JPH07502896A - Multifunctional surface proteins of streptococci - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] ・1 -'s Tom knee ■Nonpa The present invention is directed to the National Institutes of Health (National Institutes of Health). h) with Government support under Grant No. Al-11822. the government All rights reserved.

League melon dish The present invention is jointly filed with serial number 07/913,732 filed on July 15, 1992. This is a partial continuation application of a concurrent continuation application. The serial number 07/913,732 is 199 This is a continuation application with serial number 07/818,170 filed on January 8, 2017. The latter The request has now been abandoned.

The present invention relates to surface erosion of streptococci due to early colonization of the pharyngeal mucosa. Yo Specifically, on the surface of streptococci, including pathogenic streptococci such as Streptococcus pyogenes. This protein is a multifunctional protein that contains fibronectin, lysozyme, and and the ability to bind to the cytoskeletal proteins myosin and actin and their enzymatic activity. Dose-dependent dehydrogenation, especially with glyceraldehyde-3-phosphate (CADPH) Characterized by enzyme activity. This molecule is also used for AD P-lyphosylation enzymes. It also acts as an ADP-lyphosyltransferase.

The present invention also provides, for example, the whole protein and its segments, particularly those with similar activity to Tannokuku. Therapeutic compositions such as vaccines and surface tanning prepared from preserved segments with Concerning the use of Park.

Honhatsu Hada O Gyodan Diseases in mammals, especially humans, caused by streptococcal infections caused by bacteria such as Streptococcus pyogenes. Childhood diseases are an important health problem. In the United States alone, the disease primarily affects school-age children. Between 25 and 35 million cases of group A streptococcal infection are reported each year ( circle. Associated with streptococci due to the high incidence and potential severity of streptococcal infections There is an urgent need to develop effective and safe vaccines to prevent infection.

A drug such as group A type 6, which has both enzymatic power and the ability to bind to various proteins. Streptococcal surface dehydrogenase (sjr) on streptococcal surfaces from several serological groups Eptococcal 5 surface dehydrogenase: 5D H) has now been discovered. In early applications of this series, this surface protein When it was first isolated, purified and characterized, it was called MF6. This was the laboratory code given to him. This is now the main characteristic of streptococcal bacteria. Surface dehydrogenase: 5treptococcal 5surface Dehy It is called SDI because it is drθgenase. More specifically, A protein exhibiting lyceraldehyde-3-phosphate dehydrogenase (GADPH) activity. This is from Park's class.

G A D P) (Protein, as its name implies, is a protein that A class of dehydrogenase enzymes that are closely involved in the reaction. Generally this class Some are found in the cytoplasm, while others are attached to eukaryotic membranes and cytoskeleton. It will be seen together. The glycolytic enzyme of the present invention is a prokaryotic surface protein. It is believed to be unique in that respect. Such CA D P H protein has never been published before.

The CADPH protein of the present invention is similar to other GADPH family proteins. It has several similar structural features. For example, the NH2 end of three amino acids More than 80% of the end 18 molecules are identical to the CADP H family of enzymes. but However, as explained below, the CADPH proteins of the present invention differ in many respects. It is a novel product that has never been isolated and characterized.

A detailed examination of the properties of purified SDR reveals that its original conformation is probably It is a tetramer with a molecular weight of approximately 156 kDa.

The molecular weight of this protein according to mass spectrometry is approximately 35.8 kDa.

According to 5DS-PAGE, it is about 39.2 kDa.

Proteins were isolated from AlB, CXE, and GS using affinity-purified anti-3DR antibodies. Group H and L streptococci were identified on the surface of Streptococcus.

This protein consists of beta-nicotinamide adenine dinucleotide (NAD). In the presence of cellular aldehyde, 3-phosphate (G-,3-P) has a dose-dependent dehydrogenase effect. It showed drogenase activity. The multifunctional activity of SDI is associated with fibronectin, lysosin team, and by its ability to bind to the cytoskeletal proteins myosin and actin. Shown. The binding activity of SDH to myosin is determined by the globular heavy meromyosin region. It was found to be localized in SDH is streptococcal M protein, tropomyosin, Alternatively, it does not bind to the convoluted chain region of myosin. SDI is particularly important for cytoskeleton Its ability to multiplex bind to proteins, together with its GADPH activity, is important for the collection of streptococci. Demonstrates a role in desynthesis, internalization, and subsequent proliferation. all Streptococcal trypsinization of the body significantly reduced reactivity to SDH antibodies. . Even after washing in 2M NaCl or 29g SDS, the surface of streptococcus remains The reason that SDR cannot be removed is that this protein is not bound to the surrounding area. , indicating tight binding to cells. All this data is stored in this tamper. The results show that the microorganisms are surface CADPH molecules on streptococcal cells.

This novel SDI contains SDI by solubilizing selected streptococci with lysine. Obtained by making a mixture. SDR can be performed by one of ordinary skill in the art, including the methods described below. can be separated by any of a number of suitable methods known to those skilled in the art.

In addition, organisms such as Escherichia coli (E. coli) can be transformed with appropriate genes to produce large It can also be produced by allowing enteric bacteria to express SDR.

The following abbreviations are used in the description of the invention.

NAD: beta nicotinamide adenine dinucleotide PVDF: polybi Nylidene difluoride EDTA: ethylenediaminetetraacetate PMS F: paradimethylsulfonyl fluoride TLCK: N-p-tosyl-silane Zine chloromethyl ketone SDS Sodium nidodecyl sulfate Mono Q FPLC: Mono Q (product name) rapid protein liquid chromatograph Raffy 5uperose 12 FPLC: 5uperose-12 (product name) quick tap Protein liquid chromatography TSK-Phenyl HPLC:TSK-Ph enyl (product name) high performance/pressure liquid chromatography NADH: Betani Cotinamide adenine dinucleotide reduced product ELISA: Enzyme immunoassay ELH) A: Physica Inc. product name 5ephadex G-25P D-10: G-25PD-10Pharmacia-LKB Inc. trade name HEPES: (N-[2-human xyethyl]piperidine-N'-[2-ethyl] Tansulfonic acid]) RGDS: Arginine-glycine-aspartic acid-serine (Arg-Gly -Asp-8er) G-3-P Nigeblade Cellaldehyde 3-Phosphate CADPH Nigeli Cellaldehyde DO-3 ~ Phosphate dehydrogenase diagram The drawings will be explained below.

Figure 1: 5DS-polyacrylamide gel of SDH protein from M6 Streptococcus ( 1096) analysis, Lane a: D471 Streptococcus noricin extract.

Lane 1]: 65% (NH4)2SO4 saturated precipitation of ricin extract.

Lane c: -1-clear 85 S'6 (NH4) 2S 0 after 6506 precipitation 4 saturated precipitate.

Lane d: 0. Pooled Mon with 28M gradient elution.

Q Fraction.

Lane e: 5uperose 12-force ram partial purification 5DH0 lane f: Ph eny I - Purification from TSK column 5DH0 lanes a and b 50k The arrow in Da indicates the position of M protein.

Prestained marker protein mixture shown on the left.

200kDa: Myosin (H-chain) 97, -4kDa: phosphorylase b 68kDa: bovine serum albumin 43kDa: Ovalbumin 29kDa: Carbonic anhydrase 18.0kDa: Lactoku heppurine 14.3kDa: Egg white lysozyme Figure 2: (a) NH2 terminal sequence of SDH not sequenced (b) NH2 terminal amino acid sequence of SDH column and the apposition of known CADPH molecules obtained from translated gene bank databases. Comparison with amino acid sequences.

B s t GAP - Hasilus stearothermophilus GADPHEcoG AP-E. coli CADPH HumGAP-human GADPH Ch　k　G　,A　P-Chicken CADPH3mP37-Schistosoma manso ni 37kDa protein (GADPI) ZmoGAP-Zymomonas mobilis Number on the right side of GADPI diagram For residues 1-18 and 1-39, the relationship between SDH and other CA　DPH　H Shows similar percentages. The gap between the 14th and 15th positions of the chicken GADPH sequence The difference (=) is included to maximize the similarity.

Figure 3: Reinleiber-Park double reciprocal dynamics of SHD GA and DPH activities academic analysis.

pH 8,6k) in reethanolamine-phosphate EDTA-DTT buffer , 25 μg of SDI in the presence of NAD (100 μM) (, as a function of -3-P and assayed. For G-3-P, the Km is estimated to be 1.33mM.

Vmax: 0.487xlO°3M NADH/min, Y-axis intercept (1/Vmax ): 2.05, and slope (Km/Vmax): 2°73o The inset is Michaeli An analysis based on S-Menten is shown.

Km: 1.22mM and Vmax: 0.466xlO°’M NADH/min.

(b) Rheinleiber-Paak double inversion of GADPH activity of the 39 kDa protein. Numerical dynamics analysis.

In the buffer of (a), 25 μg of SDI was added to N in the presence of G-3-P (2 mM). Assayed as a function of AD. For NAD, Km is estimated to be 156.7 μM be done.

Vmax: 0.459xlO3M NADH/min, Y-axis intercept (1/Vmax) : 2.18, and slope (Km/Vrrjax): 341.74° Shown in the inset According to the Michaelis-Menten method, the Km for NAD is 148.86 μM. Estimated, Vmax is 0°445x 103M NADH/min.

Figure 4: (A) Coomassie blue staining of SDS-gel and (B) affinity Western blot analysis of SDI with purified anti-3DR antibody. These are SD This suggests a multiple bond structure of the I molecule.

Lanes a and d: crude ricin extract.

Lanes b and e: purified 5DH0 Lanes C and f: NA without sDs, D unboiled purification in saturated sample buffer. 5DH0 The arrow indicates the position of a molecule of the same size as the tetrameric form of SDI. molecular weight marker - is shown on the left. (See Figure 1 for details of each marker) Figure 5: Dot plot immunoassay with SDH placed on the surface of streptococci. this The assay was performed using affinity purified anti-3DR antibody in 2% SDS, 2M NaCl and This test examines the degree of reactivity to exposed surfaces before and after trypsin treatment. de The plots were treated with Lum1Phos-530 substrate (41) and exposed to X-ray film. Developed. The density of the image obtained on the X-ray film is read and the Dumas program (D rexel University, Phladelphia, USA) expressed as optical density in arbitrary units on an image analyzer using dot top As a final concentration analysis of the lot, the optical density of o, oos to 1.333 was determined. A linear standard curve was obtained. Each bar on the graph is the average of 4 to 8 separate readings. The upper standard deviation is shown.

Figure 6 = CADPH activity of all streptococci.

(a) Measurement of GADPH activity and conversion of NAD to NADH in the presence of G-3-P The total M6 was observed at 340 mn. For more information on buffer systems, see Materials and This will be explained in the method section. (b) Activity of M6 streptococci treated with I-lipsin. (c ) Inhibition of enzymatic activity of affinity-purified anti-3DI antibody (0.5 mg of 1:30 /ml for 3 hours at room temperature). Each bar on the graph represents the standard deviation above the average of three separate readings. show.

Figure 7: (A) Affinity purification of various Streptococcus M type Noricin extracts with anti-3DI Western blot analysis at 1:2000 dilution of 0.5 mg/ml strain with antibodies . Purified SDH and M-negative (M') streptococci are also included in the analysis.

(B) Mutagenesis of strains of various groups in the same manner as in (A) using anti-8DI antibody. Western Pro node analysis of Mutanolysin extract.

Figure 8: Competitive kinetics enzyme-linked immunosorbent assay (kin) with immobilized SDI etic enzyme-1 inked immunosorbent ass ay:　kELIsA　). Commercially available purified C from Stearothermophilus ADPH, human red blood cells, and rabbit skeletal muscle were purified using affinity-purified anti-3DR anti- (1:1000 dilution of 0.5 mg/ml strain) was used for binding competition. each curve represents the average of three independent experiments (standard deviation <5% - not shown). Also inserted (a) Streptococcus 5DR1 (b) CADPH of Stearothermophilus (c) CADPH of rabbit skeletal muscle; (d) GADPH of human red blood cells; Figure 3 shows Western blot analysis of the reactivity of initi-purified anti-3DR antibodies.

Figure 9: Binding of 1251-3DI to cytoskeletal proteins.

(A) 5 μg of various cytoskeletal proteins, myosin, and M6 protein (43 ) Coomassie blue-stained 5DS-PAGE gel (1096).

lane mini rabbit skeletal myosin.

Lane b: heaping meromyosin.

Lane C: light meromyosin.

Lane d, actin.

Lane e: M6 protein.

Lane f: S-2 fragment of heavy meromyosin.

Lane g: egg white lysozyme.

(B) Replicated gel was transferred to nitrocellulose and incubated with 125I-3DI. Park's autoradiograph. Proteins in each lane are the same as in (A). Molecular weight in left column markers (see Figure 1 for details of each marker).

Figure 10: SDI binding activity to fibronectin.

(A) Coomassie staining of an SDS gel containing 5 μg of SDH and BSA.

(B) Binding of fibronectin to SDR molecules followed by anti-fibronectin binding. Western blot analysis of replicate gels showing binding.

(C) “Similar waste membrane showing binding of 5I-fibronectin to SDR protein. Autoradiograph of tan plot. Lanes a, C and e-3DH, lanes b, d and f-BSA0 Figure 11: ADP-ribosylation of SDH. Purification SDR (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 and 12) fractions were incubated with [32P]NAD in ADPR buffer. Next Proteins were separated on a 12% SDS gel and stained with (A) Coomassie blue. . (B) Replicate gel was reacted with affinity-purified anti-3DH antibody and Western protein (C) Perform autoradiography of the same Western blot. It was. Molecular weight markers are shown in the left column.

Figure 12: ADP-ribosyltransferase activity of SDR.

SDR and various binding proteins (2740) were synthesized in the presence of [32P]NAD. cultured together. The protein mixture was precipitated, washed, and 1296 SDS-PAGE Dissolve and stain with Coomassie blue, dry replicate gels and autoradiograph I went to SDI, actin (lanes 1 and 6), chicken egg white lysozyme (lanes 2 and 7), myosin S-1 fragment (lanes 3 and 8), fib ronectin (lanes 4 and 9), and plasmin (lanes 5 and 10). Cultured. Molecular weight markers are shown in the left column.

The following Materials and Methods section is provided for convenience to facilitate understanding of the invention. belongs to.

material and method Company: Human fibronectin is manufactured by Boehringer Mannheim. Obtained from eim). Goat anti-human fibronectin and alkaline phospha Affinidi-purified heron anti-goat IgC conjugated to Sigma Obtained from a). Prestained molecular weight standards were purchased from Bethesda Laboratories (Bethesd Laboratories). It was purchased from Re5search Laboratories). PVDF membrane [Immobilon-P J is manufactured by Millipo re). Na125I is New England nucleus ( It is from New England Nuclear). Other chemistry All items were purchased from Sigma unless otherwise indicated.

mti+ Bacteria. Used for grouping various M-type group A β-hemolytic streptococcal strains and streptococci. The standard strain is from the Rockefeller University Culture Collection (The Rockefeller University culture coMection, New Y Ork, NY), and the list is shown below.

M2 (C626), M4 (C896), M5 (Manfranco), M6 (C 471), M24 (C524), M29 (D23), M41 (C101/103 /4), M57 (A995), M2S (C774), M2O (C398), M- (T28151/4): Group ASJ17A4 (M-strain): Group A degeneration, A486 var: Group 13, 090R: Group C, C74: Group DSD76, Group ESK13 1: Group F, F68C: Group G, D166B, Group H, F90A: Group LSD1 67B, group N1C559° Ishin 1 middle 6 SDH member 3゜ Crude extracts containing major surface proteins were extracted with chain cells using a previously published method (2). It was prepared using Norisin extraction treatment to remove bacterial cells. Essentially, pH 5.5 Bacteria washed in 50mM sodium acetate buffer of 3006 rough, inose and 5 MM of EI) TA in the same buffer. Add to suspension Add gin (1:100 dilution, 360 units) and incubate end-to-end for 90 minutes at 37°C. The cells were cultured with gentle rotation in contact with each other. The protoplasts to be generated are rvall) in a centrifuge at 15,000 g for 30 minutes. −”−Clear Take 25mM T r i S/'HC] (p)-i8.5.5mM Dialyzed with EDTA and concentrated with Amicon Ph, 1-10 membrane (Corp for Arniα). and used for further purification.

After lysine extraction, the pelleted protoplasts were resuspended in hypotonic buffer (2 MM of P b, x S F, ], MM of T L 2 MM acetic acid solution at pH 5°5 containing l2 and lOμg/ml DNA5e. thorium) and then subjected to three freeze/thaw cycles. The membrane was heated at 4°C, i. Precipitate at oo, 000g for 45 minutes. Cytoplasm extraction in membrane pellet and supernatant The samples were separated on an SDS gel and analyzed by coumasin blue staining. expose the membrane 1.5M sodium chloride or 100mM sodium carbonate at pH 1, 1, 3. The properties of SDH protein binding to the membrane were investigated by treating it with Bevco.

To determine whether this protein is surface exposed, trypsinized bacteria Noricine extraction was performed as previously published (3). briefly explain Then, the washed bacteria were suspended in 100mM NH, HCO3 and incubated with trypsin at 37°C. Digestion was performed with soybean trypsin (250 gg/ml) for 3 hours. Inhibitor (inactivated at 200 Ig/m2). extract of SDI protein and a comparative standard non-trypsinized bacterial paste extract. The size reduction was compared.

Dandandan O koshirae Lysine extract was used as starting material for the purification of SDR. Dialyze and concentrate The extract was precipitated with 60% saturated ammonium sulfate at 4°C. Precipitate 6,0 Centrifuged at 00g for 20 min and supernatant poured into 8566 saturated ammonium sulfate.

The resulting precipitate was dissolved in 25mM Tris/HCl (pH 8,5). 5), MonoQ dialyzed with 5mM EDTA and equilibrated with the same dialysis. FPLC column (Pharmacia LKB Biotechnology Inc.).

After washing the column with the first 5 column volumes of buffer, the bound protein was washed with 50 ml of 0. Gradient elution was carried out with 300mM NaCl. A film containing SDR The fractions were pooled and permeabilized with 35mM Tris/HC1/EDTA buffer. The sample was analyzed and chromatographed again using a MonoQ column. Next, the film containing SDR Pool the fractions and use a Cen t r 1con concentrator (cutoff molecular weight 10,000 kDa) to a volume of L Oml. Concentrate the sample to 0.3 Pre-equilibrated with 50mM Tris/HCI pH 8.5 containing NaCl of M 5uperose 12FPLC column (Pharmacia LFJ3 In (manufactured by C.). Pooling the fractions containing SDR protein, 1. 0.025M Tris/HC at pH 8.5 containing 0M ammonium sulfate TSK-phenyl HPLC column dialyzed against I buffer and pre-equilibrated with the same buffer. Bio-Racf Laboratories, Richmond CA ) was applied. Reduce protein with ammonium sulfate from 1.0M to O,OM Linear gradient elution was performed. The purity of the final product was determined by protein purification on an SDS gel. Determined by coumasin blue staining and analysis procedures. The purified product has a pH of 8.5 after dialysis with 0.025 M Tris/MCI at 4°C for various protein binding experiments. and for long-term storage at -70°C.

l-・Nl2-M"Mi': Nl2 one terminal arrangement is Matsudai It was measured by the method of ra et al. (4). To explain briefly, purified SDR is electrically charged in advance. Separated under non-denaturing conditions on a 1096 acrylamide SDS gel, Transfer to a PVDF Immobilion-P filter pre-moistened in ethanol. did. 0.0596 coumacin in a 50:40 methanol, water, acetic acid solvent mixture Visualized by blue. Pro-soto staining with methanol:water:acetic acid (50:4) 0:10) for decolorization. The part of the membrane containing the SDS band was extracted and applied. Automatic Edman disassembly with Biosystem model A470 sequencer I let it happen. Each band is about 2- It contained 3 μg of protein. Regarding amino acid composition, SDS-containing PVD F membranes were stained with 0.1% Ponceau-3 (Sigma) in 1% acetic acid. . A section of the membrane containing the protein band was excised and decolorized in water. This membrane section The mixture was hydrolyzed in 6N HCI/phenol at 110° C. for 22 hours. amino Acids were supplied using Waters Maxima software (510 pump and 490 pump). Separated using a Waters Novapek C8 column . The cysteine content was determined by the method published by Crestf 1eld (5). Analyzes were also made from PVDF-bound carboxyamidomethylated protein. all Analysis was performed by Rockefeller University's Protein BiochemistryFac ivy done with ility.

Molecular weight measurement: Molecular weight determination of purified proteins is performed using a modified matrix How to wear (modified matrix-assisted 1aser de Rockefeller University using the sorption technique (6) The department of Mass Spectrometry and Gas Phase.

1 cell Ruhi'31in-higuchi-ze CADPH box: The CADPH assay was first published by Ferdinand (7) The method was slightly modified. CADPH is the concentration of NAD+ and inorganic phosphate. Oxidative phosphorylation of DG-3-P to 1,3 diphosphoglycerate in the presence of Due to its catalytic activity, the assay solution contains triethanolamine (40mM), N a2HPO4 (50mM) and EDTA (5mM). disposable In a semi-micro 1.5 ml spectrophotometer cuvette (VWR), add 7 μl of G-3-P. (Sigma, 49 mg/ml), 100 μM NAD (Boehri Mannhe im) and the final volume after addition of the enzyme source is 1.0 ml of assay buffer was added (pH of the mixture was 8.6). 5pect NA of NAD using a ronic3000 spectrophotometer (Milton Roy) The standard absorbance at 340 nm/min is used as a measure of the conversion to DI (pro-so). Different concentrations of SDR were used to

enzyme reaction rate The enzyme reaction rate of SDR is determined by changing the NAD concentration and fixing the G-3-P concentration. For the opposite case, examine Km and V for NAD and G-3-P, respectively. max was measured. The results are NADH for each handshake for 1 minute at 340nm. It was recorded as a kinetic analysis of release. Absorbance (340nm)/min to NADHM/min The molecular extinction coefficient NADH6,22x103(8) was used for conversion. speed officer The numbers are the quadratic profile of the intercept of the linear Park plot for each substrate. Estimated based on The specific activity of the enzyme (unit/mg) is calculated using the following formula. .

Specific activity = v C1 + m/5) NAD (1 + km/5) G-3-P where, v = The specific activity of GADPH activity relative to u mol NADH/min/enzyme mg SDI is gin extract, ammonium sulfate precipitation, and fractions pooled from various purification steps. tion was measured.

゛'s IO, ``1'-'s Funnie ゛-I: New Zealand Night Rabbit , purified SDR emulsified in 200 Mg Freund's complete adjuvant. (1:1) was subcutaneously injected at multiple sites to confer immunity. Floy for rabbits One booster injection was performed with 200 μg of this protein in incomplete adjuvant. It was. All rabbits were tested 3 weeks after the first injection and 10 days after the second injection. I bled. All sera were filter sterilized and stored at 4°C.

In order to purify SDR-specific antibodies from polyclonal serum, a method was published in 2001. 2.0 mg of purified SDR was activated with glutaraldehyde using the method described in (9). was covalently bonded to the free amino groups of the affinity adsorbent. Substantially previously announced Anti-3DR rabbit serum (2-3 ml) was added to 5DH-beads using the method previously described (9). Adsorbed onto a column, eluted, dialyzed, concentrated and stored. These antibodies were It was further purified using an otein A column (manufactured by Pharmacia LKB). Anti- The monospecificity of 3DR was first checked with Westaff'Ot as described above.

SDH's stand-'do'? Nota no...fu'mouth...mu...se:SD The surface location of H was determined using a bacterial dot plot immunoassay as previously published (10). It was measured using a cell phone. Essentially, 50mM Tris/HCI buffer (pH 8,5) and 0D65. , nm l.

Strain D, 471, was cultured overnight at a controlled temperature of O. Centrifuge the aliquot from this suspension. buffer of the same volume containing either 2M NaC1 or 296 SDS resuspended in solution, rotated and centrifuged for 1 hour at room temperature, and each supernatant was saved.

After washing, the pellet was re-oxygenated with 50mM Tris/HCI buffer (pH 8,5). D65 (lnm was adjusted to 1.0. In another experiment, the Centrifuge the bacteria and suspend the bacteria in 100mM NH, HC03 at OD65on. Adjust the m to 1.0 and treat with trypsin (250 μg/ml) at 37°C for 3 hours. Ta. Trypsin activity was inhibited with trypsin inhibitors as described above and bacteria pelleted. The suspension was resuspended in Tris/HCI buffer and adjusted to 0 D65 on, ml, 0.

50 μl of each bacterial suspension was transferred to a dot plot assembly (Bio-Rad L aboratories, Richmond, CA). Transferred to paper. The reactivity of the surface-exposed epitope of the 39-kD protein was determined by Identity-purified anti-8D) I protein antibody (Hook 5mg/ml of 1 strain 1:1゜ 000 dilution). For density analysis of Dra I plots, replicate plots are Lum1-Phos'rM530 (Adamantyl-4,2-dioki Cetane phenyl phosphatase: Lumigen Inc, Detroit, Ml) and dehydrated and phosphorylated using an enzyme (alkaline phosphatase) catalyst. , generates dioxetane anion, and finally converts it into methyl methane, which is a luminescent material. It was converted to the excited state of xybenzony l anion. Next, drain the developing agent and The wet plot wrapped in plastic wrap was exposed to X-ray film for 20 minutes and then The images were developed in the following order. Image analyzer performs density analysis of each spot on X-ray film This was carried out using a conventional procedure. Density analysis of each spot on the X-ray film Dumas Program (Drexel University, Ph11aderp hia, PA) was used for interfacial whole-cell assays with an IBM computer and linked. We investigated whether SDH on the surface of cocci acts as an active GADPH enzyme. different density trypsinized streptococci and non-trypsinized streptococci of (, -3-P Triethanolamine-phosphate-1EDT with and without C,-3-P I in the presence of NAD in A-DTT buffer in a final volume of 1°0 ml for 2 min at room temperature. The bacteria were cultured, centrifuged, and pelleted as described above. Supernatant, NA The 340 nm absorbance was recorded and analyzed for the conversion of D to NADH. This enzyme power was pre-incubated with a 1+50 dilution of the purified anti-8DR antibody prepared as described above. Streptococci were also measured to determine individual inhibition of enzymatic activity using the purification step glue. Electrophoresis and Western plots of synthetic extracts and protein samples are shown below. It was performed as previously published (2, 3). Bound to nitrocellulose membrane The individual proteins were probed using affinity-purified anti-3DI antibodies (1:2. 000.0.5 mg/ml) as previously published (2,3). It became.

:j MIIi's original 1j, LC'M Serop's SDH's first:: Previously issued. As shown in Table (2), M serotype 2.4.5.6.24.29. 41.57.58.60 and M-noricin extracts were prepared. wall enzyme mutano Lysine (muralyticenzyme mutanolysin) (20μ g/ml: 5rnM ED using Sigma Chemical Co,) 50mM T containing TA, 5mM MgC12, and 30% raffinose Prepare the cell walls of each group of strains suspended in r/HCl buffer (pH 6, 8). and incubated for 60 minutes at 37°C with constant end-to-end rotation. All draws Proteins from the extract were separated using 5DS-PAGE and transferred to nitrocellulose. . Plots were generated with affinity purified anti-8D R protein antibody as follows. isolated from rabbit skeletal muscle, human red blood cells, and Stearothermophilus The cross-reactivity of G A D P H was determined by Western blot and competitive E Both LSrA were measured.

EL I S, 5 Δ White! -1! :60 affinity purified antibodies 1. min at 4.05 nm. In a diluted solution that shows an ELrSA reading of 0. It was adjusted. For ELIS, add 1μg of ELISA plate/100μ of SDR. 1/hole and coated for 3 hours at 37°C, followed by overnight at 4°C. It was carried out using a method.

Different bacteria and proteins containing cross-reactive epitopes for binding of anti-SDH antibodies The competition for GAPDH from mammalian origins is described in mI (10). It was carried out as described. Briefly, the ELISA plate is and coated as described above. Affinity purified as previously determined The optimal dilution of the antibody was used. Competitive GAPDH with 0.05% Br1j- Start with a 100-fold molar excess in antibody dilution buffer (10) at pH 7.4 containing 35 Serial dilutions were performed by gradually decreasing the molar excess over SDR. Then anti- Add SDH antibody to each well, process the plate and finally develop The binding of these proteins was detected by EL at 405 nm using IDA3 phycrotitan Rate reader, Physics Inc., described in (11) using (20) Measured by dynamic ELISA as described above.

I゛　【の　−゛　： SDH to Iodobeads (Pierce Chemical Co.) was used and labeled with 1251 by the chloramine-T method. Labeled protein 5ephadexG-25 (PD-10, PharmaciaLKB Biot Separated from free iodine by filtration on a column of ech Inc.), 10mM salt magnesium chloride, 2mM calcium chloride, 50mM potassium chloride and 15 Collected in 10mM HEPES, pH 7.4 containing 0mM sodium chloride.

Aliquots of labeled proteins containing 0.02% sodium nitride (NaN3) It was stored at -20°C. Fibronectin and fibrinolytic enzyme (plasma) in) were labeled in essentially the same manner. SDI, fibronectin and cellulin The specific activities of lytic enzymes are 2X105, 1.0X106 and 1.21x10 respectively. It was 6CPM/mg (counts/min/mg).

Combined Ω frugality: The binding activity of SDR and fibronectin is increased by using radioactive protein. It was measured. Egg white-lysozyme and/or cells, all obtained from Sigma. Skeletal proteins (myosin, H-meromyosin (HMM), light chain myosin (LMM) , tropomyosin, and actin) on 10% 5DS-PAGE (dodecyl sulfate). (sodium acid-polyacrylamide gel electrophoresis) electroblotting onto cellulose paper.

The plot was added to 15mM sodium chloride, 0.5% Tween-20, 0.04% sodium nitride and 0.04%. 5% bovine serum albumin (BSA) in lomM HEPES buffer containing pH 7.4 for 2 to 3 hours at room temperature ( block) shi, +25 I-fibronectin, 125■-fibrinolytic enzyme at room temperature in the same buffer containing at a concentration of 3 x 10 s CPM/ Time probe L.

The probed plots were then washed 3-4 times with blocking buffer.

The dried nitrocellulose plot was stained with Kodak Bl using an intensifying screen. By exposing to ue Brand film at -70℃ for 36 to 48 hours. Autoradiography was performed.

1.・Sil　2・ノタ　-i　-の゛・-／100：Overnight culture of streptococcus. Wash and cell walls in 3096 raffinose as described (2, 3). Digestion was carried out using amidase enzyme lysin at pH 6.1. Streptococcus cell wall After extraction of the lysin, the resulting cytoplasts are isolated as described in (3). ], OmM magnesium chloride and deoxyribonuclease (250 μg/m After lysis in a hypotonic buffer containing l), cytoplasm and membrane ) was further fractionated. The membrane was then ultracentrifuged (100,000 g, 45 min, 4°C ) was separated from the cytoplasmic fraction.

ADP-1 bosyl of SDR; ADP-ribosylation of SDR was performed by slightly modifying the method described in (15). carried out. Briefly, the standard reaction mixture (0.2 ml) was Lys/HCl 100mM, 10mM dithiothreitol, 1mM nicotinamide Dooradenine dinucleotide phosphate (NADP), 10mM thymidine (ADPR buffer solution). 10PM [Alpha 32P] Nicotinamide Ade After adding carrot dinucleotide (NAD) and 20 Pg of purified SDR, the reaction The reaction mixture was incubated at 37°C for 1 hour.

The reaction was then added with 50 μl of chilled 100% (w/v) trichloroacetic acid (TCA). The precipitated protein was stopped by adding water to the plate, left on ice for 30 minutes, and the precipitated protein was centrifuged. Separation was performed by separation (16,000 x g, 5 minutes, 4°C). protein pellet Wash in absolute alcohol containing 1% 5M sodium acetate and 5 peedvac. The remaining TCA was removed by drying in (Savant). 5011 dried precipitate 1 in sample buffer and then SDS as described in (2, 3). Processed with PAGE (1296 polyacrylamide). Dry the gel and sensitize it. Autotrat with Kodak x-omat film at -80°C using a clean Created a geograph.

ADP-17, GAPDH'゛ of SDH: purified SDH and The GA and PDH activities of ADP-ribosylated SDH were initially determined by Ferdinand and Described by (7), (16)i,: Improved as described It was measured by the method. Briefly, reactions were carried out in 800-850 μl of buffer ( Triethanolamine 40mM, disodium hydrogen phosphate (Na2HPO4) 50 mM, EDTA 5mM, pH 8,6), NADloomM and 1. 5 Dispensing (di) in a micro-cuvette with a volume of ml enzyme source (ADP-ribosylated and non-ADP-ribosyl) The test was carried out in a final volume of 1H containing SDI (5 μg). Pour the reaction into 7 μl Started with addition of lyseraldehyde (49 mg/ml). NAD's N A D H Absorption at 340 nm was recorded over a period of 2 minutes indicating the conversion to A.

SDH's ADP-11, 7-nitropursi...゛1mΩU- Sodium nitroprusit was added to 2mM maximum in ADPR buffer (200μl). Newly dilute to the final concentration and add 30Pg of SDR and (32P)-NAD. In addition, 11i to initiate the ADP-ribosylation reaction was preincubated for 2 min at room temperature. Cubated. Remove 40 μl aliquots at different time intervals and precipitate with TCA. I was honored.

A parallel control representing the same amount of SDR and C32P]-NAD was prepared using nitroprusidate. Incubate in the absence of aluminum and take aliquots at the same time intervals as the test samples. I started out. Separate the precipitated proteins on an SDS gel and make an autoradiograph. Ta. In a similar combination of experiments, the presence of 2mM sodium nitropridide and using streptococcal lysin extracts in ADPR buffer incubated in the absence of Further ADP-ribosylation was performed.

The results of the above approach are summarized below.

5DHc-' l-9'-(7) +r+: SDR protein is first Precipitate non-specific proteins at 60% saturation of Monium and then to 85% saturation. The lysin was precipitated from the extract by SDR is 85% ammonium sulfate precipitated. It was found in the temple (Fig. 1). Apply the dialyzed precipitate to a MonoQ FPLC column. The protein was eluted with a sodium chloride gradient from OmM to 300mM. SDH eluted at a salt concentration of approximately 280 mM.

Fractions with fibronectin binding activity were pooled, dialyzed, and further It was purified using se-12F PLC molecular sieve column. A small amount of contamination (co hydrophobic chromatography using TSK-phenyl removed by graphy. 5DS-PAGE of the final sample showed a 39kDa molecule. revealed a homogeneous protein with a large amount.

Total yield of purified protein from a 4 liter culture representing 6-8 grams wet weight of bacteria The amount was 800 μg.

N　”　”　−゛”　r1=”.

Amino-terminal amino sequence analysis of purified SDH confirmed the homogeneity of the sample and showed that almost all The result was a single amino acid at the position (Figure 2a). positions 31 and 35 A single amino acid was identified in the first 35 residues, and the remaining 4 residues were hypothetically identical. established.

The amino acid composition of the purified protein contains a high concentration of A, SP/Asn (12.1%). followed by Ala (10.7%), aty (10.3%), Val (1 0.2%) and Glu/Gin (8.4%). Laser desorption mass The mass of the purified protein (35,882 daltons) determined by optical spectrometry was was used to accurately assign the number of residues (Table 1) to 1 mole (Table 1). .

- Mi　rl゛ and Jの　： The sequence of the first 39 amino acids of SDR was translated from Gen-Ban. k) with known sequences in the database (Fig. 2b). Significant identity with nuclear GAPDH was found. The identity within the first 18 residues is , 77-83% for bacterial, eukaryotic or fungal GAPDH. This strength The homology decreases for the remaining 21 residues, with an overall identity of 41 to 56%. (Figure 2b). Comparing the amino acid compositions of various GAPDHs, the methio nin content (1,8 residues 1 mol), eukaryotic (8,4 residues 1 mol) or other It was found to be noticeably lower than for fungal GAPDH (7 residues 1 mol). (Table 1).

The amino acid composition of the remaining residues of SDI is relatively similar to that of other GAPDHs. were found to be close, but with sufficient differences that, with the exception of the amine terminal sequence, , suggesting that the SDR is different from other reported GAPD.

5DH1゛　' GAPDH゛-:: (, SDH in the presence of 3-P, in 1-liethanolamine buffer, pH 8.6) is the NAD of NAD as observed in the absorption of NADH at 340 nm. A dose-dependent conversion to ADH was demonstrated. Using 30 μg of purified SDI, G Fluctuations in the enzyme reaction rate due to changes in the concentrations of -3-P and NAD were measured. results , Lineweaver-Burk as shown in Figures 3a and 3b. According to (13), the Michaelis-Menten plot and the double reciprocal plot Both were analyzed. From these plots, km for G-3-P and NAD is estimated to be 1.33mM, Vmax is 0.487X10-3M NADH7min. determined. FIG. 5 shows an analysis based on the Michaelis-Menten method. k m, 1.22mM; and Vmax, 0°4666xlO-3M NADH/ Minutes. (b) 25 μg of 39-KD protein in G-3 in the buffer system described above. -Tested as a function of NAD in the presence of P (2mM) Ta. km for NAD is 156.7μM; Vmax, 0.459xlO-3M NADH/min; y-axis "intercept" (1/Vmax) , 2. 18; and the slope (km/Vmax) are shown in the 341.74 insert. km for NAD according to the Michaelis-Menten method is 1 48.86. czM; and Vmax is 0.445xlO-3M NADH/min It was estimated that

The 7th tongue of SDR on the top Antibodies against SDR were coupled to activated glutyraldehyde beats. Affinity purified on SDH followed by protein A column. obtained Purified anti-3DR immunoglobulin G recognized only the SDI protein band (Fig. 4). ). A dotted line (Dot-Bl) was used to confirm the position of SDR on the surface of streptococci. ot) An immunoassay was applied. The results showed that trypsin-treated streptococci were anti-8D. It was revealed that the reactivity to H immunoglobulin G was significantly reduced. SD The R protein is either superficially or tightly bound to the outer surface of the cell wall. To confirm whether the streptococcal cells are Washed with SDS.

The results showed that SDH was affected by high salt (high 5alt) or ionic detergents. It was shown that it could not be extracted.

11-Ri゛da゛no-“1”-゛: The GAPDH enzyme activity found on purified SDR is also present on the streptococcal surface. Enzymatic studies were carried out using whole streptococci to determine whether they were present. spirit The same concentrations of substrates (G-3-P and NAD) used in the prepared SDR were added. Used with whole streptococci.

The data shown in Figure 6 shows the donor layer-dependent G contacted by the whole organism. It shows APDH activity. As found for purified SDR , in the absence of the specific substrates G-3-P and NAD, intact bacteria also undergo the reaction. (Figure 6a).

Enzyme activity in the whole organism is limited (30%), but anti-3DI immunity It was also found that it can be specifically inhibited by phosphorus (Figure 6C). reaction mixture When trypsinized bacteria were used in the compound, enzyme activity decreased by 80%. It was found that (Fig. 6b). The background 2096 activity is This suggested incomplete digestion of SDH protein by psin.

I 1-11' (Riko 6 + SDH, - -: M antigen type of different streptococci The ubiquitous nature of SDR proteins in of lysin extract using affinity purified anti-3DR immunoglobulin G. Confirmed by Wesslumf 1f8 analysis. As shown in Figure 7, S DR proteins have been discovered in several serotypes. Moreover, everything changes in size. They were found to have the same molecular weight without any indication of movement.

- ``and death'' of GAPDHSDH, :Known GAPDH(!:SDH relationship using an affinity-purified anti-3DH antibody. Confirmed both in kit and competitive kinetic (k) ELISA. 5DH-specific anti- Analysis of Western blots (Figure 8, inset) for the body shows that Bacillus spp., human Did peripheral blood cells (PBC) and GAPDH from rabbit muscle react weakly? , or did not respond at all. This finding is slightly higher than 20 ~25% of the binding of anti-3DH antibodies to SHD was inhibited by 100% of these proteins. This can be achieved in molar excess and provides that rabbit muscle GAPDH exhibits the lowest activity. This was further confirmed by competitive ELISA (Figure 8). Bacillus and human G The fact that almost 20% inhibition was observed with a 20-fold molar excess of APDH suggests that this This may reflect sequence homology observed at the amino terminus of these molecules. (Figure 2).

1, / -mu -’-J (-’T, 5DI (7)-”:: Many glycolytic enzymes Since SDI has been shown to bind to cytoskeletal proteins, it is possible that SDI has similar properties. We have confirmed whether or not this is the case. 125I-8DR to some cytoskeleton It was used to probe Western blots containing proteins. The result is SDH binds myosin and its globular region (H meromyosin) and actin However, it binds to the α-helical region of myosin (L-meromyosin) or trobomyosin. It was found that the results did not match (Figures 9a and b).

The fibronectin-binding activity of 5DH (-A = SDR) Western blot] 25■-labeled fibronectin or fibronectin - Both were measured using anti-fibronectin. The results showed that both SDR proteins The first assay revealed that it could bind to fibronectin (first assay). 0b and 0 figures).

Based on all the above results, the SDR includes Streptococcus, Group A Streptococcus is the major surface protein of confirmed. Such proteins have not previously been detected, isolated, or characterized. It has not been done.

The new surface proteins are mainly composed of fibronectin, lysozyme and The ability to bind to Roskellekuhl protein and its enzyme as GA　P　DH　H characterized by elementary activity. Its molecular weight is approximately 39 kDa. Ami The first 15 amino acid residues at the end of the chain are: VaI-Val -Lys-Val -G I y-11e-Asn-Gly-Arg-11e- Gly-Arg-Leu-Ala-Phe These first 15 amino acid residues are , 10096 homologous to bacterial forms of GAPDH, and eukaryotic or fungal It is clearly shown that it has 80-90% homology with GAPDH. However, the most High homology of the first 15 amino acid residues is conserved towards the carboxy terminus of the molecule The amino acid composition is not appreciably different from that of other GAPDHs. Therefore, SDI is noticeably different from previously reported GAPDH. ing.

SDR also functions as an ADP-ribosylating enzyme, which in the presence of NAD =ADP-ribosylated. cell wall associated molecules Group A Streptococcus crude containing a mixture of citiated molecules) In the lysate extract, SDI is the only molecule that is ADP-ribosylated. has been found. ADP-ribosylation of SDR by both cytoplasmic parts of group A Processing removes the ADP-ribosye of the SDR, which The presence of SDH-specific ADP-ribosyl hydrolase in the cytoplasmic compartment It shows. Produced by sodium nitroprusdide, which naturally generates nitric oxide. Treatment of purified SDH or crude lytic enzyme extract increases the ribonation of SDR in a time-dependent manner. It was found to stimulate silation.

Both ADP-ribosylation and nitric oxide processing occur with glyceraldehyde in SDI. It inhibited the activity of 3-phosphate hydrolase. In addition to its self-ADP-ribosylation activity, However, either purified SDR or whole streptococci with surface SDH were and human lilithium, a strong SDR binding protein, specifically I was able to convert it into a file. These data indicate that SDI is associated with autoribosylation and AD This shows that it has both P-ribosyl transferase activities.

As will be understood by those skilled in the art, SDR does not refer to a single protein, but rather refers to a class of streptococcal surface proteins, all of which have similar have characteristics. Colonization and possibly internalization and proliferation of group A streptococci. Proteins are involved in reproduction. Early stages of mucosal tissue colonization by streptococci = one of the stages and subsequent infection by this bacterium It is a combination of Lysozyme is also believed to be involved in this binding step.

The enzymatic activity of SDR is due to the aldehyde reduction of tikoic acid, a polyglycerol phosphate. May be involved in binding of bacteria to endothelial cells by reaction of the original product . The function of aldehydes is to form fine particles on the tissue surface by reacting with amino groups on the tissue surface. Can bind bacteria.

Therefore, inhibition of this early binding is the primary mechanism by which streptococcal infections are inhibited or prevented. It is Noh. Therefore, fibronectin and lysothi Antibodies against SDR, which compete well with the group A streptococcal pharyngeal F11 impairs colonization of the head mucosa. Therefore, suitable antigenic determinants, e.g. SDR with an antigenic determinant containing about 20 amino acid residues and its protein antigen. The amino acid segment inhibits the binding of fibronectin, thereby inhibiting the pharyngeal mucosa. by administering an amount of a selected product effective to inhibit colonization of and are useful in inhibiting streptococcal infections in mammals, including humans.

The proteins, polypeptides and peptides of the invention can be used in any of a number of known processes. You can also get it.

Proteins can be isolated as described above. Alternatively, protein or The segment can be made by recombinant DNA technology. For example, protein or oligonucleotides for the desired segment into the plasmid. Insert the plasmid into E. coli so that the bacteria express the desired product. Can be used to convert.

For example, polypeptides of the invention containing segments of about 6 to 20 or more amino acids. Peptides and peptides are protected by protection, deprotection and cleavage techniques, each with a specific Using amino acid- or peptide-appropriate reagents, standard It can be synthesized by solid phase method. A combination of manual and automatic (e.g. Ap plied Biosystem 430 A) Solid-phase technology Can be used to synthesize peptides. Although it is a little less convenient, peptides Classical methods of synthesis can also be used. For background on solid phase technology, see An. dreu, D., Merrifield, R.B.

5teiner, H., and Boman, H.G., (1983) Pr. oc, NatlAcad, Sci USA 80. 6475-6479 ;　Andreu,　D.

Merrifield, R, B, 5teiner, H, and Bom an, H, G.

(1985) Biochemistry 24. 16 83-1 688 ;Fink, J.

Boman, A., Boman, H.G., and Merrifi eld, R, B,.

(June 1989) Int, J, Peptide ProteinR es, 33. 412-421; Fink, J., Merrifiel d, R, B, , Boman, A, and Boman, H, G, ( 1989) J. Biol, Chem, 264-6260-6267. each of which is herein incorporated by reference.

The products of the invention are bilateral. They are free base or pharmaceutically acceptable gold Existing and utilized as genus or acid addition salts. Suitable metal salts include alkali and and alkaline earth metal salts, preferably sodium or potassium salts.

Acid addition salts are suitable for use with a wide range of organic and inorganic acids, including mineral acids, such as citric acid, lactic acid, and mineral acids. It can be made from leic acid, dioxysuccinic acid, phosphoric acid and hydrochloric acid. these Salts can be made by techniques well known to those skilled in the art.

For use as a vaccine, a selected compound, conjugated to a carrier such as cholera toxin B, It is currently preferred to administer selected products. The method for preparing such a complex is Are known. One way is Be5sen and Fischetti (L4) described by. Other carriers may be utilized or the product It can also be used without a carrier.

Proteins or segments thereof can be isolated using the method of Pozzi et al. (17, 18). It can also be administered as a hybrid protein expressed on the surface of streptococci.

When immunized parenterally or orally, mice or other animals, including humans, mammals are highly susceptible to subsequent streptococcal challenges. It has strong resistance.

The currently preferred method of administering the vaccines of the invention is by the nasal route. However, the present invention is not limited thereto. Oral methods can be used.

Typically, the patient to be protected will receive an SD effective to elicit a protective immune response. treated with an amount of R or other products of the invention.

The selected drug can be alone or dispersed, dissolved or suspended It can be administered with a pharmaceutically acceptable liquid or solid carrier. for example, When the patient is to be treated intravenously, the peptide is placed in an isotonic aqueous buffer. It may be suspended as a free base or dissolved as a metal salt. other treatment methods and Pharmaceutically acceptable carriers will be apparent to those skilled in the art.

The proteins, polypeptides and peptides of the present invention and those utilized for their expression. The genes and oligonucleotides used are probes for genes and proteins. It is useful as They are antimicrobials that can identify specific strains of streptococci. It is also useful for raising the body. Testing for mammalian infections e.g. It is.

(Margin below) Table 1 The number of moles of comparative residues of the composition of G A P D H from the species and genus SDR” BSt Thaq R3M Amino acid (35,8) + (36,0) (36,0) (34,0) Set 16.8 1.7 13 19.7Ala 38.1 38 41 32.6 Pro 13.6 11 12 11.9Tyr 9.1 8 10 8.6 Val 36.5 43 29 31.2 Met 1.8 7 7 8.4 11e 22.4 19 22 15.3I, eu 23.4 26 30 1 7.8Phe 13.8 5 7 12.9 SM, rabbit skeletal muscle (20).

All publications identified herein are incorporated herein by reference.

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FTG[JRE　1.2 , ---N, PCT/us 93100082 Front page continuation (51) Int, C1,6 identification symbol Internal office reference number A61K 39109 ADZ 9284-4C//(C12N 9102 C12R1:46) (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, PR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), AU, CA, CZ, JP, UI

Claims (4)

[Claims] 1. Streptococci are treated with fibronectin, lysozyme and cycloskeletal (cy glyceraldehyde -3-phosphate dehydrogenase activity, ADP-ribosylation activity and ADP-ribosyl transfer Streptococcal surface dehydrogenases and segments thereof with transferase activity. 2. Streptococcal surface dehydrogenase or its molecules together with a pharmaceutically acceptable carrier. to inhibit colonization of mucosal tissues. The dehydrogenase contains an effective amount of Glyceraldehyde has the ability to bind to 3-phosphate dehydrogenase activity, ADP-ribosylation activity and ADP-ribosyl transfer Has enzymatic activity and is effective in inhibiting mucosal tissue colonization by streptococci. vaccine. 3. Streptococci with fibronectin, lysozyme and cycloskeletal protein Glyceraldehyde-3-phosphate dehydrogenase activity, AD Streptococcal bacteria with P-ribosylation and ADP-ribosyltransferase activities surface dehydrogenase or its segments to suppress the formation of desiccant tissue colonization. Mammals that require suppression of mucosal tissue colonization, including administration of a sufficient amount Treating mammals to inhibit mucosal tissue colonization by streptococci in Method. 4. Streptococci are solubilized with lysin to produce a mixture containing streptococcal surface dehydrogenase. and isolating the dehydrogenase from the mixture, wherein the dehydrogenase is Binds streptococci to fibronectin, lysozyme and cycloskeletal proteins glyceraldehyde-3-phosphate dehydrogenase activity, ADP-reactive Streptococcal surface with bosylation and ADP-ribosyltransferase activity How to obtain dehydrogenase.