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

1

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

2

in the NH2 terminal and in dimeric coiled-coil regions

3

Running title: NadA binding site

4 5

Regina Tavano1,2 #, Barbara Capecchi3#, Paolo Montanari3, Susanna Franzoso2, Oriano Marin2,4,

6

Maryta Ztukowska

7

Emanuele Papini

1,2,†

, Paola Cecchini1,2, Daniela Segat2, Maria Scarselli3, Beatrice Aricò

3*

and

1, 2*

8 9

1

Dipartimento di Scienze Biomediche Sperimentali, Università di Padova

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2

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

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

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3

Novartis Vaccine and Diagnostics srl, Siena

13

4

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

14

†

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

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

16

#

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

17 18

*Correspondent footnote:

19 20

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

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

22

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

23

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

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00390498276159.

1

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Abstract

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

antibodies reinforce the importance of NadA as vaccine antigen.

2

42

Introduction

43 44

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

45

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

46

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

47

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

48

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

49

meningococcal disease (5).

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

µM on Chang cells (8, 9).

60

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

61

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

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

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

64

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

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

66

membrane components (11).

67

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

68

accompanying meningococcal infection. Consequently, the comprehension of the structural

69

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

70

fatal-meningococcal sepsis.

71

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

72

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

73

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

74

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

75

COOH terminal membrane anchor, having a β-barrel structure, necessary for the export of the 3

76

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

77

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

78

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

79

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

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

81

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

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target (18).

83

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

84

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

85

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

86

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

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

4

106

Materials and methods

107 108

Plasmid construction

109

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

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

111

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

112

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

113

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

114

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

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

116

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

117

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

118 119

FACS analysis

120

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

121

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

122

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

123

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

124

FACS-Scan flow cytometer (Beckton-Dickinson).

125 126

Animals

127

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

128 129

NadA Peptides synthesis

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

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

138

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

139

mg) for 2.5 h. Crude peptides were purified by a preparative reverse phase HPLC. Molecular 5

140

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

141

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

142

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

143

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

144

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

145 146

Peptide-carrier conjugation

147

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

148

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

149

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

150

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

151

frozen in liquid nitrogen.

152 153

Immunization and production of polyclonal Abs

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

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

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

164

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

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

166 167

Fab generation and purification

168

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

169

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

170

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

171

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

172

incubated with 100 µl of ProteinA-Agarose (Sigma) for 2 hours at room temperature, under gently 6

173

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

174

recovered.

175 176

ELISA assay

177

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

178

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

179

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

180

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

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

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

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

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ELISA plate reader (Amersham Biosciences).

185 186

SDS-PAGE and Western blot

187

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

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

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

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

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

192

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

193

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

194

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

195

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

196

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

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secondary peroxidase-conjugate anti-body (DAKO).

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Adhesion assay

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

201

incubation in an antibiotic-free medium, approximately 3 x 107 [multiplicity of infection 1:100 (moi

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

203

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

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

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suspension were plated onto LB agar to calculate the cfu. 7

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

208

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

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

210

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

211

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

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

213 214

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

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

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

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

218

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

219

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

220

were determined.

8

221

Results

222 223

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

224 225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

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

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

235

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

236

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

237

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

238

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

239

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

240 241 242

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

243

NadA.

244 245

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

246

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

247

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

248

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

249

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

250

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

251

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

252

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

253

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

254

proving that anti-linear epitope antibodies cross-react with the native adhesin. NadA 52-70 was 9

255

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

256

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

257

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

258

negligible signal (not shown).

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280 281

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

282

epitopes.

283 284

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

285

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

286

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

287

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

288

405).

Antibodies and Fab fragments to NadA-peptides 25-39 and 94-110 strongly counteracted 10

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

reduced efficacy.

300

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

301

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

302

the conformation of NadA modules to exploit their functions.

303 304

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

305

cells

306 307

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

308

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

309

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

310

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

311

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

312

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

313

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

314

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

315

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

316

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

317

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

318

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

319

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

320

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

321

11

322

Discussion

323 324

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

325

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

326

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

327

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

328

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

329

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

330

NadA-NadA receptor complex.

331

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

332

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

333

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

334

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

335

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

336

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

337

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

338

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

339

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

340

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

341

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

342

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

343

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

344

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

345

target protein with their molecular partners.

346

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

347

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

348

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

349

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

350

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

351

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

352

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

353

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

354

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

355

directed to a stalk regions close to the bacterial outer membrane (208-215; 275-289) were poorly 12

356

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

357

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

358

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

359

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

360

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

361

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

362

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

363

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

364

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

365

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

366

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

367

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

368

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

369

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

370

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

371

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

372

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

373

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

374

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

375

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

376

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

377

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

378

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

379

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

380

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

381

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

382

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

383

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

384

receptors.

385

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

386

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

387

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

388

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

389

support the efficacy of NadA as an important component of an anti-menigocococcal B vaccine. 13

390

Acknowledgements

391

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

392

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

393

Diagnostics) for NadA∆351-405 purification.

14

394

References

395

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

396

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

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table of contents.

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

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meningitidis. Microbes Infect. 2:687-700.

400

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

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

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

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

404

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

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

406

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

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

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sequencing. Science 287:1816-1820.

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

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

411

Infect. Immun. 72:4217-4223.

412

6. Comanducci, M., S. Bambini, B. Brunelli, J. Adu-Bobie, B. Aricò, B. Capecchi, M. M.

413

Giuliani, V. Masignani, L. Santini, S. Savino, D. M. Granoff, D. A. Caugant, M. Pizza, R.

414

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

415

Med. 195:1445-1454.

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

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

419

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

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

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

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

424

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

425

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

426

698.

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9. Franzoso, S., C. Mazzon, M. Sztukowska, P. Cecchini, T. Kasic, B. Capecchi, R. Tavano,

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

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

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

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

432

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

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

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

435

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

436

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

437

86:143-153.

438

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

439

distinct subfamily of autotransporter proteins. Trends Microbiol. 13:199-205.

440

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

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

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Pathog. 4:e1000119.

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

444

Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of

445

adhesins. EMBO J. 19:5989-5999.

446 447

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

448

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

449

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

450

Infect. Immun. 76:5322-5329.

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451

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

452

Microbiol. 291:209-218.

453

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

454

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

455

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

456

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

457

Cell. Microbiol. 11:1044-1063.

458

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

459

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

460

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

461

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

462

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

463

17

464

Figure legends

465 466

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

467

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

468

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

469

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

470

supersecondary

471

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

472

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

473

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

474

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

475

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

476

panel A. The leader peptide is not indicated.

477

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

478

serum.

479

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

480

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

481

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

482

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

483

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

484

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

485

deviation of several experiments performed in triplicate.

structures

has

been

calculated

using

Multicoil

software

486 487

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

488

and on E. coli outer membrane.

489

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

490

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

491

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

492

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

493

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

494

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

495

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

496

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

497

relative to antibodies obtained with sequence 25-39 aa. 18

498

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

499

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

500

antibodies.

501

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

502

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

503

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

504

indicated.

505 506

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

507

associated native NadA.

508

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

509

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

510

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

511

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

512

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

513

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

514

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

515

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

516

OD of 0.75 was calculated from the graphs and plotted

517 518

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

519

abs/Fab fragments.

520

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

521

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

522

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

523

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

524

deviation from several experiments run in triplicate.

525 526

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

527

adhesion to Chang cells.

528

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

529

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

530

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

531

standard deviation of one representative experiment performed in triplicate. p.i.: pre-immune serum. 19

532 533

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

534

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

535

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

536

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

537

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

538

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

539

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

540

oligomer, is here shown for clarity.

541

20

C

B 24 |

88 |

133 182 | |

219 |

252 287 | |

316 351 | |

405 |

88-150

NadA NadAF30-87 NadAF88-150 NadAF180-218 NadAF219-288 NadAF270-315

E

NadA

NadA

N° of bacteria

A

NadA

180-218

NadA

270-315

NadA

219-288

1,E+07

1,E+06 CFU/well

Fluorescence intensity

D

1,E+05

88 AF

15 18 2 88 50 -3 -2 -1 70 80 2 19 F2 F1 F

dA T ad adA adA adA Na pE N N N N

191 –

1,E+04 T pE

dA Na

Ä dA Na

30

87 Ä dA Na

-1 88

50 Ä dA Na

18

2 0-

18 Ä dA Na

21

2 9-

88 Ä dA Na

27

0-

31

5

97 – 64 –

Figure 1

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.

b

A

a

c

d e

I II III

| 24

f

g

ccD | 88

ccT | 133

Globular domain I: 24-42 II: 43-70 III: 71-87

B

h

| 182

ccD

| 219

ccT | | 252 287

Stalk a 25-39[33]*: b 41-53: c 52-70: d 74-87: e 94-110: f 109-121: g 208-215: h 275-289:

[TNDDDVKKA]ATVAIA AYNNGQEINGFKA KAGETIYDIDEDGTITKKD ADVEADDFKGLGLK TKTVNENKQNVDAKVKA KAAESEIEKLTTK TAEETKQ VYTREESDSKFVRID

| | | 316 333 351

| 405

Linker Anchor region

C +

IgG binding to NadA E. coli

400

100000

10000

1000

-53 41

-70 52

globular domain

0 15 21 89 -87 -11 8-2 9-1 5-2 74 94 10 20 27

dA Na

200

100

peptide

dA -39 1-53 2-70 4-87 -110 -121 -215 -289 4 25 5 Na 7 5 9 8 94 27 10 20

globular domain

colied-coil stalk

colied-coil stalk leu-z

-39 25

300

leu-z

antiserum titre (to NadAF351-405)

500

1000000

Figure 2

peptide

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

A

4

5

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 matrixlinked 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).

3A Figure 3

NadAF351-405

B

NadA-E. coli

1

antibody

1/nM(OD=0.75)

10

0,1

0,01

1E-3

-39 25

-53 41

-70 52

0 89 21 15 -87 -11 5-2 9-1 74 8-2 94 27 20 10

-39 25

-53 41

-70 52

-8 74

dA Na

0,1

0,01

Fab

1/nM (OD=0.75)

1

1E-3

1E-4

7

-1 94

10

89 21 15 9-1 5-2 8-2 27 20 10

dA Na

(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.

Figure 3

A

cell adhesion (cfu % of control)

antibody Fabfragments

100

50

0 c E. dA Na

ol 405 -39 1-53 2-70 4-87 -110 -121 -215 -289 ntr 5 4 7 25 co 351 94 109 208 275 D A d Na

oli

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

Figure 4

75

75

50

50

25

25 0 40

125

125

100

100

75

75

50

50

25

25

0

0

10

20

30

40

0

125

125

100

100

75

75

50

50

25

25

0

0

10

20

30

40

0

125

125

100

100

75

75

50

50

25

25

0

0

10

20

30

40

0

125

125

100

100

75

75

50

50

25

25

0

0

10

20

30

40

0

10

20

30

40

0

10

20

30

40

0

10

20

30

40

74-87

30

0

0

nM

10

20

30

40

30

40

94-110

20

25-39

10

NadAF351-405

100

0

Fab fragments

125

100

0

cell adhesion (% of control)

antibody

125

52-70

B

0

10

20

50

Figure 4

cell adhesion cfu (% of control)

140 120

serum dilution

100 80 60

1:5

40

1:20

20

1:100

0 l ro nt Co

i. pp. .i.

N

A ad

F

4 135

05

9 -3 25

3 -5 41

0 -7 52

7 -8 74

1 5 9 10 28 12 21 -1 98594 10 20 27

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

R

II+III

ccD Leu-zip

88-133

R

I

ti An 8 8 42 -

24-42 43-87

Anti 25-39 94-120

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.