Transcellular passage of Neisseria meningitidis across a polarised

1

Transcellular passage of Neisseria meningitidis across a polarised respiratory

epithelium 2

Thomas C. Sutherland, Paola Quattroni, Rachel M. Exley, Christoph M Tang* 4

Centre for Molecular Microbiology and Infection, 6

Flowers Building,

Imperial College London, 8

London,

SW7 2AZ. 10

*For correspondence: Christoph Tang 12

Telephone: +44 (0)20 7594 3072 14

Fax: +44 (0)20 7594 3095

E-mail: [email protected] 16

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Running Title: Epithelial traversal by N. meningitidis

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Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01377-09 IAI Accepts, published online ahead of print on 28 June 2010

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Abstract

Neisseria meningitidis is a major cause of sepsis and meningitis but is also a 22

common commensal, present in the nasopharynx of between 8 to 20% of

healthy individuals. During carriage, the bacterium is found on the surface of 24

the nasopharyngeal epithelium and in deeper tissues, while to develop disease

the meningococcus must spread across the respiratory epithelium and enter 26

the systemic circulation. Therefore investigating the pathways by which N.

meningitidis crosses the epithelial barrier is relevant for understanding 28

carriage and disease, but has been hindered by the lack of appropriate models.

Here, we have established a physiologically relevant model of the upper 30

respiratory epithelial cell barrier to investigate the mechanisms responsible for

traversal of N. meningitidis. Calu-3 human respiratory epithelial cells were 32

grown on permeable cell culture membranes to form polarised monolayers of

cells joined by tight junctions. We show that the meningococcus crosses the 34

epithelial cell barrier by a transcellular route; traversal of the layer did not

disrupt its integrity and bacteria were detected within the cells of the 36

monolayer. We demonstrate that successful traversal of the epithelial cell

barrier by N. meningitidis requires expression of its Type four pili (Tfp) and 38

capsule, and is dependent on the host cell microtubule network. The Calu-3

model should be suitable for dissecting the pathogenesis of infections caused 40

by other respiratory pathogens as well as the meningococcus.


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Introduction 42

Neisseria meningitidis is a leading cause of fatal sepsis and meningitis worldwide

(61). Despite being an important human pathogen, the bacterium is also a common 44

commensal, found in the nasopharynx of between 8 and 20% of healthy individuals

(25, 62). The human nasopharynx is lined by a columnar epithelium which forms the 46

first cellular barrier encountered by the meningococcus following its acquisition. This

cell layer consists of differentiated, polarised, respiratory epithelial cells joined by 48

tight junctions that form a barrier which excludes mucosal pathogens. The majority

of cells in the layer are ciliated, resulting in a brush border, although areas of un-50

ciliated cells are also present along with mucus secreting goblet cells (54). To cause

disease the meningococcus must spread from the nasopharynx, its only natural 52

reservoir, penetrate the upper respiratory epithelial cell barrier and enter the systemic

circulation. Furthermore, during asymptomatic colonisation bacteria are not confined 54

to the epithelial surface of the nasopharynx, but are also found in clusters beneath

the epithelial cell layer in tonsillar tissue (57). Therefore defining the route and 56

mechanisms of passage of the respiratory epithelial cell layer by N. meningitidis are

relevant for understanding both meningococcal carriage and disease. 58

Traversal of an epithelial barrier by a pathogen can be considered to consist of a 60

number of steps, which include i) adhesion to the apical surface, ii) invasion of

epithelial cells, iii) survival within cells, iv) movement to the basal side of cells, and iv) 62

escape from the basolateral aspect of the epithelium. The initial step in traversal by

N. meningitidis involves attachment of bacteria to epithelial cells. Work with 64

nasopharyngeal tissue in an organ culture model indicates that meningococcal

attachment is largely limited to non-ciliated cells of the respiratory epithelium (63). 66


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For encapsulated meningococci, adhesion is mediated by bacterial surface-

associated filamentous structures called Type four pili (Tfp) (42, 46, 50, 64) which are 68

also required for colonisation of human nasopharyngeal tissue (16). Tfp perform

several important functions including DNA uptake (77), twitching motility (43) and 70

bacterial aggregation (28), and have been proposed to mediate meningococcal

attachment to host cells by binding to CD46 (33). After initial adhesion, N. 72

meningitidis forms microcolonies on the apical surface of epithelial cells and the

microvilli of infected cells becoming elongated and interweave through the 74

microcolonies (51, 63). Bacteria replicate within the microcolonies then disperse

across the cells in a Tfp-dependent manner (51). Following dispersal, the pili retract 76

and bacteria become tightly associated with the host plasma membrane; at this stage

bacteria may be internalised (50, 60, 63). 78

While the mechanisms that mediate attachment of N. meningitidis to epithelial cells 80

are well understood, there is a paucity of information about the subsequent steps

involved in traversal of the epithelial cell barrier. Indeed, there are even conflicting 82

data regarding the route of traversal with both paracellular and transcellular routes

identified in experiments with endometrial and gastrointestinal cells (3, 42, 50). 84

Although the related pathogen, Neisseria gonorrhoeae migrates through cells (76), it

is not possible to extrapolate findings from this unencapsulated bacterium to the 86

meningococcus, given the proposed role of capsule for survival within cells (60).

Therefore the aim of this study was to determine the route of traversal of N. 88

meningitidis across the respiratory epithelial cell barrier and define host and

pathogen factors involved in this key step in pathogenesis. We used Calu-3 cells to 90

establish a model for meningococcal traversal of the respiratory epithelial barrier.


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Calu-3 human bronchial epithelial cells are one of the few respiratory cell lines that 92

differentiate into polarised monolayers when grown on porous membranes in vitro

(14, 20, 21, 56). Growing the cells in this way allows bacteria to escape from the 94

basolateral surface and provides them with the epithelial cell barrier encountered in

the nasopharynx without the need for organ culture models, which are not well suited 96

for defining mechanisms of cell entry and passage. Calu-3 monolayers display

features of differentiated human airway epithelium with junctional complexes (24, 56), 98

apical-basal differentiation, and possess transport and metabolism functions (20).

These characteristics have led to Calu-3 cells becoming widely used for in vitro 100

studies of drug delivery and toxicology of the human respiratory epithelium (5, 7, 14,

17, 38, 49). We show that N. meningitidis crosses the respiratory epithelial 102

monolayer by a transcellular route without disrupting the barrier function of the layer.

We also demonstrate that the bacterial capsule and Tfp are important for this 104

process. These structures are also required for passage when cells are grown with

an air interface. On the host side, an intact microtubule network is necessary for 106

efficient traversal while we observed no effect of the proposed Tfp receptor, CD46.


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Materials and Methods 108

Bacterial strains and growth conditions

N. meningitidis was cultured in Brain Heart Infusion (BHI) broth (Oxoid) or on BHI 110

agar (1.5% wt/vol, Agar No.1, Oxoid) with 5% Levinthal’s supplement. Escherichia

coli was grown in Luria-Bertani (LB) broth (Gibco) at 37°C with shaking, or on LB 112

agar. Details of the strains used in this work are shown in Table 1. Antibiotics were

used at the following concentrations: kanamycin, 50 and 100 µg ml-1 and 114

erythromycin, 200 and 2 µg ml-1, for E. coli and N. meningitidis respectively, and

carbenicillin at 50 µg ml-1. 116

Cell culture 118

Calu-3 epithelial cells were cultured in DMEM:F12 (Invitrogen), while Chang epithelial

cells were propagated in RPMI 1640 (Invitrogen). Media were supplemented with 120

GlutamaxTM (Invitrogen) and 10% foetal calf serum (PAA Laboratories). All cells were

grown at 37°C in 5% CO2. Calu-3 monolayers were grown on approximately 1 cm2, 1 122

µm pore size, BD Falcon™ cell culture inserts (BD Bioscience) containing

polyethylene terephthalate membranes in 12 well tissue culture companion plates 124

(BD Bioscience). Cells (4 x 105) were seeded onto the apical side of membranes.

For assays performed with liquid covered culture (LCC), layers were maintained with 126

1 ml of culture medium in the apical chamber and 1.5 ml in the basolateral chamber.

Cells were allowed to grow and differentiate for five days, with the media changed 128

every second day. Air interface culture (AIC) monolayers were seeded and

maintained as above except that the apical media was removed after two days. The 130

trans-epithelial electrical resistance (TEER) across monolayers was measured with


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an EVOM TEER meter attached to STX chopstick electrodes (World Precision 132

Instruments); only monolayers with TEER of over 1400 Ω were used in experiments.

134

Texas Red labelled 70,000 MW dextran (final concentration 0.1 µM, Invitrogen) was

added to the apical chamber of cell culture wells. Samples were taken from the 136

basolateral chamber at times afterwards, and fluorescence analysed with a

spectrophotometer (Varian Cary Eclipse). For microscopy, cover slips were seeded 138

with Calu-3 cells at a density of 4 x 105 cells/well and Chang cells at a density of 5 x

104 cells/well in 24 well plates. Cells were grown to semi-confluency overnight. 140

Challenge of cells with bacteria 142

All inocula consisted of 1 ml of a bacterial suspension per tissue culture well except

for infection of AIC layers where a volume of 200 µl per well was used. Bacteria 144

were harvested following overnight culture on solid media and re-suspended in 400

µl of PBS. The concentration of the bacterial suspension was adjusted in culture 146

media to give the desired multiplicity of infection (MOI). For traversal assays, media

in the basolateral chambers of monolayers was replaced immediately prior to 148

challenge. For competitive infections (2), inocula consisting of a 1:1 ratio of mutant

to wild-type bacteria with a final MOI of 40 were prepared and added to the apical 150

chamber. The ratio of bacterial strains in the inoculum was determined by serial

dilution and plating to media with and without antibiotics. After seven hours, cell 152

culture inserts containing infected monolayers were washed then transferred to new

wells containing 1.5 ml of culture media in the basolateral chamber and left for one 154

hour. The ratio of strains in the basolateral chamber was determined by plating to

solid media. This procedure was repeated at 23 hours post-challenge. The 156


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competitive index was calculated by dividing the ratio of mutant to wild-type in the

output by the ratio in the input, and was expressed logarithmically as the logCI. 158

Adhesion assays were performed by inoculating polarised monolayers with bacteria 160

at an MOI of 40. After three hours monolayers were rinsed with Hank’s buffered

saline solution (HBSS) (Invitrogen) then treated with 1% saponin to lyse cells and 162

release all cell associated bacteria. Numbers of cell associated bacteria were

enumerated by plating to solid BHI media. The influence of purified CD46 on 164

adhesion was assessed by adding the purified protein to bacteria prior to infection of

cells. The inoculum was prepared at the required MOI, and 6.25 µg of purified CD46 166

(consisting of the extracellular domain alone) were added per 107 bacteria. Bacteria

and CD46 were incubated together at 37oC for 30 minutes, and then added to cells. 168

When required, different anti-CD46 mAbs or an isotype control at a concentration of

2 µg/ml were added to the infection medium for three hours. Details of antibodies 170

used in this study are given in Table 2.

172

For recovery of intracellular bacteria, Calu-3 monolayers were challenged with

bacteria at an MOI of 40. At eight or 24 hours after inoculation, monolayers were 174

washed three times with PBS on their apical and basolateral surfaces then incubated

with 200 µg/ml gentamicin for 30 minutes, again in both the apical and basolateral 176

chambers. Next, cell culture inserts were washed three times with PBS, and cells

were lysed by a 5 minute treatment with 1% saponin in PBS. The number of 178

intracellular bacteria was established by serial dilution of lysates and plating to solid

media. 180


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Chemicals and Inhibitors 182

Taxol and nocodazole (Sigma) were dissolved in DMSO and used at a final

concentration of 2 µM. Nocodazole was added to media on the apical surface of 184

cells which were then incubated at 4oC for 45 minutes, then left at 37⁰C for 45

minutes prior to challenge. Taxol was added to the cells at the time of the challenge. 186

The number of bacteria traversing monolayers was expressed as the proportion of

the number of bacteria recovered from the basolateral chamber of wells containing 188

no cells. Latrunculin A (Sigma) was used at a final concentration of 1 µM. In all

cases the final concentration of DMSO in culture wells never exceeded 0.1%. 190

Fixation, labelling and staining 192

Antibodies were diluted in 10% horse serum in PBS. Samples for confocal

microscopy were washed twice with PBS, fixed for 15 minutes at room temperature in 194

3% paraformaldehyde (PFA) in PBS, then washed three times with PBS. For

labelling of intracellular and extracellular bacteria, cells were incubated for one hour 196

with an α lipopolysaccharide (LPS) antibody (αL3,7,9) after which cells were washed

twice in PBS then incubated for 30 minutes with an α-mouse RRX conjugated 198

secondary antibody to label extracellular bacteria. Cells were permeablised by

treatment with 0.2% Triton X-100 (T X-100) in PBS and the labelling process was 200

repeated except a Cy2 conjugated α-mouse secondary antibody was used to label

both intracellular and extracellular bacteria. Host cell structures were labelled after 202

permeabilisation; actin was stained with fluorescently labelled phalloidin. After

labelling, cover slips were washed twice in PBS, then in ddH2O, mounted in ProLong 204

Gold (Invitrogen) and analysed by fluorescence microscopy (BX40; Olympus) or a


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confocal laser scanning microscopy (LSM510, Zeiss). Images were imported into 206

Adobe Photoshop CS2 for processing.

208

For TEM, samples were washed three times on ice with ice cold PBS then immersed

in EM grade glutaraldehyde (Agar Scientific) diluted to 0.5% in 200 mM sodium 210

cocodylate (Agar Scientific) at room temperature for 30 minutes. Next samples were

washed three times in 200 mM sodium cocodylate buffer and post-fixed in 1% 212

osmium tetroxide-1.5% potassium ferrocyanide at room temperature for one hour.

Cells were then washed in water, incubated in 0.5% magnesium-uranyl acetate 214

overnight at 4°C, washed again in water, dehydrated in ethanol, and embedded in

Epon. Cross sections were cut through fixed monolayers and placed on EM grids. 216

Lead citrate was added for contrast and grids were then examined using a Biacore

2000 transmission electron microscope. 218

Tfp and capsule expression 220

Monolayers were infected with MC58 at an MOI of 40 as described above. After 24

hours bacteria were recovered from the apical and basal chambers. A total of 10 222

colonies from the apical and 10 from the basal chambers were grown overnight on

solid media then harvested and resuspended in 0.5 ml PBS (Sigma, U.K.). For slot 224

blot analysis of Tfp expression, samples were combined with an equal volume of 2x

SDS-PAGE sample buffer and boiled for 10 minutes, then transferred to immubilon P 226

polyvinylidene fluoride (PVDF) membranes (Millipore) using a MINIFOLD slot blot

system (Whatman). The membranes were blocked in 5% milk in PBS for one hour at 228

room temperature with gentle shaking. Relevant antibodies were diluted in PBSTM

(PBS, 0.05% Tween 20, 0.5% skimmed milk), and incubated with the PVDF 230


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membrane for 1-2 hours, followed by three washes with PBSTM for ten minutes

each. Following incubation with the primary mAb (SM1 which recognises class I pilin, 232

(70)), the PVDF membrane was washed with PBSTM three times for ten minutes

each. A goat anti-murine immunoglobulin antibody conjugated to horse radish 234

peroxidase (Dakocytomation) was used as the secondary antibody. Antibody binding

was detected using ECL Western Blotting detection reagents (GE Healthcare). 236

Capsule expression was analysed by ELISA. Suspensions of bacteria were 238

prepared in PBS as described above, and diluted to a concentration of 1x109.

Bacteria were heated at 56oC for 1 hour and then re-suspended in ELISA coating 240

buffer (Sigma). Samples (100 µl) were added to wells of 96 well plates and left

overnight at 4oC. Wells with coating buffer alone or those coated with MC58∆siaD 242

were included as controls. Next plates were washed with water, then incubated for

two hours with 2% bovine serum albumin (BSA) in PBS. Following blocking, plates 244

were washed three times with PBS/Tween 0.1% then incubated with α-serogroup B

capsule mAb for one hour at 37 oC. Plates were washed three times in PBS/Tween 246

0.1% and incubated with α murine Ig goat pAb conjugated to horse radish peroxidase

(αM HRP) for a further hour at 37 oC. After further washes, 100 µl of Sigmafast OPD 248

substrate (Sigma) was added to plates for six minutes, and the reaction stopped with

100 µl of 0.3 M HCl. Absorbance was read at 492 nm using Ascent software. 250


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Results 252

Interactions between Calu-3 respiratory epithelial cells and N. meningitidis

Initially we compared the interactions of the meningococcus with Calu-3 and Chang 254

cells grown to semi-confluency then challenged with MC58, a clinical serogroup B

isolate of N. meningitidis, at a multiplicity of infection (MOI) of 100, which is in the 256

range of MOIs employed in other studies. This MOI represents a midpoint in a large

range of MOIs used to study the interaction of N. meningitidis with Chang cells (4, 258

73). MC58 adhered to and invaded into Calu-3 and Chang cells at similar levels

(average no. of adherent bacteria per cell 17.4 and 20 for Chang and Calu-3 cells, 260

respectively; average no. of internalised bacteria per cell 2.18 and 2.21 for Chang

and Calu-3 cells, respectively, Fig. S1). Furthermore adhesion of N. meningitidis to 262

Calu-3 cells was facilitated by Tfp (Fig. S1), in keeping with results using other

epithelial cell lines (47, 50, 69), and Calu-3 cells express CEACAMS (not shown), 264

which are receptors for Neisseria Opas (75).

266

As N. meningitidis adheres to and enters into Calu-3 cells, we next grew the cells on

permeable membranes in cell culture inserts so they formed a barrier between the 268

apical and basolateral chambers (Fig. S2). The development of tight junctions

between cells was monitored by measuring the TEER across layers. The level of 270

TEER across Calu-3 cells was influenced by a number of factors. Reducing the size

of pores in membranes led to higher levels of TEER but pores of less than 1 µm 272

impeded the passage of bacteria (not shown). Therefore we selected membranes

with 1 µm pores for all subsequent studies, which with seeding cells at a density of 4 274

x 105 per cm2, gave consistent levels of TEER of around 1850 Ω after 5 days (Fig.

1A). The development of the epithelial cell barrier following seeding of wells was 276


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also demonstrated by permeability of the monolayer to fluorescently labelled 70,000

MW dextran added to the apical chamber. Increasing TEER over time coincided with 278

decreasing permeability of the layer to the tracer (Fig. 1A), demonstrating that the

cells formed an effective barrier within five days of seeding. 280

Furthermore transmission electron microscopy (TEM) confirmed that Calu-3 cells 282

formed confluent monolayers with morphological features of a polarised respiratory

epithelium (Figs. 1B, C and D) consisting of a continuous monolayer of columnar 284

cells linked by junctional structures. There was apical-basal polarisation, with nuclei

located towards the basolateral side of cells, the development of tight junctions, and 286

microvilli on the apical surface (Fig. 1B). Full junctional complexes (with tight

junctions, adhesion belts and desmosomes) were present between cells (Fig. 1C), 288

and the membranes of adjacent cells were barely distinguishable (Fig. 1D). Immuno-

fluorescence microscopy of the tight junction proteins zonula occludens-1 (ZO-1, Fig. 290

1E) and occludin (Fig. 1F) showed the characteristic chicken-wire pattern formed by

their continuous presence at the margins of cells. At this time, while the tracer 292

diffused freely into the basolateral chamber in the absence of cells (Fig. S3A), no

tracer was detected in the basolateral chamber even after 24 hours (Fig. S3B). 294

Additionally, we challenged the apical surface of the monolayer with non-invasive E.

coli DH5α and monitored its appearance in the basolateral chamber over time. When 296

no cells were present on the cell culture membrane, E. coli was detected in the

basolateral chamber within two hours of challenge (Fig. S3C). In contrast, no 298

bacteria were detected in the presence of Calu-3 cell monolayers (Fig. S3D) even

after eight hours. 300


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N. meningitidis traverses the epithelial cell barrier by a transcellular route 302

Next we investigated the traversal of N. meningitidis across Calu-3 monolayers five

days after seeding. The apical surfaces of layers were challenged with MC58 at an 304

MOI of 40, and eight and 24 hours later, the inserts containing infected membranes

were washed thoroughly in media then transferred into new wells. After one hour, 306

samples were taken from the basal chambers of the new wells and were plated to

BHI. N. meningitidis was detected traversing in small numbers (equivalent to 308

0.0035% of input) at eight hours post-challenge. By 24 hours, the number of bacteria

traversing over the course of one hour had increased by around four orders of 310

magnitude (Fig. 2A). Bacteria could occasionally be found in the basal chamber prior

to 8 hours but never earlier than six hours (not shown). At 16 hours intermediate 312

levels of traversing bacteria were found (not shown).

314

Traversal of the monolayer by N. meningitidis might result from loss of integrity of the

tight junctions or cell death. Therefore, the TEER across the monolayer was 316

monitored during the course of an infection. Although the TEER across the layer fell

following bacterial challenge, levels never dropped to below 40% of initial readings 318

and also fell in wells infected with E. coli and the uninfected control wells (Fig. 2B).

The integrity of the layer during infection was also monitored by adding the 320

fluorescent tracer to the apical surface of the monolayer at the time of challenge.

Although bacteria successfully traversed the monolayer, there was no increase in 322

fluorescence in the basolateral chamber beneath the infected cells even after 24

hours. Indeed, there was no significant difference in the level of fluorescence in the 324

basolateral chamber of infected and uninfected monolayers (Fig. 2C).

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Infected monolayers were also fixed and examined for the presence of the tight

junction protein ZO-1. The distribution of ZO-1 in infected cells was indistinguishable 328

from uninfected monolayers (Fig. 3A). The protein was present at the margins of all

cells, with no gaps or disruption detected. Additionally, we examined infected 330

monolayers by TEM. At 8 hours following inoculation microcolonies of N.

meningitidis were present on the apical surface of the monolayer (Fig. 3B). These 332

bacteria were tightly associated with the apical surface of the cells of the monolayers,

and were often found on pedestal-like protrusions of the apical membrane (Fig. 3B 334

and C). After 24 hours, the number of adherent bacteria had increased and covered

a larger area of the apical surface of the monolayer (Fig. 3D). Meningococci were 336

still closely adherent to the apical surface (Fig. 3E) and examination of the margins of

cells revealed that the junctional structures were intact (Fig. 3F), consistent with the 338

results obtaining by ZO-1 labelling; even though gaps were evident between cells

below the tight junctions, these were present in infected and uninfected monolayers 340

(Fig. 1 and 3). Taken together these data demonstrate that there is no loss of

integrity of the Calu-3 monolayer coincident with traversal of N. meningitidis, 342

indicating that the meningococcus does not cross the epithelial barrier by a

paracellular route or following loss of cells from the layer. 344

These results suggest that N. meningitidis traverses the respiratory monolayer by a 346

transcellular route, which predicts that the cells of the monolayer should harbour a

population of intracellular bacteria during traversal. Therefore monolayers of cells 348

were challenged with MC58, then after eight or 24 hours were exposed to gentamicin

for one hour to kill extracellular bacteria. Cells were lysed by treatment with saponin 350

to release intracellular bacteria, which were recovered by plating lysates to solid


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media. A population of gentamicin-protected bacteria (representing approximately 352

0.1% of the input) was consistently present in cells at eight hours post-challenge, and

this number increased by an order of magnitude by 24 hours post challenge (Fig. 354

4A). In contrast, no bacteria were recovered from gentamicin-treated membranes

which had been infected with N. meningitidis in the absence of cells (not shown). 356

It was possible that the recovered bacteria survived gentamicin treatment by 358

occupying extracellular sites between cells in the monolayer that were inaccessible to

the antibiotic. Therefore the infected monolayers were disrupted by treatment with 360

trypsin for 10 minutes or latrunculin A for 20 minutes prior to incubation with

gentamicin. Exposure of the monolayer to trypsin for 20 minutes is sufficient to 362

remove all cells from the membrane, while treatment with latrunculin A for 15 minutes

leads to a complete loss of TEER (not shown). Treatment with latrunculin or trypsin 364

did not alter the number of CFU recovered from the layer after exposure to

gentamicin (Fig. 4B), consistent with the bacteria being in an intracellular 366

compartment. In addition, we performed confocal microscopy of cross sections of

the monolayer at 24 hours following bacterial challenge. This revealed the presence 368

of N meningitidis within cells, including bacteria located in basal regions of cells (Fig.

4C). 370

Tfp and the polysaccharide capsule are required for traversal of the monolayer 372

For several pathogens, the mechanisms mediating adhesion to and entry into cells in

monolayers are different for those described with semi-confluent cells (22, 45). As 374

Tfp have been implicated in meningococcal traversal of gastrointestinal epithelial

cells (50), we examined the ability of a mutant lacking Tfp (MC58∆pilE) to cross the 376


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respiratory epithelial cell barrier. The apical surface of Calu-3 monolayers were

challenged with a 1:1 ratio of the wild-type strain and MC58∆pilE, and the ratio of the 378

strains exiting the layer was determined at eight and 24 hours post-challenge. At

these times, inserts containing infected membranes were washed thoroughly in 380

media then transferred into new wells. Aliquots from the basolateral chambers of

new wells were taken one hour later, plated to media with and without antibiotics, and 382

the competitive index (CI) of the mutant calculated. Wells with inserts lacking cells

were challenged with the same inoculum as controls. We found that MC58∆pilE had 384

a significant defect for traversal across the Calu-3 monolayer after 24 hours with a

mean logCI of approximately -0.5 (Fig. 5A). To determine how Tfp contribute to 386

traversal, we analysed the adhesion of the pilus-deficient mutant and the wild-type

strain to the polarised respiratory monolayer in separate wells. Consistent with the 388

CI for MC58∆pilE for traversal, the Tfp mutant had a significant defect for adhesion to

the monolayer (p < 0.001, Fig. 5B). Furthermore the adhesion defect of MC58∆pilE 390

was reflected in reduced recovery of intracellular bacteria from MC58∆pilE infected

monolayers compared to MC58 infected cells (Fig. 5C). 392

It has been proposed that Tfp expression is downregulated after initial adhesion to 394

host cells (12). Therefore we examined pilus expression in bacteria following

passage through the monolayer. Slot blot analysis using the mAb SM1 was 396

performed on bacteria recovered from the apical and the basolateral chambers of

wells challenged with MC58. While all 10 colonies from the apical chamber 398

expressed Tfp, eight of 10 colonies were positive for Tfp in the basolateral chamber

(Fig. 5D). Identical findings were obtained when the experiment was performed on a 400

separate occasion.


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402

Recent work has indicated that the sialic acid polysaccharide capsule of N.

meningitidis contributes to survival within epithelial cells (60). We hypothesised that 404

the capsule is also involved in traversal. Therefore we determined the CI for

traversal of MC58∆siaD, which is unable to express a capsule due to loss of SiaD, 406

the polysialic acid transferase (26). The unencapsulated strain had a marked defect

in traversal (Fig. 6A); after eight hours the mean logCI of the MC58∆siaD mutant was 408

-1.2, while by 24 hours the logCI had fallen to almost -4. To understand the basis of

this defect we examined the adhesion of MC58∆siaD to monolayers, consistent with 410

previous work (26), we found that the capsule deficient strain had a significant

increase in adhesion (approximately five fold) compared to MC58 (p < 0.001, Fig. 412

6B). We also recovered intracellular bacteria from MC58 and MC58∆siaD infected

monolayers 24 hours after challenge. In contrast to adhesion, there was a significant 414

reduction in the intracellular population of MC58∆siaD compared with MC58 at this

later time point (p < 0.001, Fig. 6C). Furthermore, as capsule expression is 416

downregulated during adhesion (12), we examined capsule expression in bacteria

from the apical and basolateral chambers at 24 hours post-challenge. All strains were 418

found to be encapsulated by ELISA (Fig. 6D). Both the pilus- and capsule-deficient

strains grew in media and crossed membranes without cells as MC58 (not shown), 420

so their attenuation cannot be ascribed to a growth defect or failure to pass through

the pores in the membranes. 422

An intact microtubule network is required for successful traversal 424

Transcellular traversal of cells by bacteria should be dependent on host as well as

bacterial factors. As microtubules are required for long range trafficking of vesicles, 426


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including those containing pathogens, such as Salmonella enterica serovar

Typhimurium (S. typhimurium) (53) and N. gonorrhoeae (76), we next examined 428

whether disruption of the microtubule network using taxol or nocodazole affected

meningococcal traversal. Taxol inhibits the depolymerisation of tubulin subunits, 430

freezing intact microtubules (30). In contrast nocodazole de-polymerises

microtubules, leaving cells devoid of polymerised tubulin (11). Neither drug affected 432

bacterial replication in vitro (Fig. S4) or permeability of the layer to fluorescent tracer,

although some reduction in TEER was detected (Fig. S5). To confirm the effect of 434

these treatments on cells, monolayers were examined 24 hours after drug treatment

for the presence of microtubules and the tight junction protein occludin (Fig. S6). 436

Abnormalities in the microtubule networks of the cells of the monolayers, including

cells undergoing abnormal cell division, were seen in taxol treated cells (Fig. S6). In 438

contrast, β-tubulin labelling was diffuse throughout nocodazole treated cells,

consistent with microtubule depolymerisation (Fig. S5). The distribution of occludin 440

was unchanged following treatment with taxol or nocodazole, demonstrating (along

with the TEER and tracer data) that the layer remained intact despite drug treatment. 442

Treatment of cells with taxol or nocodazole led to a significant decrease in traversal

of N. meningitidis at eight and 24 hours post-challenge (p values for taxol and 444

nocodazole treated layers, < 0.05 and < 0.01 respectively, Fig. 7A). Interestingly,

treatment of the layers with taxol or nocodazole did not affect the number of 446

intracellular bacteria at eight or 24 hours (Fig. 7B). Taken together, these data

demonstrate that an intact microtubule network is necessary for successful traversal 448

of the layer by N. meningitidis.

450

Infection of Calu 3 monolayers grown under air interface culture (AIC)


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Calu-3 monolayers can be grown under an air interface culture (24). Cells were 452

seeded as described above except the media was removed from the apical chamber

two days after seeding. Monolayers were then left for a further three days. 454

Consistent with previous work (24), the TEER across monolayers with AIC was

approximately half that seen with monlolayers grown under liquid covered culture 456

(LCC, Fig. 8A). TEM examination revealed that the AIC monolayers closely

resemble LCC monolayers (Fig. 8B). Competitive infections comparing traversal of 458

MC58 with pilus-deficient or capsule-deficient mutants with MC58 were performed

using the AIC monolayers. In contrast to LCC monolayers, no traversal had occurred 460

at eight hours post inoculation. However by 24 hours some bacteria had traversed

the monolayer. Both the pilus-deficient and capsule-deficient mutants had significant 462

defects for traversal (Fig. 8C and D). These results indicate that AIC monolayers

could be adapted to investigate N. meningitidis infection, and retain their selective 464

barrier function during meningococcal traversal.

466

The role of CD46 during adhesion of N. meningitidis to polarised respiratory

epithelial monolayers 468

CD46 is the proposed receptor for meningococcal Tfp (33). Therefore we examined

whether CD46 is expressed by Calu-3 cells. Western blot analysis was performed on 470

whole cell lysates cells from Calu-3 cells, as well as COS-7 and HeLa cells, which

are both known to express CD46 (39). We found Calu-3 cells expressed CD46 at 472

higher levels than HeLa or COS-7 cells (Fig. 9A). Next we next analysed the

distribution of CD46 in Calu-3 monolayers. CD46 was localised to the basolateral 474

aspect of cells in the monolayer (Fig. 9B), consistent with findings from other cell

types and tissues (36, 41, 66). Furthermore, challenge of monolayers with N. 476


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meningitidis did not affect the basolateral distribution of CD46 (Fig. 9C), which was

confined to the lateral and basolateral margin of cells. 478

To test whether CD46 is involved in meningococcal adhesion to the monolayer, two

different approaches were used. First, MC58 was incubated with purified CD46 (6.25 480

µg CD46 per 107 bacteria) for 30 minutes prior to infection of Calu-3 monolayers, and

the adherent population compared with bacteria incubated in the absence of the 482

complement regulator. There was no significant difference in the level of adhesion of

CD46 bound bacteria compared to bacteria that were not pre-incubated with CD46 484

(Fig. 9D). Furthermore, the rate of bacterial adhesion was determined with anti-

CD46 mAbs in the infection medium (2 µg of anti- CD46 antibody per 1.6 x 107 CFU). 486

At three hours after challenge, the adherent population of bacteria was compared

with control wells without a mAb or with an isotype control. No significant difference 488

in the adhesion of meningococci to the monolayer was observed in the presence of

the mAbs (Fig.9E). These data are consistent with CD46 playing little role in the 490

adhesion of MC58 to the Calu-3 monolayers.

492


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Discussion 494

The ability of bacteria to cross cellular barriers is critical during colonisation and

disease. These barriers can be formed in the host by a variety of cell types including 496

epithelial and endothelial cells, and at mucosal surfaces they form a key component

of innate immunity against infectious agents. The first cell barrier encountered by 498

bacteria in the nasopharynx is the pseudostratified columnar epithelium of the upper

respiratory tract. Here we characterised the interaction of N. meningitidis, an 500

important human pathogen with monolayers of polarised Calu-3 cells. Although this

cell line is derived from the bronchial epithelium, it is relevant for studying the biology 502

of the meningococcus as the respiratory tract is lined by a columnar epithelium from

the posterior nares down to the distal bronchioles. 504

Semi-confluent Calu-3 cells grown on impermeable substrates exhibited similar 506

patterns of adhesion and uptake of N. meningitidis as Chang cells, which have been

widely used for studying this bacterium. Adhesion of the meningococcus to Calu-3 508

cells was largely dependent on Tfp, consistent with previous work with other cell lines

(47, 50, 69), and invasion occured at low, but appreciable levels. When propagated 510

on semi-permeable membranes, Calu-3 cells polarised and formed monolayers (24,

56), which gave high levels of TEER and were impermeable to fluorescently labelled 512

tracers and non-pathogenic E. coli. Furthermore, the distribution of the tight junction

proteins ZO-1 and occludin, as well as electron microscopy, demonstrated that the 514

cells displayed all the characteristics of pseudostratified columnar epithelial cells

linked by a full range of junctional structures. 516


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There is a distinction between pathogens which utilise a transcellular route to cross 518

epithelial cell monolayers (19), and those bacteria which cross by a paracellular route

following destruction of cells of the layer or disruption of tight junctions (1, 10, 18, 34, 520

35, 40, 55, 59, 68). There is conflicting evidence about the route that the

meningococcus takes to traverse the epithelial cell barrier in the nasopharynx. Work 522

with intestinal epithelial cells indicates that the bacterium migrates using a

transcellular route (50). In contrast, the meningococcus appeared to traverse an 524

endometrial epithelial:endothelial bilayer by both a para- and trans-cellular route (3).

Furthermore, the bacterium crosses the brain microvascular endothelial cells by a 526

paracellular route (8). We found that Calu-3 monolayers continued to display barrier

function during traversal of N. meningitidis. There was a minor reduction in TEER 528

across the monolayers during challenge but this was observed to some extent in both

infected and uninfected monolayers. However levels of TEER never fell to less than 530

40% of the initial readings, similar to levels following challenge with E. coli (which did

not cross the cell barrier), and monolayers remained impermeable to fluorescent 532

tracers with no change in the distribution of ZO-1 following infection. Additionally we

were unable to detect any cell destruction or alteration of junctional structures by 534

TEM. We also found that the monolayers allowed the selective passage of strains

during mixed infection experiments, confirming that traversal does not result from 536

loss of intergrity of the barrier. Finally, an intracellular population of bacteria was

detected in Calu-3 monolayers by recovery of viable organisms following treatment 538

with gentamicin, and by confocal microscopy. Attempts were made to obtain Z stack

images of infected monolayers but were unsuccessful because of auto-fluorescence 540

of membranes and bleaching of the signal when taking multiple images. To

circumvent this, membranes were folded back on themselves to allow acquisition of 542


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high resolution, cross-sectional images of infected monolayers without including the

membrane itself (Fig. 5E); inside:out staining was used to confirm the intracellular 544

location of bacteria. Thus we have several independent lines of evidence that that N.

meningitidis crosses the respiratory epithelial barrier by a transcellular route. 546

Work with polarised epithelial cells in monolayers has provided novel insights into the 548

mechanisms and sites of adhesion of pathogens. For instance, the entry of Shigella

flexneri was shown to be restricted to the basolateral surface of epithelial cell 550

monolayers (45), while a novel adhesin was identified in Salmonella which is

specifically involved in adhesion to polarised cells (22). Here we found that Tfp 552

mediate attachment of the meningococcus to the monolayer, as well as to semi-

confluent cells, demonstrating that the mechanism of initial adhesion is similar to 554

non-polarised and polarised cells.

556

To identify bacterial factors involved in traversal, we performed competitive assays

between the wild-type and mutant strains. The advantage of this approach is that it 558

eliminates well to well variation in the overall level of traversal, and allows direct

comparison of the extent of traversal of strains. Consistent with previous results (50), 560

we found that the mutant lacking Tfp had a significant defect for traversal across the

monolayer compared with the wild-type strain. The defect for adhesion of the pilus 562

deficient strain to polarised monolayers was similar in magnitude to the reduction in

number of intracellular bacteria, and the overall defect of MC58∆pilE for traversal. 564

These data suggest that Tfp contribute to traversal during the attachment of the

bacterium to the apical surface of cells. However a significant proportion of bacteria 566

were pilus-negative following traversal of the monolayer, which is likely to reflect


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antigenic variation from gene conversion with pilS resulting in a pilin-deficient 568

phenotype (9). It might be that Tfp are required for initial adhesion by mediating

bacterial aggregation, but subsequent cell entry requires individual (possibly Tfp-570

negative) bacteria to dissociate from microcolonies on the cells (32).

572

We found that Calu-3 monolayers express the proposed Tfp receptor CD46 (33), 574

which further widens the scope for experimentation using this cell culture model.

However other findings do not support a role of CD46 in meningococcal adhesion 576

(13, 23, 37, 52, 67). Therefore the Calu-3 monolayer offered another approach to

investigate this topic. Although there is evidence that CD46 is expressed on the 578

apical surface of the respiratory epithelium (58), CD46 was confined to the

basolateral surface of Calu-3 cells in the monolayer, similar to findings from 580

examination of the liver (36) and cell culture models (41, 66).. As expected from its

basolateral location, we did not find evidence that CD46 contributes to the adhesion 582

of N. meningitidis as addition of purified CD46 or anti-CD46 mAbs did not inhibit

bacterial attachment, in contrast to previous work (33). This discrepancy could be 584

because we used the four extracellular, complement regulatory, SCR domains of

CD46 rather than the full length molecule, while the mAbs may not be able to 586

compete effectively with interactions with the bacterium. Further studies are

necessary before it is possible to exclude a contribution of CD46 to initial binding of 588

N. meningitidis to Calu-3 cells.

590


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There was a significant reduction in the recovery of capsule deficient (∆siaD) mutant

from within polarised Calu-3 monolayers despite a five fold increase in adhesion of 592

this strain, consistent with previous work indicating that the capsule is dispensable for

cell entry (27, 72). The polysaccharide capsule has been proposed to have an 594

important role in the survival of N. meningitidis in epithelial and endothelial cells (48,

60). We found that the ∆siaD exhibited a dramatically reduced rate of traversal of 596

monolayers, far in excess of its defect for intracellular survival, suggesting that the

capsule has further roles during exit of the bacteria from the basolateral pole of cells. 598

Thus the capsule is virtually indispensible for passage across the respiratory

epithelial barrier (during intracellular survival and escape from the basolateral 600

surface). This emphasises the need to consider the traversal of the epithelial barrier

by the gonococcus (which does not possess a capsule) and N. meningitidis 602

independently, and not assume findings from one pathogen can be extrapolated to

the other. 604

We found that traversal of Calu-3 monolayers by the meningococcus was impaired 606

by interfering with the microtubule network using either taxol or nocodazole. The

only example of apical to basal traversal of monolayers using a paracellular route 608

without disrupting tight junctions is the passage of leucocytes across epithelia.

However, this pathway is not affected by inhibiting the microtubule network with taxol 610

or nocodazole (29). Therefore, the impact of microtubule disruption on the passage

of bacteria provides further evidence for a transcellular route of meningococcal 612

traversal. These data also indicate that traversal by N. meningitidis does not occur

by the same mechanism as transcytosis of polymeric immunoglobulins, which is 614

initiated by receptor-mediated, clathrin-dependent endocytosis (44) and does not


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require intact microtubules (31). We also found that the number of intracellular 616

bacteria at eight and 24 hours was not affected by microtubule disruption, suggesting

that microtubules are involved in basolateral escape of meningococci from cells 618

rather than entry.

620

To date, the intracellular niche occupied by N. meningitidis has not been defined. No

markers of intracellular compartments have been found to consistently co-localise 622

with intracellular meningococci (48, 50, 71), and the bacterium has even been

detected in the cytoplasm by some workers (65). Therefore Calu-3 monolayers could 624

be used to more precisely define the mechanisms and route of transcellular passage

of this important human pathogen. Calu-3 cells can also be grown with under AIC 626

conditions. This is a more lengthy process but it has been reported that the cells can

produce cilia and may produce an even more physiologically relevant model of the 628

nasopharyngeal epithelium (24). AIC monolayers still have barrier function and are

morphologically resemble monolayers grown under LCC. Furthermore, Calu-3 cells 630

could also be used to study the epithelial traversal of other pathogens present in the

respiratory tract including Streptococcus pneumoniae and Haemophilus influenzae. 632

The results should provide valuable information about the pathogenesis of these

pathogens, while defining the factors necessary for migration across the epithelium 634

could lead to the development of vaccines to prevent the diseases they cause.

636


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Acknowledgements 638

We are grateful to Elisabeth Kugelberg for her support and advice, and to Mike

Hollinshead for his help with TEM. Purified CD46 protein was generously provided 640

by Prof. Susan Lea. Calu-3 cells were a kind gift from Dr Clive Robinson (St

George’s Hospital, London), and pEG2 was gratefully received from Dr Myron 642

Christodoulides (University of Southampton). T.C.S. is supported by a Wellcome

Trust PhD studentship, and R.M.E. is a Leverhulme Trust Career Development 644

Fellow. Work in C.M.T.’s laboratory is supported by the Medical Research Council

and the Wellcome Trust. 646


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Figure legends

648

Fig. 1 Morphology of Calu-3 monolayers.

A. Development of the Calu-3 monolayer over time, showing increasing TEER and 650

decreasing permeability to Texas red labelled 70,000 MW dextran. Fluorescent

tracer was added to the apical chamber of monolayers at various times post seeding 652

and levels of fluorescence in the basolateral chamber were measured after six hours.

Error bars show the standard deviation from at least three separate experiments. B. 654

For TEM analysis, cells were grown for five days on cell culture inserts. Lower

magnification view (scale bar = 4 µm) shows cells organised into a monolayer of 656

columnar cells joined by tight junctions (TJ), and desmosomes (D). The cell culture

membrane (CCM) can be seen on the basolateral surface of the cells. Apical-basal 658

differentiation is demonstrated by the basal location of nuclei and the presence of

microvilli (MV) on the apical surface. C. Analysis of the cell boundaries shows the 660

formation of extensive junctional structures including full junctional complexes (JC)

composed of (from apical to basal surface) a tight junction (TJ), an adhesion belt 662

(AB), and a desmosome (D). Scale bar = 1 µm. D. Close apposition of cell

membranes within a tight junction. Scale bar = 1 µm. The integrity of the layer and 664

presence of tight junctions was further confirmed using confocal microscopy to

visualise the tight junction associated proteins, ZO-1 (E) and occludin (F). Scale bars 666

= 5 µm.

668


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Fig. 2 N. meningitidis traverses the Calu-3 monolayer without affecting barrier

function. 670

A. Polarised monolayers were challenged with MC58 at an MOI of 40. The number

of bacteria traversing the layer was established by moving inserts to a fresh well at 672

eight and 24 hours and then sampling one hour later, from the basolateral chamber

of the new well. Results are shown as the percentage of bacteria in the input. Dots 674

represent individual wells from four separate experiments; horizontal bars represent

the mean. B. TEER was monitored in the presence and absence of bacteria for 24 676

hours following inoculation. Despite bacterial passage, TEER did not fall below 40%

of initial levels. C. Texas red labelled 70,000 MW dextran was added to the apical 678

side of cell monolayers which were then challenged with MC58 at an MOI of 40 and

fluorescence in the basolateral chamber was monitored over the next 24 hours. 680

Fluorescence did not increase despite bacterial passage. Diffusion of tracer across

inserts with no cells was included as a control. Error bars show the standard 682

deviation from at least three experiments.

684

Fig. 3 Microscopic analysis of infected monolayers.

A. Monolayers were inoculated with MC58 expressing GFP then fixed 24 hours later. 686

The tight junction protein ZO-1 was labelled and the layers examined by confocal

microscopy. Despite bacterial infection the pattern of ZO-1 labelling was unaffected. 688

Scale bars = 5 µm. B, C. For TEM, monolayers were challenged with MC58 and

fixed eight or 24 hours later. After eight hours the monolayer remained intact and 690

appeared undamaged despite the presence of bacterial colonies on the apical

surface. Bacteria adhered tightly to cells and were often on pedestal like protrusions 692

from the apical membrane. D. After 24 hours, there was no apparent disruption to


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the integrity of the monolayer despite increased numbers of bacteria on the apical 694

surface. No gaps in the layer were observed (E). Adherent bacteria remained tightly

associated with the apical membrane. Enlargement of the region between cells (F) 696

revealed the presence of major junctional structures. Boxed regions in D indicate

sections enlarged in panels E and F. 698

Fig. 4 Identification of a population of intracellular bacteria in infected Calu-3 700

monolayers.

Monolayers of cells were challenged with N. meningitidis at an MOI of 40. A. After 702

eight or 24 hours, cells were treated with gentamicin to kill extracellular bacteria then

lysed with saponin to release intracellular bacteria which were enumerated by plating 704

of lysates. Each spot represents the result from an individual well from three

separate experiments. Horizontal bars represent the arithmetic mean. B. 706

Latrunculin A and trypsin were used to disrupt the monolayer before treatment with

gentamicin. Treatment with these chemicals caused no significant reduction in the 708

gentamicin-protected population of N meningitidis. No bacteria were recovered

following gentamicin treatment in the absence of Calu-3 cells. Error bars show the 710

standard deviation from at least three separate experiments. C. Representative

images showing intracellular bacteria (shown by pink arrowheads, present in the 712

monolayer at 24 hours post inoculation. Monolayers were infected with MC58

expressing GFP at an MOI of 40 and fixed after 24 hours. Extracellular bacteria were 714

labelled using an αL3,7,9 LPS mAb followed by a RRX conjugated secondary

antibody (red). Actin was stained with Alexa 647 phalloidin (blue). Scale bars, 5 µm. 716

Boxed areas are enlarged in the lower panels of i) and ii).

718


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Fig. 5 Tfp are important for traversal of the epithelial cell barrier.

A. Monolayers of Calu-3 cells were challenged with a 1:1 mixture of MC58 and 720

MC58∆pilE on the apical surface and the ratio of the strains exiting the basolateral

surface over one hour was determined eight and 24 hours later. The mutant lacking 722

Tfp had a significant defect for traversal. Results are shown as the log competitive

index (logCI). Each spot represents the result from an individual well from three 724

separate experiments. Horizontal bars represent the arithmetic mean. B.

Monolayers of Calu-3 cells were challenged with either MC58 or MC58∆pilE. After 3 726

hours monolayers were washed and then lysed with 1% saponin to release all cell

associated bacteria. Enumeration of cell associated bacteria by plating revealed a 728

large reduction in the numbers of cell associated bacteria for the pilus deficient strain.

C. Intracellular bacteria were recovered by lysis of cells with saponin after 730

gentamicin treatment. MC58∆pilE is present in reduced numbers within the cells of

the monolayer after eight and 24 hours compared with MC58. No bacteria were 732

recovered following gentamicin treatment in the absence of Calu-3 cells (not shown).

Significant differences are indicated by *, < 0.05, **, < 0.01, ***, < 0.001 (Unpaired T-734

test). D. Slot blot analysis of pilin expression in 10 randomly selected colonies from

the apical and basolateral chambers of wells after a 24 hour infection with MC58. 736

Eight of 10 colonies expressed pilus in the basolateral chamber compared to 10 of 10

in the apical chamber. 738

Fig. 6 The polysaccharide capsule is crucial to the traversal of N. meningitidis. 740

A. The apical surfaces of Calu-3 cells were infected with a 1:1 mixture of MC58 and

MC58∆siaD, and the ratio of the strains exiting the basal surface over one hour at 742

eight and 24 hours was determined. The capsule deficient strain had a significant


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defect for traversal. Data are shown as the log competitive index (logCI). Each spot 744

represents the result from an individual well from three separate experiments.

Horizontal bars represent the arithmetic mean. B. Monolayers of Calu-3 cells were 746

challenged with either MC58 or MC58∆siaD. After three hours monolayers were

washed and then lysed with 1% saponin to release cell associated bacteria. 748

Enumeration of cell associated bacteria by plating revealed a large increase in the

numbers of cell associated MC58∆siaD compared with the wild-type strain. C. 750

Intracellular bacteria were recovered by lysis of cells with saponin after gentamicin

treatment. Despite its increased adhesion, MC58∆siaD is present in reduced 752

numbers within the cells of the monolayer after 24 hours compared with MC58. No

bacteria were recovered following gentamicin treatment in the absence of Calu-3 754

cells (not shown). Significant differences are indicated by *, < 0.05, **, < 0.01, ***, <

0.001 (Unpaired T-test). D. Monolayers were infected with MC58 as described, and 756

bacteria were recovered from the apical and basolateral chambers 24 hours later.

Individual colonies (10 from each chamber) were grown overnight on solid media, 758

and analysed for capsule expression by ELISA using an anti-serogroup B mAb at a

variety of dilutions. The unencapsulated strain MC58∆siaD and the wild-type strain 760

were included as controls. All strains sampled from apical and basolateral chambers

were encapsulated. Spots represent the A492 of each strain. 762

Fig. 7 Disruption of microtubules within cells of the monolayer reduces 764

traversal of N. meningitidis.

A. Calu-3 monolayers were treated with nocodazole or taxol, then challenged with 766

MC58 at an MOI of 40. The number of bacteria in the basolateral chamber was

established by plating at eight and 24 hours post challenge. Both nocodazole and 768


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taxol caused a significant reduction in the number of bacteria in the basolateral

chamber at both time points when compared to untreated layers. B. Monolayers of 770

Calu-3 cells were treated with nocodazole or taxol, then challenged. Intracellular

bacteria were recovered by lysis of cells with 1% saponin after exposure to 772

gentamicin for 30 minutes. There was no significant difference in the number of

intracellular bacteria at eight or 14 hours for either drug treatment compared to 774

untreated layers. No bacteria were recovered following gentamicin treatment in the

absence of Calu-3 cells (not shown). Error bars show the standard deviation from at 776

least three separate experiments. Significant differences are indicated by *, < 0.05,

**, < 0.01, ***, < 0.001 (Unpaired T-test). 778

Fig. 8 Challenge of monolayers grown with an air interface (AI) 780

A. Monolayers were seeded as for liquid covered monolayers but media was

removed from the apical chamber after 2 days. After 5 days levels of TEER in the AI 782

monolayers was approximately half that seen for layers grown in media. Significant

differences are indicated by *, < 0.05, **, < 0.01, ***, < 0.001 (Unpaired T-test). B. 784

TEM of AIC monolayers at 5 days. The gross morphology of the layer is not

significantly different from that seen in monolayers grown under standard conditions. 786

Lower magnification view (scale bar, 5 µm) shows cells organised into a monolayer

of columnar cells joined by tight junctions (TJ), and desmosomes (D). The cell 788

culture membrane (CCM) can be seen on the basolateral surface of the cells. Boxed

sections are enlarged in panels i) and ii) (scale bar, 1 µm). C. Competitive index of 790

MC58∆pilE across AI monolayers. In contrast to monolayers grown under media,

there was no traversal of N. meningitidis at 8 hours. However by 24 hours some 792

bacteria had traversed the layer. The pilus deficient mutant has a defect for traversal


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compared to MC58. D. CI of MC58∆siaD for traversal across AI monolayers. The 794

capsule deficient mutant had a large defect for traversal. Results are shown as the

log competitive index (logCI). Each spot represents data from an individual well from 796

three separate experiments. Horizontal bars represent the arithmetic mean.

798

Fig. 9 The role of CD46 during binding of N. meningitidis to polarised

respiratory epithelial monolayers 800

A. CD46 expression detected by Western blot analysis of lysates of cell lines

(indicated). Full length CD46 migrates with a predicted molecular mass of between 802

50 and 79 kDa, while the purified SCR domains are detected as a band of 40 kDa.

B. Expression of CD46 in polarised monolayers examined by confocal microscopy. 804

Five day old monolayers immunolabelled for CD46 detected with a FITC-conjugated,

secondary antibody (green). Actin was counterstained with phalloidin conjugated to 806

an Alexa 595 fluorophore (red). C. Monolayers of Calu-3 cells were infected with

MC58 expressing eGFP (green) at an MOI of 40 for 8 hours, and immunolabelled 808

CD46 was detected with a RRX (red) conjugated secondary antibody; actin was

stained using phalloidin conjugated to Alexa 647 (blue). Representative images are 810

shown. Scale bars = 5 µm. D. Monolayers of Calu-3 cells were challenged with

MC58 at an MOI of 40 with or without 25 µg of purified CD46 per well (1 x 105 cells). 812

MC58 and purified CD46 were pre-incubated for 30 minutes prior to challenge.

Monolayers were also infected with MC58 pre-incubated with DMEM:F12 as a 814

control. E. Calu-3 cells were infected with MC58 at an MOI of 40 in presence or

absence of anti-human CD46 mAbs (indicated) at 2 µg/ml. Adhesion of N. 816

meningitidis to cells was calculated by recovering bacteria 3 hours later; error bars,

S.D. of experiments performed on three occasions. 818


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Table 1. – Strains used in this study

Species Strain Description Antibiotic resistance

Origin/references

N. meningitidis

MC58 Serogroup B clinical isolate, ET-

5

- Isolated in the UK in 1989

MC58 eGFP

MC58 carrying pEG2

Ery (6)

MC58∆pilE Transposon inserted in pilE. Tfp

deficient

Kan (15)

MC58∆siaD Deletion mutagenesis of

sialyl transferase. capsule deficient

Ery (74)

E. coli DH5α Invitrogen

820


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Table 2. – Antibodies used in this study.

Name Target Origin Working Dilution

Source/Reference

αL3,7,9 LPS 3,7,9 (4047)

Mouse monoclonal

4047

1/200 NIBSC

αZO-1 ZO-1 Rabbit polyclonal

1/30 Zymed

αOcc Occludin Rabbit polyclonal

1/30 Zymed

αβ-tub E7

β -Tubulin Mouse polyclonal

1/100 Developmental Studies Hybridoma

Bank, Iowa αSM1 PilE Mouse

monoclonal 1/1000 (70)

αmenB 95/750

N. meningitidis serogroup B

capsule

Mouse monoclonal

- NIBSC

αM HRP Mouse immunoglobulins

Goat polyclonal 1/1000 Dako

αβ-tub E7

β-Tubulin Mouse polyclonal

1/100 Developmental Studies Hybridoma

Bank, Iowa αM Cy2 Mouse IgG Donkey

polyclonal 1/200 Jackson

αM RRX Mouse IgG Donkey polyclonal

1/200 Jackson

αR Cy5 Rabbit IgG Donkey polyclonal

1/200 Jackson

122-2 CD46 Mouse

monoclonal 1/50 Santa Cruz

Biotechnology inc.

1.BB.442 CD46 Mouse

monoclonal 1/200 Santa Cruz

Biotechnology inc. E4.3

CD46

Mouse monoclonal

2µg/ml

Pharmingen

J4.48

CD46

Mouse monoclonal

2µg/ml

Santa Cruz Biotechnology inc.

Isotype control

IgG

Mouse monoclonal

2µg/ml

Dako

Phalloidin Alexa 595

Actin 1/100 Invitrogen

Phalloidin Alexa 647

Actin

1/100 Invitrogen


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References 822

1. Attali, C., C. Durmort, T. Vernet, and A. M. Di Guilmi. 2008. The interaction 824

of Streptococcus pneumoniae with plasmin mediates transmigration across endothelial and epithelial monolayers by intercellular junction cleavage. Infect Immun 826

76:5350-6. 2. Beuzon, C. R., and D. W. Holden. 2001. Use of mixed infections with 828

Salmonella strains to study virulence genes and their interactions in vivo. Microbes Infect 3:1345-52. 830

3. Birkness, K. A., B. L. Swisher, E. H. White, E. G. Long, E. P. Ewing, Jr., and F. D. Quinn. 1995. A tissue culture bilayer model to study the passage of 832

Neisseria meningitidis. Infect Immun 63:402-9. 4. Capecchi, B., J. Adu-Bobie, F. Di Marcello, L. Ciucchi, V. Masignani, A. 834

Taddei, R. Rappuoli, M. Pizza, and B. Arico. 2005. Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human 836

epithelial cells. Mol Microbiol 55:687-98. 5. Cavet, M. E., M. West, and N. L. Simmons. 1997. Transepithelial transport of 838

the fluoroquinolone ciprofloxacin by human airway epithelial Calu-3 cells. Antimicrob Agents Chemother 41:2693-8. 840

6. Christodoulides, M., J. S. Everson, B. L. Liu, P. R. Lambden, P. J. Watt, E. J. Thomas, and J. E. Heckels. 2000. Interaction of primary human endometrial cells 842

with Neisseria gonorrhoeae expressing green fluorescent protein. Mol Microbiol 35:32-43. 844

7. Cooney, D., M. Kazantseva, and A. J. Hickey. 2004. Development of a size-dependent aerosol deposition model utilising human airway epithelial cells for 846

evaluating aerosol drug delivery. Altern Lab Anim 32:581-90. 8. Coureuil, M., G. Mikaty, F. Miller, H. Lecuyer, C. Bernard, S. Bourdoulous, 848

G. Dumenil, R. M. Mege, B. B. Weksler, I. A. Romero, P. O. Couraud, and X. Nassif. 2009. Meningococcal type IV pili recruit the polarity complex to cross the 850

brain endothelium. Science 325:83-7. 9. Criss, A. K., K. A. Kline, and H. S. Seifert. 2005. The frequency and rate of 852

pilin antigenic variation in Neisseria gonorrhoeae. Mol Microbiol 58:510-9. 10. Cywes, C., and M. R. Wessels. 2001. Group A Streptococcus tissue invasion 854

by CD44-mediated cell signalling. Nature 414:648-52. 11. De Brabander, M. J., R. M. Van de Veire, F. E. Aerts, M. Borgers, and P. A. 856

Janssen. 1976. The effects of methyl (5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl) carbamate, (R 17934; NSC 238159), a new synthetic antitumoral drug interfering with 858

microtubules, on mammalian cells cultured in vitro. Cancer Res 36:905-16. 12. Deghmane, A. E., D. Giorgini, M. Larribe, J. M. Alonso, and M. K. Taha. 860

2002. Down-regulation of pili and capsule of Neisseria meningitidis upon contact with epithelial cells is mediated by CrgA regulatory protein. Mol Microbiol 43:1555-64. 862

13. Edwards, J. L., E. J. Brown, S. Uk-Nham, J. G. Cannon, M. S. Blake, and M. A. Apicella. 2002. A co-operative interaction between Neisseria gonorrhoeae and 864

complement receptor 3 mediates infection of primary cervical epithelial cells. Cell Microbiol 4:571-84. 866

14. Ehrhardt, C., J. Fiegel, S. Fuchs, R. Abu-Dahab, U. F. Schaefer, J. Hanes, and C. M. Lehr. 2002. Drug absorption by the respiratory mucosa: cell culture 868

models and particulate drug carriers. J Aerosol Med 15:131-9.


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

39

15. Exley, R. M., L. Goodwin, E. Mowe, J. Shaw, H. Smith, R. C. Read, and C. 870

M. Tang. 2005. Neisseria meningitidis lactate permease is required for nasopharyngeal colonization. Infect Immun 73:5762-6. 872

16. Exley, R. M., R. Sim, L. Goodwin, M. Winterbotham, M. C. Schneider, R. C. Read, and C. M. Tang. 2009. Identification of meningococcal genes necessary for 874

colonization of human upper airway tissue. Infect Immun 77:45-51. 17. Fiegel, J., C. Ehrhardt, U. F. Schaefer, C. M. Lehr, and J. Hanes. 2003. 876

Large porous particle impingement on lung epithelial cell monolayers--toward improved particle characterization in the lung. Pharm Res 20:788-96. 878

18. Figueiredo, C. C., P. M. Deccache, L. M. Lopes-Bezerra, and V. Morandi. 2007. TGF-beta1 induces transendothelial migration of the pathogenic fungus 880

Sporothrix schenckii by a paracellular route involving extracellular matrix proteins. Microbiology 153:2910-21. 882

19. Finlay, B. B., B. Gumbiner, and S. Falkow. 1988. Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer. J Cell Biol 884

107:221-30. 20. Florea, B. I., M. L. Cassara, H. E. Junginger, and G. Borchard. 2003. Drug 886

transport and metabolism characteristics of the human airway epithelial cell line Calu-3. J Control Release 87:131-8. 888

21. Foster, K. A., M. L. Avery, M. Yazdanian, and K. L. Audus. 2000. Characterization of the Calu-3 cell line as a tool to screen pulmonary drug delivery. 890

Int J Pharm 208:1-11. 22. Gerlach, R. G., N. Claudio, M. Rohde, D. Jackel, C. Wagner, and M. 892

Hensel. 2008. Cooperation of Salmonella pathogenicity islands 1 and 4 is required to breach epithelial barriers. Cell Microbiol 10:2364-76. 894

23. Gill, D. B., M. Koomey, J. G. Cannon, and J. P. Atkinson. 2003. Down-regulation of CD46 by piliated Neisseria gonorrhoeae. J Exp Med 198:1313-22. 896

24. Grainger, C. I., L. L. Greenwell, D. J. Lockley, G. P. Martin, and B. Forbes. 2006. Culture of Calu-3 cells at the air interface provides a representative model of 898

the airway epithelial barrier. Pharm Res 23:1482-90. 25. Greenfield, S., P. R. Sheehe, and H. A. Feldman. 1971. Meningococcal 900

carriage in a population of "normal" families. J Infect Dis 123:67-73. 26. Hammerschmidt, S., R. Hilse, J. P. van Putten, R. Gerardy-Schahn, A. 902

Unkmeir, and M. Frosch. 1996. Modulation of cell surface sialic acid expression in Neisseria meningitidis via a transposable genetic element. Embo J 15:192-8. 904

27. Hammerschmidt, S., A. Muller, H. Sillmann, M. Muhlenhoff, R. Borrow, A. Fox, J. van Putten, W. D. Zollinger, R. Gerardy-Schahn, and M. Frosch. 1996. 906

Capsule phase variation in Neisseria meningitidis serogroup B by slipped-strand mispairing in the polysialyltransferase gene (siaD): correlation with bacterial invasion 908

and the outbreak of meningococcal disease. Mol Microbiol 20:1211-20. 28. Helaine, S., E. Carbonnelle, L. Prouvensier, J. L. Beretti, X. Nassif, and V. 910

Pelicic. 2005. PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells. Mol 912

Microbiol 55:65-77. 29. Hofman, P., L. D'Andrea, D. Carnes, S. P. Colgan, and J. L. Madara. 1996. 914

Intestinal epithelial cytoskeleton selectively constrains lumen-to-tissue migration of neutrophils. Am J Physiol 271:C312-20. 916

30. Horwitz, S. B. 1992. Mechanism of action of taxol. Trends Pharmacol Sci 13:134-6. 918


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

40

31. Hunziker, W., P. Male, and I. Mellman. 1990. Differential microtubule requirements for transcytosis in MDCK cells. Embo J 9:3515-25. 920

32. Ilver, D., H. Kallstrom, S. Normark, and A. B. Jonsson. 1998. Transcellular passage of Neisseria gonorrhoeae involves pilus phase variation. Infect Immun 922

66:469-73. 33. Kallstrom, H., M. K. Liszewski, J. P. Atkinson, and A. B. Jonsson. 1997. 924

Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria. Mol Microbiol 25:639-47. 926

34. Katz, J., V. Sambandam, J. H. Wu, S. M. Michalek, and D. F. Balkovetz. 2000. Characterization of Porphyromonas gingivalis-induced degradation of epithelial 928

cell junctional complexes. Infect Immun 68:1441-9. 35. Katz, J., Q. B. Yang, P. Zhang, J. Potempa, J. Travis, S. M. Michalek, and 930

D. F. Balkovetz. 2002. Hydrolysis of epithelial junctional proteins by Porphyromonas gingivalis gingipains. Infect Immun 70:2512-8. 932

36. Kinugasa, N., T. Higashi, K. Nouso, H. Nakatsukasa, Y. Kobayashi, M. Ishizaki, N. Toshikuni, K. Yoshida, S. Uematsu, and T. Tsuji. 1999. Expression of 934

membrane cofactor protein (MCP, CD46) in human liver diseases. Br J Cancer 80:1820-5. 936

37. Kirchner, M., D. Heuer, and T. F. Meyer. 2005. CD46-independent binding of neisserial type IV pili and the major pilus adhesin, PilC, to human epithelial cells. 938

Infect Immun 73:3072-82. 38. Li, Y., W. Wang, W. Parker, and J. P. Clancy. 2006. Adenosine regulation of 940

cystic fibrosis transmembrane conductance regulator through prostenoids in airway epithelia. Am J Respir Cell Mol Biol 34:600-8. 942

39. Liszewski, M. K., T. W. Post, and J. P. Atkinson. 1991. Membrane cofactor protein (MCP or CD46): newest member of the regulators of complement activation 944

gene cluster. Annu Rev Immunol 9:431-55. 40. Lytton, S. D., W. Fischer, W. Nagel, R. Haas, and F. X. Beck. 2005. 946

Production of ammonium by Helicobacter pylori mediates occludin processing and disruption of tight junctions in Caco-2 cells. Microbiology 151:3267-76. 948

41. Maisner, A., M. K. Liszewski, J. P. Atkinson, R. Schwartz-Albiez, and G. Herrler. 1996. Two different cytoplasmic tails direct isoforms of the membrane 950

cofactor protein (CD46) to the basolateral surface of Madin-Darby canine kidney cells. J Biol Chem 271:18853-8. 952

42. Merz, A. J., D. B. Rifenbery, C. G. Arvidson, and M. So. 1996. Traversal of a polarized epithelium by pathogenic Neisseriae: facilitation by type IV pili and 954

maintenance of epithelial barrier function. Mol Med 2:745-54. 43. Merz, A. J., M. So, and M. P. Sheetz. 2000. Pilus retraction powers bacterial 956

twitching motility. Nature 407:98-102. 44. Mostov, K. E., and D. L. Deitcher. 1986. Polymeric immunoglobulin receptor 958

expressed in MDCK cells transcytoses IgA. Cell 46:613-21. 45. Mounier, J., T. Vasselon, R. Hellio, M. Lesourd, and P. J. Sansonetti. 960

1992. Shigella flexneri enters human colonic Caco-2 epithelial cells through the basolateral pole. Infect Immun 60:237-48. 962

46. Nassif, X., J. L. Beretti, J. Lowy, P. Stenberg, P. O'Gaora, J. Pfeifer, S. Normark, and M. So. 1994. Roles of pilin and PilC in adhesion of Neisseria 964

meningitidis to human epithelial and endothelial cells. Proc Natl Acad Sci U S A 91:3769-73. 966


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

41

47. Nassif, X., C. Pujol, P. Morand, and E. Eugene. 1999. Interactions of pathogenic Neisseria with host cells. Is it possible to assemble the puzzle? Mol 968

Microbiol 32:1124-32. 48. Nikulin, J., U. Panzner, M. Frosch, and A. Schubert-Unkmeir. 2006. 970

Intracellular survival and replication of Neisseria meningitidis in human brain microvascular endothelial cells. Int J Med Microbiol 27:27. 972

49. Pezron, I., R. Mitra, D. Pal, and A. K. Mitra. 2002. Insulin aggregation and asymmetric transport across human bronchial epithelial cell monolayers (Calu-3). J 974

Pharm Sci 91:1135-46. 50. Pujol, C., E. Eugene, L. de Saint Martin, and X. Nassif. 1997. Interaction of 976

Neisseria meningitidis with a polarized monolayer of epithelial cells. Infect Immun 65:4836-42. 978

51. Pujol, C., E. Eugene, M. Marceau, and X. Nassif. 1999. The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following 980

pilus-mediated adhesion. Proc Natl Acad Sci U S A 96:4017-22. 52. Rahman, M., H. Kallstrom, S. Normark, and A. B. Jonsson. 1997. PilC of 982

pathogenic Neisseria is associated with the bacterial cell surface. Mol Microbiol 25:11-25. 984

53. Ramsden, A. E., D. W. Holden, and L. J. Mota. 2007. Membrane dynamics and spatial distribution of Salmonella-containing vacuoles. Trends Microbiol 15:516-986

24. 54. Reznik, G. K. 1990. Comparative anatomy, physiology, and function of the 988

upper respiratory tract. Environ Health Perspect 85:171-6. 55. Sajjan, U., Q. Wang, Y. Zhao, D. C. Gruenert, and M. B. Hershenson. 2008. 990

Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells. Am J Respir Crit Care Med 11:11. 992

56. Shen, B. Q., W. E. Finkbeiner, J. J. Wine, R. J. Mrsny, and J. H. Widdicombe. 1994. Calu-3: a human airway epithelial cell line that shows cAMP-994

dependent Cl- secretion. Am J Physiol 266:L493-501. 57. Sim, R. J., M. M. Harrison, E. R. Moxon, and C. M. Tang. 2000. 996

Underestimation of meningococci in tonsillar tissue by nasopharyngeal swabbing. Lancet 356:1653-4. 998

58. Sinn, P. L., G. Williams, S. Vongpunsawad, R. Cattaneo, and P. B. McCray, Jr. 2002. Measles virus preferentially transduces the basolateral surface of 1000

well-differentiated human airway epithelia. J Virol 76:2403-9. 59. Soriani, M., I. Santi, A. Taddei, R. Rappuoli, G. Grandi, and J. L. Telford. 1002

2006. Group B Streptococcus crosses human epithelial cells by a paracellular route. J Infect Dis 193:241-50. 1004

60. Spinosa, M. R., C. Progida, A. Tala, L. Cogli, P. Alifano, and C. Bucci. 2007. The Neisseria meningitidis capsule is important for intracellular survival in 1006

human cells. Infect Immun 75:3594-603. 61. Stephens, D. S. 2007. Conquering the meningococcus. FEMS Microbiol Rev 1008

31:3-14. 62. Stephens, D. S. 1999. Uncloaking the meningococcus: dynamics of carriage 1010

and disease. Lancet 353:941-2. 63. Stephens, D. S., L. H. Hoffman, and Z. A. McGee. 1983. Interaction of 1012

Neisseria meningitidis with human nasopharyngeal mucosa: attachment and entry into columnar epithelial cells. J Infect Dis 148:369-76. 1014


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

42

64. Stephens, D. S., and Z. A. McGee. 1981. Attachment of Neisseria meningitidis to human mucosal surfaces: influence of pili and type of receptor cell. J 1016

Infect Dis 143:525-32. 65. Tala, A., C. Progida, M. De Stefano, L. Cogli, M. R. Spinosa, C. Bucci, and 1018

P. Alifano. 2008. The HrpB-HrpA two-partner secretion system is essential for intracellular survival of Neisseria meningitidis. Cell Microbiol 25:25. 1020

66. Teuchert, M., A. Maisner, and G. Herrler. 1999. Importance of the carboxyl-terminal FTSL motif of membrane cofactor protein for basolateral sorting and 1022

endocytosis. Positive and negative modulation by signals inside and outside the cytoplasmic tail. J Biol Chem 274:19979-84. 1024

67. Tobiason, D. M., and H. S. Seifert. 2001. Inverse relationship between pilus-mediated gonococcal adherence and surface expression of the pilus receptor, CD46. 1026

Microbiology 147:2333-40. 68. Vermeer, P. D., J. McHugh, T. Rokhlina, D. W. Vermeer, J. Zabner, and M. 1028

J. Welsh. 2007. Vaccinia virus entry, exit, and interaction with differentiated human airway epithelia. J Virol 81:9891-9. 1030

69. Virji, M., C. Alexandrescu, D. J. Ferguson, J. R. Saunders, and E. R. Moxon. 1992. Variations in the expression of pili: the effect on adherence of 1032

Neisseria meningitidis to human epithelial and endothelial cells. Mol Microbiol 6:1271-9. 1034

70. Virji, M., J. E. Heckels, W. J. Potts, C. A. Hart, and J. R. Saunders. 1989. Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which 1036

react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of 1038

their encoding sequences in the genomes of Neisseria spp. J Gen Microbiol 135:3239-51. 1040

71. Virji, M., H. Kayhty, D. J. Ferguson, C. Alexandrescu, J. E. Heckels, and E. R. Moxon. 1991. The role of pili in the interactions of pathogenic Neisseria with 1042

cultured human endothelial cells. Mol Microbiol 5:1831-41. 72. Virji, M., K. Makepeace, D. J. Ferguson, M. Achtman, and E. R. Moxon. 1044

1993. Meningococcal Opa and Opc proteins: their role in colonization and invasion of human epithelial and endothelial cells. Mol Microbiol 10:499-510. 1046

73. Virji, M., K. Makepeace, I. Peak, G. Payne, J. R. Saunders, D. J. Ferguson, and E. R. Moxon. 1995. Functional implications of the expression of PilC proteins in 1048

meningococci. Mol Microbiol 16:1087-97. 74. Virji, M., K. Makepeace, I. R. Peak, D. J. Ferguson, M. P. Jennings, and E. 1050

R. Moxon. 1995. Opc- and pilus-dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides. 1052

Mol Microbiol 18:741-54. 75. Virji, M., S. M. Watt, S. Barker, K. Makepeace, and R. Doyonnas. 1996. The 1054

N-domain of the human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae. Mol Microbiol 22:929-39. 1056

76. Wang, J. A., T. F. Meyer, and T. Rudel. 2007. Cytoskeleton and motor proteins are required for the transcytosis of Neisseria gonorrhoeae through polarized 1058

epithelial cells. Int J Med Microbiol 31:31. 77. Wolfgang, M., P. Lauer, H. S. Park, L. Brossay, J. Hebert, and M. Koomey. 1060

1998. PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae. Mol Microbiol 1062

29:321-30.


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Transcellular passage of Neisseria meningitidis across a polarised