1 mapping of the neisseria meningitidis nada cell-binding site

1

Mapping of the Neisseria meningitidis NadA cell-binding site: relevance of predicted α-helices 1

in the NH2 terminal and in dimeric coiled-coil regions 2

Running title: NadA binding site 3

4

Regina Tavano1,2 #

, Barbara Capecchi3#

, Paolo Montanari3, Susanna Franzoso

2, Oriano Marin

2,4, 5

Maryta Ztukowska 1,2,†

, Paola Cecchini1,2

, Daniela Segat2, Maria Scarselli

3, Beatrice Aricò

3* and 6

Emanuele Papini1, 2*

7

8

1Dipartimento di Scienze Biomediche Sperimentali, Università di Padova 9

2Centro di Ricerca Interdipartimentale per le Biotecnologie Innovative, Università di Padova, via U. 10

Bassi 58/B, I-35131, Padova, Italy 11

3Novartis Vaccine and Diagnostics srl, Siena 12

4Department of Biochemistry, University of Padova, via U. Bassi 58/B, I-35131, Padova, Italy 13

†Present address: Rzeszów University of Technology, Faculty of Chemistry, Department of 14

Biochemistry and Biotechnology, 6 Powstañców Warszawy Ave. 35-959 Rzeszów, Poland. 15

# R.T.: and B.C. share first authorship 16

17

*Correspondent footnote: 18

19

Beatrice Aricò [email protected], Novartis Vaccine and Diagnostics srl, Via Fiorentina 20

1, 53100 Siena, Tel : 00390577243088, Fax: 00390577243564; 21

Emanuele Papini [email protected], Dipartimento di Scienze Biomediche Sperimentali, 22

Università di Padova, Viale G. Colombo 3, 35121 Padova, Tel: 00390498276301, Fax: 23

00390498276159. 24

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00430-10 JB Accepts, published online ahead of print on 22 October 2010

on January 30, 2018 by guesthttp://jb.asm

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Abstract 25

NadA is a trimeric autotransporter protein of N. meningitidis belonging to the group of Oligomeric 26

Coiled-coil Adhesins. It is implicated in the colonization of the human upper respiratory tract by 27

hypervirulent serogroup B N. meningitidis strains and is part of a multi-antigen anti serogroup B 28

vaccine. Structure prediction indicates that NadA is made by a COOH terminal membrane anchor 29

(also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil 30

rich stalk and a NH2 terminal region involved in cell-interaction. Electron microscopy analysis and 31

structure prediction suggest that the apical region of NadA forms a compact and globular domain. 32

Deletion studies proved that the NH2 terminal sequence (24-87) is necessary for cell adhesion. In 33

this study, to better define the NadA cell-binding site we exploited i) a panel of NadA mutants 34

lacking sequences along the coiled-coil stalk and ii) several oligoclonal rabbit antibodies, and their 35

relative Fab fragments, directed to linear epitopes distributed along the NadA ecto-domain. We 36

identified two critical regions for the NadA-cell receptor interaction in Chang cells: the NH2 37

globular head domain together with the NH2 dimeric intrachain coiled-coil alpha helices, stemming 38

from the stalk. This raises the importance of different modules within NadA predicted structure. 39

The identification of linear epitopes involved in receptor binding and able to induce interfering 40

antibodies reinforce the importance of NadA as vaccine antigen. 41


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Introduction 42

43

Neisseria meningitidis serotype B strains are mostly responsible for septicaemia and meningitis in 44

developed countries (1-3). In silico analysis of the genome of a virulent N. meningitidis B strain 45

(MC58), allowed the identification of the 45 KDa Neisseria adhesin A (NadA) (4). NadA was found 46

to be expressed in ~50% of N. meningitidis strains isolated from patients, while only in ~5% of 47

strains from healthy individuals and therefore may be a risk factor for the development of 48

meningococcal disease (5). 49

NadA is also a good immunogen, able to induce a bactericidal immune response, and is a 50

component of a multiple anti menB vaccine at present under development (6, 7) 51

In vitro observations support that NadA may also be important in mucosal colonization by N. 52

meningitidis B: i) its expression on E. coli enhances bacteria association to Chang epithelial cells 53

(human conjunctiva cell line widely used in meningococcal pathogenesis studies) (8); ii) a NadA 54

knock-out mutant of N. meningitidis shows a partial, yet significant, decrease in cell adhesion and 55

invasion, as compared to wild type strain suggesting that NadA cooperates with other factors in 56

mediating bacterial cell interaction (8); iii) a soluble recombinant form of NadA (NadA∆351-405), 57

lacking the membrane anchor region, binds to specific receptor sites with an apparent affinity of 3 58

µM on Chang cells (8, 9). 59

Other studies suggest that NadA, beside its role at the level of the mucosa epithelium, also exerts an 60

immune-modulatory action on myeloid cells. Indeed, NadA-specific receptors were observed also 61

on monocytes, macrophage and monocyte-derived dendritic cells (9, 10). NadA may stimulate anti-62

meningococcal defenses by augmenting the immune response of dendritic cells (self-adjuvant 63

effect) and by increasing antigen presentation by macrophages engaged in antimicrobial activity (9-64

11). Immune-stimulatory effects of NadA were strongly synergised by meningococci-specific outer 65

membrane components (11). 66

For all these reasons, NadA appears to be an important determinant in the host-pathogen interaction 67

accompanying meningococcal infection. Consequently, the comprehension of the structural 68

determinants of NadA-cell interaction may help to find ways to neutralize early meningitidis and 69

fatal-meningococcal sepsis. 70

Structure prediction and homology comparison show that NadA is an Oligomeric Coiled-71

coil Adhesin (OCA), like YadA of Yersinia enterocolitica, UspA2 of Moraxella catarrhalis (12) 72

and BadA of Bartonella henselae (13) belonging to the group of homo-trimeric auto transporter 73

adhesins (TAAs) (12). OCAs are made by two main structural-functional parts: i) a conserved –74

COOH terminal membrane anchor, having a β-barrel structure, necessary for the export of the 75


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remaining part of the adhesin (passenger domain) on the cell surface ii) the extracellular passenger 76

domain generally formed by an intermediate stalk with a high propensity to form coiled-coil alpha 77

helices and by a –NH2 terminal region, predicted to have a globular structure and necessary for 78

binding to host cells factors (14-16). Important exceptions are represented by HadA of Haemophilus 79

influenzae in which the globular head is missing (17) and by UspA1 where, in addition to a binding 80

site located in the head region, there is a second binding site within the stalk, specific for another 81

target (18). 82

Concerning NadA, previous studies showed that the deletion of the region 24-87, corresponding to 83

the putative receptor binding domain, totally abolishes adhesion of NadA-expressing E. coli models 84

to Chang cells (8). Attempts to further map the region(s) necessary to cell binding were 85

unsuccessful because deletion mutants missing the predicted sub-domains 24-42, 43-70 and 71-87 86

were all defective in mediating bacterial cell binding. These results were interpreted assuming either 87

that the whole 24-87 region is involved in receptor binding, or, alternatively, that each separate 88

deletion alters the structure of the remaining parts of this compact fold. In addition, structure 89

prediction studies suggest that intra-chain coiled-coil alpha helices apparently located in the stalk 90

might be involved in the formation of the receptor binding site, cooperating with the NH2 globular 91

terminal region (18). Indeed, the possible involvement of dimeric intra-chain coiled coil regions in 92

the binding of OCA adhesins to their cell receptors was suggested by studies on HadA of 93

Haemophilus influenzae (17). HadA turned out to be an atypical OCA lacking the globular head, 94

and in which the adhesion function is performed by the NH2 terminal dimeric coiled-coil structures. 95

In the absence of a crystallographic 3-D map of NadA, in this study we built up on previous data 96

obtained on E. coli model using Chang epithelial cells, expressing high levels of NadA specific 97

binding sites (8), to provide a more exhaustive mapping of the cell-receptor binding site of NadA. 98

To do so, we developed deletion mutants devoid of various sequences distributed in the coiled-coil 99

region proximal to the NH2 terminal domain (24-87) and progressively closer to the outer 100

membrane anchor. Such mutants were analyzed in terms of their ability to form surface oligomers 101

on E. coli model and for their efficiency in promoting bacterial association to Chang conjunctiva 102

cells. This information was compared with the ability of sera, affinity purified antibodies (and of 103

their relative Fab fragments) to linear-epitopes within the NH2 terminal domain and the coiled coil 104

stalk to interfere with NadA expressing N. meningitidis and E. coli cell adhesion. 105


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Materials and methods 106

107

Plasmid construction 108

NadA full-length and NadA∆30-88 coding genes were obtained as previously described (8). The 109

mutated genes coding for NadA∆88-149, NadA∆180-219, NadA∆219-289 and NadA∆269-315, were generated 110

using the mutagenesis kit “Gene Taylor” accordingly to manufacturing’s instruction (Invitrogen). 111

Briefly, the forward primers containing the mutation site (deletion) and the reverse primers were 112

designed on NadA sequence in order to delete the region of interest. The digested DNA fragments 113

were cloned into pET21b vector (Novagen).The ligation products were transformed into E. coli 114

DH5α (Invitrogen) and E. coli BL21(DE3) was used as expression host (Novagen). DNA cloning 115

and E. coli transformation were performed according to the standard protocols. E. coli strains were 116

cultured at 37 °C in Luria Bertani broth supplemented with 100 µg/ml ampicillin. 117

118

FACS analysis 119

For surface detection of NadA in E. coli using FACS analysis, approximately 2x106 bacteria were 120

incubated for 1 h with anti-NadA∆351-405 (1:1000) and subsequently for 30 min with R-phycoerythrin 121

(PE)-conjugated goat F(ab)2 antibody to rabbit IgG (diluted 1:100, Jackson ImmunoResearch 122

Laboratories). All antibodies were diluted in PBS with 1% FBS. Samples were analysed with a 123

FACS-Scan flow cytometer (Beckton-Dickinson). 124

125

Animals 126

Male adult New Zealand white rabbits were obtained from Harlan Italy srl, Italy. 127

128

NadA Peptides synthesis 129

A hydrophobicity map of NadA protein was obtained based on the full-length amino acid sequence 130

of the molecule, using Lasergene (DNASTAR ) software . The peptides were synthesized by a solid 131

phase method on a Wang resin functionalized with the acid labile 4-hydroxymethylphenoxyacetic 132

acid linker (Novabiochem, Bad Soden, Germany), using an automatized peptide synthesizer (Model 133

433, Applied Biosystems, Foster City, CA, U.S.A.).The fluoren-9-ylmethoxycarbonyl (Fmoc) 134

strategy (19) was used throughout the peptide chain assembly, utilizing 2-(1H-benzotriazol-1-yl)-135

1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) as 136

coupling reagents. Cleavage of the peptides was performed by reacting the peptidyl-resins with a 137

mixture containing TFA/H20/thioanisole/ethanedithiol/phenol (10 ml/0.5 ml/0.5 ml/0.25 ml/750 138

mg) for 2.5 h. Crude peptides were purified by a preparative reverse phase HPLC. Molecular 139


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masses of the peptides were confirmed by mass spectroscopy with direct infusion on a Micromass 140

ZMD-4000 Mass Spectrometer (Waters-Micromass). The purity of the peptides was in the range 141

95-98% as evaluated by analytical reverse phase HPLC. 142

Purified peptides were coupled to KLH (Keyhole Limpet Hemocyanin), and used for animal 143

immunization. An aliquot of each peptide was retained for use in ELISA without coupling to KLH. 144

145

Peptide-carrier conjugation 146

2 mg of each peptide were conjugated with 2 mg of mcKLH (Pierce), in conjugation buffer, 147

following manufacturer instructions and the solutions were maintained under gently agitation for 2 148

hours at room temperature. Conjugates were subsequently purified by gel filtration, by using 149

Sephadex G-25 resin (Sigma). Fractions containing proteins were mixed, divided in aliquots and 150

frozen in liquid nitrogen. 151

152

Immunization and production of polyclonal Abs 153

New Zealand White rabbits were immunized by subcutaneous injection with an aliquot (750 µl) of 154

carrier-peptide conjugate mixed 1:1 v/v with Complete Freund's Adjuvant (Sigma). Three 155

subsequent injections were administered at 14-day intervals, with Incomplete Freund's Adjuvant 156

(Sigma). Anti-sera were taken on day 68 and antibodies were purified by affinity chromatography 157

using columns containing Sulfolink coupling gel (Pierce) linked to the different peptides through 158

sulphydrylic group. Briefly, columns were prepared with 2.5 ml of 50% Sulfolink coupling gel and 159

2 mg of each peptide, dissolved in coupling buffer (50 mM Tris-HCl, 5 mM EDTA, pH 8.5), were 160

added to the column and incubated at room temperature for 30 minutes. Columns were then washed 161

with coupling buffer and the non specific binding sites were blocked with a solution of 50 mM 162

cysteine (Sigma), for 30 minutes at room temperature. Subsequently, columns were washed again 163

extensively and antisera diluted 1:1 v/v with PBS were added to the columns; after extensive 164

washing, antibodies were eluted with 0.2 M glycine, pH 2.8. 165

166

Fab generation and purification 167

For each sample, 300 µl of Immobilized Papain-Agarose (Sigma) were activated with activation 168

buffer (50 mM Na-P pH 7.0, 20 mM cysteine, 10 mM EDTA) for 20 minutes at 37 °C.; the resin 169

was then washed and incubated with the antibody solution for 4 hours at 37 °C. To block the 170

reaction 75 mM iodoacetamide was added. To purify Fab fragments, papain-treated antibodies were 171

incubated with 100 µl of ProteinA-Agarose (Sigma) for 2 hours at room temperature, under gently 172


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shacking; the resin was washed three times and the supernatants, containing Fab fragments, were 173

recovered. 174

175

ELISA assay 176

The day before the experiment, polystyrene plates (Sarsted) were coated with 100 µl per well of 177

various peptides (20 ng/µl), or with recombinant NadA∆351-405 (0.5 µg/ml) or with E.coli-NadA 178

strain (described in (8)) (108/ml bacteria). The day of the experiment wells were washed, blocked 179

with 1% BSA-PBS and incubated for 1 hour with different anti-sera or purified anti-peptides 180

antibodies or Fab antibodies, depending on the experiment. After binding, wells were exhaustively 181

washed and treated with alkaline phosphatase conjugated anti-rabbit IgG H&L chain antibodies 182

(Chemicon). ABTS (Chemicon) was used as substrate and absorbance measured in an automatic 183

ELISA plate reader (Amersham Biosciences). 184

185

SDS-PAGE and Western blot 186

Whole and Fab antibodies were resolved by SDS-PAGE and then subjected to Coomassie staining; 187

alternatively proteins were blotted onto a nitrocellulose membrane and probed with an alkaline 188

phosphatase goat anti IgG, heavy or light chain, antibody (Chemicon). Blots were developed with 189

AP buffer (100 mM NaCl, 5 mM MgCl2, 100 mM Tris/Cl (pH 9.2)) supplemented with 1% v/v 190

BCIP and 1% v/v NBT (Sigma). 191

To check the trimer formation of the various NadA mutants expressed by E. coli, bacteria were 192

grown at 37 °C for 14 h and then recovered by centrifugation, resuspended in SDS-sample buffer 193

1X and boiled for 10 min. Equal amounts of proteins were separated using NuPAGE Gel System, 194

according to the manufacturer’s instructions (Invitrogen). Proteins were blotted onto nitrocellulose 195

membranes and Western blot was performed using an anti NadA∆351-405 serum (1:2000) and a 196

secondary peroxidase-conjugate anti-body (DAKO). 197

198

Adhesion assay 199

Chang cells were seeded on 24-well tissue culture plates (1 × 105 cells per well) and after 24 h 200

incubation in an antibiotic-free medium, approximately 3 x 107

[multiplicity of infection 1:100 (moi 201

100)] bacteria were added per well in DMEM supplemented with 1% FBS and incubated for 1 h at 202

37°C in 5% CO2. After removal of non-adherent bacteria by washing with, cells were lysed with 203

1% saponin (Sigma), to block the reaction, 800 µl of DMEM + 1% FBSi, serial dilutions of the 204

suspension were plated onto LB agar to calculate the cfu. 205


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206

Inhibition of adherence of E. coli-NadA with anti-NadA peptides abs/Fab fragments 207

Liquid cultures of E. coli-NadA were washed once in PBS and resuspended in DMEM + 1% FBSi 208

in the absence or in the presence of different doses of 45 nM abs/Fab fragments (A) or of indicated 209

concentrations of affinity purified ab or Fab fragments (B), for 1 h at 4 °C. Samples were used to 210

infect Chang cells monolayers (moi 100) for 1 h at 37 °C. From this point on, the adhesion assay 211

was performed as described above. E. coli-pET was used as negative control. 212

213

Inhibition of adherence of N. meningitidis with NadA linear peptides antisera 214

Cultures of N. meningitidis M58 strain grown on GC agar were resuspended in DMEM + 1% FBS 215

in the absence or presence of different concentrations of rabbit antisera obtained both immunizing 216

animals with the indicated NadA peptides and with NadA∆351-405. Preimmune sera were tested as 217

negative control. After 1 h incubation at 4 °C, bacteria were added to Chang cells monolayers (moi 218

100) for 1 h at 37 °C. As described above, after extensive washings, cells lysis and agar plating, cfu 219

were determined. 220


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Results 221

222

The proximal region 88-150 of NadA stalk is important for NadA-mediated adhesion 223

224

Based on previous studies, it was proposed that residues from aa 24 to 87, corresponding to the 225

predicted NH2 terminal globular head of NadA, are involved in the adhesion to epithelial cells of 226

NadA expressing bacteria (8). To further investigate the role of NadA head and stalk in the 227

adhesion process we designed a panel of deletion mutants based on the secondary structure 228

prediction and we expressed them in E. coli (Fig 1A and 1B). According to a prediction algorithm 229

the stalk region has a high propensity to form alpha helices dimeric coiled-coil tertiary structures 230

(17). FACS and Western blot analysis on whole bacteria showed that all mutants form superficially 231

exposed oligomers, suggesting that the deletion of the intrachain dimeric coiled-coil region do not 232

alter the NadA oligomeric organization, and exposed NadA on E. coli surface (Fig 1C and 1D). 233

Adhesion experiments were performed using each single mutant on Chang epithelial cells. The 234

results showed in Fig 1 E indicate that, in addition to the deletion of the head domain (from 30 to 87 235

aa), the lack of the first dimeric coiled-coil region (from 88 to 150 aa) totally abolishes NadA 236

adhesion property, whereas all the other regions appeared to be irrelevant for bacterial-cell binding. 237

These observations suggest that the dimeric coiled coil domain (88-150) of NadA is necessary for 238

bacteria-cell adhesion together with the previously identified globular NH2 domain (24-87). 239

240

241

Generation of antibodies against NadA peptides recognizing soluble and membrane-associated 242

NadA. 243

244

The deletion of the 88-150 coiled-coil region could impact the NadA adhesion properties either by 245

eliminating a portion directly binding to the NadA receptor, or by indirectly affecting the 246

conformation of the real receptor-binding domain. Therefore, we generated antisera to six linear 247

peptides covering these two critical domains (see Fig. 2A) and tested their ability to block NadA-248

mediated cell adhesion. Antisera generated against determinants in the middle of the stalk and 249

closer to the membrane surface (208-215 aa and 275-289 aa, respectively), irrelevant for cell 250

binding based on deletion studies with E. coli recombinant strains, were used as negative controls. 251

ELISA assays and FACS analysis showed that anti-peptides sera specifically recognized the 252

NadA∆351-405 and the ecto-domain of full-length NadA expressed on E. coli (Fig. 2B and 2C), 253

proving that anti-linear epitope antibodies cross-react with the native adhesin. NadA 52-70 was 254


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apparently the most immunogenic peptide, in fact the titre of this latter antiserum was comparable 255

to the one induced by native NadA, used as positive control. However, it is also possible that the 256

52-70 region is the more exposed and accessible region of the protein. All preimmune sera gave a 257

negligible signal (not shown). 258

To quantitatively compare the interaction of anti-NadA peptides antibodies, they were affinity 259

purified. In addition, we also produced Fab fragments because these reagents retain the same 260

specificity but have the advantage of being less sterically hindering ( ~ 50 KDa) and monovalent 261

(Fig. 3A). Affinity purified ab/Fab fragments effectively recognized the peptides used for their 262

generation and did not cross-react with any of the other peptides of our panel (not shown). 263

ELISA assay performed with increasing concentrations of purified anti-peptides antibodies and Fab 264

fragments allowed characterizing their efficacy in the interaction with purified soluble NadA∆351-405 265

and with the protein expressed on the surface of E. coli cells. Data (Fig. 3B) demonstrated that 266

antibodies and Fab fragments bind with a similar extent to recombinant soluble NadA∆351-405. On 267

the contrary, when abs and Fab fragments where challenged with the membrane-anchored NadA, 268

their binding capabilities were clearly differentiated. First of all, anti NadA 25-39 (and anti NadA 269

24-33, not shown) antibodies and Fab fragments reacted less efficiently to the adhesin expressed on 270

the bacterial surface, compared with the soluble protein. An even stronger decrease in immune 271

reactivity was evident with anti-NadA 41-53 ab/Fab, suggesting that the linear epitope 41-53 is 272

poorly accessible when the adhesin is in the membrane contest. The following 52-70 sequence 273

remains one of the most accessible site, confirming the immunogenicity data. On the other hand, 274

antibodies reactivity to epitopes from position 52 to 289 decreased progressively moving toward the 275

membrane surface, very likely for steric reasons. In agreement with this interpretation, the 276

corresponding Fab fragments, with a mass of 1/3 with respect of whole antibodies, showed a good 277

immune reactivity also with linear epitopes in position closer to the bacterial membrane. The only 278

exception was epitope 275-289, scarcely available by both specific ab and Fab. 279

280

Inhibition of NadA expressing E. coli adhesion to Chang cells by antibodies against NadA linear 281

epitopes. 282

283

All the Abs and Fab fragments directed against NadA peptides were used to evaluate the 284

contribution of defined linear epitopes to bacterial cell-adhesion mediated by NadA. As shown in 285

Fig. 4A, adhesion of NadA-expressing E. coli to Chang conjunctiva cells was totally abolished by a 286

polyclonal antibody and its relative Fab to the whole extracellular domain of the protein (NadA∆351-287

405). Antibodies and Fab fragments to NadA-peptides 25-39 and 94-110 strongly counteracted 288


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bacterial-cell adhesion (95% and 86% inhibition, respectively). Antibodies and Fab fragments 289

directed to NadA 109-121 and to the remaining part of the globular NH2 terminal domain (NadA 290

41-53, 52-70 and 74-87) were partially inhibitory (~ 20-30% decrease), while those directed to the 291

stalk region (NadA 208-215, 275-289) were scarcely neutralizing. 292

When the efficacy of antibodies and Fab fragments able to neutralize NadA-mediated bacterial 293

adhesion was compared with their reactivity with the same antigen in ELISA it turned out that 294

adhesion neutralization was achieved at sub-saturating antigen binding, that is 5 nM for anti NadA 295

24-39 and 25 nM for anti NadA 94-110 (Fig. 4B). These results suggest that the regions 24-39 and 296

94-110 in the NadA head and stalk, respectively, are essential for the binding activity. Abs/Fab 297

fragments to the interposed region 52-87 were confirmed to contrast E. coli-NadA adhesion with a 298

reduced efficacy. 299

Isolated peptides used to immunize animals were also tested for their ability to impair E. coli-NadA 300

to epithelial cells, but were found to be ineffective (not shown), hinting the possible involvement of 301

the conformation of NadA modules to exploit their functions. 302

303

Ability of sera raised against linear NadA epitopes to inhibit N. meningitidis B adhesion to Chang 304

cells 305

306

We tested the inhibitory effect of different dilutions of specific sera against NadA linear peptides on 307

adhesion of NadA expressing N. meningitidis B strain MC58 to Chang cells. As previously shown, 308

the depletion of nadA gene in MC58 leads to a partial reduction in the attachment to Chang cells 309

(8). According to this, here we show that a polyclonal anti NadA∆351-405 antiserum partially 310

decreased the adhesion of MC58 to Chang cells giving a maximal inhibition around 40% when 311

compared to the control (Fig. 5), suggesting that NadA contribution to cell adhesion was abolished 312

by specific antibodies. We observed that the whole panel of tested sera exerted a titration-dependent 313

inhibitory effect, although anti 25-39, 94-110 and 109-121 sera were the most effective, in 314

agreement with data obtained using the E. coli model. It is noteworthy that the 109-121 serum, 315

partially inhibitory on E. coli-NadA adhesion, is very efficacious in hampering meningococcal cell 316

adhesion. As expected, the sera-specific inhibitory actions were partial and compatible with the 317

neutralization of NadA contribution to the meningococcal cell adhesion. .Taken together, these 318

results highlight the key role of the NH2 region of NadA, comprising the head domain (24-87) and 319

the adjacent dimeric coiled-coil region (88-132) (Fig. 1A-B), in mediating cell interaction. 320

321


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Discussion 322

323

The adhesin NadA is an important virulence factor of serogroup B N. meningitidis strains identified 324

by a genomic approach. The discovery of NadA sequences responsible for direct interaction with its 325

cellular receptor may help to elucidate the function of this important N. meningitidis virulence 326

factor. Moreover, since this protein has been proposed to participate to epithelial colonization and 327

possibly to cell invasion by N. meningitidis B strains, such information may allow obtaining 328

antibodies able to effectively neutralize any biological function triggered by the formation of the 329

NadA-NadA receptor complex. 330

Previous data demonstrated that the globular NH2 terminal domain of NadA (24-88) is necessary for 331

cell adhesion, in agreement with the general assumption that the apical portion of the adhesin is 332

crucial (8). However, recent evidence obtained with HadA of Haemophilus influenzae, a OCA 333

adhesin lacking such terminal globular domain, and structure prediction suggest that other part of 334

the adhesin NadA are close to the globular domain and may participate to cell binding (17). On the 335

other hand, a more detailed mapping of the NadA sequences necessary for receptor association 336

within the 24-88 domain was without success (8). 337

In this study, to gain detailed information on the structural determinants of NadA cell binding, we 338

exploited i) E. coli expressing deletion of NadA and ii) antibodies directed against specific peptides. 339

These two ways to test the functional involvement of a limited protein region are complementary 340

being based on different principles. Indeed, the elimination of a sequence may give misleading 341

results if the structure of a protein is consequently affected or, alternatively, if the distance between 342

otherwise stable domains is modified. On the other hand, the binding of an antibody to the same 343

region is supposed to impair functions by impeding or hindering the normal interactions of the 344

target protein with their molecular partners. 345

Deletion studies showed that a sequence corresponding to the first predicted internal coiled coil 346

regions of NadA is required for cell receptor binding, while no other deletion was defective. These 347

observations, combined with previously obtained ones, point to the possibility that the NadA 348

receptor binding site is also formed by alpha helices forming coiled-coils. Indeed antibodies to a 349

more restricted sequence (94-110 and 109-121) within this area neutralized (with some difference in 350

efficacy in E. coli and in N. meningitidis) NadA-dependent bacterial adhesion to cells. On the 351

contrary, antibodies to a region immediately preceding the second predicted dimeric coiled coil 352

region were less inhibitory, again in agreement with deletion mutant studies. 353

Antibodies to the very NH2 terminal sequence (24-39) were also neutralizing, while antibodies 354

directed to a stalk regions close to the bacterial outer membrane (208-215; 275-289) were poorly 355


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effective. This fits with the commonly accepted view that the distal part of the protein is engaged 356

with cell receptor, while the proximal one serves to protrude this part towards the external of the 357

bacterial cells. Previous experiments performed with N. meningitidis showed that the expression of 358

NadA partially but significantly contributes to bacterial epithelial cell association (8). Here we 359

confirmed that also in this native meningococcal contest both α-helices in the NH2 terminal and in 360

the dimeric coiled-coil regions are important for the NadA-mediated cell adhesion. 361

Our data suggest that most of the supposed receptor binding site (NadA 42-88), may not be in close 362

contact with the NadA cell receptor. In fact, not only Fab fragments but also the most hindering abs 363

specific for peptides within this region inhibited bacterial binding to cells less effectively than 364

abs/Fab fragments to peptides 25-39 and 94-110. Given the great volume of the whole antibody 365

molecule, such result suggests that the sequence 42-88, although close to 24-42 one in the primary 366

structure, are placed in such a way that bound antibodies are oriented in a direction only partially 367

disturbing cell-receptor approach. Surprisingly, antibodies and Fab to the neighboring 94-110 and 368

109-121 sequence (this latter more efficiently in N. meningitidis B) are again able to neutralize 369

NadA mediated cell-bacterial adhesion. One possibility accounting for this observation is that the 370

polypeptide chain corresponding to 94-121 sequence returns close to the NH2 terminal (NadA 24-371

39) and that these two regions cooperate to form the complete NadA-receptor binding site. 372

Based on these data and on structure prediction, we propose a comprehensive model of the NH2 373

terminal domain of NadA (Fig. 6). The subdomain 24-39, predicted to form amphipatic alpha 374

helices, is assumed to be in direct contact with the NadA receptor, while the two other sub-domains 375

are only proximal. On the contrary also the following dimeric coiled coil region is proposed to 376

provide a surface area necessary for receptor binding, in agreement with deletion mutants 377

experiments. Hence, according to this hypothesis, three sets of 24-39 and 94-121 sequences may 378

form the NadA receptor binding site. This model accounts for the less efficient inhibition exerted 379

by antibodies directed to 40-88: this sequence is in fact proposed to be peripheral to the receptor 380

binding region. Such complex structure could also explain why the separate deletion of all three 381

head’s sub-domains of the head structure altered the proper folding of this region with subsequent 382

lack of the adhesion properties (8). Poorly neutralizing antibodies may be used to co-isolate NadA-383

receptors. 384

Our data provide further evidence on the role not only of the globular NH2 domain but also of the 385

neighboring dimeric coiled coil structures in NadA-NadA receptor interaction, suggesting that a 386

more complex structure of the protein participates to bind the cellular receptor. Moreover, data on 387

N. meningitidis, showing that anti-NadA antibodies can contrast NadA adhesive functions, further 388

support the efficacy of NadA as an important component of an anti-menigocococcal B vaccine. 389


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Acknowledgements 390

This work was supported by grants from the University of Padua (Progetto di Ateneo 2004 and 391

ex60% 2007 and 2008). We thank dr. M. Morandi and dr. E. Ciccopiedi (Novartis Vaccine & 392

Diagnostics) for NadA∆351-405 purification. 393


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1. van Deuren, M., P. Brandtzaeg, and J. W. van der Meer. 2000. Update on meningococcal 395

disease with emphasis on pathogenesis and clinical management. Clin. Microbiol. Rev. 13:144-66, 396

table of contents. 397

2. Tzeng, Y. L. and D. S. Stephens. 2000. Epidemiology and pathogenesis of Neisseria 398

meningitidis. Microbes Infect. 2:687-700. 399

3. de Souza, A. L. and A. C. Seguro. 2008. Two centuries of meningococcal infection: from 400

Vieusseux to the cellular and molecular basis of disease. J. Med. Microbiol. 57:1313-1321. 401

4. Pizza, M., V. Scarlato, V. Masignani, M. M. Giuliani, B. Arico, M. Comanducci, G. T. 402

Jennings, L. Baldi, E. Bartolini, B. Capecchi, C. L. Galeotti, E. Luzzi, R. Manetti, E. 403

Marchetti, M. Mora, S. Nuti, G. Ratti, L. Santini, S. Savino, M. Scarselli, E. Storni, P. Zuo, M. 404

Broeker, E. Hundt, B. Knapp, E. Blair, T. Mason, H. Tettelin, D. W. Hood, A. C. Jeffries, N. 405

J. Saunders, D. M. Granoff, J. C. Venter, E. R. Moxon, G. Grandi, and R. Rappuoli. 2000. 406

Identification of vaccine candidates against serogroup B meningococcus by whole-genome 407

sequencing. Science 287:1816-1820. 408

5. Comanducci, M., S. Bambini, D. A. Caugant, M. Mora, B. Brunelli, B. Capecchi, L. 409

Ciucchi, R. Rappuoli, and M. Pizza. 2004. NadA diversity and carriage in Neisseria meningitidis. 410

Infect. Immun. 72:4217-4223. 411

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Rappuoli, and M. Mora. 2002. NadA, a novel vaccine candidate of Neisseria meningitidis. J. Exp. 414

Med. 195:1445-1454. 415

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7. Giuliani, M.M., J. Adu-Bobie, M. Comanducci, B. Aricò, S. Savino, L. Santini, B. Brunelli, 417

S. Bambini, A. Biolchi, B. Capecchi, E. Cartocci, L. Ciucchi, F. Di Marcello, F. Ferlicca, B. 418

Galli, E. Luzzi, V. Masignani, D. Serruto, D. Veggi, M. Contorni, M. Morandi, A. Bartalesi, V. 419

Cinotti, D. Mannucci, F. Titta, E. Ovidi, J.A. Welsch, D. Granoff, R. Rappuoli, and M. Pizza. 420

2006. A universal vaccine for serogroup B meningococcus. PNAS USA 103:10834–10839.) 421

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8. Capecchi, B., J. Adu-Bobie, F. Di Marcello, L. Ciucchi, V. Masignani, A. Taddei, R. 423

Rappuoli, M. Pizza, and B. Arico. 2005. Neisseria meningitidis NadA is a new invasin which 424

promotes bacterial adhesion to and penetration into human epithelial cells. Mol. Microbiol. 55:687-425

698. 426

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and E. Papini. 2008. Human monocytes/macrophages are a target of Neisseria meningitidis 428

Adhesin A (NadA). J. Leukoc. Biol. 83:1100-1110. 429

10. Mazzon, C., B. Baldani-Guerra, P. Cecchini, T. Kasic, A. Viola, M. de Bernard, B. Arico, 430

F. Gerosa, and E. Papini. 2007. IFN-gamma and R-848 dependent activation of human monocyte-431

derived dendritic cells by Neisseria meningitidis adhesin A. J. Immunol. 179:3904-3916. 432

11. Tavano, R., S. Franzoso, P. Cecchini, E. Cartocci, F. Oriente, B. Arico, and E. Papini. 433

2009. The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the 434

proimmune effects of MenB OMVs on human macrophages, compared with NadA- OMVs, without 435

further stimulating their proinflammatory activity on circulating monocytes. J. Leukoc. Biol. 436

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12. Cotter, S. E., N. K. Surana, and J. W. St Geme 3rd. 2005. Trimeric autotransporters: a 438

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13. Szczesny, P., D. Linke, A. Ursinus, K. Bar, H. Schwarz, T. M. Riess, V. A. Kempf, A. N. 440

Lupas, J. Martin, and K. Zeth. 2008. Structure of the head of the Bartonella adhesin BadA. PLoS 441

Pathog. 4:e1000119. 442

14. Hoiczyk, E., A. Roggenkamp, M. Reichenbecher, A. Lupas, and J. Heesemann. 2000. 443

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adhesins. EMBO J. 19:5989-5999. 445

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15. Brooks, M. J., J. L. Sedillo, N. Wagner, W. Wang, A. S. Attia, H. Wong, C. A. Laurence, 447

E. J. Hansen, and S. D. Gray-Owen. 2008. Moraxella catarrhalis binding to host cellular receptors 448

is mediated by sequence-specific determinants not conserved among all UspA1 protein variants. 449

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16. El Tahir, Y. and M. Skurnik. 2001. YadA, the multifaceted Yersinia adhesin. Int. J. Med. 451

Microbiol. 291:209-218. 452

17. Serruto, D., T. Spadafina, M. Scarselli, S. Bambini, M. Comanducci, S. Hohle, M. Kilian, 453

E. Veiga, P. Cossart, M. R. Oggioni, S. Savino, I. Ferlenghi, A. R. Taddei, R. Rappuoli, M. 454

Pizza, V. Masignani, and B. Arico. 2009. HadA is an atypical new multifunctional trimeric coiled-455

coil adhesin of Haemophilus influenzae biogroup aegyptius, which promotes entry into host cells. 456

Cell. Microbiol. 11:1044-1063. 457

18. Hill, D. J., A.M. Edwards, H.A. Rowe, and M. Virji. Carcinoembryonic antigen-related cell 458

adhesion molecule (CEACAM)-binding recombinant polypeptide confers protection against 459

infection by respiratory and urogenital pathogens. 2005. Mol. Microbiol. 55:1515–1527. 460

19. Fields, G. B. and R. L. Noble. 1990. Solid phase peptide synthesis utilizing 9-461

fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35:161-214. 462

463


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Figure legends 464

465

Fig. 1. Analysis of NadA binding regions using deletion mutants expressed in E. coli. 466

(A) Proposed three-dimensional organization of NadA protein. Portions of the extracellular 467

passenger domain, which are predicted to form dimeric and trimeric coiled-coils are colored in blue 468

and red respectively. The profile of different propensities to form dimeric and trimeric coiled-coil 469

supersecondary structures has been calculated using Multicoil software 470

(http://multicoil.lcs.mit.edu/cgibin/multicoil). The NadA ‘globular head’ is colored in yellow. The 471

α-helix linker region (L2L1) and beta barrel parts within the integral outer membrane translocator 472

domains are colored in orange and green respectively (17). 473

(B) Schematic representation of the various NadA mutants used in this study. The dimeric and 474

trimeric coiled-coils, the α-helix linker and the beta region are indicated in colors according to the 475

panel A. The leader peptide is not indicated. 476

(C) FACS analysis on whole-cell bacteria expressing NadA mutants using anti-NadA∆351-405 477

serum. 478

(D) Western blot analysis of the expression of NadA in E. coli. Total cell lysates of indicated 479

deletion mutants were assayed using an anti-NadA∆351-405 serum. The mutant NadA∆30-87 was 480

previously demonstrated to be surface exposed in a trimeric form (8). 481

(E) Adhesion of NadA mutants. Chang monolayers were infected with E. coli expressing full-length 482

NadA or each single deletion mutant (moi 100). E. coli–pET, carrying the vector alone, was used as 483

negative control. Results are reported as cfu per well and values represent the mean and standard 484

deviation of several experiments performed in triplicate. 485

486

Fig. 2. Cross-reaction of rabbit antisera to linear epitopes of NadA with native NadA in solution 487

and on E. coli outer membrane. 488

(A) The picture reports the sequence of NadA linear peptides used to immunize rabbits, and their 489

approximate location along the primary structure. In the scheme it is indicated the tri-partite 490

structure of the protein: the COOH terminal anchor to the outer membrane with the linker region, 491

the putative coiled-coil intermediate stalk, and the NH2 terminal globular head. Within this latter, 492

the three sub-domains previously deleted with loss of cell-binding capacity (8) are also indicated as 493

I (24-42 aa), II (43-70 aa) and III (71-87 aa). 494

*Antisera to NadA (25-33) indicated within brackets were also used in this study beside antisera to 495

NadA (25-39). The two sera were very similar and data shown later are therefore prevalently 496

relative to antibodies obtained with sequence 25-39 aa. 497


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(B) Antisera to linear epitopes of NadA bind to the surface of NadA expressing E. coli as assessed 498

by FACS analysis. NadA binding antibodies were revealed by PE-labeled secondary anti rabbit IgG 499

antibodies. 500

(C) ELISA assay showing the titer of anti peptide sera, using NadA∆351-405 or wt NadA+ E. coli as 501

capturing antigens. Columns represent the reciprocal of serum dilution giving half maximal OD. 502

The location of the sequence targeted within the adhesin predicted structure is schematically 503

indicated. 504

505

Fig. 3. Binding of affinity purified ab/Fab specific for NadA peptides to soluble or membrane 506

associated native NadA. 507

(A) Characterization of affinity purified Abs and Fab fragments. Coomassie staining after SDS-508

PAGE of a representative example of affinity purified anti-peptide antibody (anti 52-70 aa), of its 509

cleavage by matrix-linked papain to produce Fc and Fab fragments (lanes 1,2 and 3) and western 510

blot of purified Fab fragments with anti H anti L chain antibodies (lanes 4 and 5). 511

(B) ELISA assays performed using purified NadA∆351-405 or NadA+ E. coli as capturing antigens and 512

different concentrations of affinity purified abs and Fab fragments to the indicated NadA peptides. 513

Bound ab/Fab fragments were revealed by secondary ab to rabbit IgG conjugated to HRP. After 514

colorimetric development, the reciprocal of the ab/Fab fragments nmolar concentration giving an 515

OD of 0.75 was calculated from the graphs and plotted 516

517

Fig. 4. Neutralization of NadA-mediated E. coli adhesion to Chang cells by anti-NadA peptides 518

abs/Fab fragments. 519

Adherence of E. coli-NadA to Chang cells was inhibited by the addition of 45 nM (A) or increasing 520

concentrations (B) of affinity purified abs/Fab fragments against the specified NadA peptides. 521

Adhesion efficacy is expressed with respect to control sample (E. coli-NadA infecting Chang cells 522

in the absence of ab/Fab fragments) arbitrarily fixed to 100. Data are the mean +/- standard 523

deviation from several experiments run in triplicate. 524

525

Fig. 5. Efficacy of antisera raised against NadA linear peptides to inhibit N. meningitidis 526

adhesion to Chang cells. 527

Adherence to epithelial cells of N. meningitidis serogroup B strain M58 was inhibited using 528

different dilutions of the indicated rabbit sera. Data are expressed as compared to control sample 529

(MC 58 strain in the absence of serum) arbitrarily fixed to 100. Values represent the mean and 530

standard deviation of one representative experiment performed in triplicate. p.i.: pre-immune serum. 531


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20

532

Fig. 6. Model of NadA-NadA receptor interaction. 533

The picture shows the predicted globular head (24-42: sub-domain I, colored in grey; 43-87: sub-534

domains II +III, colored in white) and the neighboring intrachain coiled-coil domain (88-133, ccD, 535

colored in gray). It is proposed that the regions around sequence 24-39 and 94-121 form the surface 536

interacting with the NadA receptor (R), while aa 42-88 are not directly associated with NadA 537

receptor. The possible interaction with specific Fab fragments and NadA receptor is also depicted, 538

to account for competition studies. A single polypeptide chain, of the three forming the adhesin 539

oligomer, is here shown for clarity. 540

541


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24 88 133 182 219 252 287 316 351 405

| | | | | | | | | |

NadAF270-315

NadAF219-288

NadAF180-218

NadAF88-150

NadAF30-87

NadA

A B C

Figure 1

NadA NadA 88-150

NadA 180-218 NadA 219-288

NadA 270-315

Fluorescence intensity

N°

of

bacte

ria

191 –

97 –

64 –

NadA F2

70-3

15

NadA F2

19-2

88

NadA F1

80-2

18

NadA F8

8-15

0

pETNadA

D

E

1,E+04

1,E+05

1,E+06

1,E+07

pET

Nad

A

Nad

A Ä30-

87

Nad

A Ä88-

150

Nad

A Ä180

-218

Nad

A Ä219

-288

Nad

A Ä270

-315

CF

U/w

ell


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Fig. 1. Analysis of NadA binding regions using deletion mutants expressed in E. coli.

(A) Proposed three-dimensional organization of NadA protein. Portions of the extracellular passenger domain, which are predicted to

form dimeric and trimeric coiled-coils are colored in blue and red respectively. The profile of different propensities to form dimeric

and trimeric coiled-coil supersecondary structures has been calculated using Multicoil software

(http://multicoil.lcs.mit.edu/cgibin/multicoil). The NadA ‘globular head’ is colored in yellow. The g-helix linker region (L2L1) and

beta barrel parts within the integral outer membrane translocator domains are colored in orange and green respectively (17).

(B) Schematic representation of the various NadA mutants used in this study. The dimeric and trimeric coiled-coils, the g-helix linker

and the beta region are indicated in colors according to the panel A. The leader peptide is not indicated.

(C) FACS analysis on whole-cell bacteria expressing NadA mutants using anti-NadAÄ351-405 serum.

(D) Western blot analysis of the expression of NadA in E. coli. Total cell lysates of indicated deletion mutants were assayed using an

anti-NadAÄ351-405 serum. The mutant NadAÄ30-87 was previously demonstrated to be surface exposed in a trimeric form (8).

(E) Adhesion of NadA mutants. Chang monolayers were infected with E. coli expressing full-length NadA or each single deletion

mutant (moi 100). E. coli–pET, carrying the vector alone, was used as negative control. Results are reported as cfu per well and values

represent the mean and standard deviation of several experiments performed in triplicate.


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AnchorStalk

Linker

region

| | | | | | | | | | |

24 88 133 182 219 252 287 316 333 351 405

ccD ccTccT ccDI II III

ab

cd e

fg h

Globular domain

I: 24-42

II: 43-70

III: 71-87

a 25-39[33]*: [TNDDDVKKA]ATVAIA

b 41-53: AYNNGQEINGFKA

c 52-70: KAGETIYDIDEDGTITKKD

d 74-87: ADVEADDFKGLGLK

e 94-110: TKTVNENKQNVDAKVKA

f 109-121: KAAESEIEKLTTK

g 208-215: TAEETKQ

h 275-289: VYTREESDSKFVRID

25-3941-53

52-7074-87

94-110

109-121

208-215

275-289NadA

1000

10000

100000

1000000

an

tiseru

m t

itre

(to

Na

dA

F35

1-4

05)

peptide

globular domain colied-coil stalk

leu

-z

25-3941-53

52-7074-87

94-110

109-121

208-215

275-289NadA

100

200

300

400

500

IgG

bin

din

g to

Nad

A+ E

. co

li

peptide

globular domain colied-coil stalk

leu

-z

C

B

A

Figure 2


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Fig. 2. Cross-reaction of rabbit antisera to linear epitopes of NadA with native

NadA in solution and on E. coli outer membrane.

(A) The picture reports the sequence of NadA linear peptides used to immunize

rabbits, and their approximate location along the primary structure. In the scheme it is

indicated the tri-partite structure of the protein: the COOH terminal anchor to the outer

membrane with the linker region, the putative coiled-coil intermediate stalk, and the

NH2 terminal globular head. Within this latter, the three sub-domains previously deleted

with loss of cell-binding capacity (8) are also indicated as I (24-42 aa), II (43-70 aa) and

III (71-87 aa).

*Antisera to NadA (25-33) indicated within brackets were also used in this study beside

antisera to NadA (25-39). The two sera were very similar and data shown later are

therefore prevalently relative to antibodies obtained with sequence 25-39 aa.

(B) Antisera to linear epitopes of NadA bind to the surface of NadA expressing E. coli

as assessed by FACS analysis. NadA binding antibodies were revealed by PE-labeled

secondary anti rabbit IgG antibodies.

(C) ELISA assay showing the titer of anti peptide sera, using NadA〉351-405 or wt NadA+

E. coli as capturing antigens. Columns represent the reciprocal of serum dilution giving

half maximal OD. The location of the sequence targeted within the adhesin predicted

structure is schematically indicated.

Figure 2


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3A

4 5

Figure 3

A

Fig. 3. Binding of affinity purified ab/Fab specific for NadA peptides to soluble or membrane

associated native NadA.

(A) Characterization of affinity purified Abs and Fabs. Coomassie staining after SDS-PAGE of a

representative example of affinity purified anti-peptide antibody (anti 52-70 aa), of its cleavage by matrix-

linked papain to produce Fc and Fab (lanes 1,2 and 3) and western blot of purified Fab with anti H anti L

chain antibodies (lanes 4 and 5).


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25-3941-53

52-7074-87

94-110

109-121

208-215

275-289NadA

1E-3

0,01

0,1

1

10

1/n

M(O

D=

0.7

5)

NadAF351-405

NadA-E. coli

an

tibo

dy

25-3941-53

52-7074-87

94-110

109-121

208-215

275-289NadA

1E-4

1E-3

0,01

0,1

1

1/n

M (

OD

=0.7

5)

Fa

b

Figure 3

B

(B) ELISA assays performed using purified NadA〉351-405 or NadA+ E. coli as capturing antigens and different

concentrations of affinity purified abs and Fabs to the indicated NadA peptides. Bound ab/fabs were revealed by

secondary ab to rabbit IgG conjugated to HRP. After colorimetric development, the reciprocal of the ab/Fab nmolar

concentration giving an OD of 0.75 was calculated from the graphs and plotted.


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Figure 4

A

Fig. 4. Neutralization of NadA-mediated E. coli adhesion to Chang cells by anti-

NadA peptides abs/Fab fragments.

Adherence of E. coli-NadA to Chang cells was inhibited by the addition of 45 nM (A) or

increasing concentrations (B) of affinity purified abs/Fab fragments against the specified

NadA peptides. Adhesion efficacy is expressed with respect to control sample (E. coli-

NadA infecting Chang cells in the absence of ab/Fab fragments) arbitrarily fixed to 100.

Data are the mean +/- standard deviation from several experiments run in triplicate.

NadA - E. c

oli

control

NadAD351-40525-39

41-5352-70

74-87

94-110

109-121

208-215

275-289

0

50

100

cell

adhe

sio

n (

cfu

% o

f co

ntr

ol)

antibody

Fabfragments


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0 10 20 30 400

25

50

75

100

125

0 10 20 30 400

25

50

75

100

125

0 10 20 30 400

25

50

75

100

125

0 10 20 30 400

25

50

75

100

125

0 10 20 30 400

25

50

75

100

125

0 10 20 30 400

25

50

75

100

125

0 10 20 30 400

25

50

75

100

125

0 10 20 30 40 500

25

50

75

100

125

0 10 20 30 400

25

50

75

100

125

0 10 20 30 400

25

50

75

100

125

antibody

Fab fragments

94-1

10

74-8

752-7

025-3

9

NadAF3

51-4

05

nM

ce

ll a

dh

esio

n (

% o

f co

ntr

ol)

B

Figure 4


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0

20

40

60

80

100

120

140

Cont

rol

p.i.

25-3

9

41-5

3

52-7

0

74-8

7

94-1

10

109-

121

208-

215

275-

289

cell

adhesi

on c

fu (%

of co

ntr

ol)

1:5

1:20

1:100

serum

dilution

Fig. 5. Efficacy of antisera raised against NadA linear peptides to inhibit N. meningitidis adhesion

to Chang cells.

Adherence to epithelial cells of N. meningitidis serogroup B strain M58 was inhibited using different

dilutions of the indicated rabbit sera. Data are expressed as compared to control sample (MC 58 strain in

the absence of serum) arbitrarily fixed to 100. Values represent the mean and standard deviation of one

representative experiment performed in triplicate. p.i.: pre-immune serum

Nad

AF3

51-4

05p.i.


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43-87

88-133

I

II+III

24-42

Leu-z

ip

Anti

42-8

8

Anti

25-39

94-120R

R

ccD

Fig. 6. Model of NadA-NadA receptor interaction.

The picture shows the predicted globular head (24-42: sub-domain I, colored in grey; 43-87: sub-domains II

+III, colored in white) and the neighboring intrachain coiled-coil domain (88-133, ccD, colored in gray). It is

proposed that the regions around sequence 24-39 and 94-121 form the surface interacting with the NadA

receptor (R), while aa 42-88 are not directly associated with NadA receptor. The possible interaction with

specific Fab and NadA receptor is also depicted, to account for competition studies. A single polypeptide

chain, of the three forming the adhesin oligomer, is here shown for clarity.


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1 mapping of the neisseria meningitidis nada cell-binding site

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