IMMUNOGENIC BACTERIAL VESICLES WITH OUTER MEMBRANE PROTEINS

Abstract

Knockout of the meningococcal mltA homolog gives bacteria that spontaneously release vesicles that are rich in immunogenic outer membrane proteins and that can elicit cross-protective antibody responses with higher bactericidal titres than OMVs prepared by normal production processes. Thus the invention provides a bacterium having a knockout mutation of its mltA gene. The invention also provides a bacterium, wherein the bacterium: (i) has a cell wall that includes peptidoglycan; and (ii) does not express a protein having the lytic transglycosylase activity MltA protein. The invention also provides compositions comprising vesicles that, during culture of bacteria of the invention, are released into the culture medium.

Claims

1-25. (canceled)

26. A process for preparing bacterial vesicles, comprising the steps of: (i) culturing an Escherichia bacterium in a culture medium such that the bacterium releases vesicles into said medium; and (ii) collecting the vesicles from said medium, wherein: (a) the bacterium has a cell wall that includes peptidoglycan; and (b) the bacterium does not express a protein having the lytic transglycosylase activity of MltA protein.

27. The process of claim 26, wherein the bacterium also has a knockout mutation of at least one further gene.

28. The process of claim 26, wherein the bacterium is E. coli.

29. The process of claim 28, wherein the bacterium is a pathogenic E. coli.

30. The process of claim 29, wherein the pathogenic E. coli is an extraintestinal pathogenic bacterium, a uropathogenic bacterium, or a meningitis/sepsis-associated bacterium.

31. The process of claim 27, wherein the bacterium is E. coli.

32. The process of claim 31, wherein the bacterium is a pathogenic E. coli.

33. The process of claim 32, wherein the pathogenic E. coli is an extraintestinal pathogenic bacterium, a uropathogenic bacterium, or a meningitis/sepsis-associated bacterium.

34. The process of claim 26, wherein the bacterium is a pathogenic Escherichia coli bacterium, which does not express a protein of the Tol-Pal complex and/or which has a knockout mutation of its mltA gene.

35. The process of claim 34, wherein the E. coli bacterium is a tolR-strain.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0192] FIG. 1 shows 2D-PAGE of vesicles of the invention.

[0193] FIGS. 2A-B show the gel filtration outputs with standard proteins (FIG. 2B) and with the centrifugation pellet (FIG. 2A) of the culture supernatant of the ΔmltA strain. The y-axis on both FIG. 2A and FIG. 2B shows absorbance at 280 nm.

[0194] FIGS. 3A-B show electron microscopy of vesicles of the invention. FIG. 3A is lower resolution electron microscopy (the bar shown 100 nm). FIG. 3B is higher resolution electron microscopy (the bar shows 50 nm).

[0195] FIGS. 4A-F show western blot analysis of vesicles of the invention with sic different antibodies. FIG. 4A shows mouse serum raised against OMVs prepared from the NZ strain by deoxycholate extraction. FIG. 4B shows mouse serum raised against ΔGNA33 knockout mutants. FIG. 4C shows mouse anti-PorA P1.4 monoclonals. FIG. 4D shows mouse anti-NMB2132 serum. FIG. 4E shows mouse anti-NMB1030 serum. FIG. 4F shows mouse anti-NMB1870 serum.

[0196] FIG. 5 compares the proteins released into culture supernatants by wild-type or ΔGNA33 bacteria. Lane 1: Molecular weight markers; lane 2: culture medium control; lane 3: 20 μg proteins collected by high speed centrifugation of ΔGNA33 culture medium at OD.sub.620 nm=0.5, corresponding to 6.5 ml of culture medium; lane 4: proteins collected by high speed centrifugation from 6.5 ml of wild-type MC58 culture medium at OD.sub.620 nm=0.5.

[0197] FIG. 6 shows SDS-PAGE of a wild-type MC58 total extract (lanes 2 and 4) and of vesicles released by ΔGNA33 knockout mutant (lanes 3 and 5). Lanes 2 and 3 are proteins not denatured at 95° C. prior to SDS-PAGE; lanes 4 and 5 were denatured at 95° C.

[0198] FIG. 7A-B show 1D SDS-PAGE (FIG. 7A) and 2D SDS-PAGE (FIG. 7B) of vesicles prepared from strain 394/98. In FIG. 7B the horizontal axis runs from pI 3 to 10 and the vertical axis runs from 10 to 200 kDa.

[0199] FIGS. 8A-B show 1D SDS-PAGE of vesicles prepared from tolR ExPEC knockout strains. FIG. 8A shows two preparations of E. coli CFT073. FIG. 8B shows three E. coli strain preparations: DH5a, 536, and 1HE3034.

[0200] FIGS. 9A-D show 1D SDS-PAGE (FIGS. 9A and 9C) and 2D SDS-PAGE (FIGS. 9B and 9D) of vesicles from ΔmltA knockout meningococci. FIGS. 9A and 9B are strain MC58. FIGS. 9C and 9D are strain NZ98/254.

[0201] FIGS. 10A-B shows 1D SDS-PAGE (FIG. 10A) and 2D SDS-PAGE (FIG. 10B) of vesicles from ΔmltA knockout meningococci.

[0202] The amino acid sequence (SEQ ID NO: 1) and nucleotide sequence (SEQ ID NO: 2) are of the membrane-bound lytic murein transglycosylase A (mltA) from the genome sequence of strain MC58 of serogroup B Neisseria meningitidis, taken from GenBank accession AAF40504.1[32]. SEQ ID NO: 3 is NMB0928, SEQ ID NO: 4 is NMB0109, SEQ ID NO: 5 is NMB1057, SEQ ID NO: 6 is NMB0928.sub.NEW, and SEQ ID NO: 8 NMB1057.sub.NEW, which both have shifted start codons.

MODES FOR CARRYING OUT THE INVENTION

[0203] Preparation of Meningococcal ΔmltA Knockout Strain

[0204] A meningococcal strain was prepared in which the mltA gene is replaced by allelic exchange with an antibiotic cassette.

[0205] N. meningitidis strain MC58 was transformed with plasmid pBSUDGNA33ERM. This plasmid contains upstream and downstream flanking regions for allelic exchange, a truncated mltA gene, and the ermC gene (encoding erythromycin resistance). The upstream flanking region (including the start codon) from position −867 to +75 and the downstream flanking region (including the stop codon), from position +1268 to +1744 were amplified from MC58 by using the primers U33FOR, U33RBV, D33FOR and D33REV [25]. Fragments were cloned into pBluescript™ and transformed into E. coli DH5 by using standard techniques. Once all subcloning was complete, naturally competent Neisseria strain MC58 was transformed by selecting a few colonies grown overnight on GC agar plates and mixing them with 20 μl 10 mM Tris-HCl (pH 6.5) containing 1 μg plasmid DNA. The mixture was spotted onto a chocolate agar plate, incubated for 6 h at 36° C. with 5% CO.sub.2, and then diluted in phosphate buffered saline (PBS) and spread on GC agar plates containing 7 μg/ml erythromycin. Allelio exchange with the chromosomal mltA gene was verified by PCR, and lack of MltA expression was confirmed by Western blot analysis.

[0206] As reported in reference 25, the ?mltA knockout strain does not have the correct topological organisation of the cellular membrane, has abnormal cell separation, abnormal cell morphology, undivided septa, double septa, cell clustering, sharing of outer membranes and reduced virulence. Reference 25 also reports that the knockout strain release various membrane proteins into the culture supernatant, including the PorA, PIB, class 4 and 5 outer membrane proteins.

[0207] A ?mltA knockout was also made from New Zealand strain 394/98 (lin3; B:4:P1.4), which is the strain from which the MeNZB™ product is produced.

[0208] Analysis of Released Proteins

[0209] The ?mltA strain was grown in GC culture medium in a humidified atmosphere containing 5% CO.sub.2 until OD.sub.600 min 0.5. Bacteria were collected by 10 minutes of centrifugation at 3500×g. The supernatant (i.e. culture medium) was filtered through a 0.22 μm pore size filler (Millipore), and the cell-free filtrate was subjected to high-speed centrifugation (200,000×g, 90 min). This centrifugation resulted in formation of a pellet, with about 8-12 mg protein per litre of culture medium. No such pellet was seen if wild-type MC58 bacteria were treated in the same way, and so the pellet formation is a result of the ?mltA knockout. The pellet was washed twice with PBS (centrifugation 200,000×g, 30 min) for further analysis.

[0210] In a first analysis, material from the pellet was re-suspended in PBS and applied to a Superdox 200 PC3.2/30 gel filtration column, run on a SMART system (Amersham Biosciences) that had been equilibrated in PBS. The flow rate was 40 μl/min, and eluate was monitored at 280 nm. The column was calibrated with 20 μg Blue dextran (2,000 kDa), 10 μg ferritine (440 kDa), 140 μg bovine serum albumin (68 kDa) and 200 μg ribonuclease A (15 kDa). As shown in FIGS. 2A-B, most of the proteins eluted in a major peak corresponding to a molecular weight substantially higher than 2,000 kDa. This result suggests that the various proteins are associated.

[0211] In a second analysis, the material present in the high molecular weight peak was subjected to negative staining electron microscopy. This analysis revealed the presence of well-organised membrane vesicles with a diameter of about 50-100 nm (FIGS. 3A-B).

[0212] These experiments suggest that deletion of the mltA gene perturbs the normal assembly of the bacterial membrane, and that this results in the spontaneous release into the culture supernatant of membrane structures which assemble in spherical, homogeneous vesicles.

[0213] FIG. 5 shows SDS-PAGE analysis of culture media after growth of wild-type or ?GNA33 bacteria, and shows the different protein release characteristics.

[0214] Analysis of Vesicles

[0215] The ?mltA-derived vesicles were compared to meningococcal vesicles prepared by the ‘normal’ detergent extraction method.

[0216] Meningococcal strains MC58, NZ394/98 and NZ98/254, and their respective isogenic ?mltA mutants, were grown in 20 ml or 200 ml GC culture medium in humidified atmosphere containing 5% CO.sub.2 until OD.sub.600 nm 0.5. Bacteria were collected by 10-minute centrifugation at 3500 g. Vesicles (‘DOMVs’) were prepared from the wild-type bacteria by detergent extraction as described in reference 206. Vesicles of the invention (‘mOMVs’) were prepared from knockout strains by filtration through a 0.22 μm pore size filter, followed by high-speed centrifugation (200,000 g, 90 min) of the filtrates, washing of the vesicle-contain pellets (centrifugation 200,000 g, 30 min) twice with phosphate buffer saline, (PBS), and then re-suspension with PBS.

[0217] Both of the mOMVs and the DOMVs were analysed by denaturing mono-dimensional electrophoresis. Briefly, 20 μg of vesicle proteins were resolved by SDS-PAGE and visualised by Coomassie Blue staining of 12.5% gels. Denaturing (2% SDS) and semi-denaturing (0.2% SDS, no dithiothreitol, an heating) conditions were used mono-dimensional electrophoresis. The amount of protein (20 μg) was determined by DC protein assay (Bio-rad), using bovine serum albumin as a standard protein.

[0218] The vesicles were denatured for 3 minutes at 95° C. in SDS-PAGE sample buffer containing 2% SDS. 20 μg of protein were then loaded onto 12.5% acrylamide gels, which are stained with Coomassie Blue R-250, 2-dimensional electrophoresis was also performed on 200 μg of protein brought to a final volume of 12SUI with re-swelling buffer containing 7M urea, 2M thiourea, 2% (w/v) (3-((3-cholamidopropyl)dimethylammonio)-1-propane-sulfonate), 65 mM dithiothreitol, 2% (w/v) amidosulfobetain-14, 2 mM tributylphosphine, 20 mM Tris, and 2% (v/v) carrier ampholyte. Proteins were adsorbed overnight onto Immobiline DryStrips (7 cm; pH-gradient 3-10 non linear). Proteins were then 2D-separated. The first dimension was run using a IPGphor Isoelectric Focusing Unit, applying sequentially 150 V for 35 min., 500 V for 35 min., 1,000 V for 30 min, 2,600 V for 10 min., 3,500 V for 15 min., 4,200 V for 15 min., and finally 5,000 V to reach 12 kVh. For the second dimension, the strips were equilibrated and proteins were separated on linear 9-16.5% polyacrylamide gels (1.5-mm thick, 4×7 cm). Gels were again stained with Coomassie Brilliant Blue G-250. 266 protein spots could be seen after Colloidal Coomassie Blue staining (FIG. 1).

[0219] The 1D and 2D gels were then subjected to in-gel protein digestion and sample preparation for mass spectrometry analysis. Protein spots were excised from the gels, washed with 100 mM ammonium bicarbonate/accetonitrile 50/50 (V/V), and dried using a Speed/Vac centrifuge. Dried spots were digested 2 hours at 37° C. in 12 μl of 0.012 μg/l sequencing grade trypsin (Promega) in 50 mM ammonium bicarbonate, 5 mM. After digestion, 5 μl of 0.1% trifluacetic acid was added, and the peptides were desalted and concentrated with ZIP-TIPs (C18, Millipore). Sample were eluted with 2 μl of 5 g/l 2,5-dihydroxybenzoic acid in 50% acetonitrile/0.1% trifluoroacetic acid onto the mass spectrometer Anchorchip 384 (400 μm, Bruker, Brumen, Germany) and allowed to air dry at room temperature, MALDI-TOF spectra were acquired on a Bruker Biflex III MALDI-TOF equipped with a 337 nm N.sub.2 laser and a SCOUT 384 multiprobe ion source set in a positive-ion reflector mode. The acceleration and reflector voltages were set at 19 kV and 20 kV, respectively. Typically, each spectrum was determined by averaging 100 laser shots. Spectra sere externally calibrated using a combination of four standard peptides, angiotensin II (1,046.54 Da), substance P (1,347.74 Da), Bombensia (1,619.82 Da) and ACTH18-39 Clip human (2,465.20 Da), spotted onto adjacent position to the samples. Protein identification was carried out by both automatic and manual comparison of experimentally-generated monoisotopic values of peptides in the mass range of 700-300 Da with computer-generated fingerprints using the Mascot software.

[0220] Results from the MC58 ?mltA mutant are shown in FIGS. 9A-B. From the 20 excised bands on just the 1D gel, 25 unique proteins were identified, 24 (96%) of which were predicted to be outer-membrane proteins by the PSORT algorithm (Table 1 below). 170 protein spots on the 2D gel, corresponding to 51 unique proteins, were unambiguously identified by MALDI-TOF (Table 1). 44/51 identified proteins have been assigned to the outer membrane compartment by the genome annotation [32]. The 7 remaining proteins were analysed for possible errors in the original annotation. Four proteins (the hypothetical proteins NMB1870, NMB0928 and NMB0109, and the glutamyltranspeptidase NMB1057) could be classified as outer membrane proteins using different start codons from those in ref. 32 e.g. for NMB1870, using the start codon assigned in reference 55.

[0221] The combined 1D and 2D electrophoresis experiments identified a total of 65 proteins in the MC58 ?mltA mutant-derived vesicles. Of these, 6 proteins were identified in both 1D and 2D gels, whereas 14 and 45 were specific for the 1D and 2D gels, respectively (Table 1). Moreover, 61 out of the 65 identified proteins were predicted as membrane-associated proteins by current algorithms, indicating that the ?mltA vesicles (mOMVs) are mostly, and possible exclusively, constituted by membrane proteins.

[0222] the ?mltA knockout of strain NZ394/98 was similarly subjected to 1D and 2D SDS-PAGE (FIGS. 7A-7B). Table 2 shows 66 proteins that were identified in one or both of the gels, together with the predicted location of the proteins. Again, most of the proteins were predicted as membrane-associated. The 47 proteins common to Tables 1 and 2 are shown in Table. 3.

[0223] Results from the NZ98/254 ?mltA mutant are shown in FIGS. 9C-D. 66 proteins were identified from those two gels, 57 of which were assigned to the outer membrane compartment. Again, therefore, the mOMVs are highly enriched in outer membrane proteins. 46 of the 57 proteins had also been identified in the MC58-derived mOMVs.

[0224] For comparison, FIGS. 10A-B shows the results from NZ98/254 DOMVs. Proteomic analysis revealed 138 proteins, only 44 of which were assigned to the outer membrane compartment. The remaining 94 proteins belonged to the cytoplasmic and inner membrane compartments. Of these 44 membrane proteins, 32 were also found in the 57 outer membrane proteins found in the mOMVs from the isogenic strain.

[0225] While mOMVs are largely constituted by outer membrane proteins, therefore, about 70% of DOMV proteins are either cytoplasmic or inner membrane proteins. DOMVs differ from mOMVs not only for the proportion of cytoplasmic proteins but also for the different profile of their outer membrane proteins. Of the 44 outer membrane proteins seen in DOMVs, only 32 were also seen in mOMVs.

[0226] 19 proteins seen in mOMVs from both MC58 and NZ98/254, but not in the DOMVs from NZ98/254, are listed in Table 4 below.

[0227] A total cell extract of bacteria was prepared as follows: Bacterial cells were washed with PBS, and the bacterial pellet was resuspended in 8 ml of 250 mM Tris-HCl 7.3 containing protease inhibitor cocktail (Roche Diagnostic). 2 μM EDTA and 2000 matrix of benzonase (Merck) were added, cells were disrupted at 4° C. with Basic Z 0.75V Model Cell Disrupter equipped with an “one shot head” (Constant System Ltd) by 2 cycles, and the unbroken cells were removed by centrifugation 10 min at 8,000×g at 4° C. This extract was analysed by SDS-PAGE, for comparison with a protein extract of the vesicles produced by ?GNA33 knockout mutant's vesicles (lanes 3 % 5). Moreover, these proteins are retained as stable trimers in the vesicles that do not dissociate into monomers in SDS-PAGE sample buffer with a low concentration of SDS (0.2%) under seminative conditions (no heating before electrophoresis; lanes 2 & 3), but that do denature at 95° C. (lanes 4 & 5).

[0228] LPS levels in detergent-extracted OMVs are typically 5-8% by weight, relative to protein [207]. When tested with the Limulus assay, the endotoxin content of the vesicles was about twice as high as found in detergent-extracted OMVs.

[0229] Finally, the yield of vesicles in a growing culture was assessed. It was found that up to 20 mg of OMV-associated proteins could be recovered per gram of cells (wet weight) in culture supernatants of early expotentially growing cultures (OD.sub.600 nm−0.5).

[0230] Vesicle Immunogenicity

[0231] As the ?mltA-derived vesicles are highly enriched in outer membrane proteins, their ability to elicit bactricidal antibodies capable of killing a broad panel of MenB clinical isolates was investigated.

[0232] The strain chosen for the testing was 394/98. This strain was chosen because it is the strain from which the MeNZB™ OMV-based vaccine is prepared, thereby aiding a direct comparison of ?mltA vesicles of the invention with OMVs prepared from the wild-type strain by typical prior art methods.

[0233] 10 μg of each type of vesicle was adsorbed to an aluminium hydroxide adjuvant (3 mg/ml) and injected into mice 5-week old CD1 female mice (5-10 mice per group). The vesicles were given intraperitoneally on days 0 and 21. Blood samples for analysis were taken on day 34, and were tested for SBA against 15 different serogroup B strains corresponding to 11 different sub-types, including the four major hypervirulent lineages, using pooled baby rabbit serum as the complement source. Serum bactericidal titers were defined as the serum dilution resulting in 50% decrease in colony forming units (CFU) per ml after 60 minutes incubation of bacteria with reaction mixture, compared to control CFU per ml at time 0. Typically, bacteria incubated with the negative control antibody in the presence of complement showed a 150 to 200% increase in CFU/ml during the 60 min incubation. Titers were as follows, expressed as the reciprocal of the serum dilution yielding ≧50% bacterial killing:

TABLE-US-00001 BCA titer Serogroup:Typo:Subtype mOMVs DOMVs B:4:P1.4 >8192 >32768 B:15:P1.7, 4 >65536 32768 B:4, 7:P1.7, 4 >32768 >32768 B:14:P1.4 >32768 >65536 B:4:P1.7, 4 >32768 8192 B:4,:P1.4 >8192 >8192 B:14:P1.13 16384 512 B:4, 7:P1.7, 13 >8192 128 B:4:P1.15 >8192 128 B:21:P1.9 >8192 <16 B:2b:P1.10 1024 <16 B:4, 7:P1.19, 15 1024 <16 B:2b:P1.5, 2 1024 <16 B:2a:P1.2 <16 <16 B:NT:P1.3 <16 <16

[0234] The results show that serum from ?mltA-derived vesicles were at least as bactericidally effective, and usually better than, OMVs prepared by chemical extraction, except for the homologous strain. The vesicles of the invention thus give much better cross-strain reactivity than typical OMVs. Moreover, taking a 1:1024 dilution as the threshold for bactericidal efficacy, the vesicles of the invention were effective against 87% of the strains, whereas the artificial OMVs were only 40% effective.

[0235] Thus mOMVs are better than DOMVs for eliciting complement-dependent antibody killing when tested over a panel of 15 different serogroup B strains. The anti-mOMV mouse sera showed high bactericidal activities against the homologous strain and against 14 additional strains, including 10 different PorA subtypes. In contrast, mouse sera raised against DOMVs show high bactericidal titers only against six MenB strains, belonging to two PorA subtypes. These results indicate that the protection of anti-mOMV sera was not only due to the elicitation of bactericidal antibodies against PorA, which is one of the most abundant outer membranes proteins and the most potent inducer of bactericidal antibodies, but also to other bactericidal antigens which in mOMVs are present in higher amounts them DOMVs.

[0236] Wester Blot

[0237] To confirm that the ?mltA-derived vesicles do contain conserved, protective antigens, they were run on an SDS-PAGE, transferred onto a PDF filter and immunoblotted using specific anti-sera against six proteins antigens previously shown to be protective and highly conserved, including ‘287’, ‘953’, ‘741’ (GNA1870) and ‘NadA’.

[0238] The vesicles were separated onto 10% acrylamide SDS-PAGE gels employing a Mini Protein II electorophoresis apparatus (Bio-Rad). After protein separation, gels were equilibrated with 48 mM Tris-HCl, 39 mM glycine, pH 9.0, 20% (v/v) methanol and transferred to a nitrocellulose membrane (Bio-Rad) using a Trans-Blot™ semi-dry electrophoretic transfer cell. The nitrocellulose membranes were blocked with 10% (w/v) skimmed milk in PBS containing 0.2% (w/v) sodium azide.

[0239] As shown in FIGS. 4A-B all six proteins were abundant in the vesicles. In contrast, the same six proteins were poorly represented in the DOMVs.

[0240] In conclusion, the ?mltA-derived vesicles are predominantly constituted by outer membrane proteins, whereas DOMVs are heavily contaminated by cytoplasmic proteins. When used to immunize mice, sera raised against ?mltA-derived vesicles showed a higher and wider strain coverage than DOMVs.

[0241] Extraintestinal Pathogenic E. coli

[0242] A knockout strain of ExPEC CFT073 was prepared by isogenic deletion of the tolR gene, replacing it with a kantamycin resistance marker. The knockout strain was grown to OD.sub.600 nm 0.4, and the culture was then centrifuged. The supernatant was filtered through a 0.22 μm filter and the filtrate was precipitated using TCA. The pellet was then resuspended in Tris buffer.

[0243] The same growth and purification procedure was used for the parent strain, without the knockout, and SDS-PAGE analysis of the two final preparations is shown in FIG. 8A. the right-hand band is from the knockout strain and shows enrichment of several protein bands.

[0244] Further tolR knockout ExPEC strains were prepared from strains DH5a, 536 and 1HE3034. Vesicles were prepared as before, and SDS-PAGE analysis of TCA precipitates is shown in FIG. 8B.

[0245] The knockout mutant produces high amounts of vesicles, and these vesicles were subjected to proteomic analyses, including 1D and 2D SDS-PAGE and tryptic digestion of surface-exposed proteins in the vesicles followed by sequence analysis of released peptides.

[0246] It will be understood that the invention has been described by was of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

TABLE-US-00002 TABLE 1 NMB Protein name/theoretical MW/theoretical pl/gravy index 1 d 3-10 Psort 1 NMB0018 pilin PilE/15 246/9.21/−0.571 X OM-PS 2 NMB0035 conserved hypothetical protein/40 218/4.74/−0.371 X OM-IN 3 NMB0044 peptide methionine sulfoxide reductase/55 718/6.54/−0.569 X OM-IN 4 NMB0086 hypothetical protein/34 987/4.82/−0.505 X OM-IN 5 NMB0088 outer membrane protein P1, putative/45 902/9.35/−0.428 X OM-PS 6 NMB0109 conserved hypothetical protein/43 188/6.77/−0.587 X X OM-PS(b) 7 NMB0124 translation elongation factor TU/42 909/5.07/−0.136 X cyto NMB0139 translation elongation factor TU/42.925/5.07/−0.142 cyto 8 NMB0138 elongation factor G (EF-G)/77 244/5.08/−0.293 X cyto 9 NMB0181 outer membrane protein OmpH, putative/16 829/9.07/−0.897 X OM-PS 10 NMB0182 outer membrane protein Omp85/86 254/8.37/−0.505 X X OM-PS 11 NMB0204 lipoprotein, putative/12 207/8.08/−0.446 X OM-PS 12 NMB0278 thiol: disulfide interchange protein DsbA/23 428/5.16/−0.298 X OM-IN 13 NMB0281 peptidyl-prolyl cis-trans isomerase/35 248/9.62/−0.388 X OM-PS 14 NMB0294 thiol: disulfide interchange protein DsbA/23 566/5.09/−0.477 X OM-IN 15 NMB0313 lipoprotein, putative/52 645/9.97/−0.824 X OM-PS 16 NMB0345 cell-binding factor, putative/29 448/9.13/−0.570 X X OM-PS 17 NMB0346 hypothetical protein/26439/5.15/−0.716 X OM-PS 18 NMB0382 outer membrane protein class 4/23 969/6.26/−0.456 X X OM-PS 19 NMB0407 thiol: disulfide interchange protein DsbA/21 721/9.23/−0.308 X OM-PS 20 NMB0460 transferrin-binding protein 2/75 292/5.79/−0.982 X OM-IN 21 NMB0461 transferrin-binding protein 1/99 314/9.45/−0.699 X OM-PS 22 NMB0550 thiol: disulfide interchange protein DsbC/26 451/6.93/−0.345 X OM-IN 23 NMB0554 dnaK protein/68 792/4.85/−0.357 X cyto 24 NMB0622 outer membrane lipoprotein carrier protein/19 996/9.47/−0.490 X OM-PS 25 NMB0623 spermidine/putrescine ABC transporter/39 511/5.38/−0.437 X OM-PS 26 NMB0634 iron(III) ABC transporter, periplasmic binding protein/35 806/9.60/−0.338 X OM-PS 27 NMB0663 outer membrane protein NsgA/16 563/9.49/−0.214 X OM-PS 28 NMB0700 IgA-specific serine endopeptidase X OM-PS 29 NMB0703 competence lipoprotein ComL/29 275/8.72/−0.761 X OM-IN 30 NMB0783 conserved hypothetical protein/15 029/7.05/−0.221 X OM-PS 31 NMB0787 amino acid ABC transporter/26 995/5.42/−0.287 X OM-IN 32 NMB0873 outer membrane lipoprotein LolB, putative/19 575/5.23/−0.470 X OM-IN 33 NMB0928 hypothetical protein/39 502/9.13/−0.596 X X OM-IN(b) 34 NMB1030 conserved hypothetical protein/18 700/7.16/−0.429 X OM-PS 35 NMB1053 class 5 outer membrane protein/28 009/9.68/−0.610 X X OM-PS 36 NMB1057 gamma-glutamyltranspeptidase/61 590/5.94/−0.160 X OM-IN(b) 37 NMB1126 hypothetical protein/22 025/8.03/−0.355 X X OM-IN NMB1164 hypothetical protein/22 025/8.03/−0.355 OM-IN 38 NMB1285 enolase/46 134/4.78/−0.200 X cyto 39 NMB1301 30S ribosomal protein S1/61 177/4.9/−0.240 X cyto 40 NMB1332 carboxy-terminal peptidase/53 238/9.12/−0.420 X IN 41 NMB1352 hypothetical protein/13 699/9.52/−1.397 X OM-PS 42 NMB1429 outer membrane protein PorA/40.129/8.73 X X OM-PS 43 NMB1457 transketolase/71 659/5.45/−0.183 X cyto 44 NMB1483 lipoprotein NlpD, putative/40 947/9.55/−0.266 X X OM-PS 45 NMB1533 H.8 outer membrane protein/18 886/4.61/17 X OM-IN 46 NMB1557 conserved hypothetical protein/15 419/7.34/−0.429 X OM-PS 47 NMB1567 macrophage infectivity/potentiator/26 875/5.50/−0.540 X OM-IN 48 NMB1578 conserved hypothetical protein/21 135/4.86/−0.381 X OM-IN 49 NMB1612 amino acid ABC transporter/27 970/4.87/−0.408 X OM-PS 50 NMB1636 opacity protein: authentic frameshift/27180/9.52 X X OM-PS 51 NMB1710 glutamate dehydrogenase, NADP-specific/48 490/5.98/−0.190 X cyto 52 NMB1714 multidrug efflux pump channel protein/48 482/8.38/−0.261 X OM 53 NMB1870 hypothetical protein/26 964/7.23/−0.485 X OM-IN(b) 54 NMB1898 lipoprotein/17 155/7.01/−0.709 X OM-IN 55 NMB1946 outer membrane lipoprotein/29 258/5.01/−0354 X OM 56 NMB1949 soluble lytic murein transglycosylase; putative/65 617/9.31/−0.525 X OM-IN 57 NMB1961 VacJ-related protein/27.299/4.65/−0.344 X OM-PS 58 NMB1969 © serotype-1-specific antigen, putative X cyto 59 NMB1972 chaperonin, 60 kDa/57 423/4.9/−0.052 X cyto 60 NMB1988 Iron-regulated outer membrane protein FrpB/76 823/9.42/−0.700 X OM-PS 61 NMB2039 major outer membrane protein PIB/33 786/6.54/−0.468 X X OM-PS 62 NMB2091 hemolysin, putative/19 412/9.55/−0.152 X OM-IN 63 NMB2095 adhesin complex protein, putative/11 385/9.52/−0.470 X OM-IN 64 NMB2102 elongation factor TS (EF-TS)/30 330/5.30/−0.016 X cyto 65 NMB2159 glyceraldehyde 3-phosphate dehydrogenase/35 845/5.40/−0.028 X cyto

TABLE-US-00003 TABLE 2 NMB ANNOTATION PSORT 1 D 2 D 1 NMB0035 conserved hypothetical protein OM-IM X 2 NMB0044 peptide methionine sulfoxide reductase OM-IM X 3 NMB0086 hypothetical protein OM-IM X 4 NMB0088 outer membrane protein P1, putative OM-PS X X 5 NMB0109 conserved hypothetical protein OM-PS(b) X X 6 NMB0124 cyto(c, x) X X 7 NMB0138 elongation factor G (EF-G) cyto (x) X 8 NMB0182 outer membrane protein Omp85 OM-PS X X 9 NMB0204 lipoprotein, putative OM-PS X 10 NMB0278 thiol: disulfide Interchange protein DsbA OM-IM X 11 NMB0294 thiol: disulfide Interchange protein DsbA OM-IM X 12 NMB0313 lipoprotein, putative OM X 13 NMB0345 cell-binding factor, putative OM-PS X X 14 NMB0346 hypothetical protein OM-PS X X 15 NMB0382 outer membrane protein class 4 OM-PS X X 16 NMB0460 transferrin-binding protein 2 OM-IM X 17 NMB0461 transferrin-binding protein 1 OM-PS X 18 NMB0462 spermidine/putrescine ABC transporter, periplasmic spermidine/putrescine-binding protein OM-PS(b) X 19 NMB0550 thiol: disulfide interchange protein DsbC OM-IM X X 20 NMB0554 dnaK protein LITT. X 21 NMB0604 alcohol dehydrogenase, zinc-containing IM X 22 NMB0623 spermidine/putrescine ABC transporter, periplasmic spermidine/putrescine-binding protein OM-IM X 23 NMB0631 phosphate acetyltransferase Pta IM X 24 NMB0634 iron(III) ABC transporter, periplasmic binding protein OM-PS X 25 NMB0663 outer membrane protein NspA OM-PS X X 26 NMB0669 conserved hypothetical protein OM-PS X 27 NMB0703 competence lipoprotein ComL comL OM-IM X X 28 NMB0787 amino acid ABC transporter, periplasmic amino acid-binding protein OM X 29 NMB0872 conserved hypothetical protein OM-PS X 30 NMB0873 outer membrane lipoprotein LolB, putative OM-IM X X 31 NMB0928 hypothetical protein OM-IM(b) X X 32 NMB0944 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase IM X 33 NMB0983 phosphoribosylaminolNidazolecarboxamide formyltransferase/INP cyclohydrolase IM X 34 NMB1030 conserved hypothetical protein OM-PS X X 35 NMB1040 hypothetical protein OM-PS X 36 NMB1053 class 5 outer membrane protein opc OM-PS X X 37 NMB1057 gamma-glutamyltranspeptidase OM-IM(b) X 38 NMB1124 hypothetical protein OM-IM X 39 NMB1125 hypothetical protein OM-IM X X 40 NMB1126 hypothetical protein OM-IM X X 41 NMB1285 Enolase LITT. X 42 NMB1301 30S ribosomal protein S1 LITT. X 43 NMB1309 Nbrial biogenesis and twitching motility protein, putative IM X X 44 NMB1313 trigger factor FACS+ X 45 NMB1332 carboxy-terminal peptidase IM X X 46 NMB1398 Cu—Zn-superoxide dismutase OM-PS X 47 NMB1429 outer membrane protein PorA porA OM-PS X X 48 NMB1483 lipoprotein NlpD OM-PS X X 49 NMB1497 TonB-dependent receptor OM X 50 NMB1518 acetate kinase IM X 51 NMB1533 H.8 outer membrane protein OM-PS X 52 NMB1567 macrophage infectivity potentiator OM-IM X 53 NMB1574 ketol-acid reductoisomerase CYTO X 54 NMB1612 amino acid ABC transporter, periplasmic amino acid-binding protein OM-IM X 55 NMB1710 glutamate dehydrogenase, NADP-specific LITT. X 56 NMB1812 putative, pilQ protein, authentic frameshift OM-PS X 57 NMB1870 hypothetical protein OM-IM(b) X 58 NMB1898 lipoprotein mlp OM-IM X X 59 NM81902 DNA polymerase III, beta subunit CYTO X 60 NMB1949 soluble lytic murein transglycosylase, putative OM-IM X 61 NMB1961 VacJ-related-protein OM-PS X 62 NMB1972 chaperonin, 60 kDa LITT. X X 63 NMB1988 iron-regulated outer membrane protein FrpB OM-PS X X 64 NMB2039 major outer membrane protein PIB OM-PS X X 65 NMB2091 hemolysin, putative OM-IM X 66 NMB2139 conserved hypothetical protein OM-IM X 34 66

TABLE-US-00004 TABLE 3 NMB0035 NMB0044 NMB0086 NMB0088 NMB0109 NMB0124 NMB0138 NMB0182 NMB0204 NMB0278 NMB0294 NMB0313 NMB0345 NMB0346 NMB0382 NMB0460 NMB0461 NMB0550 NMB0554 NMB0623 NMB0634 NMB0663 NMB0703 NMB0787 NMB0873 NMB0928 NMB1030 NMB1053 NMB1057 NMB1126 NMB1285 NMB1301 NMB1332 NMB1429 NMB1483 NMB1533 NMB1567 NMB1612 NMB1710 NMB1870 NMB1898 NMB1949 NMB1961 NMB1972 NMB1988 NMB2039 NMB2091

TABLE-US-00005 TABLE 4 NMB0044 NMB0088 NMB0204 NMB0278 NMB0294 NMB0313 NMB0345 NMB0346 NMB0460 NMB0550 NMB0873 NMB0928 NMB1030 NMB1057 NMB1483 NMB1870 NMB1898 NMB1961 NMB2091

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