WO1993014198A1 - Proteine de surface multifonctionnelle de streptococci - Google Patents

Proteine de surface multifonctionnelle de streptococci Download PDF

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WO1993014198A1
WO1993014198A1 PCT/US1993/000082 US9300082W WO9314198A1 WO 1993014198 A1 WO1993014198 A1 WO 1993014198A1 US 9300082 W US9300082 W US 9300082W WO 9314198 A1 WO9314198 A1 WO 9314198A1
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sdh
protein
activity
streptococci
adp
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PCT/US1993/000082
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Vincent A. Fischetti
Vijaykumar Pancholi
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The Rockefeller University
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Priority to EP93902960A priority Critical patent/EP0672123A1/fr
Priority to AU34351/93A priority patent/AU668908B2/en
Priority to JP5512544A priority patent/JPH07502896A/ja
Publication of WO1993014198A1 publication Critical patent/WO1993014198A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01009—Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (1.2.1.9)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to a surface protein of streptococci which is involved in early colonization of the pharyngeal mucosa. More specifically, it relates to a multifunctional protein which is on the surface of streptococci, including pathogenic streptococci, such as Streptococcus pyogenes and is particularly characterized by its ability to bind fibronectin, lysozyme, and the cyclosketal proteins myosin and actin as well as by its enzyme activity, specifically dose dependent dehydrogenase activity with glyceraldehyde-3-phosphate (GADPH) .
  • the molecule also functions as an ADP- ribosylating enzyme and as an ADP-ribosyl transferase.
  • compositions and uses of the surface protein including, for example vaccines prepared from the whole protein and segments thereof, particularly conserved segments having activity similar to that of the protein. 2
  • Mammalian diseases especially human diseases caused by streptococcal infection with bacteria such as Streptococcus pyogenes are a significant health problem.
  • 25 to 35 million cases of group A streptococcal infections which primarily afflict school age children are reported annually (1) .
  • the high incidence and potential severity of streptococcal infections provide impetus for development of an effective and safe vaccine to prevent streptococcal related infections.
  • SDH streptococcal surface dehydrogenase
  • GAPDH proteins are a class of dehydrogenase enzymes intimately involved in mammalian physiological reactions. Generally, members of the class are found in the cytoplasm, but some have been found associated with membranes and cellular cyclosketal structures of eukaryotes.
  • the glycolytic enzyme of this invention s believed to be unique n that it s a surface protein of prokaryotes. No other such GAPDH protein has previously been described.
  • the GAPDH protein of this invention has some structural characteristics similar to other proteins of the GAPDH family. For example, over 80% of the NH - terminal 18 of 39 amino acids are identical to the GAPDH family of enzymes. However, it differs in many other respects, as will be explained hereinafter. It is, therefore, a novel product which has not heretofore been isolated and characterized.
  • a detailed characterization of purified SDH has disclosed that its native conformation is probably a tetramer with a molecular weight of about 156 kDa.
  • the molecular weight of the protein by mass spectrometric analysis is about 35.8 kDa.
  • SDS PAGE it is about 39.2 kDa.
  • the protein has been identified on the surface of Groups A, B, C, E, G, H and L streptococci utilizing affinity purified anti-SDH antibodies.
  • the protein exhibited a dose dependent dehydrogenase activity on glyceraldehyde-3-phosphate (G-3-P) in the presence of beta nicotinamide adenine dinucleotide (NAD) .
  • G-3-P glyceraldehyde-3-phosphate
  • NAD beta nicotinamide adenine dinucleotide
  • the multifunctional activity of SDH was revealed by its ability to bind fibronectin and lysozyme as well as the cytoskeletal proteins myosin and actin.
  • the binding activity of SDH to myosin was found to be localized to the globular heavy meromyosin domain.
  • SDH did not bind to streptococcal M protein, tropomyosin or the coiled- coil domain of myosin.
  • the multiple binding capacity of SDH especially in connection with cycloskeletal proteins, in conjunction with its GAPDH activity indicates a role in the colonization, internalization and the subsequent proliferation of streptococci. Trypsin treatment of whole streptococci resulted in a marked reduction in their reactivity to SDH antibodies.
  • the inability to remove SDH from the streptococcal surface after washing in 2 M NaCl or 2% SDS indicates that the protein is not peripherally associated but tightly bound to the cell.
  • the novel SDH is obtained by solubilizing the selected streptococcus with lysin to produce a mixture containing SDH.
  • the SDH may be isolated from the mixture by any of a number of convenient methods known to the skilled artisan including the method illustrated below.
  • It may also be produced by transforming an organism such as E. coli with an appropriate gene so that the E. coli will express SDH.
  • NAD beta nicotinamide adenine dinucleotide
  • PVDF polyvinylidine difluoride
  • EDTA Eyhylenediamine tetra acetic acid
  • PMSF Paradimethyl sulfonyl fluoride
  • TLCK N-p-tosyl-L-lysine chloro-methyl ketone
  • SDS Sodium dodecyl sulfate
  • Mono Q FPLC Mono Q(Trade Name) Fast protein liquid chromatography
  • Superose 12 FPLC Superose-12(Trade Name) Fast protein liquid chromatography
  • TSK-Phenyl HPLC TSK-Phenyl (Trade Name) high performance/pressure liquid chromatography
  • NADH beta-nicotinamide adeninedinucleotide
  • reduced ELISA Enzyme linked im unosorbent assay
  • ELIDA Trade name of Physica Inc. Sephadex G-25 PD-10: G-25 PD-10: trade name of
  • HEPES (N-[2-hydroxyethyl]piperizine-N , -[2-ethansulfonic acid])
  • RGDS Arginine-Glycine-Aspartic acid-Serine (Arg-Gly- Asp-Ser)
  • G-3-P Glyceraldehyde-3-phosphate
  • GAPDH Glyceraldehyde-3-phosphate dehydrogenase
  • Fig. l SDS-polyacrylamide gel (10%) analysis of
  • Lane a Lysin extract of D471 streptococci.
  • Lane b Precipitate of 65% (NH 4 )_S0 4 saturation of the lysin extract.
  • Lane c Precipitate of 85% (NH.J-SO. saturation of the supernatant after 65% precipitation.
  • Lane d Pooled Mono Q fractions at 0.28 M gradient elution.
  • Lane e Partially purified SDH from the Superose 12 column.
  • Lane f Purified SDH from Phenyl TSK column. Arrow marks on lanes a and b at 50 kDa indicate the position of M protein. Prestained marker protein mixture with molecular mass as indicated on the left margin.
  • Fig. 2 (a) The NH_-terminal sequence of SDH. (b) Comparison of the NH_-terminal amino acid sequence of SHD with the amino acid sequences of the known GAPDH molecules obtained fr the translated Genbank database.
  • Fig. 3 Lineweaver-Burk's double reciprocal kinetic analysis of GAPDH activity of SHD. 25 ug of SDH was assayed as function of G-3-P in the presence of NAD (lOOuM) in triethanolamine-phosphate-EDTA-DTT buffer at pH 8.6. The Km for G-3-P was estimated to be 1.33 uM,
  • the Km for NAD was estimated to be 156.7 uM, Vmax:0.459 X l ⁇ "3 M NADH min ⁇ , Intercept on Y axis(1/Vmax) 2.18, and slope (Km/Vmax) 341.74.
  • Km for NAD by the method of Michaelis-Menten as shown in the inset was estimated to be 148.86 uM and Vmax: 0.445 X 10 ⁇ 3 M NADH min "1 .
  • Fig. 4 (A) Coomassie Blue stain of SDS-gel and (B) Western-blot analysis of SDH with affinity purified anti-SDH antibodies suggesting a multimeric structure for the SDH molecule. Lanes a and d: Crude lysin extract.
  • Lanes b and e Purified SDH. Lanes c and f: Unboiled purified SDH in sample buffer without SDS and saturated wit NAD. Arrow mark indicates the position of a molecule of the size consistant with a tetrameric form of SDH. MW markers are indicated on the left margin (Details on each marker-see Fig. 1) .
  • Fig. 5 Dot blot immunoanalysis to locate SDH on the streptococcal surface. The assay determines the extent of reactivity of affinity purified anti-SDH antibodies to surface exposed protein before and after 2% SDS, 2M NaCl and trypsin treatments. Dot blots were treated with LumiPhos-530 substrate (41) and developed on X-ray film. Densitometric reading of the image obtained on the X-ray film was expressed as an optical density in terms of arbitrary units measured on an image analyzer using the Dumas program (Drexel University, Philadelphia, USA) . An internal linear standard curve for the optical density 0.008 to 1.333 was obtained for final densitometric analysis of the dot blot. Each bar representes the mean of four to eight separate readings + S.D.
  • Fig. 6 GAPDH activity of whole streptococci.
  • the GAPDH activity was observed at 340 mn of whole M6 streptococci by determining the conversion of NAD to NADH in the presence of G-3-P. Details of the buffer system is described in materials and methods.
  • Fig. 7 (A) Western-blot analysis of lysin extract of various streptococcal M types with affinity purfied anti-SDH antibodies at a 1:2000 dilution of 0.5 mg/ml stock. Purified SDH and an M negative (M ⁇ ) streptococci are also incuded in the analysis. (B) Western-blot analysis of mutanolysin extract of various grouping strains of streptococci using anti-SDH antibodies as described in (A) .
  • Fig. 8 Competition kinetic enzyme-linked immunosorbent assays (kELISA) with immobilized SDH.
  • GAPDH commercially available purified GAPDH from E ⁇ stearothermophilus, human erythrocytes and rabbit skeletal muscle were used to compete for the binding of affinity purified anti-SDH antibodies (1:1000 dilution of 0.5 mg/ml stock).
  • Each curve represents the mean of three separate experiments with less than 5% standard deviaiton (not shown) .
  • Inset shows the Western blot of the reactivity of affinity purified anti-SDH antibodies with (a) streptococcal SDH and GAPDHs of (b) bacterial (B. stearothermophilus) , (c) rabbit skeletal muscle and (d) human erythrocytes.
  • Fig. 9 Binding of 125I-SDH to cytoskeletal proteins.
  • A Coomassie Blue stained SDS-PAGE gel (10%) containing 5 ug protein of various cytoskeletal proteins, lysozyme and M6 protein (43) .
  • Lane a rabbit skeletal myosin.
  • Lane b heaving meromyosin.
  • Lane c light meromyosin.
  • Lane d actin.
  • Lane e M6 protein.
  • Lane f M6 protein.
  • Fig. 10 Binding activity of SDH to fibronectin.
  • A Coomassie stain of an SDS gel containing 5 ug of SDH and BSA.
  • B Western-blot analysis of a duplicate gel showing the binding of fibronectin followed by anti- fibronectin to the SDH molecule.
  • C Autoradiograph of a similar Western blot showing the binding of 125I- fibronectin to the SDH protein. Lanes a, c & e - SDH.
  • Fig. 11 ADP-ribosylation of SDH.
  • Purified SDH (lanes 1, 5 and 9) , crude streptococcal cell wall extract (lanes 2, 6 and 10), cytoplasm (lanes 3, 7 and 11) and membrane (lanes 4, 8 and 12) fractions were incubated with [ 32P]NAD in ADPR buffer. The proteins were then separated on a 12% SDS gel and stained with (A) Coomassie blue.
  • B Western blot analysis of a duplicate gel reacted with affinity purified anti-SDH antibodies and
  • Fig. 12 ADP-ribosyl transferase activity of SDH.
  • Human fibronectin was obtained from Boehringer Mannheim. Goat anti-human fibronectin and affinity purified rabbit anti-goat IgG coupled to alkaline phosphatase were obtained from Sigma. Pre-stained molecular weight standards were purchased from Bethesda
  • PVDF membrane ' Immobilon-P' l, was from Millipore. Na 125I was from New England Nuclear. All other chemicals and reagents unless otherwise indicated were purchased from Sigma.
  • Group A 5-hemolytic streptococcal strains of various M types and standard strains used for streptococcal grouping were from The Rockefeller University culture collection (New York, NY) and are listed as follows: M2(D626), M4(D896), M5(Manfrando) , M6(D471), M24(CS24), M29(D23), M41(C101/103/4) , M57(A995), M58(D774), M60(D398), M ⁇ (T28/51/4) ; group A, J17A4 (an M- strain) ; group A variant, A486var; group B, 090R; group C, C74; group D, D76; group E, K131; group F, F68C; group G, D166B; group H, F90A; group L, D167B; and group N, C559. Lysin extraction and location of SDH protein:
  • a crude extract containing the major surface proteins was prepared using the procedure of lysin extraction to remove the streptococcal cell wall as described before (2) .
  • bacteria washed in 50 mM sodium acetate buffer, pH 5.5 were suspended in the same buffer containing 30% raffinose and 5 MM EDTA. Lysin is added to the suspension (1:100 dil; 360 Units) and incubated for 90 min at 37°C with end-to-end slow rotation.
  • the resulting protoplasts sedimented at 15,000 X g for 30 min in a Sorvall centrifuge. The supernatant was saved, dialyzed against 25 mM Tris/HCl, pH 8.5,5mM EDTA, concetrated on Amicon PM-10 membrane (Amicon Corp) and used for further purification.
  • the pelleted protoplasts were resuspended and lysed in hypotonic buffer (2 MM sodium acetate, pH 5.5, containing 2 mM PMSF, 1 MM TLCK, 10 MM MgCl, and 10 ug/ml DNAse) followed by three freeze/thaw cycles.
  • hypotonic buffer (2 MM sodium acetate, pH 5.5, containing 2 mM PMSF, 1 MM TLCK, 10 MM MgCl, and 10 ug/ml DNAse
  • the membranes were sedimented at 100,000 x g for 45 min at 4°C.
  • the membrane pellet and cytoplasmic extract in the supernatant were analyzed with Coomassie blue stain after separation on SDS gel.
  • Membranes were further treated with 1.5 M sodium chloride or 100 mM sodium carbonate, pH 11.3 to determine the nature of association of SDH protein with the membrane.
  • lysin extraction of trypsinized bacteria was carried out as described earlier (3). Briefly, washed bacteria were suspended in 100 mM NH.HC0 3 and digested with trypsin (250 ug/ml) at 37°C for 3 hr, after which the trypsin was inactivated by the addition of soybean trypsin inhibitor (200 ug/ml) . Lysin extracts of trypsinized and control non-trypsinized bacteria were compared for the loss or reduction in the size of SDH protein.
  • Lysin extraction was used as the starting material for the purification of the SDH.
  • the dialyzed, concentrated, lysin extract was precipitated at 60% saturation of ammonium sulf te at 4°C.
  • the precipitates were centrifuged at 6,000 X g for 20 min and the supernatant was brought to 85% saturation of ammonium sulfate.
  • the resulting precipitate was dialyzed against 25 mM Tris/HCl buffer pH 8.5, 5 mM EDTA and passed over to Mono Q FPLC column (Pharmacia LKB Biotechnology Inc.) equilibrated with the same dialyzing buffer.
  • NH_-terminal amino acid sequence was determined according to the method of Matsudaira et al (4) . Briefly, the purified SDH was separated on a pre- electrophoresed 10% acrylamide-SDS gel under non- denaturing condition and then transferred to PVDF Immobilon-P filter pre-wetted in ethanol. Protein was visualized by 0.05% Coomassie blue in 50:40 methanol, water, acetic acid solvent mixture. The blots were destained in methanol: water: acetic acid (50:40:10).
  • the portion of the membrane containing the SDH band was excised and subjected to automated Edman degradation on an Applied Biosystem model A470 sequenator. Each band contained about 2-3 ug protein as determined by BCI protein estimation method (Pierce) .
  • the PVDF membrane containing the SDH was stained with 0.1% Ponceau-S (Sigma) in 1% acetic acid.
  • the section of membrane containing the protein band was excised and destained with water. This section of membrane was hydrolyzed in 6N HCl/phenol at 110°C for 22 hr. Amino acids were separated on Waters Novapek C8 column analyzed with Waters Maxima software, 510 pump and 490 detector. Cysteine content was analyzed also from the PVDF bound carboxyamide methylated protein as described by Crestfield (5) . All analyses were performed by Protein Biotechnology Facility of the Rockefeller University. .
  • Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Activity is:
  • GAPDH assay was carried out according to the method originally described by Samuel (7) with a minor modifications. Since GAPDH catalyzes the oxidative phosphorylation of D-G-3-P to form 1,3-diphosphoglycerate in the present of NAD and inorganic phosphate, the assay solution was made of triethanolamine (40 mM) , Na.HPO. (50 mM) and EDTA (5 mM) .
  • Disposable semi-micro 1.5 ml capacity spectrophotometer cuvette contained 7ul G-3-P (Sigma, 49 mg/ml) , 100 uM NAD (Boehringer Mannheim) and assay buffer to a final volume of 1.0 ml after the addition of enzyme source with pH of the mixture being 8.6. Different concentrations of SDH were used to plot the standard curve for the absorbance at 340 nm per minute as a measure of conversion of NAD to NADH using Spectronic 3000 spectrophotometer (Milton Roy) .
  • GAPDH activity for SDH was measured in the lysin extract, ammonium sulphate precipitate and pooled fraction at various purification stages.
  • New Zealand white rabbits were immunized subcutaneously with 200 ug of purified SDH emulsified in Freund's complete adjuvant (1:1) at multiple sites. Rabbits were boosted once with 200 ug of this protein in Freund's incomplete adjuvant (1:1). All rabbits were bled 3 weeks after the first and 10 days after the second immunization. All sera were filter sterilized and stored at 4°C.
  • the surface location of SDH was determined with the monospecific antibodies using a bacterial dot-blot immunoassay as previously described (10) .
  • an overnight culture of strain D471 was adjusted to OD g5 _ n 1.0 with 50 mM Tris/HCl buffer, pH 8.5. Aliquots of this suspension were centrifuged and resuspended to the same volume of buffer containing either 2 M NaCl or 2% SDS, and rotated at room temperature for 1 h, centrifuged, and the respective supernatants were saved. After washing, the pellets were again adjusted to 0D g50 nm 1.0 with 50 mM Tris/HCl buffer, pH 8.5.
  • the bacterial suspension in the Tris/HCl buffer was centrifuged, and the bacteria were suspended in 100 mM NH 4.HCO3- to nm 1.0 and treated with trypsin (250 ug/ml) for 3 h at 37 C. Trypsin activity was inhibited with trypsin inhibitor as described above, and the bacteria were pelleted and resuspended in the
  • Tris/HCl buffer to OD 6 c 50 n nm 1.0 50 ul of each bacterial suspension was transferred to nitrocellulose paper using dot-blot assembly (Bio-Rad Laboratories, Richmond, CA) . Reactivity of surface-exposed epitopes of the 39-kD protein was determined using affinity-purified anti-SDH protein antibodies (1:1,000 dilution of 0.5 mg/ml stock).
  • a duplicate blot was developed wi .th Lumi.-PhosTm530 (Adamantyl-1,2- dioxetane phenylphosphate; Lumigen Inc., Detroit, MI), which undergoes enzyme (alkaline phosphatase)-catalyzed dephosphorylation to form a dioxetane anion that is converted ultimately into an excited state of the methyl meta-oxybenzoate anion, the light emitter.
  • the developer was then drained off, and the wet blot wrapped in Saran Wrap was exposed to x-ray film for 20 min and developed using conventional procedures.
  • Densitometric analysis of each spot on the x-ray film was carried out on an image analyzer using the conventional procedures. Densitometric analysis of each spot on the x-ray film was caried out on an image analyzer using the Dumas program (Drexel University, Philadelphia, PA) interfaced with IBM computer.
  • a whole cell assay was developed to determine whether SDH on the surface of streptococci serves as an active GAPDH enzyme.
  • Different concentrations of trypsinized and non-trypsinized streptococci were incubated with and without G-3-P in presence of NAD in triethanolamine-phosphate-EDTA-DTT buffer as described above in a final volume of 1.0 ml for a period of 2 min at room temperature and centrifuged to pellet out the bacteria.
  • the supernatants were analyzed for the conversion of NAD to NADH by recording absorbance at 340 nm.
  • This enzymatic activity was also determined on streptococci preincubated with 1:50 dilution (1 mg/ml) of purified anti-SDH antibodies as prepared above to determine specific inhibition of enzymatic activity.
  • Electrophoresis, Western blotting of lysin extraction and protein samples at different purification stages were carried out as described earlier (2,3).
  • Lysin extracts of M serotypes 2, 4, 5, 6, 24, 29, 41, 57, 58, 60, and M ⁇ were prepared as described (2).
  • the muralytic enzyme mutanolysin (20 ug/ml; sigma Chemical Co.) was used to prepare cell wall extracts of each grouping strain suspended in 50 mM Tris/HCl buffer, pH 6.8, containing 5 mM EDTA, 5 mM MgCl_, and 30% raffinose, and incubated at 37°C for 60 min under . constant end-to-end rotation. Proteins in all the extracts were separated on SDS-PAGE and transferred to nitrocellulose. The blots were probed with affinity- purified anti-SDH protein antibodies as described above.
  • B.stearothermophilus were determined both on Western blot and competitive ELISA as described below.
  • Affinity purified antibodies were adjusted to a dilution that gave an ELISA reading of 1.0 at 405 nm after 60 min.
  • ELISA was performed following standard procedures except that ELISA plates were coated with 100 ul/well of 1 ug/ml SDH for 3 hr at 37°C followed by overnight at 4°C. Competition of GAPDH from different bacterial as well as mammalian origin containing cross reactive epitopes for the binding of Anti-SDH antibodies was performed as described previously (10) . Briefly, ELISA plates were coated as described above with SDH. Optimum dilution of affinity purified antibodies as determined above was used.
  • Competing GAPDH were serially diluted in antibody diluting buffer containing 0.05% Brij-35 pH 7.4 (10) at decreasing molar excess relative to SDH starting with 100 X molar excess.
  • Anti-SDH antibodies were then added in each well and the plates were processed and finally developed and binding of these proteins was determined by kinetic ELISA as described (11) using ELIDA 5 microtitre plate reader Physics Inc. (20) at 405 nm.
  • SDH was labeled with 125I by the chloramine-T method using Iodobeads (Pierce Chemical Co.).
  • the labeled protein was separated from free iodine by filtration over a column of Sephadex G-25 (PD-10, Pharmacia LKB Biotech Inc) and collected in 10 mM HEPES buffer saline pH 7.4 containing 10 mM MgCl_, 2 mM CaCl_, 50 mM KC1 and 150 mM NaCl.
  • the labeled protein was stored at -20°C in aliquots containing 0.02% NaN_.
  • Fibronectin and plasmin were labeled essentially by the same method.
  • the specific activities of SDH, fibronectin and plasmin were, respectively, 2X10 5 , 1.0X10 6 and 1.21X10 6 CPM/ g.
  • the Binding activity of SDH and fibronectin was determined by the use of radioactive proteins. Egg white-lysozyme and/or cytoskeletal proteins (myosin, heavy meromyosin (HMM) , light chain myosin (LMM) , tropomyosin, and actin) all of which obtained from Sigma, were electrophoresed on 10% SDS-PAGE gels and electroblotted on nitrocellulose paper.
  • cytoskeletal proteins myosin, heavy meromyosin (HMM) , light chain myosin (LMM) , tropomyosin, and actin
  • the blots were blocked in 10 mM HEPES buffer containing 15 mM NaCl, 0.5% Tween-20, 0.04% NaN 3 and 0.5% BSA pH 7.4 for 2-3 hr at room temperature and probed for 3-4 hr at room temperature in the same buffer contai .ni.ng 125I- fibronectin, 125I-plasmin at 3X105CPM/ml.
  • the probed blots were then washed 3-4 times with blocking buffer.
  • Autoradiography were prepared by exposing the dried nitrocellulose blots to Kodak Blue Brand film with an intensifying screen for 36-48 hour at -70 C.
  • the ADP-ribosylation of SDH was performed as described (15) with slight modification. Briefly, the standard reaction mixture (0.2 ml) contained 100 mM Tris/HCl at pH 7.4, 10 mM dithiothreitol, 1 mM NADP, 10 mM thymidine (ADPR buffer) . After the addition of 10 uM
  • the GAPDH activity of purified SDH and the ADP- ribosylated SDH was measured by the method originally described by Fritz (7) and modified as described (16) . Briefly, the reaction was performed in a final volume of 1 ml containing 800-850 ul of buffer (40 mM triethanolamine, 50 mM Na.HP0 4 , 5 mM EDTA, pH 8.6),
  • Sodium nitroprusside was freshly diluted in ADPR buffer (200 ul) to a final concentration of 2 mM and preincubated for 2 minutes at room temperature before the addition of 30 ug of SDH and [ 32 P]-NAD to start the ADP- ribosylation reaction. At different time intervals, 40 ul aliquots were removed and precipitated with TCA. A parallel control representing the same quantity of SDH and [ 32P]-NAD were incubated in the absence of sodium nitriprusside and aliquots were taken at the same time intervals as the test samples. Precipitated proteins were separated on SDS gel and autoradiographed. In a similar set of experiments, ADP-ribosylation was also performed using 200 ul of a streptococcal lysin extract in ADPR buffer incubated in the presence and absence of 2 mM sodium nitroprusside.
  • SDH protein was precipitated from the lysin extract by first precipitating non-specific proteins at 60% saturation of ammonium sulfate followed by 85% saturation. The SDH was found in the 85% ammonium sulfate precipitate (Fig. 1) .
  • the dialyzed precipitate was applied to a Mono Q FPLC column and the proteins eluted with an NaCl gradient from 0 mM to 300 mM. SDH eluted at a salt concentration of about 280 mM. Fractions with fibronectin binding activity were pooled, dialyzed and further purified on a Superose-12 FPLC molecular sieving column.
  • NH,-terminal amino acid sequence analysis of the purified SDH confirmed the homogeneity of the preparation resulting in a single amino acid at nearly all positions (Fig. 2a) . Except for positions 31 and 35, a single amino acid was identified in the first 35 residues with the remaining four tentatively identified.
  • the amino acid composition of the purified protein indicated a high content of Asp/Asn (12.1%), followed by Ala (10.7%), Gly (10.3%), Val (10.2%), and Glu/Gln (8.4%).
  • the mass of the purified protein 35,882 daltons as determined by laser desorption mass- spectrometry was used to more precisely assign the number of residues/mol (Table 1) .
  • Antibodies to SDH were affinity purified on SDH- bound to activated gluteraldehyde beads followed by a protein A column.
  • the resultant purified anti-SDH IgG recognized only the SDH protein band (Fig. 4) .
  • Dot-Blot immunoassay was applied to determine the location of SDH on streptococcal surface. Results revealed that trypsin treated streptococci were markedly reduced in their reactivity to anti-SDH IgG (Fig. 5) . To determine if the
  • SDH protein is peripherally bound to the cell wall or tightly bound, the streptococcal cells were washed with 2M NaCl and 2% SDS. The results revealed that the SDH was not extractable by the high salt or ionic detergent.
  • Fig. 6 Data presented in Fig. 6 revealed a dose dependent GAPDH activity catalyzed by the whole organisms. As found with the purified SDH, the intact bacteria also did not catalyze the reaction in absence of the specific substrates G-3-P and NAD (Fig. 6a) . The enzymatic activity on the whole organisms was also found to be partially (30%) but specifically inhibitable by anti-SDH IgG (Fig. 6c) . Enzymatic activity was found to be decreased by 80% when trypsinized bacteria were used in the reaction mixture (Fig. 6b) . The background 20% activity suggested an incomplete digestion of SDH protein by trypsin.
  • SDH protein ubiquitous nature of the SDH protein in different streptococcal M serotypes was determined by Western blot analysis of lysin extracts using affinity purified anti-SDH IgG. As shown in Fig. 7, SDH protein was found in several serotypes. Furthermore, all were found to be of same molecular weight without any indication of size variation.
  • the fibronectin binding activity of SDH was determined both by using 125I-labeled fibronectin or fibronectin-anti-fibronectin on a Western blot. The results revealed that the SDH protein was able to bind fibronectin in both assays (Figs. 10b and c) .
  • SDH is a major surface protein of streptococci, including group A streptococci and has both enzyme activity and multiple binding activity. No such protein has previously been detected, isolated and characterized.
  • the novel surface protein is principally characterized by its ability to bind fibronectin, lysozyme and cycloskeletal protein as well as by its enzymatic activity as a GAPDH. Its molecular weight is approximately 39 kDa. The first fifteen amino acid residues at the amino terminal are:
  • GAPDH 100% homology with the bacterial form of GAPDH and 80-90% homology with eukaryotic or fungal GAPDH.
  • SDH is, however, significantly different from previously recorpted GAPDHs, because the high homology of the first fifteen amino acid residues is not preserved towards the carboxy end of the molecule and the amino acid composition varies appreciably from other GAPDHs.
  • SDH also functions as an ADP-ribosylating enzyme which, in the presence of NAD, is auto-ADP-ribosylated. It has been found that in a crude lysin extract of group A streptococci containing a mixture of cell wall associated molecules, SDH is the only molecule that is
  • ADP-ribosylated Treatment of ADP-ribosylated SDH with the cytoplasmic fraction of Group A removed the ADP- ribosye of SDH which indicates the presence of SDH specific ADP-ribosyl hydrolase in the cytoplasmic compartment. Treatment of purified SDH or the crude lysin extract with sodium nitroprusside, which spontaneously generates nitric oxide, was found to stimulate the ADP-ribosylation of SDH in a time dependent manner. Both ADP-ribosylation and nitric oxide treatment inhibited the glyceraldehyde-3-phosphate dehydrogenase activity of SDH.
  • SDH does not represent a single protien, but rather a class of surface proteins of streptococci, all of which have similar properties.
  • the protein is involved in the colonization and probably in the internalization and proliferation of group A streptococci.
  • the enzyme activity of SDH may be involved in the binding of the bacteria to endothelial cells by reaction of an aldehyde reduction product of teichoic acid which is a polyglycerol phosphate.
  • the aldehyde function could bind the bacteria to the tissue surface by reaction with amino group on that surface.
  • proteins, polypeptides and peptides of this invention may be obtained by any of a number of known processes.
  • the protein can be isolated as described above. Alternativley, the protein or segments thereof can be prepared by recombinant DNA techniques. For example, the gene for the protein or an oligonucleotide for the desired segment can be inserted into a plasmid and the plasmid used to transform E. coli so that the bacteria will express the desired product.
  • Polypeptide and peptides within the scope of the invention containing, for example from about 6 to 20 or more amino acid segments may be synthesized by standard solid phase procedures with appropriate amino acids using the protection, deprotection and cleavage techniques and reagents appropriate to each specific amino acid or peptide.
  • a combination of manual and automated (e.g.. Applied Biosystem 430A) solid phase techniques can be used to synthesize the novel peptides of this invention.
  • Altough less convenient, classical methods of peptide synthesis can also be employed.
  • the products of the invention are amphoteric. They can exist and be utilized as free bases or as pharmaceutically acceptable metallic or acid addition salts.
  • Suitable metallic salts include alkali and alkaline earth metal salts, preferably sodium or potassium salts.
  • Acid addition salts may be prepared from a wise variety of organic and inorganic acids including mineral acids, for example citric, lactic, aleic, tartaric, phosphoric and hydrochloric acids. These salts can be prepared by procedures well known to those skilled in the art.
  • cholera toxin B For use as a vaccine, it is presently preferred to administer the selected product conjugated to a carrier such as cholera toxin B.
  • a carrier such as cholera toxin B.
  • the protein or segment thereof may also be administered as a hybrid protein expressed on a streptococcal surface utilizing the procedure of Pozzi et al (17,18).
  • the presently preferred method for the administration of the vaccines of the invention is by the intranasal route, but the invention is not so limited. Other parenteral or oral prcoedures may be employed.
  • the patient to be protected will be treated with an amount of SDH or other product of the invention which is effective to elicit a protective immune response.
  • the selected agent may be administered alone or in a pharmaceutically acceptable liquid or solid carrier in which it may be dispersed, dissolved or suspended. If, for example, the patient is to be treated intravenously, the peptide may be suspended as a free base or dissolved as a metallic salt in isotonic aqueous buffer.
  • Other methods of treatment and pharmaceutically acceptable carriers will be apparent to the skilled artisan.
  • proteins, polypeptides and peptides of this invention and the genes or oligonucleotides which are employed in their expression are useful as probes for genes and proteins. They are also useful to raise antibodies by which specific strains of streptococci can be identified. For example in tests for mammalian infections.
  • D-glyceraldehyde-3- phosphate dehydrogenase the purification and characterization of the enzyme from the thermophiles Bacillus stearothermophilus and Thermus aquaticus. Eur. J. Biochem. 108:535.

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Abstract

L'invention concerne une nouvelle protéine de surface streptococcique se caractérisant principalement par son pouvoir à lier les fibronectines et les lysozymes, mais qui possède également une activité glycéraldéhyde-3-phosphate déshydrogénase, une activité de ribosylation d'ADP et une activité ADP-ribosyl transférase. Cette protéine est notamment utile dans la préparation des vaccins pour assurer une protection contre les infections streptococciques.
PCT/US1993/000082 1992-01-08 1993-01-07 Proteine de surface multifonctionnelle de streptococci WO1993014198A1 (fr)

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EP93902960A EP0672123A1 (fr) 1992-01-08 1993-01-07 Proteine de surface multifonctionnelle de streptococci
AU34351/93A AU668908B2 (en) 1992-01-08 1993-01-07 Multifunctional surface protein of streptococci
JP5512544A JPH07502896A (ja) 1992-01-08 1993-01-07 連鎖球菌の多官能性表面タンパク

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US81817092A 1992-01-08 1992-01-08
US07/818,170 1992-01-08
US91373292A 1992-07-15 1992-07-15
US07/913,732 1992-07-15

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006732A2 (fr) * 1993-09-01 1995-03-09 The Rockefeller University Proteines bacteriennes exportees et vaccins acellulaires realises a partir de ces proteines
WO1995012410A1 (fr) * 1993-11-05 1995-05-11 Eli Lilly And Company Conception et production de vaccins
WO1997026008A1 (fr) * 1996-01-22 1997-07-24 Regents Of The University Of Minnesota VACCIN A BASE DE PEPTIDASE C5a DU STREPTOCOQUE
US6245335B1 (en) 1996-05-01 2001-06-12 The Rockefeller University Choline binding proteins for anti-pneumococcal vaccines
EP1140994A1 (fr) * 1998-12-22 2001-10-10 Microscience Limited Proteines de surface exterieure, leurs genes, et leur utilisation
US6355255B1 (en) 1998-12-07 2002-03-12 Regents Of The University Of Minnesota Streptococcal C5a peptidase vaccine
US6660270B2 (en) * 2000-06-12 2003-12-09 University Of Saskatchewan Immunization of dairy cattle with chimeric GapC protein against Streptococcus infection
US7256265B2 (en) 1999-12-03 2007-08-14 Regents Of The University Of Minnesota Streptococcal C5a peptidase vaccine
US7419672B2 (en) 1998-12-22 2008-09-02 Emergent Product Development Uk Limited Genes and proteins, and their use
US7592011B2 (en) 1998-12-22 2009-09-22 Emergent Product Development Uk Limited Genes and proteins, and their use
US8563001B2 (en) 2008-11-05 2013-10-22 Regents Of The University Of Minnesota Multicomponent immunogenic composition for the prevention of beta-hemolytic streptococcal (BHS) disease
CN111198271A (zh) * 2018-11-16 2020-05-26 山东泽济生物科技有限公司 一种用于甘油醛-3-磷酸脱氢酶检测的化学发光酶联免疫试剂盒

Citations (1)

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WO1990015872A1 (fr) * 1989-06-21 1990-12-27 The Rockefeller University Poxvirus de recombinaison et vaccin streptococcique contenant une proteine m

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WO1990015872A1 (fr) * 1989-06-21 1990-12-27 The Rockefeller University Poxvirus de recombinaison et vaccin streptococcique contenant une proteine m

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Title
ABSTRACTS OF THE ANNUAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY vol. 91, no. B-30, May 1991, WASHINGTON US R.LOTTENBERG ET AL 'A group A Streptococcal plasmin receptor demonstrates homology with Glyceraldehyde-3-phosphate dehydrogenase' *
THE JOURNAL OF EXPERIMENTAL MEDICINE vol. 176, no. 2, 1 August 1992, NEW-YORK US pages 415 - 426 V.PANCHOLI AND V.A.FISCHETTI 'A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate dehydrogenase with multiple binding activity' *

Cited By (28)

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US5981229A (en) * 1993-09-01 1999-11-09 The Rockefeller University Bacterial exported proteins and acellular vaccines based thereon
WO1995006732A3 (fr) * 1993-09-01 1995-06-01 Univ Rockefeller Proteines bacteriennes exportees et vaccins acellulaires realises a partir de ces proteines
WO1995006732A2 (fr) * 1993-09-01 1995-03-09 The Rockefeller University Proteines bacteriennes exportees et vaccins acellulaires realises a partir de ces proteines
US5928900A (en) * 1993-09-01 1999-07-27 The Rockefeller University Bacterial exported proteins and acellular vaccines based thereon
WO1995012410A1 (fr) * 1993-11-05 1995-05-11 Eli Lilly And Company Conception et production de vaccins
US5679349A (en) * 1993-11-05 1997-10-21 Eli Lilly And Company Vaccine design and production
CN1087953C (zh) * 1993-11-05 2002-07-24 伊莱利利公司 疫苗的设计和生产
WO1997026008A1 (fr) * 1996-01-22 1997-07-24 Regents Of The University Of Minnesota VACCIN A BASE DE PEPTIDASE C5a DU STREPTOCOQUE
US8440205B2 (en) 1996-01-22 2013-05-14 Regents Of The University Of Minnesota Streptococcal C5A peptidase vaccine
US6270775B1 (en) 1996-01-22 2001-08-07 Regents Of The University Of Minnesota Streptococcal C5a peptidase vaccine
US7635483B2 (en) 1996-01-22 2009-12-22 Regents Of The University Of Minnesota Streptococcal C5a peptidase vaccine
US6951653B2 (en) 1996-01-22 2005-10-04 Regents Of The University Of Minnesota Streptococcal C5a peptidase vaccine
US5846547A (en) * 1996-01-22 1998-12-08 Regents Of The University Of Minnesota Streptococcal C5a peptidase vaccine
US6784164B2 (en) 1996-05-01 2004-08-31 The Rockefeller University Choline binding proteins for anti-pneumococcal vaccines
US6245335B1 (en) 1996-05-01 2001-06-12 The Rockefeller University Choline binding proteins for anti-pneumococcal vaccines
US7425327B2 (en) 1996-05-01 2008-09-16 The Rockefeller University Choline binding proteins for anti-pneumococcal vaccines
US6355255B1 (en) 1998-12-07 2002-03-12 Regents Of The University Of Minnesota Streptococcal C5a peptidase vaccine
US7419672B2 (en) 1998-12-22 2008-09-02 Emergent Product Development Uk Limited Genes and proteins, and their use
US7592011B2 (en) 1998-12-22 2009-09-22 Emergent Product Development Uk Limited Genes and proteins, and their use
EP1140994A1 (fr) * 1998-12-22 2001-10-10 Microscience Limited Proteines de surface exterieure, leurs genes, et leur utilisation
US7256265B2 (en) 1999-12-03 2007-08-14 Regents Of The University Of Minnesota Streptococcal C5a peptidase vaccine
US7258992B2 (en) 2000-06-12 2007-08-21 University Of Saskatchewan Immunization of dairy cattle with chimeric GapC protein against Streptococcus infection
US6875853B2 (en) 2000-06-12 2005-04-05 University Of Saskatchewan Immunization of dairy cattle with chimeric GapC protein against streptococcus infection
US6660270B2 (en) * 2000-06-12 2003-12-09 University Of Saskatchewan Immunization of dairy cattle with chimeric GapC protein against Streptococcus infection
US8563001B2 (en) 2008-11-05 2013-10-22 Regents Of The University Of Minnesota Multicomponent immunogenic composition for the prevention of beta-hemolytic streptococcal (BHS) disease
US9127050B2 (en) 2008-11-05 2015-09-08 Regents Of The University Of Minnesota Multicomponent immunogenic composition for the prevention of beta-hemolytic streptococcal (BHS) disease
CN111198271A (zh) * 2018-11-16 2020-05-26 山东泽济生物科技有限公司 一种用于甘油醛-3-磷酸脱氢酶检测的化学发光酶联免疫试剂盒
CN111198271B (zh) * 2018-11-16 2023-04-28 山东泽济生物科技有限公司 一种用于甘油醛-3-磷酸脱氢酶检测的化学发光酶联免疫试剂盒

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JPH07502896A (ja) 1995-03-30
AU668908B2 (en) 1996-05-23

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