12 cells was investigated by 395

differential staining and laser scanning confocal microscopy (LSCM). Differential 396

staining with TAMRA (internal and external bacteria red) and Alexafluor488 (external 397

bacteria yellow / green) permitted the discrimination and visualisation of bound and 398

internalised bacteria after the mucus layer was removed. Using this approach, C. jejuni 399

was visualised both adhering to and internalised into E12 cells (Fig 2A). In agreement 400

with previous studies (42), internalised C. jejuni were visualised in close proximity to and 401

around cell nuclei (Fig 2A). In addition, C. jejuni remained bound and internalised 48 402

hours postinfection without affecting the viability of E12 cells as shown by trypan blue 403

exclusion assays (data not shown), indicating that the E12 cell line tolerates prolonged 404

host-pathogen interactions. 405


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The interaction of C. jejuni with E12 cells was also visualized by scanning electron 406

microscopy (SEM). Organisms were not evenly distributed on the surface of E12 cells 407

but appeared to cluster around cell-cell junctions (Fig 2B). In keeping with a recent report 408

(32), microvilli appeared effaced on cells heavily colonized with bacteria compared to 409

areas on the surface of cells lacking bacteria (Figs 2B and 2C). 410

411

C. jejuni translocation and effect on monolayer resistance 412

413

As observed in Fig 1B(iii), C. jejuni 81-176 translocated to the basolateral compartment 414

following apical infection of E12 cells. Enumeration of bacteria in the basolateral 415

compartment using plate assays revealed that modest numbers had translocated by 5 416

hours postinfection (with the exception of 21 day E12 cell cultures) (Fig 3). The reason 417

for the lack of translocation at 5 hours in day 21 E12 cells compared with day 21 HT29 418

cells is not clear but may reflect the particularly high TEER of E12 monolayers at 21 419

days (Fig 1A (ii)). By 24 hours, as many as 108 organisms were present in the basolateral 420

compartment. As C. jejuni did not reproduce when introduced directly into the basolateral 421

compartment of control wells (with overlying E12 monolayers) and given a lack of any 422

translocation by the probiotic L. helveticus (data not shown), these results suggested a 423

specific translocation mechanism for C. jejuni. Very high translocation levels at later time 424

points indicates either a very efficient translocation mechanism or the possiblility that 425

some disruption of the monolayer has started to occur. It is also noteworthy that there 426

were similar levels of C. jejuni translocation in E12 and HT29 cells. 427

428


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There was a discernible ovoid or circular pattern of C. jejuni adherence to E12 cells, 429

particularly at lower magnification (Fig 4A). The ovoid or circular shapes rendered 430

suggested that the organisms were preferentially adherent to cell-cell junctions. On this 431

basis, and in the light of previous studies (27, 40), we hypothesised that bacteria were 432

preferentially attaching to cell-cell junction structures in order to translocate the 433

monolayer paracellularly. To investigate this further, we examined if C. jejuni colocalised 434

with the tight junction protein occludin. Following infection and removal of the mucus 435

layer, C. jejuni could be found aligning with the tight junctions of E12 cells and 436

colocalising with occludin as early as 3 hours postinfection (Fig 4B). 437

438

To investigate the effect of C. jejuni on the barrier properties of E12, 21 day cultured 439

cells were infected with bacteria and the TEER measured over a period of 48 hours in 440

infected compared to uninfected cells. By 24 hours postinfection, the TEER had not 441

fallen despite translocation of large numbers of organisms to the basolateral compartment 442

(Fig 5). Only by 48 hours postinfection, was there a significant reduction in the TEER 443

values of infected cells indicating that tight junctions were disrupted. These results 444

indicate that early translocation of C. jejuni occurs without disrupting the tight junctions 445

of E12 cells and the microscopy results support the contention that the organisms 446

translocate via a paracellular route. 447

448

449

450

451


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Comparison of C. jejuni pathogenicity in E12 cells with 452

non mucus adherent cells 453

454

We exploited the presence of an adherent mucus layer on E12 cells to determine the 455

effect of mucus on the virulence of C. jejuni using total association assays and the 456

gentamycin protection assay. Polarised and non polarised HT29 parent cell lines which 457

do not harbour or secrete mucus, and non polarised E12 cells which secrete mucus into 458

the culture supernatant but do not harbour an adherent mucus layer were used as controls. 459

Not surprisingly (given the thick mucus layer), there was an increase in C. jejuni total 460

association (colonised, adherent and internalised bacteria) to E12 cells by more than a 461

hundred fold in the presence of a mucus layer (7, 14 and 21 day cultured E12 cells) 462

compared to non polarised E12 and HT29 cells, and by ten fold in the presence of mucus 463

compared to 21 day polarised HT29 cells (Fig 6A). 464

465

C. jejuni binding to underlying E12 cells after penetrating the mucus layer was also 466

investigated using 7, 14 and 21 day cultured E12 cells. Cells were infected with C. jejuni 467

and the mucolytic agent N-acetylcysteine was used to remove the E12 mucus layer, after 468

which CFU of adherent and internalised bacteria were quantified (see Materials and 469

Methods). C. jejuni binding to E12 cells increased from 7 day (1.6 x 105 cfu / well), 470

through 14 day (5.6 x 105 cfu / well) to 21 day cultured E12 cells (2.5 x 106 cfu / well) 471

(Fig 6B). In 7, 14 and 21 day cultured E12 cells the relative proportion of mucus 472

colonised bacteria that had adhered to E12 cells increased from 2.6%, to 2.8% to 15.2%, 473

respectively. 474


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Internalisation also was enhanced in mucus adherent cells (E12), compared to non mucus 475

adherent parental HT29 cells (Fig 6C). Invasion was approximately 100 fold higher in 476

polarised E12 cells (with mucus) compared to non polarised E12 and HT29 cells (without 477

a mucus layer) and ten fold higher than polarised HT29 cells (21 days in culture). As a 478

control, the probiotic bacterium L. helveticus R0052 did not invade E12 cells (data not 479

shown). Thus C. jejuni total association and internalisation increased when E12 and 480

HT29 cells became polarised, and further increased in the presence of mucus. The ratio of 481

internalised to adherent bacteria was approximately a log fold higher in E12 compared 482

with HT29 cells, i.e. between 1:27 and 1:200 in E12 cells compared with approximately 483

1:2000 in HT29 cells. Taken together these results suggest that both mucus and cell 484

polarity promote the efficiency of C. jejuni total association and internalisation. 485

486

II: The Effect of probiotics on the pathogenicity of C. jejuni 487

488

A) Probiotics colonised E12 mucus and adhered to underlying cells 489

490

Having established E12 cells as a suitable model of Campylobacter infection, we used it 491

to examine the effect of probiotics on the pathogenicity of C. jejuni. Immunofluorescence 492

and confocal microscopy were used to demonstrate probiotic colonisation of E12 mucus 493

and binding to underlying cells. Briefly, E12 cells were precolonised with TAMRA (red) 494

stained L. rhamnosus for 4 hours followed by infection with C. jejuni for a further 24 495

hours. After the incubation period, C. jejuni was labelled green with an in-house, whole-496

cell anti-C. jejuni antibody (see Methods). To detect probiotic organisms (L. rhamnosus) 497


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and C. jejuni that adhered to underlying E12 cells, the infection process was repeated as 498

above, except that the mucus layer was removed (see Methods), prior to labelling 499

adherent C. jejuni green with antibody. L. rhamnosus and C. jejuni were visualised both 500

colonising E12 mucus and bound to underlying cells by confocal microscopy (data not 501

shown). Colocalisation of C. jejuni and L. rhamnosus also could be observed in mucus on 502

merged confocal images. Colocalisation of at least some L. rhamnosus and C. jejuni 503

raises the possibility of either a direct interaction between these organisms or a shared 504

affinity for a common receptor in intestinal mucus. In contrast, there was little evidence 505

of colocalisation of organisms when adherent to cells and the circular adhesion pattern 506

seen for C. jejuni (Fig 4A) was not evident for either organism when used to coinfect E12 507

cells (data not shown). Total association assays indicated that approximately 107 508

organisms were present in mucous / adherent to cells after 24 hours for each of the 509

probiotic strains tested. 510

511

B) Probiotics attenuated C. jejuni total association to E12 cells 512

513

Individual probiotic isolates and combinations thereof were investigated for their effect 514

on the pathogenicity of C. jejuni using E12 cells. Three strains of Lactobacillus (L. 515

rhamnosus R0011, L. helveticus R0052 and L. salivarius AH102), and B. longum 516

AH1205 were used in addition to a commercial combination of L. rhamnosus and L. 517

helveticus (‘Lacidofil’ TM), and an in house combination comprising L. rhamnosus, L. 518

helveticus and L. salivarius (RhHeSa) mixed equally (~ 109 cfu / well each) (see 519

Materials and Methods). These probiotics were used to initially colonise 14 day old E12 520


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cells for 4 hours prior to C. jejuni infection. Some of the isolates and combinations 521

reduced total association of C. jejuni to E12 cells (Fig 7A). However, other preparations 522

had no effect (e.g L. helveticus) and in general, the relative reduction was modest (Fig 523

7A). 524

525

However, when probiotic strains were allowed to ‘precolonise’ monolayers for 15 hours, 526

the effect on C. jejuni pathogenicity was considerably enhanced (Fig 7A). Probiotic 527

colonisation numbers did not increase significantly at 15 hours compared with 4 hours 528

(data not shown). Only L. salivarius and ‘Lacidofil’ did not show more marked inhibition 529

of total association of C. jejuni after 15 hours compared with 4 hours precolonisation. 530

Many of the preparations reduced total C. jejuni association 1-1.5 logfolds with the most 531

effective reduction induced by B. longum where C. jejuni recovery was reduced from 9.2 532

x 106 to 3.3 x 105. Given the accentuation of the effect following prolonged 533

precolonisation, further experiments were performed at both 4 and 15 hours after 534

colonisation with probiotic isolates. 535

536

C) Precolonisation of E12 cells with probiotics inhibited C. jejuni 537

internalisation 538

539

Only the RhHeSa probiotic combination attenuated C. jejuni internalisation after 4 hours 540

precolonisation (Fig 9B). However, with the exception of L. helveticus, precolonisation of 541

E12 cells with probiotic preparations for 15 hours prior to infection with C. jejuni 542

significantly attenuated C. jejuni invasion (Fig 7B). Precolonisation of E12 cells with 543


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heat treated L. rhamnosus for 15 hours had no effect on C. jejuni invasion. The 544

preparations combining more than one isolate tended to have the greatest effect on 545

attenuation of invasion with approximately a 90% reduction in recovered C. jejuni 546

compared to baseline (Fig 7B). 547

548

D) Precolonisation of E12 cells with probiotics attenuated C. jejuni 549

translocation to the basolateral medium 550

551

With the exception of RhHeSa, no probiotic strain or combination of strains affected C. 552

jejuni translocation after a 4 hour preincubation period with E12 cells (Fig 7C). However, 553

after 15 hours precolonisation, L. rhamnosus on its own or when combined with L. 554

helveticus and L. salivarius markedly attenuated C. jejuni translocation (Fig 7C). L. 555

helveticus and Lacidofil reduced C. jejuni translocation after preincubation for 15 hours, 556

but this difference was not statistically significant. L. salivarius and B. longum did not 557

affect C. jejuni translocation despite their attenuation of C. jejuni total association and 558

internalisation. In addition, following precolonisation of E12 cells with L. rhamnosus for 559

15 hours and infection with C. jejuni for a further 48 hours, L. rhamnosus did not prevent 560

the previously described C. jejuni mediated disruption of tight junctions (data not shown). 561

562

563

564

565

566


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DISCUSSION 567

568

Campylobacter research has been hampered by the lack of an easily accessible animal 569

model that mimics human Campylobacter infection. Therefore conventional tissue 570

culture has been of major importance in exploring C. jejuni pathogenicity (14, 21, 27, 571

42). The E12 cell line provides an important additional tool for Campylobacter research 572

by mimicking in vitro, a fundamentally important feature of mucosal infection, the 573

overlying mucus layer. In this study, the interaction of C. jejuni with E12 cells was 574

characterised in depth. We believe that E12 cells provide an important novel technique to 575

complement recent seminal studies looking at the effect of specific mucin molecules on 576

C. jejuni pathogenicity (26, 39). C. jejuni colonisation and reproduction in mucus, 577

binding to and invasion of underlying cells, and translocation to the basolateral aspects of 578

cells was demonstrated, validating the E12 model as suitable for the study of 579

Campylobacter-host interactions. 580

581

The route of C. jejuni translocation to the basolateral aspects of cells is a subject of 582

debate. While some studies have described C. jejuni transcellular transcytosis (5, 15), 583

others have implicated the paracellular route of translocation (4, 27). Disruption of tight 584

junctions by C. jejuni also has been shown in other studies (8, 24). In this study, C. jejuni 585

aligned with tight junctions and colocalised with occludin as early as three hours 586

postinfection, suggesting paracellular passage of organisms. Although many bacterial 587

pathogens affect tight junctions and the related pathogen, Helicobacter pylori attaches 588

near intercellular boundaries (12), this appears to be the first report providing evidence of 589


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physical alignment of a bacterial species with tight junctions and colocalisation with 590

occludin. Other important non-bacterial human pathogens including Toxoplasma gondii 591

(2) and Hepatitis C (23) are known to interact directly with tight junction proteins. 592

Indeed, passage of C. jejuni paracellularly without affecting the integrity of tight 593

junctions is reminiscent of T. gondii. Further experiments examining the nature of the 594

interaction of C. jejuni with occludin and other cell junction components are required. 595

596

In previous studies, C. jejuni motility has been shown to increase in viscous solutions 597

(11) and in media mimicking the viscosity of mucus (38). In addition, in vitro studies 598

using conventional cell lines have demonstrated increased C. jejuni binding and 599

internalisation into intestinal cells following preincubation in media of increased 600

viscosity (38), crude human mucus (6) and purified mucins (10). In this study we confirm 601

that the overlying mucus layer supports C. jejuni reproduction and enhances adhesion to 602

and invasion of the underlying epithelium. C. jejuni replication in mucus may ensure 603

bacterial persistence in the host since it is rich in nutrients and is essentially 604

microaerophilic. Whether the increased pathogenicity in the presence of mucus is caused 605

by increased viscosity or other factors is not clear. However, it is unlikely that the 606

observed increase in C. jejuni total association and invasion into E12 cells was due to 607

increased bacterial numbers secondary to reproduction in mucus alone, because 608

increasing the innoculum did not result in an increase in C. jejuni total association and 609

invasion in other experiments (data not shown). 610

611


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In vivo, commensal bacteria establish themselves in the gut shortly after birth and 612

subsequently maintain a population in dynamic equilibrium with the intestinal mucosa 613

(13, 31). Animal and human studies of the effects of probiotics on the gut involve at least 614

a period of persistent colonisation for the effects to be observed (33). Despite this, most 615

in vitro studies of probiotic action typically involve short exposure of model epithelia to 616

the probiotic strains used (1, 19, 25, 46). It is possible that such brief pretreatment of cells 617

with probiotics fails to reproduce at least some of the effects that would be seen in vivo. 618

The E12 model is suited to the study of more persistent colonisation and in this manner, 619

more accurately reflects conditions in vivo. We observed a striking difference in the 620

effectiveness of nearly all the probiotic strains used when cells were precolonised for 621

fifteen hours compared to four hours. In nearly all cases, prolonged precolonisation 622

allowed for much more effective attenuation of C. jejuni virulence. In future studies, it 623

will be useful to explore even longer time periods (e.g twenty four or even fourty eight 624

hours) to see if further reduction in C. jejuni pathogenicity occurs. 625

626

One of the potential mechanisms by which probiotics work is to reduce luminal pH (33, 627

36). All the organisms used in this study are lactic acid producing. C. jejuni is inactivated 628

at low pH (pH 5) (38) and during co-culture with L. salivarius we observed that C. jejuni 629

lost viability at a pH of 4.7 (unpublished results). It is possible that probiotic-induced 630

acidity contributes to the effects on C. jejuni virulence in our study. However, in order to 631

avoid a drastic reduction in pH that would affect cell and organism viability, we 632

repeatedly replenished the E12 cell culture medium during co-culture of probiotics with 633

C. jejuni. In addition, complex effects of different probiotic strains on various aspects of 634


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C. jejuni pathogenicity (invasion, adhesion and translocation) are unlikely to be explained 635

by the effects of changes in pH alone. 636

637

Mechanisms of probiotic-induced alteration in pathogenicity may be generalised and non-638

specific (e.g. consumption of local nutrients, interference with host binding sites). 639

Alternatively, a wide variety of more specific mechanisms of action have been ascribed 640

to probiotics during protection of the mucosa from infecting organisms in vitro and in 641

animal models (31, 33, 36). It is not clear what mechanisms of action were involved in 642

probiotic-induced attenuation of C. jejuni pathogenicity in our experiments. In order to 643

avoid a non-specific effect of low pH on C. jejuni viability (38) we repeatedly 644

replenished the E12 cell culture medium during co-culture. In addition, we feel that 645

probiotic-induced changes in epithelial barrier function are unlikely to have caused 646

reduced translocation of C. jejuni as there was no concomitant effect of these 647

commensals on TEER (data not shown). However, a noteworthy feature of our 648

experiments was the heterogeneity in the ability of probiotics to attenuate different 649

aspects of C. jejuni pathogenicity. Probiotic strain-specific variation in attenuation of C. 650

jejuni invasiveness also was recently reported using conventional cell lines (46). These 651

findings suggest different mechanisms of action for the different probiotic strains and 652

cocktails used. These data also suggest that the mechanisms by which an organism such 653

as C. jejuni colonises mucus, invades epithelial cells and translocates to the basolateral 654

aspects of cells, are, at least to some extent, mutually exclusive and independent of one 655

another. 656

657


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These studies point to the potential for a number of well characterised probiotic isolates 658

to prevent and / or attenuate C. jejuni disease in humans. The possibility that such an 659

approach could also be used to reduce chicken broiler colonisation is also attractive as an 660

effective means to reduce Campylobacter infections in humans (29). Our studies provide 661

a rational basis for the further investigation of specific probiotic strains for future in vitro 662

and clinical studies (for example, the prevention of C. jejuni infection in travellers). Our 663

data point to the likelihood that probiotic treatment may have a greater potential as a 664

method of prophylaxis against C. jejuni infection rather than as a method of treatment of 665

an acute illness. 666

667

In summary, we describe a novel model of Campylobacter infection. E12 cells are unique 668

in harbouring an adherent overlying mucus layer providing an opportunity to study the 669

interaction of enteric pathogens with mucus, mucin, endogenous flora and related luminal 670

factors in a manner that more accurately represents conditions in vivo compared to 671

conventional cell culture methods. This model confirms the contribution of mucus to 672

enhancing C. jejuni pathogenicity, provides strong supportive evidence for paracellular 673

passage of C. jejuni through interaction with tight junction proteins and establishes the 674

potential efficacy of probiotic treatment for the prevention and attenuation of C. jejuni 675

related disease in humans. 676

677

678

679

680


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Acknowledgements 681

682

This work was supported by a principal investigator grant from Science Foundation 683

Ireland (SFI). We also acknowledge funding support from the Children’s Research 684

Centre, Our Lady’s Hospital for Sick Children, Crumlin, Dublin 12, Ireland. We thank 685

Brendan Dolan, Anne Cullen and Dr Cormac O’Connell for guidance in fluorescence 686

microscopy, confocal and electron microscopy respectively. 687

688

689

690

691

692

693

694

695

696

697

698

699


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REFERENCES 700

701

702

1. Altenhoefer, A., S. Oswald, U. Sonnenborn, C. Enders, J. Schulze, J. Hacker, 703

and T. A. Oelschlaeger. 2004. The probiotic Escherichia coli strain Nissle 1917 704

interferes with invasion of human intestinal epithelial cells by different 705

enteroinvasive bacterial pathogens. FEMS Immunol Med Microbiol 40:223-229. 706

2. Barragan, A., F. Brossier, and L. D. Sibley. 2005. Transepithelial migration of 707

Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 708

(ICAM-1) with the parasite adhesin MIC2. Cell Microbiol 7:561-568. 709

3. Behrens, I., P. Stenberg, P. Artursson, and T. Kissel. 2001. Transport of 710

lipophilic drug molecules in a new mucus-secreting cell culture model based on 711

HT29-MTX cells. Pharm Res 18:1138-1145. 712

4. Beltinger, J., J. del Buono, M. M. Skelly, J. Thornley, R. C. Spiller, W. A. 713

Stack, and C. J. Hawkey. 2008. Disruption of colonic barrier function and 714

induction of mediator release by strains of Campylobacter jejuni that invade 715

epithelial cells. World J Gastroenterol 14:7345-7352. 716

5. Bras, A. M., and J. M. Ketley. 1999. Transcellular translocation of 717

Campylobacter jejuni across human polarised epithelial monolayers. FEMS 718

Microbiol Lett 179:209-215. 719

6. Byrne, C. M., M. Clyne, and B. Bourke. 2007. Campylobacter jejuni adhere to 720

and invade chicken intestinal epithelial cells in vitro. Microbiology 153:561-569. 721


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

33

7. Chaveerach, P., L. J. Lipman, and F. van Knapen. 2004. Antagonistic 722

activities of several bacteria on in vitro growth of 10 strains of Campylobacter 723

jejuni/coli. Int J Food Microbiol 90:43-50. 724

8. Chen, M. L., Z. Ge, J. G. Fox, and D. B. Schauer. 2006. Disruption of tight 725

junctions and induction of proinflammatory cytokine responses in colonic 726

epithelial cells by Campylobacter jejuni. Infect Immun 74:6581-6589. 727

9. Corry, J. E., D. E. Post, P. Colin, and M. J. Laisney. 1995. Culture media for 728

the isolation of campylobacters. Int J Food Microbiol 26:43-76. 729

10. de Melo, M. A., and J. C. Pechere. 1988. Effect of mucin on Campylobacter 730

jejuni association and invasion on HEp-2 cells. Microb Pathog 5:71-76. 731

11. Ferrero, R. L., and A. Lee. 1988. Motility of Campylobacter jejuni in a viscous 732

environment: comparison with conventional rod-shaped bacteria. J Gen Microbiol 733

134:53-59. 734

12. Guttman, J. A., and B. B. Finlay. 2009. Tight junctions as targets of infectious 735

agents. Biochim Biophys Acta 1788:832-841. 736

13. Holzapfel, W. H., P. Haberer, J. Snel, U. Schillinger, and J. H. Huis in't Veld. 737

1998. Overview of gut flora and probiotics. Int J Food Microbiol 41:85-101. 738

14. Hu, L., and D. J. Kopecko. 1999. Campylobacter jejuni 81-176 associates with 739

microtubules and dynein during invasion of human intestinal cells. Infect Immun 740

67:4171-4182. 741

15. Hu, L., B. D. Tall, S. K. Curtis, and D. J. Kopecko. 2008. Enhanced 742

microscopic definition of Campylobacter jejuni 81-176 adherence to, invasion of, 743


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

34

translocation across, and exocytosis from polarized human intestinal Caco-2 cells. 744

Infect Immun 76:5294-5304. 745

16. Jandu, N., Z. J. Zeng, K. C. Johnson-Henry, and P. M. Sherman. 2009. 746

Probiotics prevent enterohaemorrhagic Escherichia coli O157:H7-mediated 747

inhibition of interferon-gamma-induced tyrosine phosphorylation of STAT-1. 748

Microbiology 155:531-540. 749

17. Janssen, R., K. A. Krogfelt, S. A. Cawthraw, W. van Pelt, J. A. Wagenaar, 750

and R. J. Owen. 2008. Host-pathogen interactions in Campylobacter infections: 751

the host perspective. Clin Microbiol Rev 21:505-518. 752

18. Keely, S., A. Rullay, C. Wilson, A. Carmichael, S. Carrington, A. Corfield, D. 753

M. Haddleton, and D. J. Brayden. 2005. In vitro and ex vivo intestinal tissue 754

models to measure mucoadhesion of poly (methacrylate) and N-trimethylated 755

chitosan polymers. Pharm Res 22:38-49. 756

19. Konkel, M. E., B. J. Kim, V. Rivera-Amill, and S. G. Garvis. 1999. Bacterial 757

secreted proteins are required for the internalization of Campylobacter jejuni into 758

cultured mammalian cells. Mol Microbiol 32:691-701. 759

20. Korlath, J. A., M. T. Osterholm, L. A. Judy, J. C. Forfang, and R. A. 760

Robinson. 1985. A point-source outbreak of campylobacteriosis associated with 761

consumption of raw milk. J Infect Dis 152:592-596. 762

21. Krause-Gruszczynska, M., M. Rohde, R. Hartig, H. Genth, G. Schmidt, T. 763

Keo, W. Konig, W. G. Miller, M. E. Konkel, and S. Backert. 2007. Role of the 764

small Rho GTPases Rac1 and Cdc42 in host cell invasion of Campylobacter 765

jejuni. Cell Microbiol 9:2431-2444. 766


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

35

22. Lesuffleur, T., A. Barbat, E. Dussaulx, and A. Zweibaum. 1990. Growth 767

adaptation to methotrexate of HT-29 human colon carcinoma cells is associated 768

with their ability to differentiate into columnar absorptive and mucus-secreting 769

cells. Cancer Res 50:6334-6343. 770

23. Liu, S., W. Yang, L. Shen, J. R. Turner, C. B. Coyne, and T. Wang. 2009. 771

Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and 772

are downregulated during infection to prevent superinfection. Journal of virology 773

83:2011-2014. 774

24. MacCallum, A., S. P. Hardy, and P. H. Everest. 2005. Campylobacter jejuni 775

inhibits the absorptive transport functions of Caco-2 cells and disrupts cellular 776

tight junctions. Microbiology 151:2451-2458. 777

25. Mack, D. R., S. Ahrne, L. Hyde, S. Wei, and M. A. Hollingsworth. 2003. 778

Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to 779

intestinal epithelial cells in vitro. Gut 52:827-833. 780

26. McAuley, J. L., S. K. Linden, C. W. Png, R. M. King, H. L. Pennington, S. J. 781

Gendler, T. H. Florin, G. R. Hill, V. Korolik, and M. A. McGuckin. 2007. 782

MUC1 cell surface mucin is a critical element of the mucosal barrier to infection. 783

J Clin Invest 117:2313-2324. 784

27. Monteville, M. R., and M. E. Konkel. 2002. Fibronectin-facilitated invasion of 785

T84 eukaryotic cells by Campylobacter jejuni occurs preferentially at the 786

basolateral cell surface. Infect Immun 70:6665-6671. 787


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

36

28. Mooney, A., C. Byrne, M. Clyne, K. Johnson-Henry, P. Sherman, and B. 788

Bourke. 2003. Invasion of human epithelial cells by Campylobacter upsaliensis. 789

Cell Microbiol 5:835-847. 790

29. Morishita, T. Y., P. P. Aye, B. S. Harr, C. W. Cobb, and J. R. Clifford. 1997. 791

Evaluation of an avian-specific probiotic to reduce the colonization and shedding 792

of Campylobacter jejuni in broilers. Avian diseases 41:850-855. 793

30. Nachamkin, I., J. Engberg, M. Gutacker, R. J. Meinersman, C. Y. Li, P. 794

Arzate, E. Teeple, V. Fussing, T. W. Ho, A. K. Asbury, J. W. Griffin, G. M. 795

McKhann, and J. C. Piffaretti. 2001. Molecular population genetic analysis of 796

Campylobacter jejuni HS:19 associated with Guillain-Barre syndrome and 797

gastroenteritis. J Infect Dis 184:221-226. 798

31. Neish, A. S. 2009. Microbes in gastrointestinal health and disease. 799

Gastroenterology 136:65-80. 800

32. Nemelka, K. W., A. W. Brown, S. M. Wallace, E. Jones, L. V. Asher, D. 801

Pattarini, L. Applebee, T. C. Gilliland, Jr., P. Guerry, and S. Baqar. 2009. 802

Immune response to and histopathology of Campylobacter jejuni infection in 803

ferrets (Mustela putorius furo). Comparative medicine 59:363-371. 804

33. Ng, S. C., A. L. Hart, M. A. Kamm, A. J. Stagg, and S. C. Knight. 2009. 805

Mechanisms of action of probiotics: recent advances. Inflammatory bowel 806

diseases 15:300-310. 807

34. O'Hara, A. M., and F. Shanahan. 2007. Mechanisms of action of probiotics in 808

intestinal diseases. TheScientificWorldJournal 7:31-46. 809


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

37

35. Pope, J. E., A. Krizova, A. X. Garg, H. Thiessen-Philbrook, and J. M. 810

Ouimet. 2007. Campylobacter reactive arthritis: a systematic review. Seminars in 811

arthritis and rheumatism 37:48-55. 812

36. Sherman, P. M., K. C. Johnson-Henry, H. P. Yeung, P. S. Ngo, J. Goulet, and 813

T. A. Tompkins. 2005. Probiotics reduce enterohemorrhagic Escherichia coli 814

O157:H7- and enteropathogenic E. coli O127:H6-induced changes in polarized 815

T84 epithelial cell monolayers by reducing bacterial adhesion and cytoskeletal 816

rearrangements. Infect Immun 73:5183-5188. 817

37. Smith, J. L., and D. Bayles. 2007. Postinfectious irritable bowel syndrome: a 818

long-term consequence of bacterial gastroenteritis. Journal of food protection 819

70:1762-1769. 820

38. Szymanski, C. M., M. King, M. Haardt, and G. D. Armstrong. 1995. 821

Campylobacter jejuni motility and invasion of Caco-2 cells. Infect Immun 822

63:4295-4300. 823

39. Tu, Q. V., M. A. McGuckin, and G. L. Mendz. 2008. Campylobacter jejuni 824

response to human mucin MUC2: modulation of colonization and pathogenicity 825

determinants. Journal of medical microbiology 57:795-802. 826

40. van Alphen, L. B., N. M. Bleumink-Pluym, K. D. Rochat, B. W. van Balkom, 827

M. M. Wosten, and J. P. van Putten. 2008. Active migration into the subcellular 828

space precedes Campylobacter jejuni invasion of epithelial cells. Cell Microbiol 829

10:53-66. 830

41. Wagner, R. D., S. J. Johnson, and D. Kurniasih Rubin. 2009. Probiotic 831

bacteria are antagonistic to Salmonella enterica and Campylobacter jejuni and 832


http://iai.asm.org/

Dow

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38

influence host lymphocyte responses in human microbiota-associated 833

immunodeficient and immunocompetent mice. Molecular nutrition & food 834

research 53:377-388. 835

42. Watson, R. O., and J. E. Galan. 2008. Campylobacter jejuni survives within 836

epithelial cells by avoiding delivery to lysosomes. PLoS pathogens 4:e14. 837

43. WHO. 2000. Campylobacter. Fact Sheet No 255. 838

44. WHO. 2001. Health and nutritional properties of probiotics in food including 839

powder milk with live lactic acid bacteria. Available at: 840

http://www.who.int/foodsafety/publications/fs_management/en/probiotics.pdf. 841

45. Willis, W. L., and L. Reid. 2008. Investigating the effects of dietary probiotic 842

feeding regimens on broiler chicken production and Campylobacter jejuni 843

presence. Poultry science 87:606-611. 844

46. Wine, E., M. G. Gareau, K. Johnson-Henry, and P. M. Sherman. 2009. 845

Strain-specific probiotic (Lactobacillus helveticus) inhibition of Campylobacter 846

jejuni invasion of human intestinal epithelial cells. FEMS Microbiol Lett 847

300:146-152. 848

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Figure legends 861

862

Fig 1: Interaction of C. jejuni with E12 cells. (A) Change in E12 cell mucus layer 863

thickness and transepithelial resistance over time. The deep pink Schiff’s reagent-stained 864

mucus layer clearly increases in thickness between seven, fourteen and twenty one days. 865

(i) Cells are seen adherent to the transwell filter. Remnants of liver tissue cells used for 866

sample preparation can be visualised both above and below the filter. (ii) There is a 867

marked increase in TEER compared to 21 day cultured HT29 cells (21D HT29). Data 868

presented as means ± standard deviations (error bars). n = 10 for each group. Mag: x400. 869

Scale bars: 150 µm. (B) Immunofluorescent micrograph of C. jejuni colonising 21 day 870

cultured E12 cells. (i) cell nuclei are visible following staining with DAPI on the 871

transwell filter. (ii) same field as (i), showing extensive colonisation by C. jejuni stained 872

green within the mucus layer and predominantly located at the periphery of underlying 873

cells. (iii) some organisms (arrows) can be seen below the cell layer and above the 874

transwell filter (translocation). Bringing organisms into focus results in some lack of 875

definition of the filter and mucus in (ii) and (iii). Mag: x400. Scale bars: 10 µm (C) C. 876

jejuni reproduction in association with E12 cells. Three sets of 21 day cultured E12 cells 877

were infected with C. jejuni for 3 hours. Medium was replaced with bacteria free medium 878

and two sets of wells further incubated for 24 and 48 hours. A total association assay (n = 879

7) was performed after washes at each time point. * denotes significant difference 880

(p<0.05) between 24 and 48 hrs compared to 3 hrs. Error bars indicate standard 881

deviations. 882

883


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Fig 2: Confocal and electron micrographic images of C. jejuni on E12 cells. (A) 884

Differential immunofluorescence distinguishes internalised (red stained) and external 885

adherent (yellow-green) C. jejuni organisms following infection of E12 cells. Internalised 886

organisms commonly coalesce around cell nuclei (arrows) which are stained blue with 887

DAPI. Mag: x400. Scale bar: 10 µm. Typically, some areas of the monolayer harboured 888

mainly internalised bacteria (as seen to left of image) while in other areas both 889

internalised and external adherent bacteria could be visualised (right side of micrograph) 890

(B) SEM of C. jejuni adhering to E12 cells. Organisms typically coalesced around cell-891

cell interfaces (yellow arrows). White arrows indicate relative effacement of microvilli on 892

cells that are heavily colonised with C. jejuni compared to those in the upper part of the 893

micrograph with intact microvilli and few organisms. Mag: x3500. Scale bar: 2 µm. C) 894

SEM of two adjacent uninfected cells (slightly separated at the cell-cell junction during 895

processing) with intact microvilli. Mag: x3500. Scale bar: 2 µm. 896

897

Fig 3: Recovery of C. jejuni translocated to the basolateral medium after 5 hours and 24 898

hours of apical infection using 7, 14 and 21 day cultured E12 cells compared to 21 day 899

cultured HT29 polarised cells (n = 8). Error bars indicate standard deviations. Results are 900

presented as CFU / ml. 901

902

Fig 4: Pattern of C. jejuni adhesion to E12 cells. (A) Two different magnifications are 903

shown indicating a circular or ovoid pattern of distribution of C. jejuni as it clusters 904

towards cell-cell junctions. Scale bars: 10 µm, 5 µm. Mag: x200, x400 respectively. (B) 905

Confocal microscopy of the tight junction protein occludin and C. jejuni. There is clear 906


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pericellular distribution of C. jejuni organisms (stained red) and colocalisation with green 907

stained occludin protein (yellow spots). Mag: x630. Scale bars: 10 µm. 908

909

Fig 5: Transepithelial resistance values of 21 day E12 cells following C. jejuni infection 910

remain unaffected despite very high C. jejuni translocation efficiency at 24 hours. There 911

is no evidence of loss of TEER until later time points (48 hours). Pink shaded bars 912

represent TEER of E12 cells infected with C. jejuni compared to the blue shaded 913

uninfected controls. * denotes significant difference (p<0.00005) compared to 48 hour 914

uninfected E12 cells (n = 12). Error bars indicate standard deviations. 915

916

Fig 6: Colonisation, adhesion and invasion of E12 cells (A) C. jejuni total association 917

(mucus colonised, adherent and invaded organisms) to polarised E12 cells compared to 918

parental cell lines. Day 7, 14 and 21 day cultured E12 cells are indicated as 7DE12P, 919

14DE12P, and 21DE12P respectively. 2DE12NP, 2 day cultured non polarised E12 cells. 920

2DHT29NP, 2 day non polarised HT29 cells. 21D HT29P, 21 day polarised HT29 cells 921

(no mucus layer). * denotes significant difference compared to 21DE12P. 7DE12P 922

p<0.005, 2DE12NP p<0.005, 2DHT29NP p<0.0005, 21DHT29P p<0.05 (n = 11). Error 923

bars indicate standard deviations. (B) Adhesion of C. jejuni to E12 cells after removal of 924

the mucus layer. * denotes significant difference compared to 14 and 21 day cultured E12 925

cells. 14 day E12 p<0.05, 21 day E12 p<0.05 (n = 8). Error bars indicate standard 926

deviations. (C) C. jejuni internalisation into polarised E12 cells compared to parental cell 927

lines. * denotes significant difference compared to 21DE12P. 2DE12NP p<0.005, 928

2DHT29 NP p<0.05, 21DHT29P p<0.05 (n = 8). Error bars indicate standard deviations. 929


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Fig 7: Effect of probiotics and probiotic cocktails on C. jejuni total association to (A), 930

invasion of (B) and translocation across (C) 14 day cultured E12 cells. (A) E12 cells 931

were pre-colonised with probiotics and probiotic combinations for 4 and 15 hours prior to 932

infection with C. jejuni (C.j) for a further 24 hours. Heat trt Rh, heat treated L. 933

rhamnosus. Rh, L. rhamnosus. He, L. helveticus. Sal, L. salivarius. Bifi, B. longum. L. 934

rhamnosus and L. helveticus, ‘Lacidofil’. Error bars indicate standard deviations. * 935

denotes significant difference compared to C. jejuni only (n = 9). P<0.005 for all groups. 936

(B) E12 cells were pre-colonised with probiotics for 4 and 15 hours prior to infection 937

with C. jejuni for a further 24 hours. The gentamycin protection assay was used to 938

quantify internalised C. jejuni. * denotes statistical difference compared to C. jejuni only 939

(n =8). P<0.05 for all groups. Error bars indicate standard deviations. (C) E12 cells 940

cultured for 14 days, were pre-colonised with probiotics for 4 and 15 hours prior to 941

infection with C. jejuni for a further 24 hours (n = 8). Serial dilutions of the basolateral 942

medium were made in PBS and plated onto solid medium. Error bars indicate standard 943

deviations. * denotes statistical difference compared to C. jejuni only. Rh p< 0.05, 4 hour 944

Sa+He+Rh p<0.05, 15 hour Sa+He+Rh p< 0.05. 945

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Figures 953

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Fig 2 970

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Fig 3 989

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Fig 5 1013

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