CA2127550A1 - Multifunctional surface protein of streptococci - Google Patents

Abstract

A novel streptococcal surface protein principally characterized by fibronectin and lysozyme binding activity, but also having glyceraldehyde-3-phosphate dehydrogenase activity, ADP-ribosylating activity and ADP-ribosyl transferase activity are described. It is particularly useful for the preparation of vaccines to protect against streptococcal infections.

Description

~ 093/14198 2 1 2 ~ 5 5 0 P~T/US93/0~82 MULTIFUNCTIONAL SURFACE PROTEIN OF STREPTOCOCCI
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Thi~ invention was made with goYernment support under Grant Number AI-11822 awarded by the National Institutes of Health. The GoYernment ha~ certain rights in the invention.

REL~TED APPLICATTON
. . _ This application is a continuation in part of commonly owned and cop~nding application ~erial number 07/913,732 filed July 15, 1992 which is, in tu~n, a continuation of applica*ion ~erial number 07/818,~70 filed January 8, ~992. The latter applicatlon is now abandoned.

Thi~ invention relates to a surfac~ protein of streptococci which is involved in early c~lonization of the pharyng~al mucosa. More sp~cifically, it relate~ to a ~ultifunctional p~otein which is on the surface of : streptococci, includin~ pathogenic streptocosci, such as Streptococcus pyoqenes and is particularly characteriæed `` ~ by its ability to bind fibronectin, lysozyme, and the ~o cyclosketal prot~in myosin and ac~in as well a~ by its enzy~e activity, specific~lly dose dependent dehydrogenase activity with gly~eraldehyde-3-phosphatP
~G~DPH~. The molecu~e also functions ~s an ADP-ribosylating enzy~e and as an ~DP ribosyl transferase.
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2 5 I It is concerned also wi~h thexapeutic compositions and uses of the surf ace protein including, ~or example vaccines prepared ~from the: whole protein and segments thereof, E~articularly~ conserved segments ha~ring ac:tivity similar to that of ehe protei-. ~ .

WO93/14198 P~T/US93/ ~ 82 2127~50 ~2 BACKGROUND OF THE INVENTION

Mammalian diseases, especially human di~eases caused by streptococcal infection with bacteria ~uch as Streptococcus pyogenes are a ~ignificant health problem.
In the United States alonej 25 to 35 million cases of group A strep~ococcal inf~ctions, which primarily afflict school age children are reported annually (l). The high inciden~e and potential severity of str~ptococcal infections provide impetu~ ~or development of an e~fective and safe vaccine to prevent streptococcal related infec~ions.

It has now been discovered that there is a streptococcal surface dehydrogenase (SDH) protein on the surface of streptococci from several serological groups such as ~roup A type 6 strep~ococci which has both `~ enzymatic activity and a binding capacity for a variety of proteins. In the earlier applications in this series, this ~Ur~aze protein was referred to as MF6, that b~ing the laboratory code designation assigned to it when it was initially isolated, purified and characterized. It is now referred to as SDH ~ince one of its principal characteristics is that it is Streptococc~l Surface Dehy~rogenase.~ ~ore specifical~y, it is a member of the clas of proteins which manife~t glyceraldehyde-3-~
~5 phosphate de~ydrogenase (GAPDH) activity.

G~PDH proteins,~as~the name implies, are a class of dehydrogenase enzymes in~imately involved in ma ~ alian physiological r~actions. Generally, mem~ers of thelclass are~found in the~cytoplas~r but some have been found ~ associated with membranes and cellular cyclosketal : structur~s o~ eukaryotes. Th~ g~ycolytic enzyme of this ::

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, WO93/14t98 ~12 7 53 Q PCT/US93/~082 invention is believed to be unigue ln that it is a sur~ace protein of prokaryot~s. No other such G~PDH
prot~in has previously b~en described.

The GAPD~ protein of this invention ha~ some structural characteristics similar to other proteins of the GAPDH family~ For example, over 80% of the NH2-terminal 18 of 39 amino acids are identical to the GAPDH
family of enzymes. ~o~ever, it dif~ers in many other respects, as will be explained hereinafter~ It is, therefore, a novel product which has not hereto~ore been isolat~d and characterized.

A detailed characterization of puri~ied SDH has disclos~d that its native conformation is probably a tetramer wlth a molecular weight of about 156 kDa~ The molecular weight of the protein by mass pectrometric analysis is about 35.~ kDa. By sns PAGE, it is about 3g.2 kDa.
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; The prot~in has been identified on the sur~ace of Group~ A, B, C, E~ G, ~ and L streptococci utiliæing ~o affinity purified anti-SD~ antibodies. The protein :: :
exhibited a dose dependent dehydrogena~e actiYity on glyceraldehyde-3-phosphate (G-3~P) i n the presence of.
bet~nicotinamide adenine dinucl~otide (N~D). The ~ultifun¢tional~ activi~y of SDH was revealed by its ability to b$nd~fibronec~in::~and lysozyme as well as the ytoskeletal proteins myosin and actin. The binding acti:~ity o SDH~to~myo6in;was found to be localized to t~e globular heavy merom~o~in domain. SDH did not bind to~streptococcal M protein,~tropomyosin: or the coiled~
~coi~l ~omain of myoæin:~ me multiple binding capacity of SDH e pecially in connection with:~ycloskeletal proteins, in~conjunction with its GAPDH~acti~ity indicates a role W093/14198 PCT/US93/OQ~82 in the coloniæation, in~ernalization and the subsequent proliferation of streptococci. Trypsin tr~atment of whole streptococci result~d in a marked reduction in their reactivi~y to SDH antibodi~s. Th2 inability to remove SD~ from the streptococcal surface after washing in 2 M NaCl or 2% SDS indicates that the protein is not p~ripherally associated but tightly bound to the cell.
These data all indicatQ *hat the prok~in i~ a sur~ace G~PDH mol~cule on the streptococcal ¢~

The novel SDH is obtained by solubilizing the selected streptococcus with lysin to produce a mixture containing SDH~ The 5DH may be isolated from the mixture by any of a number of con~enient methods known to the skilled artisan including the method illustrated below.

It may al50 be produced by tranæformin~ an organism such as E. coli with an appropriate gene so that the E.
coli will express SDH.

The following abbreviations are employed in the description oP this invention: ~

NAD: beta nicotinamide adenine dinucleotide : PVDF: polyvinylidine difluoride ~DT~: Eyhylenediamine tetra acetic acid : P~SF: Paradimethyl sulfonyl fluoride TLCK: ~-p-tosyl~L-lysine chloro methyl ke~one 2~ SDS: Sodium~dodecyl sulfate : Mono Q FPLC: Mono Q(Trade Name) Fast protein liquid chromatography Super~se 12 FPLC: Superose-l2(Trade Name~ Fast protein liquid hromatography TSK-Phenyl HPLC: TSK-Ph~nyl lTrade Name) high performance/pre~sure liquid : ~

~W093/1~198 ~ 2 7 S ~ ~ PCT/US93/ ~ 82 chromatography NADH: beta-nicotlnamide ad~nined~nucl20tide, reduced ELISA: Enzyme linked immunosorbent assay ELIDA: Trad~ name of Physica Inc.
Sephadex ~-25 PD-10: G-25 PD-10: trade name of Pharmacia-LKB Inc.
HEPES: ~N-[2-hydroxyethyl]piperizine-N'~2-ethansulfonic acid~) RGDS: Arginine~Glycine-A partic acid-Serine (Axg~-Gly-~sp-Ser) G-3-P: Glyceraldehyde-3-phosphate G~PDH: Glyceraldehyde-3~phosphate dehydrogenase THE FIGURES

There follows a description of ~he figures.

Fig. 1: SDS-polyacrylamide gel (10%) analysis o~
SDH protein ~rom M6 e~reptococci. Lane a: Lysin extra~t of D47~ streptococci. Lane b: Precipikate of 65%
(N~4)2S04 satu~ation o~ ~he;ly~in extract. Lane c:
Precipitate of 85% (NH4~2S04 saturation o~ ~he : 20 supernatant after 65% precipitati~n. Lane d: Pooled Mono Q fractions at 0028 M gradient elution. Lane e: ~
; : Partially purified::SDH fxom the Superose 12 col ~ . ~ane f: Puri~ied SDH~from Ph~nyl ~SR::aolumn. Arrow marks on lan~ a and b at 50 kDa indicate the position of M
protein~ Prestained marker protein~mixtur~ ~ith : ~ molecular mass:as indica~ed on the left margin. 200 k~a:~yosin(H-ch~in)~, 97.4 kDa-: Phosphorylas~ b, 68 kDa:bo~ine~j~serum albumin, 43 kDa: ~valbumin, 29 kDa:i : ca~bonic~anhydrase~, ~18.0 kDa: lactoglobulin, 14.3 kDa:
~ 30~ egg-white ly80z~me.

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WO93/14198 PCT/US93/~82 ~ 1 2 ~ 5 5 6 Fig. 2: (a) The NH2-terminal sequence of SDH.
(b) Comparison of the NH2-terminal amino acid seguence of SHD with the amino acid sequences o~ the known GAPDH
molecules obtained frm the translated Genbank database.
BstG~P- Bacillus stearothermoPhilus GAPDH, EcoGAP-Escherichia coli GAPDH, ~umGAP- h~man GAPDH, G~PDH, ChkGAP- Chicken GAPDH, SmP37- 5chi~tosomia manæoni 37 kDa .
protein (GAPDH) (54)~ ZmbGAP- zYmomon2s mobilis GAPDH.
Numb~rs on the right side of the figure indic~te the percentage similarity of SDH with other GAPDH molecules with residues 1-18 and 1-39. The gap(-) between the 14th and 15th residue of the chicken GAPDH se~uence was introduced to maximize homology.

Fig. 3: Lin~weaver-Burk's double reciprocal kinetic analysis of GAPDH acti~ity of SHD. 25 ug o~ SDH
was assayed as function of G-3-P in the presence of NAD
~: (lOOuM) in triethanolamine-phosphate-EDTA-D~T buffer at : pH 8.6. The Km fo~ G-3-P wa~ estimated to be 1.33 uM, Vmax: 0.487 X 10 3 M NADH min-l, intercept Y axis (l/Vmax): 2.05 and slope (Rm/Vmax): 2.73. The inset shows the analysis based on ~ichaelis-Menten~. Km: 1.22 mM and Vmax: 00 466 X 10 3 M NADH min 1, (b) Lineweaver-Burk's double reciprocal kinetic analysis o~ G~PDH
activity of the 39~kDa protein. 25 ug of SDH was assayed as a function of ~AD in the presence of G-3-P (2 mMj in : the buffer system as described in (a). The ~m for NAD
was esti~a~ed to be 156.7 uM, Vmax:~.459 X 10 3 M NADH
min 1, Intercept::on Y axi~ Vmax) 2.18, and slope (Km/Vmax~ 341.74. K~ ;for NAD by the m~thod of . 3Q Nichaelis-M~nten as shown in :the inset was estimated!to be 148.86 uM and Vmax:~ 0.445 X lO 3 M ~ADH min 1.

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,~.~93/14198 212 7 5 5 a PCT/US93/00082 ~ .

Fig. ~: (A) Coomassie Blue stain o~ SDS~gel and (B) Western-blo~ analysis of SD~ with affinity purified anti-SDH antibodies sugge~ting a multimeric structure for the SDH molecule. ~ane~ a and d: Crude ly~in extract.
Lanes b and e: Puri~ied 5DH. Lanes c and f: Unboiled purified SDH in sample buffer without SDS and æaturated wit NAD. Arrow mark indicateæ the position of a molecule of the size consistant with a tetrameric fcrm of SDH. MW
markers are indicated on the le~t margin (Detail~ on each marker-see Fig. 1).

Fig. 5: Dot blot i~munoanalysis to locate SDH on the streptococcal ~urface. The assay determin2s the extent of reactivity o~ a~f inity purified anti-SDH
antibodies to surface ~xposed protein before and after 2%
SDS, 2M ~aCl and trypsin treatments. Dot blots were treated with LumiPhos-530 substrate (41) and de~eloped on X ray filmO Densitometric reading of the image obtained o~ the X-ray film;was expressed a~ an optical density in t~rms of arbit~ary units~m~asured on an image analyzer using the ~umas progra~:(Drexel University, Philadelphia, US~). An internal }inear standard curve for~the optical density ~0.008 to 1.333 was:obtained ~ox final : de~sitometric ~nalysis of the:dot blot. Each bar representes the mean of four~to eight separa~e readings S.D. ~ ~

Fig. 6: G ~ DH~activity of whale streptococci. (a) : ~ The GAP~H acti~lty was observed at 340 mn of whole M6 :
:: s~reptococci by detennining the conversion of 2~AD to NADH
. inl the presence o* G-3-P. I:setails of the bu~fer cys~em is deæcri~ed in materials ~and m~thods. (b) Activity of tryp inized M6~:streptococci. (c)~ I~hi~ition of enzyme :
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' WO93/14198 8 PCT/US93/~82 ~, ~ 2 7 activity of affinity purifi~d anti SD~ antibodies (1:30 of 0.5 mg/ml for 3 hr at room temperature). Each bar represent the mean o~ three separate readings ~ S~Do Fig. 7: (A) Western-blot analysis of lysin extract s of various strep~ococcal ~ types with affinity purfied anti-SDH antibodies at a 1:2000 dilution of 0.5 mg/ml stock. Purified SDH ~nd an ~ nega~ive (M ) streptococci are also incuded in the analysis. (B) Western-blot analysis of mutanolycin ~xtract of various gxouping strains of streptococci using anti-SDH antibodies as described in (A).

Fig. 8: Competition kinetic enzyme-linked immunosorbent assays tkELISA) with immobilized SDH.
Commercially a~ailable purified GAPD~ from B.
stearothermop~llus, human erythrocytes and rabbit skeletal muscle were used to compete for the binding of affinlty purified anti-SDH antibodies (1:1000 dilution of 0.5 mgtml stock). Each curve repre~ents the mean of three ~epara~e experiments with le s than 5% standard deviaiton ~not shown). Ins~t ~hows the W~stern blot of ~: the r~act.i.~ity of af~init~ purified anti-SDH antibodies : with (a~ streptococcal SDH and GAPDHs of (b) bacterial ' (B. stearothermophilus), (c) rabbit skeletal mus~le and (d) human erythrocytes.
' ~5 Fig. 9: Binding o~ 125I-SDH to cytos~eletal proteins. (A) ~oomassie Blue s~ained~SDS-PAGE gel (10%~
containing 5 ug protein of various cytoskeletal proteins, ysozyme ahd M6 pro~ein (43~. Lane a: rabbit skeletal myosin. Lane b: heaving meromyosinO Lane c: light m@romyosin. Lane d: actin~ Lane e: M6 protein. Lane f:
S-2 ~ragment of heavy meromyosin. Lane g: egg WhitP
lysozyme. (Bj autoradiograph of proteins in a duplicate ~ :

WO 93/1419g 212 7 5 !j Q PC~/US93/00082 9 .
i gel after transfer onto nitrocellulose and incubation with the 12~1-S~H. Tha proteins in eac:h lane are as describe~ in Fig. 9 (A) . MW markers on the lert margin (Detail of each marker - ee Fig. l).

Fig. lO: Binding activity of SDH to fibronectin.
(A) Coomassie stain of an SDS gel containirlg 5 ug of SDH
~nd 13SA. (B) Western-blot analysis of a duplicate gel showing the binding of fibrone::tin followed by anti-~ibronectin to the SD~ ~ol~cule. (C) Autoradiograph of a similar W~stern blot showing the binding o~ l25I-~ibronect.i n to the SDH protein . Lanes a, c & e - SDH.
Lanes b, d, & f - BSA.

Fig . ll: ADP-ribosylation o~ SDH . Purif ied SD~I
( lanes l , 5 and 9 ), crude streptococcal cell wall extract ~lanes 2 , ~ and lO), cytoplasm (lanes 3 , 7 and ll) and membrane (lanes 4, 8 and 12~ fractions were incubated with ~32P~NAD in ~PR buffer. The proteins were then parated on a 12~6 ~ SDS gel and stained with tA~ Coomassîe blue. (B~ We~;tern blot analysis of a duplicate gel reac~ed with af~inity purified anti-SDH antil~odies and (C~ autoradiography of a si~ilar Western blot. MW marker is indicated on the le~t side.

Fig . 12: ADP-ribosyl transf exase activity of SD~I .
SDH and various~ bindig proteins (~740~ were incubated ~; 25 tolaether in the presence of ~32P]N~D. Proteirl mixtures :~ w~re precipit~:ted, washed and r~solved on 12~ SDS-PAGE:
and stained with Coomassie;blue and a duplicate gel was ;: dried and autoradiographed. SDH was incubated with actin ~(lanes, 1 an~ 6~, chioken egg white Lysozyme (lanes, 2 and 7~, Sol fragment of myosin ~lanes, 3 and 8), : fib~onec~in :~lanes~ 4 and 9), and:plasmin (lanes, 5 and ~ lO)o ~W markers are indicated~o~ the left side~

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W~ 93/14198 PCr/US93/~2 The f ollowing Materials and Methods section is provlded f or convenience and ease o~ understanding the invention .

MAT13RIA~S AND METHODS

5 Materials:
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n f ibronectian was ob~ain~d ~om Boehringer ~lannheim ~ Goat anti-human :~ibronectin and af f inity purif ied rabbit anti-goat I~; coupled to alkaline phosphata~3e wer~ obtained from Sigma. Pre stained molecul~r w~ight standards w~re purcha~;ed from Bethesda Research Laboratories. PVDF meDIbrane ~Immobilon-Pn was ~r~m Millipore. Na125I was from New England Nu~l~ar. All other ch~micals and reagents unles; otherwise indicated were purchased from Sigma.

Bact~ria:

Bac:teria. Group A ~ hemolytic streptoaoccal strains o~ variou ~ 3s ~and standard stra~ins used for streptococc:al ~grouping w~re from The P~ocksfeller Universi~:y cultur~ colIection~ (New York, NY~ and are ~a listad ~ as foll~ow~;: M2 (I: 626~ 4 tD896), M5 (~an~rando), .
M6(~4~71), ~24(CS241,~M29(DZ~3~, M41(ClOl/~03/4), M57:(A995), 2~58(D774), M60(1:398), ~ (T28/51/4); group A, : J17~4 ~:an M- strain)~, group ~ A va~iant, A486var; group Br : : :: : :` ::
; 09OR; group C,: C74;~: group :~, D76; group E, Kl3l; group F, ~: 25 F68C; ~group G,~ D166B;; grou~ :H, ~F9OA; group L, Dl67:B; and group N,~:C~59~

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,~O9~/14198 21 ~ 7 5 ~ a PC~/US93t~082 Lysin_extraction and locat on of_SDH protein:

A crude extract containing the major surface proteins was prepared using the procedure of lysin extraction ~o remove the streptococcal cell wall as described before (2). Esæentially, bacteria washed in 50 mM sodium acetate buffer, pH 5.5 were suspended in the same buffer containing 30% ra~finose and 5 NM EDTA.
Lysin is added to the su~;pen~ion (l: loo dil; 360 Urlits and incubated ~or 90 min at 37C with end-to-en~ slow rotation. The resulting pr~toplasts ~edimented at 15,000 X g ~or 30 min in a Sorvall centrifuge. The supernatant was saved, dialyzed again~t 25 mM Tris/HCl, pH 8.5,5mM
EDT~, concetrated on ~micon PM-10 membrane (Amicon Corp) and used for further puri~ication.

lS After lysin extraction, the pelleted prot~plasts were resuspended and lysed in hypotonic buffer (2 MM
sodium ac~tate, pH 5.5, ~ontaining 2 mM P~SF~ 1 MM TLCK, 10 MM MgC12 and 10 ug/ml DNAse~ followed by three fre~ze/thaw cycles. ~he membranes were sedimented at 100,000 x g ~or 45 min at 4C. The me~brane~pellet and cytoplasmic extract in the supernatant were analyzed with~
: Coomassie blue stain~a~ter separation on SDS gel.
Membranes were further treated with 1.5 M sodium ~hloride or 100 mM sodium carbonate, pH 11. 3 to determine lthe natura of associa~ion o~ SDH protein with the membran~.

To determine whether this protein is surf ace exposed, lysin extra~tion:of trypsinized bacteria was rried outias:déscribed earlier~3). Bri fly, washed : bacteria were suspended in 100 mM NH4HC03 and digested : 30 : with trypsin ~250 ug/ml) at 37C ~or 3 hr, after which the trypsin was inactivated by thP addition of soybean trypsin inhibitor (200~ug~ml). Lysin extracts of WO93/14198 ~1 2 PCT/US93/~82 trypsinized and control non-trypsinized bacteria were compared for the loss or reduc~ion in the size of SDR
protein.

Purification of S~H:

Lysin extraction waæ used as the starting material for the purification o~ the SDH. The dialyzed, concentrated, lysin extract wa~ precipi~ated at 60%
saturation o~ ammonium sulfate at 4C. The precipitates were centrifug d at 6,000 X g for 20 min and the ~upernatant was brought to 85% satur~tion o~ ammonium sulfate. The re~ulting precipitate was dialyzed against ~5 mM Tris/HCl bu~fer pH 8.5, 5 mM ~DTA and passed over to Mono Q FPLC column (Pharmacia LKB Biot~chnology Inc.) e~uilibrated wi~h t~e same dialyzing buffer. After`the col~mn was washed with 5 column ~olumes of starting buffer, bound proteins were eluted with a 50 ml linear NaCL gradient from O to 300 mM. Fractions containing 5DH
were pooled and~dialyzed against the 35 ~M Tri~/~Cl/EDTA
buffer and rechroma~ographed on th2 ~ono Q column.
Fractions containing SDH w~re then pool~d, and coneen~rated ~o a volume l.O ml cn~Centricon : concentrator (cuto~ mw lOjOOO kDa). The concentrated sampl~ was applied:~o a Superose 12 FPLC column (Pharmacia ~RB Inc): pr~-e~uilibrated with 50 ~M Tris/~Cl ~: : 25 pH 8.5:containing 0.3 M:NaCl and 5 mM EDTA. Fractions : ~ ~ containing SDH~protein were pooled~ dialyzed against 0.025 N Tris-tHCl buffer pH 8.5 containing l.O M (NH4)2S04 and applied to a~TSK-phenyl HPLC column ~Bio-Rad aboratories,~Richmond~CA) preequilibrated in the same buff:~r. Theprotein was~ elu~ed by a decr~asing linear : gradient of ~H~zS04 from 1.0 M to 9.0 M~ The purity of the ~inal pr~duct;:was determined by Coomasie hlue stain of the purified~protein on SDS gel and ~y the analytical :: : ~:

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,-~093/1~1~8 2 1 ~ 7 ~ 5 o PCT/US93/00~2 procedures. Purified material wa~ stored at 4C after dialyzing against 0.025 M ~ris/HCl pH 8.5 ~or various protein binding experiments or at -70C ~or longer storage.

Anal~tical Procedures (NH -terminal se~q~ e and amino acid com~osition~:

NH2-terminal amino acid sequence was determined according to the method of ~atsudaira et al ~4~.
Briefly/ the purified SDH was separated on a pre-electrophorese~ 10% ~crylamide-SDS gel under non-denaturing condition and th2n transferred to PVDF
Immobilon-P filter pre-wetted in methanol. Protein was visualized by ooos% Coomassie ~lue in 50:40 methanol, water, acetic acid solvent mixture. The blots w~re destained in methanol: water: acetic acid ~50:40:lO).
The portio~ of the membrane containing the SDH band was exciced and 5ub; ected to automated Edman degradation on an ~pplied Biosystem model A470 sequenator. Each band contained about 2-3 ug protein as determined by BCI
pxotein e~timation method (Pierce). ~or ami~o acid composition, the PVDF membr~ne containing the SDH was s~ained with 0.1% Ponceau-S (Sigma~ in 1% acetic acid.
The ~ection of me~brane containing the prot in ~and was exci ed and destained~with water~ This ~ection ~f : 2~ m~mbrane was hydrolyzed:in 6N HCljpheno7 at llOC for 22 hr. Amino acids were separated on Waters No~apek C~
column analyæed with;Waters Maxima so~tware, 510 pump and 490 de~ector. Cy~teine content was analyz~d also from the PVDF boun~ carboxyamide methylated protein as des~ri~ed by Cres~field ~(53. All analyses were performed by Protein Biotechnology Facility of the Rockefeller ~:; University~ ~
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W~93/14198 ,1 4 PCT/US93/0~2 olecular Mass Determination:

~ olecular ma~s of the puri~ied protein was determined in the department of ~ass Spectrometry and Gas Phase ion chemistxy of The Rockefeller University using the modified method of matrix-a~sisted laser desorption technique (6).

Glyceraldehyde-3-phosphate D~ydr~ nase (G~PDH~
Activity: .

GAPDH assay wa carried out according to the method originally describ~d by Ferdinand (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 as~ay solution was made of t~iethanolamine (40 mM~/ Na2HP04 (50 ~ 15 m~) and EDTA (5 mM). Disposable semi-micro 1.5 ml capacity spectrophotom~ter cuvette (VWR) contained 7ul G-3-P (Sigma, 49 mg/ml), 100 uM~NAD ~Boehringer Mannheim~
and as.ay buffer to a final volume of 1.0 ml after the a~dition o~ ~nz ~ e source with pH of th~ mixture being :20 8.6. Different:c:oncsntrations of SDH were used to plot the standard curve ~or~he absorbance at 340 nm per q ~; minute as a measure of conversion:of~NAD to NADH using.
; ~ : Spectronic 3000 speotrophotometer tMilton Roy).

: Enzy~e Kinetlcs~

Kinetics ~of enzymatio~r~action of SDH were made with v~rying;concentra~ions of NAD and a ~ixed concentration of~G-3-P and~vice:versa to determine respectively the Km and Vmax ~or NAD and G-3-P. The results were recorded as rate~analysis o~ NADH;releas~ at:~every half second for a period of:l min at 340 nm. The molar extinction : `

,-3~,0 93/14198 1 ~, 2 1 ~ 7 .rj 5 QPCr/VS93/00082 coefficient of NADH 6.22 X 103 (8) was used to convert absorbance (340 nm~/min to NADH ~/min. The kinetic co-e~ficient were estimated from the ~econdary plots of intercept of primary Lineweaver-Burke plot~ with respect to each sub~trate. The ~pecific activity of th0 enzyme (units/mg) was measured using the equation:

Sp activity = v(l~m/S)NAD(l+Km/S)~ 3 pwhere ~ = u moles NADH/min/mg of enzyme.

Specific activity of GAPDH activity for 5DH wa~
10 measured in the lysin extract, ammonium sulphate precipitate and pooled ~raction at Yarious purification stages.

Rabbit Immunization and affinity purification of immune sera:

Naw Zealand white rabbits were immunized ~ su~cutaneously with 200 ug:of purified SDH emulsified in ;:~ Fr~und's compl~te ~djuvant (1:1) at multiple sites.
Rabbi~s:were boosted once with 200 ug of this protein in Freund's inc~mplete ad~uvant~ 1). All ra~bits were bled 3 w~eks after the first and 10 days after the secon~
` - : immuniza~ion. All sera were filter:~cterilized and stor~d . .
~ at 4C. ~ ~
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To purify~SDH specific antibodies from the : polyclonal sera,~2.0:mg:of purified:SDH was linked : 75 co~alently with~free àmino group:of gluteraldehyde-ac~i~ated a~finity adsorbent 2S described before (9).
nti-:~DH: rabblt æera (2-3 ml):was adsorbed to and eluted rom ~he:SDH-bea~ds column:,~ dialyz~d, concentrated and stored~a~ describe~before~(9)0~ These antibodies wexe ~: :30 fùrther purified:on~:Pro~ein A column (Pharmacia LKB) , :

W093/~4198 1 ~ P~/USg3/~18:Z!

essentially using the same buffer described ~bove (9).
The monospecificity of anti SDH was first checked on Western blot as described above.

Dot-Blot Immunoassay to determin2 location of SDH:

~he surf ace locati~n o~ SDH was determined with the monospecific antibodieæ using a bacterial dot-blQt immunoaæ~ay as pr~viously d~scribad (lO). E~sentially, an overnight culture oP strain D471 was adjusted to OD650 nm 1.0 with 50 m~ Tris/HCl bufer, pH 8.5. Aliguots of this suspension were centrifuged and resuspende~ to the same volume of buffer containing either 2 ~ NaCl or 2%
SDS, and rotated at room temperature for 1 h, centrifuged, and the respective supernatants were saved.
Aftex washing, the pellets were again adjusted to OD650 l; nm 1.O with 50 ~M Tris/HCl buffer, pH 8.5. In a separate experiment, the bacterial suspension in the ~ris/HCl buf*er was centrifuge~, and~the bacteria were suspended in 100 mM NH4Hco3 to OD650 ~m 1.O and ~rea~ed with trypsin (250 ug/ml~ for 3 h at 37C. Tryp~in activity was i~hibit~ with t~ypsin inhibitor as described above, , ~
~: ~ and the bacteria were pelleted and:resuspended in the Tris/HCl buffer~to:;OD650~nm~l.0 50 ul of each bacterial .
suspe~sion was trans~erred to nitroc llulose paper using dot-bl~t ass~mbly~(Bio-Rad Laboratories, ~ichmond, C~).
Reactivity of~ ~urface-exposed epitopes of the 39-kD
;protein Wa8 determined using àffinity-purified anti-S~H
: ~ protein ant:ib~odies (l:l,000 dilution of 0.5 mg/ml stock).
: For densitom~ric~analysis~of the dot blot, a duplicate lot was:develop~d with Lumi-Pho6T~530 (Adamantyl-lt2-; 30 dioxetane ph~nylphosphate; Lumigen Inc., netroit , ~I), which under~o~s:enzyme:;(alk line~phospha~ase)-catalyzed depho~phorylation:to~form a dioxetane anion that is conver~ed ultimately into an excited~state o~ the methyl :

, :

~093/14198 212 7 ~ S Q PCT/US93/00082 meta-o~y~enzoate anion, ~he 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 dev~loped using conventional procedures. Densitometric analysis of each spot on the x-ray ~ilm was carried out on an image analyzer using the conventional procedures.
Densitometric an~lysis of each spot on the x--ray film was caried out on an image analyzer using the Dumas program (Drexel Univ~rsity, Philad~phia, PA) inter~aced with IBM
1 0 computer .

GAPDH en~ymatic activity of in.act_SDH protein on whole streptococci:

A whol~ cell assay was developed to determine whether SDH on the surface of streptococci ser~es as an active GAPDH enzyme. Different concentrations of trypsinized and non-trypsiniæed ctreptococci were incubated with and without G-3~P in presence of NAD in triethanolamine-phosphate-EDT~-DTT buffer as described above in a ~inal volume of l.0 ml for a period of 2 min ~ 20~ at room .témperature ~nd centrifuged to pellet ou the :~ : bacteria. The supexnatants were analyzed for the conversion of NAD to N ~ by:recording absorbance at 340 nm. This enz~mati~ activity was also determined on streptococci preinoubated with 1:50 dilution (l mg~ml) of purified anti-SDH:antibodies as prepared above to :~ ~ : determine specific inhibit:ion of enzymatic activity.
:
Po1yacrylamide-~qel elec~roPh-oresis and _ :

: ~ Electrophoresis, Western blotting o~ lysin extraction and protein samples at di~ferent purification : stages:werie carried out as de~cribed earlier t2,3).
Spe~ific prote:ins:~bound to the nitrocellulose membrane WO9~/14~98 PCT/US93/OQ~
~ ~ 5~ ~ 18 were pro~ed and visualized with affinity purified anti~
SDH antibodies (1:2000, 0.5 mg/ml) as d~scribed previously ~2,3).

Presence of SDH_on heteroloqous streptococcal M
serotypes:

Ly~in extracts of M serotypes 2, 4, 5, 6, 24, 29, 4l, 57, 5~, ~0, and ~ were prepared as descri~ed (2).
The muralytic enzyme mut~nolyæirl (20 ug/ml; siqma Chemical Co.) was used to prepare cell wall extracts of each grouping str~in suspended in 50 mM Tris/HCl bu~r, pH 6~8, containing 5 mM EDTA, 5 mM MgC12, and 30%
raffinose, and incubated at 37C for 60 min under .
constan~ end-to-end rotation. Proteins in all the extracts were sep~rated on SDS-PAGE and transferred to nitro~ellulose. The blots were probed with affinity-purified anti-SDH protein antibodies as described above.

Relationshi~ o~ ~PHDs from bace~rial and ~ammalian origins with SD~:

The cross reactivity of GAPDHs isolatad from rabbit skele al mu:scl~,~human erythrocytes and B.~tearothermoE_i-us were ~ete~mined both on Western blot .. . .
and comp~titi~e ELISA as described~below.
: :
~ ELISA and Competitive i~ibltion:
: : ~ :
:A~inity purified antibodies~were ~djusted to a dllution that:;ga~e an ELISA reading of l.0 at 405 nm aft~r 60 min. ELISA was performed following standard : proc~dures except that:ELISA:plates were coated with lO0 ; ul/well of l ug/ml SDH f~r 3 hr at 37C followed by overnight at 4~C.
: ~ , : ~

~w~ g3,l4l9~ 2 1 2 7 ~ 5 ~ PCT~US93/~0~2 Competition of GAPDH from different bacterial as well as mammalian origin containing cross reactive epit~pes ~or the binding of Anti SDH antibodie~ was per~ormed as describ~d pr~viously ~0~. Briefly, ~LISA
plates were c~ated as described above with SDH. Optimum dilution of affinity purified antibodies as determined above wa~ u~ed. Competing GAPDH were ~erially diluted in antibody dilu~ing bu~er containing 0~05% Brij-35 p~ 7.4 (10) at decreasing molar excess rel~tive to SDH starting with 100 X molax exces O Anti-SDH antibodies were then added in ~ach w~ll and the plates were processed and ~inally developed and binding of these proteins was determin~d by kinetic ELISA as de~cribed (11) u~ing ELIDA
5 microtitre plate reader Physics Inc. ~20) at 405 nm.

Radioiodination of Proteins:

SDH was lab~led with 125I by the chloramine~T method using Iodobeads (Pierce Chemical C~.). The labeled protein was s~parated from free:iodine by filtration over a column of Sephadex G-25 (PD-10, Pha~acia LKB Biotech ;~20 Inc)~and collected in 10 mM HEPES buffer saline pH 7.4 containing 10 m~ MqCl2, 2 mM CaCl , 50 mM KCl and 150 mM
NaCl. The lab~led pxotein wa~ stored at -20C in aIiquots containing:0.02% NaN3. Fibronectin and plasmin ` ~ were labeled~essentially:by the same m~thod. The:: 25 ~peci~ic activities of SDH, fibronectin and plasmin were, : ; re:sp~ctively~, 2X1~05, 1:.0X106 and 1.21X106 CPM/mg.
, Bindinq Studies:

:The Binding acti~ity of~SDH and fibrone~tin was :~ determin~d by:the~use of radioaative proteins. Egg : white-lysozym~ and/or cytoskeletal protei~s (myosin, heaYy~meromyosin~(HMM), light chain myosin (LMM3, .

WOg3/1419X PCT/US93/~ ~2 tropomyosin, and actin) all of which ~btained ~rom sigma, were electrophoresed on 10% SDS PAGE gels and electroblo~ted on nitroce~lulo~e paper. The blot~ were blocked in 10 mM H~PES buffer containing 15 mM NaCl, 0.5%
Tween-20, 0.04% NaN3 and 0.5% ~SA pH 7.4 for 2 3 hr at room temperature and probed for 3-4 hr at room tempera~ure in th~ same buf~er containing 1~5I-fibxonectin, 125I-pla min at 3X105CPM/ml~ The probed blots were then washed 3-4 times wi~h bloaklng bu~fer.
Autoradiography were prepared b~ exposing the dried nitrocellulose ~lots to Kodak Blue Brand film with an int~nsifying screen for 3~-48 hour at -70~.

Lysin trac~ion~eion of Stre~tococ i for ribosylation study:

An overnight culture of streptococci was washed and the cell wall was digested using the amidase enzyme lysin in 30% raffinose at pH 6.1 as described t2,3). After lysin extraction, which represents the cell wall fraction of ~he str~ptococci, the resulting protoplasts were further fractionatQd into cytoplasm and membrane after lyisng in a hypotonic buffer cont~ining 1~ mM MgC12 and DNAse (250 uglml~as described (3). :Membranes were then -separated from the cyt~plaamic fraction by ultracen~rifuga~ion (100,00 X:g, 45 minn, 4C~.

~: 25 ADP-Ribosylation of SDH: ~ ~
: ` :~ :
~: ~he ADP-ribo~ylation of SDH was performed as dels~ribed (15~ with slight modification. Briefly, the standard reaction mixture (0~2 ml~) contained 100 mM
TrisfH~l at pH 7.:4,~ 10 mM dithiothreitol, 1 mM NADP~ 10 :~ 30 mM::th~midine (ADPR~bu~Per~ ;A~ter the addîtion of 10 uM
~lpha 32P]N~D and 20 ug of:purified SD~, the reaction -~ : :

~ :: : : :
.

_.WO93/1419~ 21 2 7 5 ~ ~ PCT/US93J00082 mixture was incubated for 1 hour at 37C. The reaction was then stopped by the additicn of 50 ul of 100% (w/v) chilled trichloroacetic acid (TCA), and allowed to stand for 30 minute~ on ice after which time the precipitat~d pr~teins were separated by centrifugation ~16,000 X g, 5 minutes at 4C~. The protein pellet was wash~d in absolute alcohvl containing 1% of 5 M sodium acet~te and dri~d in a Speedvac (Savant~ to remove remaining TC~.
The dried precipitates w~re di~sclved in 50 ul of sample buf~er and then subjected t~ SDS PAGE tl2%
polyacrylamide) as described (2,3~. The gel was dried, and autoradiograms wsre made with Kodax X-omat film using an intensifying screen at -80C.

GAP~H ACtlV ty of ADP-ribosylated SDH:
, The GAPDH activity of purified SDH ~nd the ~DP-ribosylat~d SDH was measured by the method originally described by Ferdinand (7~ and modified as described 16). ~riefly,:the rsaction was performed in a final volume o~ 1 ~1 containing 809-850 ul o~ buffer (40 mM
triethanolamine, 50 mM Na2HP04, 5 ~ EDTA, p~ 8.6), 100 uM NAD and~the enzyme source (ADP ribosylated and non-ADP-rihosylat~d SDH, :5 ug~ ~dispensed in a 1.5 ~1 capacity microcu~ette.~: The reaction was initiated with .
the addition:of ~7 ul glyceraldehyde:(49 mg/ml) 7 ~ sorbanc~ at~A340~ nm~showing the conversion of N~D to ~ NADH was recorded o~er~a period of 2 m.in.
:
Eff SDH~

: Sodium nitroprusside~was fre~hly diluted in ADPR
30: ~ ~uffer (200~ul) ~o a final concentration of 2 mM and preincubated for:2 minutes at:~room t~perature bef~re the ~:
, : : :
: ~ :

WO93/i4198 PCT/lJSg3~0~82 ~ r 0 22 a addition of 30 ug of SDH and [32p] NAD to start the ADP-ribosylation reaction. At different time intervals~ 40 ul aliguots were removed and precipitated with TC~ A
parallel control repres~nting the same quantity of S~H
and ~32P]-NAD were incubated in the absence of sodium nitriprusside and aliguots were taken at the same time interval~ as the test samples. Precipitat~d proteins were ~eparated on SDS gel and autoradiographed. In a similar set of experime~ts, ADP-rib~ylation was also performed u~ing ~00 ul of a ~treptococcal ly~in extrac~
in ADPR buf~er incubated in the presence and absence of 2 mM sodium nitroprussi~e.

The results o~ the foregoing procedures are summarized below.

Purif1cation of SDH ~rotein r~gEt~r:

SDH protein, was precipitated from the lysin extract by fir~t preaipitating non~specific proteins at 60%
. saturation o a ~ onium sulfate ~ollowed by 85%
saturation. The SD~ was found in the 85% ammonium sulfate precipitate (Fig, l~. The dialyzed precipitate was appli~d to a Mono Q FPLC column and the proteins elut~d with an NaCl gradient~ from 0 mM to 300 mM. SDH
eluted at a salt~concentration of about 280 mM.
~:~ Fractions with fibroncctin binding activity were pooled, dialyzed and~urther purified on a Superose-l2 FPLC
molecular sieving co}~n. The small amount of contaminating~proteins was removed by hydrophobic chromatography using a TSK-pheny} column. SDS-PAGE of the final preparation~reveal~d a h~mogeneous pro~ein with ~ -30 a molecular weight~of 39 kDa. The total yield of : puri~ied protei~ from f:our liters of culture representing ~: : 6-8 gms wet weight of bacteria:was ~00 ug.
~: ;

:
.

WO 93/14198 21 2 7 5 ~ ~ P~/US93/OOOX2 N--terminal Seauence an~ amino acid coml~osition anal~rses NH2-terminal amino acid sequence analysi; o the purif ied sDH c:onf irmed the homogeneity of the preparation resulting in a single amino ac:id at nearly all positions (Fig. 2a3. Ex~ept ~or positions 31 and 35, a single amino acid wa~3 id~nti~i~d in the ~irst 35 residues with the remaining~ f our tentativel}r id~ntif ied .

The am~no acid co~po~ition of the puri~ied protein indicated a high content of Asp/Asn (12 . 1%~, followed by Ala (10.7~6), Gly (10.3%), Val (10.2%~, and Glu/Gln (8.4%). The ma~3s s~ the puri~ied protein ~35,~82 daltons ) as determirled by laser desorption ma~s-spectrometry was used to more precisely assign the number of residue~/mol (Table 1).

Amino Ac:id Sequen e and Composition Comparison:
:
,~
When the sequence of the f irst 3 9 ~mino acids of SD~I
was cs:~mpared to known æequences i n the translat:ed Gen-Bank data~ase; (Fig, 2b~, significant identity was ~ound ,~
: with: bact rial and e~aryotic: GAPDHs. The identity within the first 18 residlaes was 77-83% with bacterial~
eukaryotie,~: or fungal G~PDHs. This strong homoïogy decreas~ed ov~ the remaining 21 residues with an overall idQntity of :from 41-56% ~(Fig. `2b)~, ; ~ en the~amino~ acid compo itions of the Yarious : ~ 25 GAPDHs w~re~ c:o31lpared, th~ methionine content of SDH was ound to~ be signi~ia~antly low (I.8 residue/mol) with relation to the eukaryotic ( 8 . 4 residue jmol ) or other baclterial ~DHs ~(~7;~ residues/mol) ~;(Table 1). Although ~: ~: the a~in~ aci~l~compo :itions :of the rem;~inirlg residu~s of 30 ~ SDN ;were found~ to: be rèlatively close to that of the 1` ~

~093/14198 PCT/US93~ ~ 2 other GAPDHs, sufficient d~ff~rences were found that suggest that, except for the NH2-terminal sequence, SDH
is di~ferent from other repoxted G~PDHs.

GAPDH activity of SDH ~rotein:

In the pr~sence of G-3-P in triethanolamine buffer at pH 8.6, SDH ~howed a do~e dependent conversion of N~D
to N~DH a~ ob~erved by absorbanc~ of the lattex at 3~0 nm. Using 30 ug of puri~ied SDH, ~ariation of enæyme reaction rates with varying concentrations of G-3 P and 1 a N~D was detexmined. ThP results were analyzed bo~h as Michaelis~Menten plots aæ well as double reciprocal plots according to Lineweav@r~Burk (13) as shown in Figs. 3a and b. From the e plots the Km for G-3-P and N~D was estimated to be l.33 m~ Vmax 0.487 X lO 3 M NADH min l.
Fig. 5 shows the analysis based on the method of Micha~lis-~ent~n. ~ , 1.22 m~; and Vmax, 0.466 x lO 3 M
: ~ NADH/min. ~b) 25 ug of the 3~-kD protein was assayed as a ~: function of NAD in the pr~sence of G-3-P (2mM) in ~he buffer y~tem described above. The Km for NAD was estimated ~ be 156.7 ~ ; Vmax, 0.459 x l~ 3 ~ NADH/min;
~-: intercept on y-axis~1/Vmax), 2.l8; and slope (Km/Vmax), 34~.7~ Km for N~D by the meth~d of Michaelis-Menten as q : shown in the inset was estimated to be 148.86 uM; and

-3 ~ Vmax, 0~445~x::10 ~ ~:N~H/min.

: ~ ~ 25 Determina~lon of~:loca~ion of SD~ on cell:

: Antibodies~to SDH wer~af~inity puri~ied on SDH-~ b~und to activa~ed gluteraldehyde beads ~ollowed by~a : prot~:in A column. ~he resultant purified anti-SDH IgG
: r~cognized only ~he SD~ protein band~ig. 4). Dot-~lot 30~ immunoa~say was::applied to determine the location of SDH
on ~treptococcal~-ur~ace. Reiults revealed tha~ trypsin ::
: :

~_W0 93/14198 2 ~ i 5 a PCr/U~3/00082 treated streptococci were markedly reduced in their reactivity to anti-S~H IgG; (Fig~ 5~. To determine if the SDH protein is peripherally bound to the cell wall or tightly bound, the streptococGal cellæ w~re washed wi1:h 2M NaCl and 2% SDS. The results revealed that the SDH
was not extractable by the high s~lt or ionic detergent.

_urface En~e activi~ of streptococci:

To d¢termille if the G~PDH enzymati~ activity f ound with the puri~ied SD~ protein was also present on the stxepto~:os:cal surface, enzymatic studies were carried out using wh~le streptococci. The same concenkration of substrates (G 3-P and NAD) used with the purif ied SDH
were us~d with whole streptococci.

Data presQnted în FigO 6 revealed a dose dependent GAPI~H ac:ti~rity catalyzed by the whole organi~3m~:. As found with the purified SDH, the intact b~c~eria also did not catalyze the reaction in absene:e of th~ specif ic substrates Go3-P and ~AD (Fig. 6a). The enzymatic activi~:~ on the whole org~ ;ms was also f ound to be partially: (30%) but specifically inhibitable by anti-SDH
IgG (~ig. 6c). ~ Enzymatic activity was ~ound to be d~cEea ed by B0% when trypsinized bac:teria were used in the reaation mixture (Fig. 6b). The background 20%
acti-.rity ~;uggested an incompl~te digestion of SDH protein by ;trypsin Preval~nc_ of SDH ~teil~ In oel~r I( serotypes:

The ubi~itous nature of the SDH protein in different ~;treptococs::al M serotypes was determined by Western blot analysis of lysin extrac~s using affinity puri~ied anti-SDH: IgG. As ~hown in Fig~ 7, SDH pro~ein ~ ' -.

W093/14198 P~T/US93/0~82 was found in several ~erotypes. Furthermore, all were found to be of same molecular weight without any indication of ~ize variati~n.

Relationship of SDH with GAPDHs of bacterial, animal and human ori i~:

The relationship of SDH with known GAPDHs was determined ~y both Western blot and competitive kin~tic (k) E~ISA using af~inity-p~rified anti-SDH antibodies.
Western blot (Fig. 8, insert) analysis with with SDH-specific antibodies revealed that GAPDH from bacillus, human RBCs, and rabbit muscle reacted weakly or not at all~ This ~indin~ wa~ further confirmed by competitoin ELIS~ ~Fig. 83 showing that only a maximum of 20 25~
inhibition of binding of anti-SDH antibodiee to SHD,could be achieved with 100 molar exce~s of these proteins~ with the rabbit muscle~ GAPDH exhibiting the least actiYity.
The fact that almost 20% inhibition is o~served wikh 20 times mola~ excess~of bacillus and h~man GAPDH may reflect the 3e~uence homology ob~erved at the Nff2 termini ~:20 ~ ~ of these mole~les~(Fig. 2jO : r ~: Bindi~nq ~ro~rt~_o~ SDH with lysoz ~ e and cytoskel~tal , roteins: ~ . ;: : : :

:: Sinc~:many:~glycolyti~ enzymes have been shown to bind cytoske~etal:proteins a d~termination was made as ~o ;~ 25 whether SD~ ~as:a~similar property.: 1~5I-SDX w~s used to pr~be a:We~tern~blo~:containing~severa:l cytosk~lçtal ~ prstein The results rev~aled that SDH bi~ds to myosin :~: :: : and~its globular~domain (heavy~meromyosin) and actin but not to~the ~-helica~ d~main o~myosin ~light meromyosin) ~ or tropom~osin.~(Figs.~9a:and b): :

, `: :
' ,_~093/14198 2 1 2 7 ~ ~ ~ PCT/US93/OOOX2 Binding of fibronectin to the SDH prot~in:

The fibronectin binding activity of SDH was determined both by using l25I~labeled fibronectin or fibronectin-an~i-fihronectin on a West~rn blot. The results revealed that the SDH protein wa8 able to bind fibronectin in both assay (Figs. lOb and c).

Based on the result~ of all of the ~boYe, it has b~n determined that SDH i~ a major ~urace pro~ein of str~ptococci, including group A streptococci and has both enzyme activity and multipl~ binding activity. No su h protein has previously been detected, isolated and charact~rized. The novel surface protein is principally characterized by its ability to bind fibronectin, lysozyme a~d cyclo~keletal protein as well as by i~s enzymatic activity a-~ a GAPDH. Its molecular weight is approximately 39 kDa. The first fifteen amino acid r~sidues at the amino te~minal are:

~ Val-Val-Lys-Val-Gly-Ile-Asn-Gly-Arg~ Gly-Arg-Leu : -Ala-Phe Theæe flrst ~i~teen amino acid residues mani~est 100% homolo~ with the bacterial form of GAPDH and 80-9096 . . - ~
~: homology with eukaryo c~or ~ungal GAPDX. 5DH is, : however, significant1y difSer~nt from preYiously recorpt~d GAPDHs, because the high homol~y of the first : 25~: fifteen amino acid residues is no~ pxeserved ~owards the : car~oxy end of the molecule and th~ amino acid omposition varies appr~ciably from other GAPDHs.

SDH also functions as an~ADP-ribosylating enzyme :~ which, in the presence of NAD, is auto-ADP-ribosylated.
~ : 30 It has been found that in a crude lysin extract of group :

W093/14198 PCT/US93/0~2 A streptococci containing a mixture of cell wall associat~d ~olecules, SDH is the only molecule that is ADP-ribo~ylated. Treatment of ~DP-ribosylated SDH with the cytopla~mic fraction of Group A removed the ADP-ribosye of SDH which indicates the pre~ence of SDH
specific ADP-ribosyl hydrolase in the cytoplasmic compartment. Treatment of purified SD~ or the crude lysin extract with sodium nî~roprusside, which spontaneously generate nitric oxide, was found to stimulate the ADP-ribo~ylation of SDH in a time dependent manner. Both ADP-ribosylation and nitric oxide treatment inhibited the ~lycer~ldehyde-3-phosphate dehydrogenase activity of SDH. In addition to its auto ADP-ribosylation activity, either purified SDH or whole streptococci with surfac~ SDH were able to ADP-ribosylate specifically both chicken and human lysozyme, strong SDH
binding proteins. These data show that SDH has both autoribosylation and ADP-ribosyl trans~era6e acti~ities.

SDH, as will be reco~nized by those skilled in the art, does not represent~a single protien, but rather a class of surface~proteins of streptococci, ~ll of which have simil~ properties.: The protein is involv~d in the ~ coloniæation and probably:in the int~rnalization and : proliferation of:group A~streptococci. One of the : 25 initial steps in~th~ colonization of mucosal tissue by ~; streptococci and ~ubsequent infection by this bacteria, ;: ~ is the binding of the~bacteria to fibxonectln~ Lysozyme : is alæo believed to~ be involved in this binding step.

The enzyme activity of SDH may be involved in the ' I' . i ` I ` ~ ,. .
binding of the bacteria to endothelial cells by reaction o~ an aldehyde reduction product;of teichoic acid which : : :

, ~ 93~1~198 2 1 2 7 ~ ~ ~ PCT/US93/~82 is a polyglycerol phosphate. The aldehyde function could bind the bacteria to the tissue surface by reaction with ~mino group ~n that surfaae.

Inhibition of this initial binding is, therefore, a major fuction in inhibiting ~r preventi~g streptococcal infectionO Accordingly, antibodies to SDH which successfully ~ompete with ~i~ronectin and ly30zyme for binding sites on the bacteria will inhibi~ the colonization of the pharyngeal muaosa by group A
tréptoGocci. Therefor~ the SDH and amino acid segments o~ the protein containing the appr~priate antigenlc determlnanks, for example those containing from about 6 to about 20 amino acid residues, are useful to inhibit streptococcal infection o~ ~a~mals, including humans, by administering an amount of the selected product which will be effectiv~ to inhibît fibronectin binding and thereby inhibit colonization of~the pharyngeal mucosa.
:: :
The proteins, pol~ eptides and peptides of this invention may be obtained by any::of a number of known : : 20 processes. : -The protein can be~isolated~as described above.
; Alternativley, rthe protein or se~ments thereof can be prepared by~recQmbinant~DNA t~chniques. For example, the :~ : g~ne for the prot~in or an oli~onucleo~ide for the ~25 desired eg~ent~can be inserted into a plasmid and the plasmid:used~to transf~rm E. coli so that the ba teria will ~xpresæ:~he desired product.

Polypeptide and~eptides within the scope of the invention c~ntaining, for example~rom abou~ 6 to 20 or more amino aci~ ~eg~ents, may be sy~thesized by standard ~ ~ solid phase procedures with appropriate ~mino acids using :~: : ::

W093/14198 PCT/US93/O,Q.~.8Z
~r~ 30 the protection, deprotec~ion and cleavage techniques and reagents appropriate to ~ach speciPic amino acid or peptide. A combination of ~anual and auto~ated (e.g., Applied Biosystem 430A) solid phase techniques can be u~ed to synthesize the novel peptides of this invention.
Altough less convenient, classical methods of peptide synthesis can also be emplo~ed. For background on solid phase techni~ues, rePerence is made to Andreu, D~, Merri~i~ld, R.B.~ Steiner, H. and Boman, ~I.G., (1983) Proc~ Natl. Acad. Sci US~ 80, 6475-6479; ~ndreu, D., Merrifield, R~B., St~iner, H. and Boman, H.G./ (1985) Biochemistry 24, 1683-1688; Fink, J., Boman, A., Boman, H . G., and Merrifield, R~B., (June l~9) Int~ J0 Peptide Protein Res. 33, 412-42~; Fink, J., Merrifield, R.B., Boman, A. and ~oman, H.G., (1989~ J. Biol. Chem. 264-6260-6267; each of which is hereby incorporated herein by reference.
~, :
~: The products ~f the invention are amphoteric. They can exist and be utilized as free bases or as ~: ~ 20 pharmaceutically acceptabl~ metallic or acid addition alts. Suitable metallic salts include alkali and ;` : alkaline earth~etal salts, preferably sodium or ~: ~ potassium sal~s.~ Acid addition salts may be prepared from a wise va~iety~of organic and inorganic acids including mineral acids, for example ci~ric, lacti~, maleic, tartaric, phosphor;ic:and hydrochloric acids.
: These sal~s~can be pr~pared~by procedures well known to those skilled in~the~art.~

` ~
For use~as:a vaccine, it i8 presently pre~erred to admini~ter the selected p~oduct conjugat~d to a carrier such as cholera~toxi~ B~: Methods for preparing such ~ ; ~

: ~

WO93/141g8 ~12 7 ~ ~ Q PCT/US93/00082 co~juga~es are known. One procedure is de~cribed by B~ssen and Fischetti ~14~. Oth~r carriers can be employed or the products can be used without a carrier.

The protein or segmen~ thereof may al~o be administered as a hybrid protein expres~d on a streptococcal surface utilizing ~h~ procedure of Pozzi et al (17,18).

Mice or other m ~ als including human6 wh~n immunized parenterally or orally have significant resistance to subsequent streptococcal challenges.

The presently preferr~d method for the administration of the vaccines of the invention is by the intranasal route, but the invention i~ not so limited.

Other parenteral or oral prcoedures may be employed.

Typicallyt the patient to be protected will be tr~ated with an a~ount o~ 5DH or other product of the invention which is e~ective to elicit a protective : immune response.: The sel~cged agen~ may b~ a~ministered alone or in a pharmaceutically acceptabl~ liguid or solid ~ carri~r in which~it;may~be:dispersed, dissolved or ~: suspend~d. If, for~xample, the patie ffl is to be treated intravenously,~he p~ptid ay be ~u~pended as a free : bas@ or dis olved ~as a: metallic salt in i50tonic a~ueous ~ uffer. Other methods:of:treatment and pharmaceuti~ally :~ 25 ~acceptable carriers will be apparent to the skilled artisan.
:
Ths proteins,:~polypeptida~ and peptides of this invention an~:the~ genes or oligonucleotides which are employed in their expression~are useful as probe~ ~or ~30 genes and protein~ ~:They~ ~re~also ~seful to rai~e ~ , :~:~ : : :

WO 93/141~8 PCI/lJS93/0~0~2 antibodies by which specif ic strains of ~;treptococci can be identif ied . For example in t2g;ts f or mammalian inf ection~ .

~,, :,~ , : , :
.

, ~: : :: : : :~

: ~ :

wo 93/l4lg8 2 i 2 7 ~ 5 ~ PCTJUS93/~082 ~BLE 1 Comparison of Amino ~cid Composition of SDH Protein from M Type 6 Streptococci wi~h That of GAPDH from Different Species ~ ~ _ . _ __ _ No. o~ residues/mol .... _ . _ . ... .. . ... ....... ., . , , . _ S~ BSt Tha~ RSM

~sn/Asp 43.3 41 36 35~5 G1u/G1n 29.9 26 24 18.7 Ser 16.~ 17 13 1~.7 G1y 36.6 24 25 31.7 ~is 7.2 9 10 9.8 Arg ~5.5 15 15 10.1 Thr 27.0 ~ 22 21.6 Ala 3~.1 38 41 3~.6 Pro 13.6 11 12 11.9 : Tyr ~ 9.1 ~ 10 8.6 2a~ Val 36.5 43 ~9 ~31.
M~t 1.8~ : 7 7 8.4 ~ Ile~ ~ 22.4 19 ~ :22 lS.3:~ Leu ~ ~23~.4 ~ 26 30 17.8 : ~ Phe ;~13.~ ~ ~ 7 12.9 -2S ~ ~Ly8 21.:4 :~: 23 23 23.9 C~sS~ : 3:.1~ ;: 2~ .0 Trp~ ND ~ 2: ~ 3 ND

~ Bst, B.stearothe~ophi1us (19~; Thaq, Th~rmus aquaticus : 30~ ~52); RSM, Rab~it:~skeletal:;mu~c1~e~(203.
* Nean~of threè~determinati~ns.
Mo1ecu1ar ~ass of~SDH:protein ~35,882~ was measured by :

WO93/~4198 P~T/US93/00082 . ..

15~ laser desorption ma~s ~pectrometry.
Determined by carboxy amidomethylation ~e hod (5).

The publications id~ntified in thi~ ~pecification are all incorporated herein by r~ference.

~EFER~NCES
1. Wannamaker, L. W. 1973. The chain~ that link the throat to the beart. Circulation 48:9.

2. ~ancholi, V. and V.A. Fischetti. 1989.
Identif~catiDn o~ an endogeneous m~mbrane anchor-cleaving ~nzyme for group A streptococcal M protein. J. Exp~ Med.
: 17~:211g.

3. Pancholi, V. and V.A. Fishcetti. 19~8. Xsolation and characterization of the c~ ass~ciat~d r~gion of group A
streptococcal M6 protein~ J. Bact~riol. 170:2618.

I5 4. Matsudaira, P. 19~7. Sequence ~rom picomole ; quantities of prot~ins ~lectroblotted onto polyvinylidene difluoride:me~brane~. J. Biol. Chem. 26~:10~035.

5. Cre tfiPld, A.M., S. Moore, and W.H. Stein. 1963. , : ~ The preparation and~enæymatic hydrolysi~ of reduced and ~ S.: carboxy~ethylated proteins~J. Biol. Ch~m: 238:~22.

; 6. ~Beavis~ R.C.:~and B. ~. Chait. 1990. High aacuracy ma~s~de~rmination of proteins:using matrix assisted : ~ ~ lasor~desorption~mass spectrometry. Anal. ~iochem.
2:1~36.

: ~ Ferdinand,~;W. 1964.~ The:isolation~and specific activity of rabbit-muscle glyceraldehyde phosphate dehydrogena e.~Biochem. J 9~:578.~: ~

: -. :

WO 93/1419B 2 ~ ~ 7 ~ 5 G PCl/VS~3/00082 .

8. Horecker, Bo L., and ~. Kornberg. 1948. The extinction co f f icient of the reduced band of pyridine nucleotides ., J. Biol . Chem. 175: 385 .

9. Jones, K. F. and V.A. Fischetti. 1988. The S importance of the location of antibody binding on the M6 protein for opsonization and phagocytosi~i o~ group ~ M6 streptoc:occi. J. Exp. M~d. 167: lll4 .

10. Joale;, K.F., S.A. ~, B.W. Erickson, S~X.
Hollingshead, J.R. Scott, and ~I.A. Fischetti. 1986.
Immunochemic:al localization and amino acid sequence of cross-reac:tive ~pitope~; within the group A streptocoGcal M6 protein. J., Exp. Med. l64: l226.

11. Fischetti,: V.~. and M. Windels. 19~8. Mapping the i~munodeterminants of the comple~e strepts:~coccal M6 l 5 protein molecule: Identif ication of an immunodominant region. J. Immunol. ~ 41: 3592 .

2 . Beavis, R. C:~, and B. T. Chait. l990 . l~apid, sensitive analysis of protein mixkure by mass spectro~n~try. Proc.
Na~}. Acad. Sai. U~ S.A. 87: 6873 ., ~ ~ 9 13. Lineweaver, H. and D. ~urk. 1934. The det~rmination of enzy~e dissociat~ on constants, J. Am. Chem. So~:.
5~:658. ~:
, :
~: ~ 14. ~Be sen~ D. and Y.A. ~Fischetti:. l988. Influerlce of ` int~anasal immNnization with synt:hetic peptides 2 5 clorrespo~ding to aonserved epitope~; of M protein on mucosa~ colonization by group A streptococci. Infect.
Immun. 56:2666. ~ ~

: ~`: : ` : :

:
`:` ~ : : :
: : :

WO 93/14198 PCI~/US93/00082 ~! '"

15. Kot~, ~.Y., Skurat, A.~r., Sergienko, E.A., Bulargirla" T.V. & Severin, E.S. (1992) FEBS Lett. 300, 9-12 .

16. Pancholi" V. & Fi~;chetti, V.P.. 1992. A l~ajor Surf ace Protein On Group A Streptococc~ A
Glyceraldehyd~-3~Pho~phate-Dehydrogenase with Multiple Binding Activity. J. Exp. Med. 176~ 415~42~o 17. Pozzi9 G., M. Contorni, M.R. Oggioni~ R. Manganelli, M. Tomma~ino, F. Cavalieri, and V.A~ Fischetti. 1992.
The delivery and expression o~ a heterologous antigen on th~ surface of streptococcî. Infect. Immun. 60:1902-19~7.

18. Pozzi/ G., ~. Contorni, M.R. Oggioni, R. Manganelli, and V~A. Fischetti. 1992. Expression of ~6 protein gene of Streptococcus pyo~enes in Streptococcus gordonii after chromosomal integration and transcriptional fusion. Res.
Microbiol. 143:449-45~. :

: 19. Harris, J.I., J.D. ~oc~ing, M.J. ~unswick, K.
Suzuki, and J.E. Walker. 1980. D-glycer~ldehyde-3-phosphate dehydrogenase: the purification ~nd char~cteriz~tion of the enzyme from the thermophiles 13acillus tearothermophiluc and ~hermus aquatic:us. Eur.
J. Biochem. 108: 535.

: 20. Caswell, A~H., and ~.M. Corbett. 1985. Interaction of glyceraldehyde-3-phosphate dehydrogenase with isolated micros~mal subfractions of skeletal muscl~. J. Biol.
Chem. 260: 6892 .

.

Claims (4)

WHAT IS CLAIMED IS:

1. Strptococcal surface dehydrogenase and segments thereof capable of binding streptococci to fibronectin, lysozyme and cycloskeletal proteins, having glyceraldehyde-3-phosphate dehydrogenase activity, ADP-ribosylating activity and ADP-ribosyl transferase activity.

2. A vaccine effective to inhibit colonization of mucosal tissue by streptococci containing, together with a pharmaceutically acceptable carrier, a streptococcal surface hydrogenase or a segment thereof in an amount which is effective to inhibit said colonization, said hydrogenase being capable of binding streptococci to fibronectin, lysozyme and cycloskeletal proteins, having glyceraldehyde-3-phosphate dehydrogenase activity, ADP-ribosylating activity and ADP-ribosyl transferase activity.

3. A method of treating a mammal to inhibit colonization of mucosal tissue by streptococci in a mammal in need of such inhibition which comprises administration of a streptococcal surface hydrogenase on a segment thereof capable of binding streptococci to fibronectin, lysozyme and cycloskeletal proteins, having glyceraldehyde-3-phosphate dehydrogenase activity, ADP-ribosylating activity and ADP-ribosyl transferase activity in an amount sufficient to effect such inhibition.

4. A method of obtaining a streptococcal surface dehydrogenase which comprises solubilizing streptococci with lysin to produce a mixture containing the dehydrogenase and isolating the dehydrogenase from the mixture said dehydrogenase being capable of binding streptococci to fibronectin, lysozyme and cycloskeletal proteins, having glyceraldehyde-3-phosphate dehydrogenase activity, ADP-ribosylating activity and ADP-ribosyl transferase activity.