Identification of Neisseria meningitidis Outer Membrane Vesicle

Identification of Neisseria meningitidis Outer Membrane Vesicle Complexes Using 2-D High Resolution Clear Native/SDS-PAGE Juan Marzoa, Sandra Sańchez, Carlos M. Ferreiro ´ s, and Marıá Teresa Criado* Departamento de Microbiologıá y Parasitologıá, Facultad de Farmacia, Campus Sur, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain Received July 20, 2009

Abstract: The identification and characterization of meningococcal outer membrane vesicle complexes can be important for gaining an in-depth understaining of their structure and functionality. Analysis of the vesicle complexome by ‘traditional’ 2-D analysis, in which isoelectrofocusing is used for separation in the first dimension, is hampered by the high hydrophobicity and extreme isoelectric points of many relevant proteins. Analysis of the meningococcal outer membrane vesicle complexome using Blue Native (nondenaturing) electrophoresis instead of isoelectrofocusing in the first dimension showed several porin complexes, but their composition could not be clearly resolved after separation by SDS-PAGE in the second dimension. In this work, using a recently described native separation technique shigh resolution Clear Native Electrophoresissand different bidimensional approaches, we were able to demonstrate the presence of relevant outer membrane complexes which could be resolved with a higher resolution than in previous analysis. The most relevant were nine porin complexes formed by different combinations of the meningococcal PorA, PorB and RmpM proteins, and comparison with the complexes formed in specific knockout mutants allowed us to infer the relevance of each porin in the formation of each complex. Keywords: Neisseria meningitidis • outer membrane proteome • high resolution clear native electrophoresis • outer membrane complex.

Introduction The search for effective meningococcal antiserogroup B vaccines is currently focused in outer membrane (OM) proteins that can show high antigenic cross-reactivity and a low interstrain variability. The main antigenic OM proteins, PorA and PorB1-4 have been extensively studied, but up to date, their antigenic variability is an important drawback for the design of a widely effective vaccine based on them. Attempts to obtain vaccines using conserved minor surface antigens5 have not resulted in a good vaccine candidate, and the search for effective antigens (or antigen combinations) still continues.6 The detection and identification of epitopes of conformational * To whom correspondence should be addressed. E-mail: mteresa.criado@ usc.es. Tel. no.: +34 981 563100, ext 14946. Fax no.: +34 981 594631. 10.1021/pr9006409

 2010 American Chemical Society

and/or shared nature that can form in OM complexes, which could be important or even critical for immune responses,7-10 has been hampered by the use of denaturing analytical techniques, such as 1-D SDS-PAGE or 2-D IEF/SDS-PAGE, which are the most employed methods for protein separation, previous to Western blotting, in almost all studies for antigen detection in Neisseria meningitidis outer membranes. Blue native polyacrylamide gel electrophoresis (BN-PAGE)11 is one of the best gel systems for the analysis of protein complexes under nondenaturing conditions. In this technique, the anionic dye Coomassie G-250 is used to confer, under physiological conditions, a negative charge to the complexes proportional to their size, avoiding the dissociation of subunits and, thus, retaining their native structure. Nevertheless, although we found that 1-D BN-PAGE produces a good resolution of OM meningococcal complexes,12 the high amount of Coomassie stain associated to them hinders a good transfer and detection of the antigenic proteins by Western blotting. Also, our attempts to obtain 2-D proteome maps from the BNPAGE gels, using SDS-PAGE in the second dimension, resulted in a poor resolution of the components of some relevant complexes due to an important lateral diffusion of many protein spots after the second dimension run, particularly the porins.12 A recent work by Wittig et al.13 described a new technique that they named high resolution clear native electrophoresis (hrCNE) in which the good resolution of complexes attained in BN-PAGE is combined with the excellent preservation of the structure and function of complexes brought off by clear native electrophoresis (CNE), also overcoming the interferences produced by the Coomassie dye in subsequent analyses, and allowing an excellent separation of membrane complexes in native conditions, making it optimal for functional proteomic analysis. In hrCNE, the Coomassie dye is omitted from the sample and substituted in the cathode buffer by a mixture of two detergents, one of them neutral and the other anionic. The mixed micelles formed by both detergents confer the charge needed for the electrophoretic migration of the complexes, which are then separated on the basis of their size by the acrylamide gradient in the gel. Using different anionic and neutral detergent mixtures allows the analysis of complexes with differing stability. So, the alternative use of n-dodecyl-βD-maltoside (DDM), Triton X-100 or digitonin could result in different complex patterns, even allowing the detection of supercomplexes in the membrane structure. The technique has Journal of Proteome Research 2010, 9, 611–619 611 Published on Web 11/04/2009

technical notes been highly successful for the separation of physiologically active mitochondrial complexes14 and we think that it could be also applied to obtain a high resolution analysis of the OM complexome of N. meningitidis, as a precursor step in the study of conformational and/or shared epitopes that could form due to the interaction of the components of the complexes. The aim of this work is the analysis of meningococcal outer membrane vesicles (OMVs) using hrCNE/SDS-PAGE 2-D electrophoresis. An in-depth knowledge of the outer membrane complexome will help to advance in the knowledge their structure and physiology, which could help to understand the possible role of interactions between the OM proteins (the main meningococcal surface antigens) in the immune response against this pathogen.

Material and Methods Strains and Culture Conditions. The strain N. meningitidis H44/76 (B:15:P1.7,16:F3-3) and its homologous mutants lacking PorA, PorB, RmpM or FetA were kindly donated by Dr. Ian Feavers (National Institute for Biological Standards and Control, Great Britain). Strain maintenance and culture were carried out as described previously.15 All cultures were done under iron restriction in Mueller-Hinton broth with 100 µM Desferal added (MH-Desferal), and cells were recovered for extraction of OMVs when in exponential growth phase (12 h). Outer Membrane Vesicle Preparations. Meningococcal OMVs were obtained using a modification of a previously described protocol.15 Bacteria recovered from MH-Desferal cultures were suspended in distilled water at 250 mg/mL (wet weight) and processed three times in a French pressure cell at 1.1 × 108 kPa. Cellular debris was removed by centrifugation at 10000g for 10 min at 4 °C, and OMVs were recovered at 200 000g (10 min, 4 °C), suspended in distilled water and stored at -80 °C. Concentration of OMVs in samples was estimated from the protein content calculated using the Bicinchoninic acid (BCA) protein assay.16 High Resolution Clear Native Polyacrylamide Gel Electrophoresis (hrCNE). hrCNE analyses were done following a modification of a previously described protocol.17 Gradient gels (5-15% or 8-11% polyacrylamide in 50 mM Bis-Tris/500 mM 6-aminohexanoic acid) were used for separation of the complexes in a Mini-Protean 3 Cell (BioRad Laboratories S.A., Spain). Gels can be precast and maintained for up to 3 days at 4 °C in sealed bags containing 50 mM Bis-Tris/500 mM 6-aminohexanoic acid. Just before running, a 4% polyacrylamide stacking gel was cast over the gradient gels. Anode buffer was 50 mM Bis-Tris-HCl (pH 7.0). Cathode buffer (pH 7.0) contained 50 mM Tricine, 15 mM Bis-Tris-HCl, 0.05% (w/v) sodium deoxycholate and 0.02% (w/v) n-dodecyl-β-D-maltoside (DDM). For some experiments, 0.05% (v/v) Triton X-100 was used instead of DDM in the cathode buffer. For the analyses, OMV stocks were diluted to 1 mg/mL in water; then, 100 µL of 50 mM Bis-Tris-HCl (pH 7.0)/1 M 6-aminohexanoic acid and 45.5 µL of 10% (w/v) DDM were added to each milliliter of OMVs (final concentrations were 4.4 mM Bis-Tris-HCl, 87.3 mM 6-aminohexanoic acid, 0.4% DDM, 873 µg/mL OMVs; 4.6:1 DDM/protein ratio). Complexes were solubilized by incubation at room temperature for 30 min; thereafter, insoluble material was removed by centrifugation at 100 000g for 15 min at 4 °C. Before loading the gel, 5% (v/v) glycerol and 0.01% (w/v) Ponceau red were added to each sample. Each lane was loaded with 20 µg (24 µL) of solubilized OMVs and electrophoretic separation was done at 4 °C. Power 612

Journal of Proteome Research • Vol. 9, No. 1, 2010

Marzoa et al. was set to 50 V constant voltage for the first hour and 100 V constant voltage for about 6 h. Because of differences in the mobility of the complexes depending on the detergent used in the cathode buffer (DDM or Triton X-100), Kaleidoscope Standards (Bio-Rad Laboratories S.A., Spain) were used to standardize the total running time, which was considered complete when the front of the standards reached the bottom of the gel. High molecular weight markers (HMW Native; 66-669 kDa; GE Healthcare Espan ˜ a S.A., Spain) were used to estimate the molecular weight of the complexes. Gels were fixed with trichloroacetic acid and stained with Coomassie blue G-250 using a standard protocol. Bidimensional Analyses: hrCNE/SDS-PAGE and hrCNE/ hCNE Diagonal Electrophoresis. Bidimensional analyses to identify the components of membrane complexes were done using SDS-PAGE in the second dimension (hrCNE/SDS-PAGE). Lanes cut from hrCNE gels (first dimension) were incubated in SDS sample buffer for 10 min at 95 °C and then placed on top of 12% polyacrylamide gels in a Mini-Protean 3 Cell (BioRad Laboratories S.A., Spain). Electrophoretic separation was carried out at 150 V constant voltage, and gels were stained with Coomassie and analyzed using PDQuest software (BioRad Laboratories S.A., Spain). To determine the influence of detergents in the separation and stability of the complexes, 2-D hrCNE/hCNE diagonal analyses were done using DDM in the cathode buffer for the first dimension run and Triton X-100 for the second, and vice versa. Conditions for separation in both dimensions was done as described above for the 1-D hrCNE analyses. Image Analysis and Protein Identification. The analysis of complexes in 1-D separations and that of proteins in 2-D maps, and calculation of their molecular weights were carried out using the Quantity One (1-D) and PDQuest (2-D) analysis software (Bio-Rad Laboratories S.A., Spain) after scanning of stained gels at high resolution (1200 dpi). Identifications of the components of the complexes resolved by 1-D hrCNE and that of the components of the complexes resolved by 2-D diagonal hrCNE/hCNE were done at the CIC bioGUNE (Technology Park of Bizkaia, Derio, Spain) by NanoLC/MS-MS analysis of the bands cut from the gels. Identification of the proteins associated to each complex after 2-D hrCNE/SDS-PAGE was done at the Servicio de Espectrometrıá de Masas (University of Valencia, Spain) by MALDI-TOF fingerprinting analysis of the spots cut from Coomassie-stained gels. Mass spectrometry (MS) analyses were done using a Reflex IV MALDI-TOF-MS analyzer spectrometer (Bruker Daltonics, Spain). FlexControl 2.4 software (Bruker) was used for control of the instrument and FlexAnalysis 2.4 software (Bruker) for evaluation of the spectra. After trypsin digestion, each sample (0.5 µL) was deposited on an AnchorChip MTP384-600 plate (Bruker), and consecutively covered with 0.5 µL of an R-cyano4-hydroxycinnamic acid (HCCA) matrix. Peak lists were generated from a minimum of 200 shots for acquisition of data. Signal to noise (S/N) ratio was 0.1, and the smoothing Savitzky Golay algorithm (one cycle, width 0.2 m/z) was applied. Background threshold for subtraction of baseline was set using a median algorithm (flatness 0.8), and the percentage peak height used for centroiding was determined using the SNAP algorithm (included in the FlexAnalysis software). Mascot v. 2.2.06 (www.matrixscience.com) was used as the search engine. Peptide tolerance was set to 50-100 ppm and resolution for all MS modes was m/z 1619.82 > 10 000. All

Meningococcal Outer Membrane Complexome

technical notes

Figure 1. Diagonal hrCNE/hCNE analysis of meningococcal OMVs using DDM in the cathode buffer for native separation in the first dimension, and Triton X-100 in the second one (left panel, top; DDM/TX) and vice versa (left panel, bottom; TX/DDM). Right panel shows the Gaussian representations of the main porin complexes, obtained by image analysis. Bottom image (B) was flipped horizontally and then turned 90° clockwise before superposition (C) over upper image (A). White ellipses show the porin spots in the DDM/TX diagonal gel, and gray ellipses indicate those found in the TX/DDM diagonal gel. Numbers correspond to complexes Cx1-Cx9 in Table 1. D1-D10 and T1-T10 designate the main complexes detected in the 1-D hrCNE analyses using DDM or Triton X-100, respectively.

fragmentation spectra were searched against the MSDB and Swiss-Prot databases (7 078 992 and 510 076 entries, respectively).

Results and Discussion In a previous work, we analyzed the N. meningitidis outer membrane vesicle proteome using a 2-D approach in which native BN-PAGE was used for separation in the first dimension and denaturing SDS-PAGE in the second to investigate the presence and subunit composition of putative complexes.12 Several complexes were detected after native separation in the first dimension, but resolution of their components after the second dimension run was highly impaired due to a strong lateral diffusion that limited the assignation of individual spots (proteins) to each complex. Consequently, in this work, we used a recently described high resolution native technique13 instead of BN-PAGE for separation in the first dimension run. The outer membrane vesicles used for this study were obtained from cultures done under iron restriction to induce the expression of some important proteins that are regulated by this metal. The vesicles were obtained from the cells using a French Press

and ultracentrifugation to eliminate cytosolic proteins. Analyses done in previous works to compare the composition of these vesicles with those obtained by treatment with sodium deoxycholate (the method employed for preparation of OMV-based meningococcal vaccines) demonstrated that OMVs produced using the French Press contain substantially less cytoplasmic proteins and are similar to native OMVs released during growth.12,18-21 Detection of Complexes Using High Resolution Clear Native Electrophoresis. Analysis of the meningococcal OMVs using hrCN allowed the detection of apparently different bands depending on the detergent (DDM or Triton X-100) used in the cathode buffer for electrophoresis (Figure 1). Three bands of 740, 540, and 470 kDa (D1-D3, and T1-T3) showed similar apparent weights independently of the detergent used. Seven relevant bands (D4-D10 and T4-T10) with lower molecular weights seemed to be different on the basis of their apparent mobility (between 140 and 220 kDa when using DDM, and between 230 and 470 kDa when using Triton X-100). In both cases, a variable number of lower molecular weight bands were also observed. These bands were cut and analyzed by LC/MSJournal of Proteome Research • Vol. 9, No. 1, 2010 613

technical notes

Marzoa et al.

Table 1. LC/MS-MS Identification of the Components of the Main Complexes Revealed Using 2-D Diagonal hrCNE/hCNE Electrophoresisa DDM MW (kDa)

Triton X-100

complex

band

band

CxChap CxGlu CxKet

D1 D2 D3

740.0 540.0 470.0

T1 T2 T3

740.0 540.0 470.0

Cx1 Cx2 Cx3

D10 D9 D8

145.0 154.0 170.0

T10 T10 T9

390.0 390.0 360.0

Cx4

D9

154.0

T8

320.0

Cx5

D7

175.0

T8

320.0

Cx6

D6

186.0

T7

310.0

Cx7

D5

200.0

T6

300.0

Cx8

D5

200.0

T5

250.0

Cx9

D4

216.0

T4

390.0

ID code components of the complex

MW (kDa)

60 kDa haperonin glutamine synthetase ketol-acid reductoisomerase PorB PorB PorA PorB PorA PorB PorA PorB PorA PorB PorA PorB PorA PorB RmpM PorA PorB RmpM

peptides

score (XC)

∆ CN

sequence coverage (%)

19 4 4

5.34 3.41 5.26

0.37 0.35 0.47

35 9 34

NMB_1972 NMB_0359 NMB_1574

AAF42301.1 AAF40802.1 AAF41927.1

24 20 3 16 5 10 12 15 6 16 11 13 3 15 3 11 15 3

5.27 5.09 4.70 5.51 4.94 4.47 5.46 4.60 4.62 4.76 4.36 3.99 4.02 4.87 3.65 5.32 5.61 2.32

0.36 0.47 0.55 0.48 0.49 0.41 0.48 0.46 0.46 0.36 0.45 0.42 0.42 0.58 0.23 0.50 0.48 0.38

68 76 12 64 18 39 41 57 22 50 39 48 11 48 17 33 46 12

NMB_2039 NMB_2039 NMB_1429 NMB_2039 NMB_1429 NMB_2039 NMB_1429 NMB_2039 NMB_1429 NMB_2039 NMB_1429 NMB_2039 NMB_1429 NMB_2039 NMB_0382 NMB_1429 NMB_2039 NMB_0382

AAF42360.1 AAF42360.1 AAF41790.1 AAF42360.1 AAF41790.1 AAF42360.1 AAF41790.1 AAF42360.1 AAF41790.1 AAF42360.1 AAF41790.1 AAF42360.1 AAF41790.1 AAF42360.1 AAF40822.1 AAF41790.1 AAF42360.1 AAF40822.1

TIGR

GenBank

a Naming of the complexes corresponds to that used in Figure 1. Sequence coverages and database IDs are those corresponding to the homologous genes in the N. meningitidis MC58 strain. Correspondence with the bands detected in 1-D hrCNE analyses is shown in columns 2 and 3.

MS to determine the composition and identify the putative complex components. As shown in Table 1 the first three bands were identified as homomeric protein complexes formed by the 60 kDa chaperonin (MSP63), the glutamine synthetase and the ketol-acid reductoisomerase, respectively (independently of the detergent used in the cathode buffer). The other seven relevant complexes mentioned above all contained meningococcal porins. The major outer membrane protein PIB (PorB) was present in all of them, most also contained the major outer membrane protein PorA, and the outer membrane protein class 4 (RmpM) was detected in two of them. To determine the possible equivalence between bands D4-D10 and T4-T10, we analyzed the OMVs in a 2-D diagonal hrCN/ hrCN electrophoresis using DDM for separation in the first dimension and Triton X-100 in the second one, and vice versa. The results obtained (Figure 1) showed that some of the bands are formed by mixtures (or associations) of at least two complexes, resulting in nine different individual complexes. They were named Cx1-Cx9 and the identification of their component proteins (by LC/MS-MS) is also shown in Table 1. As can be seen, all these complexes had different mobilities depending on the sequence of detergents used for the diagonal hrCN/hCN diagonal electrophoresis analyses (they appeared either above or below the diagonal in the gels). Image analyses and the results of the LC/MS-MS analyses of these complexes (Figure 1, right side; Table 1) allowed to demonstrate that they are exactly the same despite the differences in their mobility and, consequently, in their apparent molecular weight. In the first dimension analysis, some of these individual complexes are coincident in only one band, depending on the detergent used. For instance, when the hrCN separation in the first dimension is done using DDM, complexes Cx2 and Cx4 appear in band D9, whereas Cx3 and Cx5 appear in band D8. On the contrary, when Triton X-100 is used 614


for the first dimension hrCN separation, complex Cx2 appears with Cx1 in band T10, and Cx4 appears with Cx5 in band T8. Identification of the components of complexes Cx1-Cx9 by LC/ MS-MS showed that all them contain the PorB protein (Cx1 and Cx2 being homomeric), seven contain also the PorA protein (Cx3-Cx9), and the RmpM protein is also present in two of them (Cx8 and Cx9). Notwithstanding the LC/MS-MS results did not show the presence of other proteins associated to these complexes, we think that this possibility should not be discarded in view of the results obtained in the 2-D hrCN/SDS-PAGE analyses that will be commented below; furthermore, LC/MS-MS identifications done in replicates of the 2-D gels using different gradients and/or variations in the concentrations of the detergents in the cathode buffers (results not shown) suggest the association of other proteins to some of the porin complexes, such as the transferrin-binding protein A or the Fe-regulated protein B. This has not been reflected in Table 1 due to the low significance of the scores for the corresponding peptides in the LC/MS-MS analyses. Analysis of Complexes Using 2-D hrCN/SDS-PAGE Electrophoresis. The 2-D electrophoretic analysis of OMVs using nondenaturing hrCN in the first dimension and denaturing SDS-PAGE in the second allows the attainment of proteomic maps in which, theoretically, the spots aligned vertically correspond to the individual proteins forming each complex. Figure 2 shows the proteomic maps obtained using either DDM or Triton X-100 in the cathode buffer for separation under native conditions in the first dimension. Identification by MALDI-TOF fingerprinting analysis of the main spots observed is shown in Tables 2 and 3. Table 2 lists the spots identified as proteins whose predicted location is the outer membrane, the periplasm, and outer membrane-associated proteins, or proteins that have been consistently found in OMV preparations

Meningococcal Outer Membrane Complexome

technical notes

Figure 2. Bidimensional hrCNE/SDS-PAGE analysis of OMVs using DDM (top; DDM/SDS) or Triton X-100 (bottom; TX/SDS) in the cathode buffer for native separation in the first dimension. All spots identified by MALDI-TOF and recognized as proteins associated to the outer membrane are indicated with numbers and their identification is referenced in Table 2. Proteins appearing as multiple spots are boxed.

in other studies18-21 Table 3 lists the spots identified as proteins that are described to be cytoplasmic in public databases (SwissProt). We analyzed a total of 151 spots selected on the basis of the results obtained from five replicates and on our previous results obtained in BN/SDS-PAGE analyses.12 Eighty of the 151 spots were unequivocally identified using MALDI-TOF (45 from the 2-D gels obtained using DDM and 35 from the gels obtained using Triton X-100). Because of the coincidence of several of the spots in the two gels and to the presence of multiple spots (trails) for some of the proteins, a total of 46 different proteins were distinguished. Twenty-nine of them (marked in Figure 2 and listed in Table 2) are those located or associated to the outer membrane. Only 17 of them were found in both gel types,

nine only in gels obtained with DDM and three only in those with Triton X-100. Some proteins that have been reported to be associated forming complexes could not be shown as such depending on the detergent used for separation in native conditions, which demonstrates the importance of a thorough search for an appropriate selection of the detergents used in the cathode buffer for the analysis of particular complexes. One example is that formed by the transferrin-binding proteins A and B (TbpA and TbpB), which were detectable as a complex when using DDM, but not (only the TbpA could be identified) when using Triton X-100. The only complexes that could be invariably detected independently of the detergent used are those that we named CxChap, CxGlu, CxKetol, as well as all porin Journal of Proteome Research • Vol. 9, No. 1, 2010 615

technical notes

Marzoa et al. a

Table 2. MALDI-TOF Identification of the Main Spots Detected Using hrCNE/SDS-PAGE Electrophoresis spot

identification

score


m/ub

TIGR IDc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Conserved hypothetical protein Trasferrin-binding protein A Trasferrin-binding protein B Outer membrane protein P64k Glutamate-ammonia ligase Fe-regulated protein B 60 kDa chaperonin Copper-containing nitrite reductase Glutamine synthetase PorA Ketol-acid reductoisomerase PorB RmpM Macrophage infectivity potentiator pilS cassette Outer membrane lipoprotein ATP synthase F0, B subunit Class 5 outer membrane protein H.8 outer membrane protein Opacity protein Opacity protein Thiol:disulfide interchange protein dsbA (dsbA-2) Outer membrane lipoprotein Opacity-related protein POM1 IgA-specific metalloendopeptidase Conserved hypothetical protein Periplasmatic s/p-binding protein Thiol:disulfide interchange protein dsbA (dsbA-1) Multidrug efflux pump channel protein

83 185 58 88 135 281 103 43 193 54 157 144 120 156 58 44 75 60 46 107 63 119 53 111 134 74 75 103 127

32 30 15 20 31 41 23 20 43 22 37 57 45 50 32 24 50 27 32 41 39 45 22 39 49 29 21 52 31

10/35 23/25 10/38 12/25 14/26 27/21 11/9 6/25 23/37 6/28 16/25 12/22 12/33 17/31 7/47 5/30 7/36 8/53 7/28 11/39 8/60 14/36 5/19 11/36 13/30 11/37 9/27 12/40 12/18

NMB_0035 NMB_0461 NMB_0460 NMB_1344 NMB_0359 NMB_1988 NMB_1972 NMB_1623 NMB_1710 NMB_1429 NMB_1574 NMB_2039 NMB_0382 NMB_1567 NMB_0024 NMB_1946 NMB_1938 NMB_1053 NMB_1533

GenBank IDc

NMB_0294 NMB_1946

NMB_1652 NMB_0462 NMB_0278 NMB_1714

molecular weight (kDa)

AAF40506.1 AAF40898.1 AAF40897.1 AAF41719.1 AAF40802.1 AAF42315.1 AAF42301.1 AAF41975.1 AAF42057.1 AAF41790.1 AAF41927.1 AAF42360.1 AAF40822.1 AAF41921.1

42.3 102.1 77.4 61.8 52.1 79.1 57.4 40.7 48.5 42.1 36.4 35.7 26.2 28.9 22.3 31.2 17.1 29.9 18.5 27.0 26.3 25.4 31.2 28.9 175.9 46.4 44.4 25.2 50.5

AAF42275.1 AAF42267.1 AAF41451.1 AAF41888.1 (O30756_NEIME) (O30753_NEIME) AAF40745.1 AAF42275.1 (OPR1_NEIMC) (AAW89025.1) AAF42001.1 AAF0899.1 AAF40732.1 AAF42061.1

a Numbers correspond to those maked in Figure 2. Only spots reported to be located or associated to membranes are listed. Sequence coverage, database IDs and molecular weights are those corresponding to the homologous genes/proteins in the N. meningitidis MC58 strain. b m/u, matched/unmatched. c Missing IDs indicate no annotation for a homologous gene in strain MC58 in public databases. IDs in parentheses indicate homologous genes in strains different from MC58 and not described in that strain.

Table 3. MALDI-TOF Identification of Spots Detected Using hrCNE/SDS-PAGE Electrophoresis That Are Not Described to Be Associated to the Meningococcal Outer Membranea identification

biopolymer tranport protein ExbB hypothetical protein phosphatidylserine decarboxylase precursor-related protein lacto-N-neotetraose biosynthesis glycosyltransferase integrase/recombinase XerC hypothetical protein transposase alcohol dehydrogenase, zinc containing glycosyltransferase lactate dehydrogenase aminopeptidase A argyninosuccinate lyase IMP dehydrogenase pyruvate dehydrogenase hypothetical protein polyribonucleotide nucleotidyltransferase

score


m/ub

TIGR IDc

GenBank IDc

molecular weight (kDa)

48 70 63

25 30 33

7/34 8/36 8/31

NMB_1729 NMB_1870 NMB_0963

AAF42074.1 AAF42204.1 AAF41369.1

24.1 28.9 28.9

56

36

9/82

NMB_1926

AAF42255.1

32.8

45 103 67 104 56 71 49 56 273 95 112 100

26 36 34 35 31 22 20 22 64 31 33 28

8/53 10/23 10/65 11/22 10/76 7/10 7/30 9/36 24/18 11/26 15/28 17/47

NMB_1868 NMB_0086 NMB_1399 NMB_0604 NMB_0218 NMB_1377 NMB_1569 NMB_0637 NMB_1201 NMB_1342 NMB_1345 NMB_0758

AAF42202.1 AAF40549.1 AAF41763.1 AAF41031.1 AAF40674.1 AAF62327.1 (CAB84986) AAF41060.1 AAF41583.1 AAF41717.1 AAF41720.1 AAF41171.1

34.0 36.8 37.3 37.9 42.1 43.1 49.7 51.2 52.4 55.2 57.1 76.4

a Sequence coverage, database IDs and molecular weights are those corresponding to the homologous genes in the N. meningitidis MC58 strain. matched/unmatched peptides. c ID in paranthesis indicate homologous genes in strains different from MC58.

complexes, despite the different mobility observed for the last, already commented above, and suggests that the assignation of proteins to complexes after 2-D electrophoresis, contrary to the commonly accepted, must not be done only under the assumption that spots of the same shape and located in the same vertical are components of a single complex. The exist616


b

m/u,

ence of these complexes agrees with our previous results obtained using BN/SDS-PAGE12 and SDS/SDS diagonal electrophoresis.22 The different mobility of the porin complexes when using DDM or Triton X-100 is difficult to explain. Results obtained by other authors already demonstrated that different detergents

Meningococcal Outer Membrane Complexome can affect the mobility of individual pure proteins (and consequently their apparent molecular weight) in native electrophoresis.13 Although a rational explanation was not given for this phenomenon, it is conceivable that the association of a protein (or a complex) with different mixtures of detergents could produce micelles with differing sizes and/or charges. When compared with analyses done by other authors,18-21 we found less cytoplasmic proteins in our OMV preparations (less than 30% of the total spots identified), which confirms our previous observation that OMVs obtained using the French Press results in a lower presence of cytoplasmic proteins. It is noteworthy that only two of the 17 cytoplasmic proteins identified were coincident in the 2-D analyses done using DDM and Triton X-100 for separation in the first dimension (Table 3), which suggests that the choice of detergents for native electrophoresis also produces different effects on the presence of cytoplasmic proteins, and these effects are more pronounced than on membrane proteins. Our results also show that some of the proteins, especially the porins, appear in multiple locations forming spot trails. Either they are forming complexes in which the stoichiometry of the subunits must be different, or are associated with other proteins to build different complexes. In view of the evidence that some bands in the 1-D native analyses contain more than one complex, assignation of spots to a particular complex after a single 2-D analyses cannot be done only on the basis of their location in the same vertical and a similar shape of the spots, as already commented. In our study, this would lead to the erroneous conclusion that band D9 is a single PorA/PorB complex, whereas the diagonal analyses shown in Figure 1 demonstrate that band D9 contains a homomeric (PorB) and a heteromeric (PorA/PorB) complexes. The three spots consistently observed, with the same mobility, when using either DDM or Triton X-100 for native separation in the first dimension (CxChap, CxGlu and CxKetol) also behaved similarly in the 1-D hrCN (bands D1-D3 and T1-T3) and in the 2-D diagonal hrCN/hCN analyses, and also in analyses done using OMVs obtained from the four knockout mutants. Complex CxChap, according to its molecular weight (740 kDa), and similar to their orthologues in some other bacteria, could be formed by multimers of the MSP63 protein that aggregate forming two superimposed heptameric rings.23 Although the quaternary structure of the chaperonin complexes can be different in other species such as Escherichia coli, in which the Skp chaperonin associates forming trimers,24,25 its apparent molecular mass is very close to the theoretical mass (882 kDa) of a double heptameric ring structure. Its location in the outer membrane is consistent with the need for a correct conformation of outer membrane proteins after transport and with the fact that sera from individuals convalescent from meningococcal meningitis contain anti-chaperonin antibodies.26 Orthologues of the complex CxGlu have been also analyzed in other bacterial species, finding that the glutamine synthetase molecules associate forming two superimposed hexameric rings27 and are cotranscribed and/or associate with integral membrane proteins implicated in the transport of nitrogenous compounds;28 the molecular mass calculated for our CxGlu complexes suggests that they could have a similar quaternary structure. Orthologues of the CxKetol complex have been also found in other species, being formed by associations of six dimers that produce complexes containing 12 ketol-acid reductoisomerase subunits; again, this structure agrees with the

technical notes

Figure 3. Bidimensional hrCNE/SDS-PAGE analysis of OMVs from knockout mutants lacking some of the pore components (PorA, PorB or RmpM). Analyses in left panel were done using using DDM in the cathode buffer for native separation in the first dimension. Those in right panel were done using Triton X-100. Only the regions containing the pore components are shown.

molecular mass calculated in our experiments. Although the association of the CxGlu and CxKetol complexes with the outer membrane is not in agreement with their reported cytoplasmic location and function, we found them consistently in all 1-D and 2-D analyses, independently of the detergent used for separation in native conditions, and in OMVs from all the homologous mutants analyzed, as can be seen in Figure 3. It is important to remark that these three complexes are present in relatively high amounts and that all our OMV preparations showed a null NADH oxidase activity, which suggests a minimal contamination with cytoplasmic components or inner membrane. Some other spots identified by MALDI-TOF in the 2-D analyses (Table 2) and known to form complexes in N. meningitidis and other species are spot 15, which corresponds to the PilS cassette, implicated in cellular adhesion, and spot 8 (copper-containing nitrite reductase), which has been described as an outer membrane protein that forms homotrimers and is implicated in the growth of bacteria in oxygen-deficient environments. Finally, among the cytoplasmic proteins identified (Table 3), it has been reported that the polyribonucleotide phosphorylase forms homotrimers. All the above data about formation of complexes by the different proteins mentioned has been obtained from the ExPASy Proteomics Server (UniProtKB/Swiss-Prot databases) (www.expasy.ch). It is important to have into account that information about quaternary structures formed by many of the proteins or their subcellular location in the databases is reported ‘by similarity’. For example, for the meningococcal polyribonucleotide nucleotidyltransferase (Q9K062; PNP_NEIMB), the server reports ‘Homotrimer. Organized into a structure containing a number of Journal of Proteome Research • Vol. 9, No. 1, 2010 617

technical notes RNA-processing enzymes (by similarity)’ for its structure, and ‘Cytoplasmic (by similarity)’ for its location. Analysis of Complexes in OMVs from the Homologous Knockout Mutants. The analysis of OMVs from the homologous mutants lacking either PorA, PorB, or RmpM is shown in Figure 3. As can be seen, the main alterations are produced in the porin complexes. In the mutant lacking PorA, an increased formation of homocomplexes containing PorB can be observed, and only one of the two heterocomplexes that contained the RmpM was detectable. In OMVs from the mutant lacking PorB, the effect on the porin complexes was much more marked: the PorA-containing complexes visible in the wild-type strain showed a much lower intensity and, in turn, two high molecular mass PorA homocomplexes (>700 kDa) were formed. This behavior was also observed in a previous study in which BNE was used for the analyses, although only one high molecular mass PorA complex could be resolved in that work.12 In the mutant lacking RmpM, the two RmpM-containing complexes detected in the wild-type strain disappeared, and an increment of the PorB homocomplexes and PorA/PorB heterocomplexes was observed. Also, two new high molecular mass complexes were detectable: a PorA/PorB heterocomplex and a PorB homoclomplex that were much better detected when Triton X-100 is used for separation in native conditions in the first dimension. Finally, in the mutant lacking FetA, the only effect observed in the complexome maps was a loss of the Feregulated protein B, with no effect on the main complexes; apart from this, the complexome map was identical to that of the wild-type strain. The results obtained in this work confirm our previous hypothesis that meningococcal porins associate in multiple complexes, both homomeric and heteromeric, and the participation of the RmpM protein in some of them.12,22 Also, our results agree with our previous observations that PorB has a predominant role in the correct formation of porin complexes in the outer membrane. We were not able in this work to consistently demonstrate the association of other proteins to the porins, so we cannot propose an explanation to the existence of that variety of porin complexes, and more in-depth analysis must be done in order to find an appropriate explanation for that.

Conclussions In this work, we used 1-D hrCN native electrophoresis in combination with LC-MS/MS, and 2-D diagonal hrCN/hCN and hrCN/SDS-PAGE in combination with LC-MS/MS and MALDI-TOF analysis, to identify the main outer membrane vesicle meningococcal complexes. The results obtained allowed the analysis of the complexes with a resolution much higher than that obtained in previous studies performed using BNE for separation in native conditions. We have been able to confirm that the most abundant outer membrane complexes are those formed by the main porins (PorA and PorB), which consistently show forming different associations. The relevance of each porin in the formation of the different complexes could be deduced from the modifications observed in the analysis of outer membrane vesicles from knockout mutants. We think that the hrCN technique performed using different detergents and combined with other separation or analysis techniques can significantly contribute to the study of membrane complexes, not only in N. meningitidis, but also in other bacterial species and cell types. 618


Marzoa et al.

Acknowledgment. We wish to acknowledge CIC bioGUNE (Technology Park of Bizkaia, Derio, Spain; member of CIBER-ehd and Proteored) for the LC-MS/MS and MALDI-TOF analysis. This work was supported by grants PGIDIT05PXIB20302PR from the Xunta de Galicia, and PI050178 from the Fondo de Investigacioń Sanitaria (FIS, Ministerio de Sanidad y Consumo), Spain. References (1) Ferreiro´s, C. M.; Criado, M. T.; Va´zquez, J. Emerging strategies in the fight against meningitis. Mol. Cell. Aspects 2002, 135–150. (2) Girard, M. P.; Preziosi, M. P.; Aguado, M. T.; Kieny, M. P. A review of vaccine research and development: meningococcal disease. Vaccine 2006, 24 (22), 4692–4700. (3) Urwin, R.; Russell, J. E.; Thompson, E. A.; Holmes, E. C.; Feavers, I. M.; Maiden, M. C. Distribution of surface protein variants among hyperinvasive meningococci: implications for vaccine design. Infect. Immun. 2004, 72 (10), 5955–5962. (4) Derrick, J. P.; Urwin, R.; Suker, J.; Feavers, I. M.; Maiden, M. C. Structural and evolutionaryinference from molecular variation in Neisseria porins. Infect. Immun. 1999, 67 (5), 2406–2413. (5) Perrett, K. P.; Pollard, A. J. Towards an improved serogroup B Neisseria meningitidis vaccine. Expert Opin. Biol. Ther. 2005, 5 (12), 1611–1625. (6) Weynants, V. E.; Feron, C. M.; Goraj, K. K.; Bos, M. P.; Denoe¨l, P. A.; Verlant, V. G.; Tommassen, J.; Peak, I. R.; Judd, R. C.; Jennings, M. P.; Poolman, J. T. Additive and synergistic bactericidal activity of antibodies directed against minor outer membrane proteins of Neisseria meningitidis. Infect. Immun. 2007, 75 (11), 5434–5442. (7) Troncoso, G.; Sańchez, S.; Criado, M. T.; Ferreiro´s, C. M. Analysis of Neisseria lactamica antigens putatively implicated in acquisition of natural immunity to Neisseria meningitidis. FEMS Immunol. Med. Microbiol. 2002, 34 (1), 9–15. (8) Ito, H. O.; Nakashima, T.; So, T.; Hirata, M.; Inoue, M. Immunodominance of conformation dependent B-cell epitopes of protein antigens. Biochem. Biophys. Res. Commun. 2003, 308 (4), 770–776. (9) Arockiasamy, A.; Murthy, G. S.; Rukmini, M. R.; Sundara Baalaji, N. Conformational epitope mapping of OmpC, a major cell surface antigen from Salmonella typhi. J. Struct. Biol. 2004, 148 (1), 22– 33. (10) Exner, M. M.; Wu, X.; Blanco, D. R.; Miller, J. N.; Lovett, M. A. Protection elicited by native outer membrane protein Oms66 (p66) against host-adapted Borrelia burgdorferi: conformational nature of bactericidal epitopes. Infect. Immun. 2000, 68 (5), 2647–2654. (11) Wittig, I.; Scha¨gger, H. Features and applications of blue-native and clear-native electrophoresis. Proteomics 2008, 8 (19), 3974– 3990. (12) Marzoa, J.; Abel, A.; Sańchez, S.; Chan, H.; Feavers, I.; Criado, M. T.; Ferreiro´s, C. M. Analysis of outer membrane porin complexes of Neisseria meningitidis in wild-type and specific knock-out mutant strains. Proteomics 2009, 9 (3), 648–656. (13) Wittig, I.; Karas, M.; Scha¨gger, H. High resolution clear native electrophoresis for in-gel functional analysis and fluorescence studies of membrane protein complexes. Mol. Cell. Proteomics 2007, 6 (7), 1215–1225. (14) Wittig, I.; Carrozzo, R.; Santorelli, F. M.; Scha¨gger, H. Functional assays in high-resolution clear native gels to quantify mitocondrial complexes in human biopsies and cell lines. Electrophoresis 2007, 28 (21), 3811–3820. (15) Gomez, J. A.; Criado, M. T.; Ferreiro´s, C. M. Bactericidal activity of antibodies elicited against the Neisseria meningitidis 37 Kda ferric binding protein (FbpA) with different adyuvants. FEMS Inmunol. Med. Microbiol. 1998, 20 (1), 23–30. (16) Smith, P. K.; Krohm, R. I.; Hermason, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurent of protein using Bicinchonic acid. Anal. Biochem. 1985, 150 (1), 76–85. (17) Jansen, C.; Wiese, A.; Reubsaet, L.; Dekker, N.; de Cock, H.; Seydel, U.; Tommassen, J. Biochemical and biophysical characterization of in vitro folded outer membrane porin PorA of Neisseria meningitidis. Biochim. Biophys. Acta 2000, 1464 (2), 284–298. (18) Ferrari, G.; Garaguso, I.; Adu-Bobie, J.; Doro, F.; Taddei, A. R.; Biolchi, A.; Brunelli, B.; Giulliani, M. M.; Pizza, M.; Norais, N.; Grandi, G. Outer membrane vesicles from group B Neisseria meningitidis ∆gna33 mutant: Proteomic and immunological comparison with detergent-derived outer membrane vesicles. Proteomics 2006, 6 (6), 1856–1866.

technical notes

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PR9006409

Journal of Proteome Research • Vol. 9, No. 1, 2010 619

Identification of Neisseria meningitidis Outer Membrane Vesicle

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