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
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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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14
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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15
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18. Hill, D. J., A.M. Edwards, H.A. Rowe, and M. Virji. Carcinoembryonic antigen-related cell 458
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463
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18
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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19
(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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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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