High Resolution Clear Native Electrophoresis (hrCNE) Allows a

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High Resolution Clear Native Electrophoresis (hrCNE) Allows a Detailed Analysis of the Heterotrimeric Structure of Recombinant Neisseria meningitidis Porins Inserted into Liposomes Paula Freixeiro, Ernesto Diéguez-Casal, Liliana Costoya, Juan Marzoa, Carlos M. Ferreirós, María Teresa Criado, and Sandra Sánchez* Departamento de Microbiología y Parasitología, Facultad de Farmacia, Campus Sur, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain S Supporting Information *

ABSTRACT: Three recombinant proteins of Neisseria meningitidis, rPorB, rPorA, and rRmpM, were purified and incorporated into liposomes prepared by dialysis-extrusion. The protein complexes formed using different combinations of recombinant proteins were studied by high resolution clear native electrophoresis (hrCNE) and 2-D hrCNE/SDS-PAGE, analyzing the influence of the stoichiometry of the two porins in the formation of complexes and comparing them with native porin complexes present in OMVs from five different N. meningitidis strains. Insertion of the recombinant proteins into liposomes allowed a complete refolding of porin complexes, and the electrophoretic analyses showed that, when the three recombinant proteins are present, the pattern of porin complexes obtained is similar to that observed in native OMVs. We could show homocomplexes of each individual porin and PorA/PorB, RmpM/PorB, and PorA/PorB/RmpM heterocomplexes. Our results suggest that RmpM binds only to PorB, confirm the trimeric structure of N. meningitidis pores, and demonstrate that insertion into liposomes restores the native structure of porin complexes. KEYWORDS: Neisseria meningitidis, porin, liposome, conformational epitope, high resolution clear native electrophoresis, membrane complex, refolding

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INTRODUCTION Some of the vaccine candidates currently in study against serogroup B meningococcal meningitis are based on OM porins included in OMVs in which porin complexes are presented in their native conformation. This is the case for HexaMen1 and NonaMen,2 which use a combination of antigens from different serogroups, and the vaccines used to control outbreaks in Cuba, Norway, and New Zealand, VA-MENGOC-BC,3 MenBvac,4 and MeNZBTM,5 respectively. Consequently, we think that a detailed analysis of the conformation of porin complexes could be interesting for the improvement of the efficacy of these vaccines. The meningococcal porins PorB and PorA are the main OM proteins and, to form pores, acquire a β-barrel structure and associate, forming homo- or heterotrimers.6−8 Their transmembrane regions are highly conserved, but many of their exposed loops have hypervariable regions that are responsible for their differentiation in serotypes and serosubtypes. The associations of porins to form the pores lead to the formation of nonlinear epitopes that can have an important role in the development of protective immune responses.9 The reduction-modifiable protein M (RmpM) got its name because its electrophoretic mobility is different in the presence or absence of reducing agents. It is highly conserved and is © XXXX American Chemical Society

considered a structural protein that associates to the peptidoglycan layer forming noncovalent bonds through a OmpA-like domain present in its C-terminal end,10 being also associated to OM proteins (OMPs) such as LbpA, TbpA, and FrpB11 and to the porin complexes,6 leading to the formation of heterooligomeric complexes in which RmpM is dimeric. Consequently, it has been postulated that this protein plays an important role in the stabilization of the OM complexes and is present in the current OMVs-based vaccines.12 Antibodies to this antigen do not show bactericidal or opsonic activity and do not interfere with the bactericidal activity of antibodies against major antigens.13 Recombinant PorA, PorB, and RmpM have been successfully expressed in E. coli,10,14,15 and several studies showed that, in order to achieve an appropriate immune response (bactericidal antibodies), it is essential to recover their native structure, which can be accomplished by incorporation into liposomes.16,17 Besides recovering the ability to produce bactericidal antibodies, liposomes also play an important role as adjuvants, especially when other molecules such as modified LPS and mannose are also included in the liposome Received: September 7, 2012

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dx.doi.org/10.1021/pr3008573 | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

formulations.18,19 Proteoliposomes are good tools for the renaturation of OMPs because they mimic the natural environment present in the OMVs, thus being suitable for the study of the activity, function, and lipid−protein and protein−protein interactions, and have a consistent and welldefined composition.20 For the analysis of protein complexes in liposomes, it is essential to maintain their conformation. High resolution clear native electrophoresis (hrCNE) is a native electrophoresis technique in which one anionic detergent, such as sodium deoxycholate (DOC), and one or more neutral detergents, such as n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-glucopyranoside (OG) digitonin, or Triton X-100 (TX-100) are added both in the cathode and in the sample buffers. The mixed micelles formed by the detergents confer to the proteins the net negative charge necessary for their solubilization and migration to the anode, allowing the separation of complexes with a very high resolution.21 Further analysis in a second dimension using denaturing SDS-PAGE electrophoresis (2-D hrCNE/SDSPAGE) allows the separation of the components of the protein complexes to determine their composition. If a 2-D analysis is done using again hrCNE in the second dimension (2-D hrCNE/hrCNE), changing the detergents added to the cathode buffer, the mobility of some protein complexes can change. This allowed us to further improve resolution of meningococcal porin complexes8 and to confirm that the pores formed by recombinant porins in liposomes show similar conformations as those found in native OMVs in a previous study using Blue Native Electrophoresis.15 In this work, we analyzed the formation and structure of protein complexes obtained when different recombinant proteins (PorA, PorB, and RmpM) are incorporated into liposomes and the influence of the stoichiometry of both porins in the formation of the complexes, comparing them with native porin complexes found in OMVs from several N. meningitidis strains.

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Table 1. Characteristics of the Neisseria meningitidis Strains Used

a

strain

serogroupa

serotype:subtype

clonal complex

NZ98/254 3061 3063 Nm26 NmP27

B B B B Aa

15:P1.7-2,4 4:P1.7-2,4 1:P1.22,14 4:P1.16 15:P1.7,16

ST-41 ST-41 ST-213

Aa, autoagglutinating.

2. Purification of Recombinant Porins

Expression of the recombinant proteins was done using a modification of the method described by Sambook et al.22 The E. coli clones were grown for 24 h at 37 °C in 2xYT agar plates containing 30 μg/mL kanamycin for the rPorA and rPorB clones and 50 μg/mL ampicillin for the rRmpM clone. Two colonies were then inoculated in tubes containing 10 mL of 2xYT broth with the corresponding antibiotics, grown for 18 h at 37 °C with 180 rpm constant shaking, transferred to flasks with 500 mL of the same medium, and incubated for 1 h at 37 °C. Expression of the recombinant proteins was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma, USA) to a final concentration of 0.2 mM and incubated for 4 h in the same conditions. Cells were recovered by centrifugation at 10000 × g for 20 min, suspended in 8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCL, and 20 mM imidazol, pH 8 (lysis buffer), and sonicated ten times in ice (10 s pulses at 30 s intervals). Insoluble material was removed by centrifugation at 20000 × g for 1 h at 4 °C, and the supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA) column (5 mL) equilibrated with 25 mL of lysis buffer. The column was washed with 75 mL of 8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCL, and 20 mM imidazol, pH 6.3, and the recombinant proteins were eluted with 8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCL, and 500 mM imidazol, pH 5.9. Fractions collected were analyzed using SDS-PAGE and dot-blotting, and those containing the recombinant porins were pooled. The proteins were concentrated by precipitation with ethanol (80% v/v) for 18 h at 4 °C and collected by centrifugation at 15000 × g for 10 min at 15 °C.

MATERIALS AND METHODS

1. Bacterial Strains

Strains N. meningitidis 3061 and 3063 (NIBSC codes) and two E. coli clones (derived from strain E. coli One Shot TOP 10 with pTrcHis2 TOPO expression vector; Invitrogen, USA) for the rPorA and rPorB proteins (E. coli 01447 and E. coli 01508, respectively) were kindly donated by Dr. Ian Feavers (National Institute for Biological Standards and Control, U.K.). The plasmid has a gene for resistance to kanamycin and appends a His-tagged tail and a c-may epitope (used for tracking) to the recombinant proteins. Strain NZ98/254 (used for production of the MeNZBTM vaccine) was donated by Dr. Martin (Institute of Environmental Science and Research, Kenepuru Science Center, Porirua, New Zealand). Strains Nm26 and NmP27 are from our laboratory collection and were obtained from meningitis patients in our community. Characteristics of the meningococcal strains are shown in Table 1. The expression plasmid (pET-20b) containing the gene for rRmpM was kindly donated by Dr. Buchanan (Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, U.K.). This plasmid was cloned in strain E. coli BL21 for expression of the recombinant protein. The plasmid has a gene for resistance to ampicillin and appends a His-tagged tail and a c-may epitope to the protein.

3. Preparation of Proteoliposomes

Liposomes were prepared using a modification of the method described by Humphries et al.23 Briefly, 0.5 mg of purified recombinant protein were dissolved in 0.5 mL of 10 mM HEPES, pH 7.2, containing 2% (w/v) SDS. The solution was then added to 2 mL of 10 mM HEPES, pH 7.2, containing 50 mg of octyl-β-D-glucopyranoside (Fluka, Slovakia), and incubated for 3 h at room temperature. Lipid membranes were made by mixing L-α-phosphatidylcholine and cholesterol (Sigma, USA) in a 7:2 molar ratio and solubilized in the protein solution. Unilamellar vesicles were obtained by dialysis of the liposomes in PBS containing 0.1% thimerosal for 72 h at 4 °C and extrusion through 100 nm membranes. Proteoliposomes were collected by ultracentrifugation at 200,000 × g for 20 min at 4 °C, and the protein incorporated was indirectly quantified by measuring the amount remaining in the supernatant, using the bicinchoninic acid (BCA) protein assay.24 Finally, proteoliposomes were resuspended in PBS, stored at 4 °C, and used within 3 days. The different proteoliposomes containing combinations of the recombinant proteins were named LipA (rPorA), LipB (rPorB), LipR B



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Figure 1. (A) SDS-PAGE of purified recombinant proteins, (B) transmission electron microscopy picture of liposomes, and (C) 1-D hrCNE of proteoliposomes with different ratios of the recombinant proteins PorA, PorB, and RmpM (A, B, R in the lane headings).

10000 × g for 10 min at 4 °C, and OMVs in the supernatant were recovered by ultracentrifugation at 200000 × g for 10 min at 4 °C, suspended in distilled water and stored at −80 °C. The yield of OMVs was estimated from their protein content, determined using the BCA protein assay.24

(rRmpM), LipAB 1:1, LipAB 1:2, LipAB 2:1, LipAR 2:1, LipBR 4:1, and LipABR 2:4:1 (numbers indicate the ratios between the amounts of each protein). 4. Particle Size Measurements

Determination of the mean diameter and PDI of the liposomes was done by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, U.K.). The angle for detection of scattered light was set to 173°, and measurements were done at 25 °C. Sizes (mean ± SD) were calculated from three measurements in which at least 300 particles were analyzed per sample.

7. High Resolution Clear Native Electrophoresis

hrCNE was done following a modification of the method described by Wittig et al.26 Proteoliposome and OMV preparations were suspended in water to 1 mg/mL, and then 6-aminohexanoic acid in Bis-Tris-HCL was added (final concentrations of 87.3 mM and 4.4 mM, respectively, pH 7.0). Finally, n-dodecyl-β-D-maltoside (DDM) was added to the samples at a 1:4 DDM/protein ratio for proteoliposomes or 1:2 for OMVs. Complexes were solubilized for 30 min at room temperature, and insoluble material was removed by ultracentrifugation at 100,000 × g for 15 min at 4 °C. Polyacrylamide gradient gels (5−15% or 8−11% in 50 mM Bis-Tris, 500 mM 6-aminohexanoic acid, pH 7.0) were used for separation of the solubilized complexes in a Mini-Protean 3 Cell (BioRad Laboratories S.A., Spain). Just prior to running the experiments, a 4% polyacrylamide stacking gel was cast (in the same buffer) over the gradients, and 5% (v/v) glycerol and 0.01% (w/v) Ponceau red were added to each sample before loading.

5. Morphological Study

Morphology of the liposomes was checked using transmission electron microscopy (TEM). Liposome samples were dialyzed against PBS for 12 h at 4 °C, centrifuged at 200,000 × g for 20 min at 4 °C, resuspended in water, and stained with 2% phosphotungstic acid. 6. Outer Membrane Vesicles

Meningococcal OMVs were obtained using a modification of a previously described protocol.25 Bacteria grown in MHDesferal were recovered by centrifugation at 10000 × g for 10 min at 4 °C, suspended in distilled water at 250 mg/mL (wet weight), and processed three times in a French press at 1.1 × 108 kPa. Cellular debris was removed by centrifugation at C



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Anode buffer used was 50 mM Bis-Tris-HCL, pH 7.0, and cathode buffer was 50 mM Tricine, 15 Mm Bis-Tris-HCL, 0.05% DOC, 0.02% DDM, pH 7.0. Each lane was loaded with 25 μg of protein for proteoliposomes and 20 μg for the OMVs. Electrophoretic separation was done at 4 °C, and Kaleidoscope Standards (Bio-Rad Laboratories S.A., Spain) were used to standardize the total running time. Voltage was initially set to 50 V and raised to 100 V when the sample entered the separating gel, and separation was stopped when the lower molecular weight standard reached the bottom of the gels (about 5 h). High Molecular Weight Markers (HMW Native; 66−669 kDa; GE Healthcare, Spain) were used to estimate the molecular weight of complexes. Gels were fixed with trichloroacetic acid (TCA) and stained with Coomassie Brilliant Blue G250 (CBB).

Table 2. Measurement of Particle Sizes of the Different Proteoliposomes Using a Zetasizer nano ZS (Malvern Instruments Ltd, Worcestershire, U.K.)a

8. Bidimensional Electrophoresis

Figure 1C shows s a 1-D hrCNE analysis of the complexes formed when the recombinant proteins were incorporated into liposomes, and Figure 2 shows the composition of these

proteoliposomes LipC (blank liposomes; control) LipA LipB LipAB 1:1 LipAB 2:1 LipAB 1:2 LipAR 2:1 LipBR 4:1 LipABR 2:4:1 a

Bidimensional electrophoresis analyses were done using either SDS-PAGE or hrCNE in the second dimension (2-D hrCNE/ SDS-PAGE or 2-D hrCNE/hrCNE). For 2-D hrCNE/SDSPAGE, lanes cut from 1-D hrCNE gels were incubated with SDS sample buffer for 10 min at 95 °C and then placed on top of 12% polyacrylamide SDS-PAGE gels in a Mini-Protean 3 Cell (BioRad Laboratories S.A., Spain), and separation was done at 200 V constant voltage. For 2-D hrCNE/hrCNE, lanes cut from 1-D hrCNE gels were directly placed on top of another hrCNE gel, and separation was done as described for 1D hrCNE but using Triton X-100 in the cathode buffer. Gels were fixed with TCA, stained with CBB, scanned, and analyzed using Bio-Rad’s Quantity Two software.

size (nm) 95.4 140.3 113.7 138.3 130.2 138.2 124.2 114.4 129.7

± ± ± ± ± ± ± ± ±

0.1 1.4 3.9 0.8 0.2 0.3 1.0 1.8 2.4

Each value shows the mean ± SD of three measurements.

9. Western Blot Assays

Western blot analyses were done following standard protocols and using an anti-rRmpM serum obtained following a previously described protocol.27 Complexes formed by the recombinant proteins in proteoliposomes were separated using 1-D hrCNE and transferred to nitrocellulose membranes for 1 h at 350 mA. Membranes were blocked for 1 h with 5% Blotto (Bio-Rad Laboratories, Spain) in Tris-buffered saline (TBS) and then incubated overnight with anti-rRmpM serum (working dilutions were 1:1000 for OMVs and the LipAR 2:1 and LipBR 4:1 complexes, and 1:100 for the LipABR 2:4:1 ones). Finally, membranes were incubated with goat anti-mouse polyvalent immunoglobulins-peroxidase (Sigma, Spain; 1:1000 working dilution) for 90 min and revealed with 4-chloro-1naphthol.

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Figure 2. Bidimensional hrCNE/SDS-PAGE analysis of proteoliposomes with different ratios of the recombinant proteins PorA, PorB, and RmpM (marked as A, B, R respectively).

complexes analyzed using 2-D hrCNE/SDS-PAGE. In all cases in which PorA or PorB were present, the corresponding homocomplexes were formed; when both porins were present, PorA/PorB heterocomplexes were also formed, and when RmpM was also incorporated, it associated only to PorB, thus forming BR and ABR complexes but not AR ones. Recombinant PorA and PorB homocomplexes showed 205 kDa and 142 kDa apparent molecular weights respectively (lanes LipA and LipB), rPorA/rPorB heterocomplexes showed 179 and 160 kDas (lanes LipAB). The incorporation of rRmpM in the formulations resulted in different complexes depending on the other proteins used (lanes LipAR, LipBR, and LipABR). It is noteworthy that a faint band of 104 kDa, which seems to be formed by only rPorB, can be also observed in all proteoliposomes in which rPorB was incorporated. Proteoliposomes in which only the rRmpM protein was incorporated did not show any complex (not shown). It must be remarked that the molecular weights estimated in hrCNE gels are not accurate due to the differences in the mobility of the complexes depending on the detergents used for separation. This is

RESULTS

Formation and Characterization of Proteoliposomes

Figure 1 shows the three purified recombinant proteins (A), a transmission electron microscopy image of proteoliposomes (B), and the complexes formed when different combinations of the recombinant proteins are inserted into liposomes (C). As can be seen, recombinant proteins were obtained with a very high purity. The faint bands observed in the gels in Figure 1A are degraded fragments of the respective proteins, as was checked by Western blot with the corresponding antiprotein sera (not shown). The mean sizes obtained for proteoliposomes were very homogeneous but slightly variable, depending on the different protein combinations tested (see Table 2). Total protein incorporated into proteoliposomes was about 80% in all cases. D



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Figure 3. Coomassie staining of hrCNE (left) and Western blot (right) analysis of proteoliposomes and OMVs from the N. meningitidis H44/76 strain. Proteoliposomes were analyzed using a 5−15% gradient gel, whereas an 8−11% gradient gel was used for analysis of the OMVs. The antirRmpM serum was used at a working dilution of 1:1000.

specially evident for porin complexes as it has been already demonstrated in a previous study.8 Identification of the components of the complexes using 2-D hrCNE/SDS-PAGE (Figure 2) also showed some faint spots with molecular weights higher than those of the rPorA and/or rPorB monomers, which must be dimers of the porins with different stoichiometries (rPorA−rPorA, rPorA−rPorB, and rPorB−rPorB). Analysis of Outer Membrane Vesicles Using 2-D hrCNE/hrCNE and hrCNE/SDS-PAGE

Figure 3 shows the profile of complexes found in native OMVs (from strain H44/76) separated using hrCNE and analyzed using an anti-rRmpM serum. As can be seen, this protein seems to be a component of three complexes, agreeing with the results obtained when LipABR 2:4:1 liposomes are analyzed in the same way (lane LipABR). Coomassie staining of the gels for the analysis of LipAR proteoliposomes showed two bands (205.0 and 292.0 kDa), although none of them was recognized by the anti-rRmpM serum in Western blots. Figure 4 shows a 2-D analysis of native OMVs from the strain N. meningitidis 3061. The complexes formed by our recombinant proteins in the proteoliposomes (Figure 2, lane LipABR) showed a composition similar to that of some of the native complexes found in the OMVs (rPorA and rPorB homocomplexes and different rPorA/rPorB/rRmpM heterocomplexes). The other four N. meningitidis strains analyzed showed similar profiles both in hrCNE/hCNE and in hrCNE/ SDS-PAGE (not shown). The results obtained from the 2-D hrCNE/hCNE analyses showed that some bands observed after 1-D hrCNE are further resolved in at least two spots (two different complexes that have the same mobility when using one detergent in the buffer and different mobility when using the other8).

Figure 4. Analysis of the main outer membrane complexes of the N. meningitidis 3061 strain by 1-D hrCNE (top), 2-D hrCNE/SDS-PAGE (middle), and 2-D hrCNE/hCNE (bottom). The main proteins and complexes are indicated. Complexes with mobility depending on the detergent used in hrCNE are encircled.

recombinant proteins into liposomes has been shown to lead to the restoration of their native conformation and, consequently, to the formation of conformational epitopes.15,27 Proteoliposomes also show adjuvant activity and can be produced with a more homogeneous composition than native OMVs, which led us to their investigation as possible substitutes for the last. The method used for preparation of the proteoliposomes in this study, using dialysis and extrusion, rendered homogeneous-size particles, did not affect the reconformation of our proteins, and allowed a good control for the amount of proteins incorporated, which showed it better than other methods27 for our study. The results obtained using 2-D hrCNE/SDS-PAGE after analysis of the different proteoliposomes showed that rPorA in

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DISCUSSION Most vaccines against group B meningococci currently under research are based on the immune response against OMVs, in which the main determinant antigens are the porins PorA and PorB that, in their native state, associate to form either homotrimeric or heterotrimeric pores.8 Some of the studies of the porin structures, in which these vaccines are based, were done using recombinant porins14,15,17 that could obviate the response against conformational epitopes that form when the proteins associate to build native pores. The incorporation of E



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PorB heterocomplexes, and those from strain 3063 showed two different PorB homocomplexes. This suggests that other proteins could associate to some porin complexes in native OMVs. High resolution clear native electrophoresis does not allow an accurate determination of the molecular weights of complexes,28 due to their modification during the formation of detergent micelles. Therefore the apparent molecular weights of complexes do not correlate with the sum of those of their constituent proteins, and we can not deduce the number of subunits at the complexes based on their electrophoretic mobility. Nevertheless, it has been already reported that N. meningitidis porins associate to form trimeric complexes in which other proteins (RmpM, TbpA, FetA) were also found.6−8 In our proteoliposome complexes, the trimeric structure agrees with the profiles of dimers shown in the 2-D hrCNE/ SDS-PAGE analyses (Figure 3), in which partial denaturation of homocomplexes results in a single type of dimers (rPorB or rPorA homodimers, respectively), whereas that of heterocomplexes results in two types of dimers: rPorA or rPorB homodimers and rPorA-rPorB heterodimers. So, the 205 kDa and 142 kDa complexes shown in Figure 1C should be rPorA and rPorB homotrimers, the 179 kDa one a heterotrimer formed by two rPorA and one rPorB units, and the 160 kDa complex a heterotrimer formed by two rPorB and one rPorA units. In the same way, we suggest that the 220 kDa complex observed in the LipABR 2:4:1 and LipBR 4:2 proteoliposomes could be a rPorB heterotrimer bound to RmpM, and the 240 and 265 kDa complexes, PorA/PorB heterotrimers bound to rRmpM. The analysis of the complexes formed in LipAR proteoliposomes showed two bands, which suggests that rRmpM/rPorA complexes (197 kDa) could be also formed. Nevertheless, contrary to what happened in all other rRmpMcontaining complexes, specific anti-rRmpM serum did not react in Western blots with that band, which suggests that it can be a rPorA aggregate. The analysis of that band using 2-D electrophoresis also failed to detect rRmpM in that complex, agreeing with our suggestion that RmpM only associates to complexes through binding to the PorB protein.

the LipA liposomes formed only one type of homocomplexes, presumably trimers (205.0 kDa), whereas two types of rPorB complexes could be found in LipB liposomes, presumably dimers (104.0 kDa) and trimers (142.0 kDa). When the two porins were used in the preparation of proteoliposomes, the two main homocomplexes of each porin were detected, in contrast with our previous results with liposomes prepared using sonication and analyzed by 2-D BN/SDS-PAGE.15 This could be explained because sonication is a more aggressive procedure than extrusion, which could lead to the disruption of homocomplexes. On the other hand, it is also possible that the strongest conditions and lower resolution of Blue Native Electrophoresis21 could produce the disruption of the porin homocomplexes. With respect to the AB heterocomplexes, when the ratio of rPorA/rPorB was 1:1 (LipAB 1:1), all bands (complexes) showed similar intensity, whereas when that ratio was different (LipAB 1:2 and LipAB 2:1), the bands corresponding to the homotrimeric complexes of the more abundant porin were more intense, suggesting that, in vitro, porins associate spontaneously. In order to check the association of RmpM to the porins, the protein ratios used were those that we could infer from the analysis of native OMV complexes, in which the amount of PorB is about twice that of PorA and RmpM is lower than that of PorA, so we chose to produce and analyze LipAR1:2, LipBR4:1, and LipABR2:4:1 proteoliposomes. In the LipABR 2:4:1, incorporation of rRmpM did not change the formation of the main homo- and heterocomplexes and induced the formation of one rPorB/rRmpM and two rPorA/rPorB/ rRmpM new heterocomplexes. These complexes are poorly seen in the electrophoretic analyses by 2-D hrCNE/SDS-PAGE due to the low amount of rRmpM, but Western blot assays with an anti-RmpM serum revealed them neatly (Figure 3). Previous works already suggested that RmpM can associate with both Neisseria meningitidis porins6 playing a role in the stabilization of porin complexes, but our immunoassays using an anti-rRmpM serum suggest that this protein binds to porin complexes through the PorB. The association through PorB is further suggested by the presence of rPorB/rRmpM heterocomplexes both in the LipABR 2:4:1 proteoliposomes and in the five native OMVs strains analyzed, in which no PorA/ RmpM complexes could be found. Our electrophoretic analysis of OMVs from wild-type strains show that the patterns and electrophoretic behaviors of complexes from all the strains used in this study are consistent with those found in previous studies using the strain H44/768. When 2D hrCNE/hCNE is used for the analysis (using different detergents for each dimension), the porin complexes are displaced from the diagonal because their solubilization and migration depends on the formation of mixed micelles that have different sizes and charges. These differences in the electrophoretic mobility allow the resolution of complexes that co-migrate in the first dimension run and are resolved after separation in the second dimension. The porin complexes observed in wild-type strains are similar to those found after incorporation of both recombinant porins and rRmpM into liposomes (LipABR 2:4:1), which suggests that we were able to reproduce the native conditions in OMVs, thus allowing proteins to recover their native conformation and associations. However, while we found only two types of rPorA/rPorB heterocomplexes in the proteoliposomes, OMVs from strains Nm26 and NmP27 showed three different PorA/

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CONCLUSION

The dialysis-extrusion method proved to be very suitable for the preparation of proteoliposomes in which the patterns and structures of the complexes formed resemble those found in native OMVs, suggesting a correct refolding of the proteins involved in the formation of membrane pores. Also, the use of recombinant proteins instead of native ones does not seem to interfere in the reshaping process and in the formation of the complexes, which makes them useful for obtaining large amounts of antigenic preparations. These findings, together with the ability of proteoliposomes containing porins to induce bactericidal immune responses and with the easy standardization of the proteoliposome composition, make these vehiculization systems very interesting for the preparation and testing of new vaccine formulations that could be useful against N. meningitidis. F



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ASSOCIATED CONTENT

S Supporting Information *

Figure showing the 2-D hrCNE/hCNE and 2-D hrCNE/SDSPAGE analysis of OMVs from five hererologous strains. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +34 881814946. Fax: +34 881815106. E-mail: sandra. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants 2007/XA063, 08CSA025203P, and INCITE09E1R203016ES from the Xunta de Galicia (autonomic government), Spain. P.F. holds a predoctoral grant by the Xunta de Galicia (Plan I2C).

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REFERENCES

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High Resolution Clear Native Electrophoresis (hrCNE) Allows a

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