Analysis of Outer Membrane Protein Complexes and Heat-Modifiable

Analysis of Outer Membrane Protein Complexes and Heat-Modifiable Proteins in Neisseria Strains Using Two-Dimensional Diagonal Electrophoresis Sandra Sa´ nchez, Jesu´ s Arenas, Ana Abel, Marı´a-Teresa Criado,* and Carlos M. Ferreiro´ s Departamento de Microbiologı´a, Facultad de Farmacia, Universidad de Santiago, 15782 Santiago de Compostela, Spain Received August 23, 2004

Two-dimensional diagonal SDS-PAGE was used to resolve membrane complexes and identify proteins with temperature-dependent mobility in Neisseria meningitidis and N. lactamica. The main membrane complexes were composed of porins and were formed by heteromers of PorA, PorB and RmpM in N. meningitidis, and by PorB and RmpM in N. lactamica. Also, other proteins, including Opa, with temperature-dependent mobility were clearly demonstrated. The method allows improved detection of the components of membrane complexes and proteins with temperature-dependent mobility which is difficult to resolve with other analytical approaches. Keywords: Neisseria meningitidis • Neisseria lactamica • porins • membrane complexes • mobility shift • diagonal electrophoresis

Introduction Meningococal meningitis and septicaemia disease represents a serious health problem, specially for serogroup B. Vaccines against serogroup B disease have been developed which are based on outer membrane vesicles,1 in which PorA and PorB porins (class 1 and 2/3 proteins) are the main surface antigens. A number of other antigens are present, including Opa (class 5 proteins), Omp85 and RmpM. Most analyses of the composition, structure and function of neisserial porins are based on artificial constructions made using recombinant proteins (either PorA or PorB, usually obtained as inclusion bodies in E. coli) inserted in synthetic lipid membranes, with very few studies of natural porins isolated in native conditions.3-5 In these studies, analyses have been performed using one-dimensional SDS-PAGE and western blotting, with identification of the antigens using specific monoclonal antibodies. Characterization of proteins with temperature-dependent mobility is difficult using one-dimensional SDS-PAGE, especially when the proteins are present at low concentrations and/ or have midrange molecular masses (15 to 50 kDa), a range in which many interfering proteins exist, hindering their detection.2,6 In this study, we have used two-dimensional diagonal SDSPAGE and different conditions for the treatment of the samples before running in each dimension (presence or absence of β-mercaptoethanol in the sample buffer and different incubation temperatures) in order to improve the resolution of membrane protein complexes and the detection and location of heat-modifiable proteins. * To whom correspondence should be addressed. Tel: +34 981 563 100. Fax: +34 981 594 912. E-mail: [email protected]. 10.1021/pr049846i CCC: $30.25

 2005 American Chemical Society

Table 1. Characteristics of the Neisseria Strains Used in This Study origina

strain

B16B6 M982 Nm17 Nm20 Nm30 NmP0 NmP3 NmP5 NmP27 NlP2 NlP3 a

N. meningitidis CSF, reference strain CSF, reference strain blood CSF CSF oropharynx oropharynx oropharynx oropharynx N. lactamica oropharynx oropharynx

serogroup/serotypeb

B:2a:P1.2 B:9:P1.2 C:2b:P1.2,5 C:2b:P1.2,5 C:2b:P1.2,5 W135:nt:P1.3,6 B:2a:P1.2,5 B:nt:P1.3,6 A:15:P1.7,16 n.a. n.a.

CSF, cerebrospinal fluid. b nt, nontypeable; n.a., not applicable.

Experimental Section Bacterial Strains and Growth Conditions. Five Neisseria meningitidis invasive strains, four N. meningitidis carrier strains and two N. lactamica strains were used in this work (Table 1). Culture and maintenance of the strains were done as described previously.7 Cultures in iron-restricted conditions were done in Mueller-Hinton (MH) broth with 100 µM Desferal added. Outer Membrane Vesicles. Outer membrane vesicles (OMVs) were obtained as described previously.8 Briefly, bacteria from MH-Desferal cultures were recovered by centrifugation (10 000 × g/10 min/4 °C), suspended in 0.1 M acetate buffer, pH 5.8, with 0.2 M LiCl (5 mL per gram of cells) and incubated for 2 h at 45 °C in a shaking water bath. The suspension was then processed three times in a French press at 1.1 × 105 kPa. Cellular debris was removed by centrifugation (10 000 × g/10 Journal of Proteome Research 2005, 4, 91-95

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research articles min/4 °C), and OMVs in the supernatant were recovered by ultracentrifugation (200 000 × g/10 min/4 °C) and suspended in distilled water applying two 5 s ultrasonic pulses. OMV concentrations were estimated by the protein content, determined using the bicinchoninic acid (BCA) protein assay.9 Immune Sera. Mouse immune sera were obtained using OMV preparations from iron-restricted cultures and following an immunization protocol described previously.10 Briefly, five one-month-old CBA mice were injected intraperitoneally, each with 20 µg of OMVs in complete Freund’s adjuvant (100 µL) on day 0, and same dose in incomplete Freund’s adjuvant was administered on days 14 and 28 (boost dose). Mice were bled 5 days later through the retro-orbital plexus and the blood samples pooled, allowed to clot at room temperature and centrifuged at 2000 × g for 10 min. All sera were heatinactivated (56 °C/30 min) and stored in 250 µL aliquots at -20 °C until use. Monoclonal antibody against conserved epitopes in the PorA (mab 9-1-P1.C) was kindly provided by Drs. W. Zollinger and E. Moran (Walter Reed Army Institute, U.S.A.), anti-PorB (mab F1.9H10/1B3) by Dr. A. Silva (Adolfo Lutz Institute, Brazil), anti-RmpM (mab 185-H8) by Drs. E. Rosenqvist and J. Kolberg (National Institute of Public Health, Norway), and anti-Opa (mab 4B12/C11) by Dr. G. Morelli (MaxPlanck Institut fu ¨ r Infektionsbiologie, Germany). Diagonal Electrophoresis. SDS-PAGE in 7.5-15% gradient gels and standard electrophoretic settings were used for both the first and second dimension in diagonal electrophoresis.11 In all assays, samples contained 200 µg of OMVs diluted 1:1 with double-strength sample buffer without β-mercaptoethanol and were heated at 37 °C for 20 min just before running for the first dimension. After running, sample strips were cut, placed into containers with sample buffer (with or without β-mercaptoethanol, depending on the experiment), and incubated either at ambient temperature or at 95 °C for 5 min. The strips were then placed on top of the second dimension gels, overlayed with 0.5% agarose and run as usual for separation. Proteins in the gels were stained with Coomassie Brilliant Blue R-250 for image analysis or silver nitrate for MALDI-TOF. Immunoblotting. Proteins were transferred from unstained gels to poly(vinylidene difluoride) (PVDF) membranes using a Bio-Rad Mini-Trans Blot Electrophoretic Transfer Cell (Bio-Rad Chemical S. A., Spain) according to the manufacturer’s instructions. Membranes were blocked with 5% skimmed milk in TBS for 2 h at room temperature, washed three times with TBSTween (0.05% Tween-20 in TBS) and incubated overnight at 37 °C with 600 µL of a previously determined working dilution of the immune sera (1:1000 in all cases). After washing again, the membranes were incubated with HRP-conjugated goat antimouse immunoglobulins (Sigma-Aldrich Quimica S. A., Spain), washed, and developed with 4-chloro-1-naphthol.12 Protein Identification. Identification of some proteins was done by molecular mass estimation, blotting with monoclonal antibodies and MALDI-TOF peptide map fingerprinting analysis. For MALDI-TOF, individual spots were cut from silverstained diagonal electrophoresis gels and sent to the Centro Nacional de Biotecnologı´a (Madrid, Spain) for identification. Image Analyses. Analysis of the proteins and antigens, and calculations of their molecular weights were done using the Bio-Rad Quantity One software (Bio-Rad Chemical S. A., Spain). The gels or Western blot membranes were scanned at high resolution (1200 dpi) and the images obtained filtered to remove noise and streaking. The images presented are the final Gaussians produced by the software used. 92

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Figure 1. Diagonal SDS-PAGE analysis of outer membrane proteins from N. meningitdis M982. Second dimension was run after nondenaturing (A) or denaturing (B) treatment of first dimension strips. One-dimension gels run after denaturing treatment are located at right. In (A), putative complexes are pointed inside rectangles. In (B), spots derived from dissociation of complexes are pointed inside circles and those with temperature-dependent mobility are pointed inside rectangles. Molecular weights (MW) and identification of some proteins are indicated.

Results Analysis of the outer membrane proteins from the N. meningitidis strains by diagonal electrophoresis after incubation at ambient temperature for the second dimension (nondenaturing even in the presence of β-mercaptoethanol) shows that all detectable proteins are located in the diagonal, with the exception of only three spots with molecular weights of 190, 95, and 72 kDa respectively, that appear at the leftmost side of the gels (Figure 1A). When incubation for the second dimension was performed at 95 °C, these spots appear to partially dissociate giving rise to three new ones with 43, 38, and 36 kDa molecular masses (Figure 1B). Also, a large 95 kDa spot observed under nondenaturing conditions (Figure 1A) dissociates into three spots with exactly the same masses (43, 38, and 36 kDa) as those above-mentioned. Some other spots are also observed below the diagonal (70, 60, 55, and 20 kDa), whereas 95, 50, 43, and 30 kDa spots can be clearly seen over the diagonal (Figure 1B). The same dissociation and temperature-dependent mobility patterns (with some minor variations) were observed in the other N. meningitidis strains analyzed (not shown), and were independent of the presence of β-mercaptoethanol in the second dimension sample buffer. Figure 2 shows the same analysis performed with outer membrane proteins obtained from the N. lactamica NlP3 strain. As it can be seen by comparing Figure 2, parts A and B, three proteins with molecular weights of 50, 38, and 31 kDa seem to be derived from complexes, and a 30 kDa spot shows temper-

Proteins in Neisseria Strains

Figure 2. Diagonal SDS-PAGE analysis of the outer membrane proteins from N. lactamica NlP3. Second dimension was run after nondenaturing (A) or denaturing (B) treatment of first dimension strips. One-dimension gels run after denaturing treatment are located at right. In (A), putative complexes are pointed inside rectangles. In (B), spots derived from dissociation of complexes are pointed inside circles and those with temperature-dependent mobility are pointed inside rectangles. Molecular weights (MW) of some proteins are indicated.

ature-dependent reduction in mobility. The same pattern was observed for the other N. lactamica strain analyzed (not shown). Detection of the antigenic proteins with anti-OMV serum after transferring from the SDS-PAGE gels to PVDF membranes revealed that the main meningococcal complex-forming antigens are the 43, 38, and 36 kDa proteins (Figure 3A), whereas the most noticeable antigen showing temperature reduced mobility corresponds to the 30 kDa protein. Identification of some of these antigens with monoclonal antibodies demonstrates that the 43 kDa antigen is PorA, the 38 kDa antigen is PorB, the 36 kDa antigen is RmpM, and the 30 kDa antigen is an Opa-family protein (Figure 3). Note that several antigenic spots (located in horizontal rows, except for the Opa) can be observed for each protein after electrophoresis in denaturing conditions. Analysys of these individual spots by MALDI-TOF peptide mass fingerprinting confirmed their identification as PorA, PorB, RmpM, and Opa.

Discussion Porins are the most abundant neisserial outer membrane proteins, forming trimeric associations to build membrane pores whose main physiological role is the exchange of ions and probably some other molecules. In the pathogenic Neisseria, porins also act as adjuvants, activating antigenpresenting cells, interact with some components of the complement cascade, and seem to modulate apoptosis through interaction with the mitochondrial membranes.13

research articles Most studies on the structure of neisserial porins to date have been done using artificial systems using recombinant porins (either PorA or PorB) and synthetic membranes, or with mutants defective in one of the two porins,5,14,15 and very few studies have been done with native porins.3,4 There is a general agreement that neisserial porins occur as homotrimeric structures formed by either PorA or PorB (stabilized by association with RmpM), and scarcely ever has been suggested that pores could be formed by heterotrimeric associations of PorA and PorB subunits.16 Our results in this study suggest that the main detectable protein complexes in the meningococcal membranes have a molecular mass of 95 kDa and are formed by the proteins PorA (43 kDa), PorB (38-42 kDa) and RmpM (36 kDa) in N. meningitidis, and by PorB (38 kDa) and RmpM (31 kDa) in N. lactamica. The lower molecular mass of N. lactamica RmpM has been reported in a previous study.17 Although it has been reported that the most abundant meningococcal porin complexes are formed by PorB trimers, their high susceptibility to denaturation by SDS even at low temperatures hampers their detection in our experiments.4,15 We did not detect complexes formed by only PorA in any of the N. meningitidis strains tested, which is in disagreement with the proposed structure of the pores formed by either PorA or PorB homotrimers. Theoretically, PorA homotrimers should have a 129 kDa molecular mass, whereas PorB homotrimers sould be between 114 and 126 kDa (molecular mass of PorB is strain-dependent). If only one RmpM molecule were associated to stabilize the pores, then the molecular masses shoud increase by 36 kDa, resulting in 165 kDa for PorA complexes, and between 150 and 162 kDa for PorB complexes, but we did not find spots with these masses that dissociate after denaturation, which suggests that the mobility of complexes must be higher due to compactness of their structure. It is possible that the 95 kDa complex detected in nondenaturing conditions could be a mixture of PorA homotrimers and PorA/PorB heterotrimers (associated with RmpM), but this can be only true if, due to conformational peculiarities, both complexes have a very compact structure and the same apparent electrophoretic mobility despite their different molecular masses. In any case, it is not possible that PorB homotrimers locate in the same 95 kDa spot because, as already commented, they dissociate in our experimental conditions. Consequently, we suggest that some meningococcal outer membrane porin complexes are formed by PorA/PorB heterotrimers. However, our results agree with the existence of PorB as the only pore-forming protein in N. lactamica,18 and suggest that PorB pores in this species are more stable to SDS denaturation than in N. meningitidis. It is noteworthy that two different porin complexes, formed by the same proteins, can be detected with our system in the N. meningitidis strains. The first ones correspond to the 95 kDa spot, whereas the second ones have higher molecular masses and, probably, are formed by aggregates of porins and other proteins located in “membrane islands” in the native membranes (or formed by autoaggregation during the solubilization process) that do not enter the gel in nondenaturing conditions. The four spots with 230, 190, 95, and 72 kDa molecular masses detected at the leftmost side in the nondenaturing gels could correspond to these aggregates in a partially dissociated state. It is also interesting to note that the monoclonal antibodies used to detect PorA, PorB, and RmpM do not react with the complexes, suggesting that the target epitopes are not externally Journal of Proteome Research • Vol. 4, No. 1, 2005 93

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Figure 3. Western blots of meningococcal outer membrane antigenic proteins separated by diagonal electrophoresis after treatment of first dimension strips in nondenaturing (A and B) or denaturing (C and D) conditions for the second dimension run. A and C blots were revealed with anti-N. meningitidis M982 OMVs mouse antiserum. B and D blots were sequentially revealed with anti-PorA, antiporB, anti-RmpM, and anti-Opa monoclonals.

accessible. We can also deduce from our results that, besides the porins, some other proteins must be forming complexes that resolve in the 70, 60, 55, and 20 kDa spots located under the diagonal after denaturation. These proteins have not been identified in this study. With regard to the proteins that appear over the diagonal, they must be produced by either breakage of intramolecular disulfide bonds or by temperature-induced structural relaxation, leading to molecules with lower mobility and, consequently, an apparently higher molecular weight.11 The best known proteins of this type in the Neisseria are a family of virulence-associated outer membrane opacity proteins (Opa), specialized in host-cell recognition and tropism, attachment, colonization of mucosal surfaces and, possibly, invasion of specific host-cells during infection. They are integral outer membrane components whose expression is controlled by phase-variation mechanisms in which a variable number of genes are implicated (three or four opa genes in the meningococci and up to twelve genes in the gonococci). Frequent phase shifts result in natural populations in which individual cells can express different combinations of Opa proteins, whose molecular weight varies between 20 and 32 kDa. They have a basic isoelectric point, a trimeric structure, and expression and antigenic hypervariability.2 We have shown in this study that diagonal electrophoresis is very useful for the detection these proteins. It can be seen that, besides the Opa, the meningococci have at least two other proteins (95 and 50 kDa spots) with temperature-dependent behavior in SDS-PAGE. The 43 kDa spot detected over the diagonal are folded monomers of PorA,3 as can be demonstrated by its reaction with the anti-PorA mAb and MALDITOF analysis. It is very interesting that detection of Opa with the monoclonal antibody reveals four Opa forms in the strain N. 94

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meningitidis M982, some of which do not experiment temperature-induced mobility shifts and whose molecular masses are coincident with some other minor proteins, which makes their identification difficult using one-dimensional SDS-PAGE. The number of Opa forms in the other N. meningitidis strains studied was found to be variable from none to four (data not shown). In conclusion, diagonal two-dimensional SDS-PAGE in which different sample treatments are used for each dimension has demonstrated to be an improved, reliable, easy and quick method for the detection of multiprotein complexes, temperature-induced dissociation of proteins, or temperature-driven protein conformation changes. In our case, this technique allowed the demonstration of PorA/PorB heteromeric porin complexes, resulted in a good resolution of the Opa proteins, and evidenced some other proteins with temperature-induced mobility variations.

Acknowledgment. We wish to thank Dr. Andrew Gorringe, from the Health Protection Agency (Porton Down, Great Britain) for his helpful suggestions for the improvement of the work and for his aid in the revision of the manuscript. This work was supported by Grant PGIDT01BIO20301PR from the Consellerı´a de Innovacio´n, Industria e Comercio from the Xunta de Galicia, Spain. References (1) Poolman, J. T.; Feron, C.; Dequesne, G.; Denoe¨l, P. A.; Dessoy, S.; Goraj, K. K.; Janssens, D. E.; Kummert, S.; Lobet, Y.; Mertens, E.; Monnom, D. Y.; Momin, P.; Pe´pin, N.; Ruelle, J. L.; Thonnard, J. J.; Verlant, W. G.; Voet, P.; Berthet, F. X. Outer membrane vesicles and other options for a meningococcal B Vaccine. In: Ferreiro´s, C. M., Criado, M. T., Va´zquez, J., Eds.; Emerging Strategies in the Fight Against Meningitis: Molecular and Cellular Aspects; Horizon Scientific Press: Norfolk, UK, 2002, 135-150.

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Proteins in Neisseria Strains (2) Hauck, C. R.; Meyer, F. Small talk Opa proteins as mediators of Nesseria-host-cell communication. Curr. Opin. Microbiol. 2003, 6, 43-49. (3) Jansen, C.; Wiese, A.; Reubsaet, L.; Dekker, N.; de Cock, H.; Seydel, V.; Tommassen, J. Biochemical and biophysical characterization of in vitro folded outer membrane porin PorA of Neisseria meningitidis. Biochim. Biophys. Acta 2000, 1464, 284-298. (4) Minetti, C.-A.; Blake, M. S.; Remeta, D. P. Characterization of the structure, function and conformational stability of PorB class3 protein from Neisseria meningitidis. A porin with unusual physicochemical properties. J. Biol. Chem. 1998, 273, 25329-25338. (5) Song, J.; Minetti, C. A.; Blake, M. S.; Colombini, M. Successful recovery of the normal electrophysiological properties of PorB (Class 3 porin) from Neisseria meningitidis after expression in Escherichia coli and renaturation. Bochim. Biophys. Acta 1998, 1370, 289-298. (6) Malorny, B.; Morelli, G.; Kusecek, B.; Kolberg, J.; Achtman, M. Sequence diversity, predicted two-dimensional structure and epitope mapping of neisserial Opa proteins. J. Bacteriol. 1998, 180, 1323-1330. (7) Pintor, M.; Ferro´n, L.; Go´mez, J. A.; Gorringe, A.; Criado, M. T.; Ferreiro´s, C. M. Blocking of iron uptake by monoclonal antibodies specific for the Neisseria meningitidis transferrin binding protein 2. J. Med. Microbiol. 1996, 45, 252-257. (8) Go´mez, J. A.; Criado, M. T.; Ferreiro´s, C. M. Bactericidad activity of antibodies elicited against the Neisseria meningitidis 37 kDa ferric binding protein (FbpA) with different adjuvants. FEMS Immunol. Med. Microbiol. 1998, 20, 23-30. (9) Smith, P. K.; Krohn, R. I.; Hermason, G. T.; Mallia, A. K.; Gartner, F. M.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Clenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 76-85. (10) Brodeur, B. R.; Larose, Y.; Tsang, P.; Hamel, J.; Ashton, F.; Ryan, A. Protection against infection with Neisseria meningitidis group

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