Note: Descriptions are shown in the official language in which they were submitted.
2192175
Wo 9513~65a Pcrlus9alo7loo
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SURFACE PROTEIN OF STAPHYLOCOCCUS AUREUS
FI~LD OF TH~ lNV~ L~
This invention relates to methods and compositions for
preventing ini~ections by Staphylrcocc~1c aureus ~S. aureus),
iAl ly those triggered by the use of catheters prosthetic
devices and heart valve rep~ ore specirically, it
relates to methods and compositions useful ror inhibiting the
ability of a surface protein of S. aureus to i, l- t a&esion
of the organism to endoth~ ic~l cells or to catheters at the
skin-catherter junction thereby initiating infection.
BA~ KUUNL~ OF THE lr~ v~;h L10~
S. aureus is one of the most ~requently encountered
pathogens in infections acquired in the hospital. It accounts
for 25% of all hospital acquired infections resulting from the
use of catheters or similar :~LLU-LUL~:5. Since a great majority
of patients entering a hospital re~uire some sort of intravenous
device, there is a very high probability of infection. The same
type of risk applies to the use of prosthetic devices such a as
hip and other joint rPrl~r ~ because the ability of
staphylococci to a&ere to such devices.
.
One of the initial reactions of the r~ n host to the
presences of a catheter is to coat the object with fibrinogen and
other matrix proteins as a prelude to the systemic reaction which
is intended to expel the invasion. In a hospital setting, this
provides an U~U~U~Lity of infection by s. aureus which attaches
--1--
2 1 9 2 1 ~ 5 ~ oo
wo s~l3~6s~
itself to the fibrinogen at the s3cin-catheter injunction and
thereafter worXs its ~ay through the skin and into the blood.
Since the strains prevalent in hospitals, nursing hcmes and other
patient care facilitieS are o~ten antibiotic resistant, these
types of infections are ~L-~ -ly serious and very ~ iclllt to
contain. Accordingly, the art has t~Ypt~nt~Pd much effort to
prevent such uyyU~ L-l..istic infections.
BRIEF S~RY ûF T~IE INVENTION
A surface protein, and the gene which expresses it, have now
been discuv,~ d. This protein enables the invading bacteria to
Adhe're to the f ibrinogen . Antibodies to f ibrinogen or to the
surface protein will prevent bacterial ~hP~i~n and thereby
inhibit infection. The protein and segments of the protein are
useful as vaccines or for the pro~ n of antibodies useful for
passive protection of patients prior to the use of a catheter or
equivalent device.
This inYention, therefore, comprises the protein itself and
segments thereof, the gene and segments thereof which produce
such products, vectors for the gene and its useful segments,
organisms transformed by such vectors, monoclcnal and polyalonal
antibodies to the protein and its useful s~ Ls, vaccines
produced utilizing the protein and its segments and methods of
preventing s. aureus infections utilizing such products. The
invention also includes diagnostic probes utilizing the gene
products described herein.
--2--
2 ~ ~ 2 i 7 ~ L~ ~ ~ ~ Ioo
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1~} FIGURES
There follows a brief descripticn of the figures.
Fig. 1 Western blot of cellular i~ractions of clone number 14
probes with fibrinogen l~ollowed by anti-fibrinogen antibody
cu..j, ~y~t~. The control contA i nC the lysate of an E. coli clone
with pBR322 insert in ~ Zap. The arrows indicate a 34 kl~
reactive band together with an upper band which may be a dimer.
Results of a duplicate blot probed with 125I fibrinogen were
similar.
Fig. 2 Nestern blot (A~ and silver-stained gel (B) of the
pe_iplasmic extract of clone number 14 fractionated in a
fibrinogen column. The Western blot was probed with
fibrinogen/anti-fibrinogen conjugate. The short arrow indicates
the fibrinogen-reactive band from crude lysate positive control.
The long arrow marks a 34 kD protein that reacts with fibrinogen
t4th lane in (A) ] and is eluded with 3 M potassium thiocyanate,
but not with PBS with o . s M ~2Cl nor with acid elusion.
Fig. 3 Is the complete sequence of clone 36 (C36~ ~n'-n~;n':T
fibrinogen reactive protein.
Fig. 4 Se~uence comparison of strain DB to that of
coagulases from strains 8325-4, 213 and BB with the Pileup
prosram under the GCG package. The arrows indicate the 11 amino
acid sequence that is unique to the protein. This sequence
shares homology with a cell wall anchor motiJ found in other gram
--3--
WO 9al316aa 21~ 21~ S ~ c ]
pcsitive Yall protein Eowever, there i5 no ~oto identify to
t~is mctif i~ the Genban3c. The arrows at rosiues 4~9 and 419
indicate a unique secuenc2 segrlent of this protein.
The aminc half (residues 59-325) c~ this protein is
primarily helical as predictsd by the Gar~ier analysis. Two
areas (residues 58-194 and 264-2g7) reveal a 7 residue
periodicity in which residues in positicn 'a' and 'd' in a heptad
mcti1' 'abcdefg' are either ~y~ u~hobic cr ncnpolar. This pattern
is consistent with a coiled-coil c~n1'n~-tional ~ L~. The
sa~ is analyzed by the Matcher ProSrall ~22).
Fig. 5 Se~uence comparison of cur protein (strain DB) to
that o~ coagulases from strains 8325-4, 213 and BB with the
Pileup program under the GCC package. The arrows indicate the 11
amino acid sequence that is unicue to cur protein. ~his se~uence
shares hcmclogy with a cell wali anchor mctif found in cther gram
pcsitive cell wall prctein. Xo~ever, there is no ~ let~
identi~y to this mctif in the Genbank.
The follcwin5 abbreviations are employed in the description
cf this invention:
Strain DB - a ~ild strain of S. aureus
N2Y broth - a co~mercially available
gro~th medium
I i3 - a commercially available
growth medium
IPTG - Isopropyl-beta-D-
th i o~ tu~yLunoside
BS~ - bcvine serum albumin
--4--
~ 2 1 7 ~ PCrlUssslo~loo
~ woss/3463~ ~1
.
X-gal - 5 bL 4-chlero-3-indclyl-
beta-D-~lA~ ~ vl~yL~:ncside
525 - sodium dodecyl sulfate
NP40 - a com~ercially available
nvn i r~n; C deterge~:t
pBR322 - a conmercially available
pla5mid ol' known ~rLLl ~_-UL'~
rRInos~ipt - a commercially available
~hArJamiri of known '--~LU I_ULC
SSPE _ 0.l5~aCl, 10~ ~a~2P4'
Im ~EDTA pH7 . 4
'rSE buffer - 0.1~ ~ris, 20% sucrose,
5~ EDTA p}I8
PBS - Fhosphate buffered saline
Strain DB has been deposited at the A~erican Type Colture
Collection under the A~cracsir~n nu~ber
The ~ollowing Materials and ~ethods section is provided for
convience and ease o~ understanding of this invention.
M~ ?T~r C ANI? MET~ICDS
Bacteria, plasmids and pha~e
Strain DB which has been phenotypically characterized (5
was used in the construction of a geno~ic library. E. coli
strain Sure (stratagene) was the host cell for the~~ Zap vector.
Clones 14 and 3 6 were phagemids derived ~rom ~ibrinogen-reactiVe
--5--
~ ~ n~ r pcrlus9slo7loo
wo ss/346s i ~ J
.
pla~ues containing the insert. A pBluescript rh~g~m;~, pAC8,
whic'~ cont2ined a protsin A clone was derived from a Zap gencmic
library of DB.
Media and Antibiotics
Unless otherwise indicated, the following media wew used:
NZY broth and LB (23) were used for the growth of E. coli
strains .
Prer~arations of affinity purified qoat anti-fibrinoqen conjusate
Goat 2nti-human fibrinogen antibody obtalned commercially
~Cappel, West Chester PA) was a~finity-purified on a fibrinogen
column as previously descri~ed (7~. The fibrinogen column was
LlL 2~C~ ed with glutardialehyde activ2ted beads (Boehringer
M~nnhoim, Tn~;anaroliS, IN) as déscribed in the manufacturer's
insert (4). The monospecificity of the affinity purified goat
anti-human fibrinogen IgG was verified by an irlmunoblot using
purified fibrinogen and plasma as antigens (7). The protein
concentration of the affinity purified antibody was detarm;np~ by
the BCA protein assay reagent (Pierce ChF~m~ c~ Rocke~ord, IL).
Affinity purified goat anti-fibrinogen antibody was conjugated to
bovine intestinal alkal;no phosphates (Sigma, St. Lousis, MO) as
described by Vo ller ( 3 2 ) .
Construction and screeninq of staphylococcal qeno_ic library
A genomic library of strain DB was constructed with the Zap
vector that has been digested with EcoRI and dephosphorylated
(Stratagene cloning kit). DB ~ LI _ ~1 DNA was extracted from
--6--
WO 9513'~6~5 f~
l~a~a~phin-lysed cells as pr2viously described (5,29). Genomic
DNA was sheared w ith a 2 6 gauge syringe and sub j ect to gel
filtration on Sepharose C~23 to re ove fL~ q s~aller than
Lkb. Fractions c~nt:~;n;ng 4~5 kb fL, Ls were po~led, treated
with T4 polymerase to produce blunt ends, methylate with EcoRI
~:ethylase (New Eng7and Biolabs, Boston, MA), insert into the
EcoRI site of the Zap vector with EcoRI linkers and packaged in
vitro with ~:i g~raC-k packing extracts (statagene) . Over 90% of
the r~ ~;n~nt phages were r~-or;7F~ as white plaques when
plated on lac host strain Sure in the presence of IPTG and X-
gal.
For the screening of f ibrinogen reactive plaques,
rF 'in~nt phage was incubated with the E. coli host on NZY agar
(23) at 42C for gene expression. Following transfer to
duplicate nitrocellulose filters (82 h7m in diameter, Schleicher &
Schuell, Keene, N}~), the filters were blocked with lOml of TNT
buffer (10 m~ Tris with 0.15 M NaCl and 0.05% Tween 20)
containing 1% BSA for 1 hour at RT and then incubated at 37C for
3h with 25 ug of fibrinogen (Sigh7a ~4883). This fibrinogen
preparation had been further purified over a protein A sepharose
column to re~ove contaminated IgG and was found to be easentially
free of contaminants as determined by a silver-stained SDS-gel
containing this protein . The f ilters were then washed once with
TNT containing O .1% BSA and O . l~c NP40, and twice with TNT with
0.1~ BSA for 5 min. each. Affinity purified goat anti-fibrinogen
antibody ~lk;.lin~ phosphatase conjugate diluted 1:1000 in TNT
buffer with 1% BSA was then incubated with the filter for 1 hour
at RT. After washing the filters twice with TNT with 0.1% BSA
and O.1% NP40 and three times with TNT with O.1% BSA for 5 min
each, fibrinogen-reactive plaques were visualized with 5-bromo-
--7--
r~,~l1.J,.,.~l~CO
,~ Wo 9sl3~6~s 2 1 9 2 1 7 5
4-cloro-3-indolyl phosphate as a substrate as described by Blake
et al (1). A~Zap vector w~th a pB~322 insert plated on E.coli
or E. coli cells alone served as negative controls. Positive
clones detected by fibronogen/anti-~ibrinogen conjugate were
cnrtf i ~ by allowing plaques on duplicate filters to react with
125I fibrinogen. This screeni~g p~ du~ was similar to the
method described above except that 125 fibrinogen (lO0,000 cpm)
was used in place of the cold llnl:~h l~d fibrinogen. Following
reaction, the nitroc~lllllose filters were washed twice with TNT
with 0.1% BSA and 0. l~c NP40 and three times with TNT with ~.1%
BSA for 5 min each, and finally subjected to autoradiography.
Purif ied plaques were isolated by rescreening each positive clone
at least 4 times.
DNA seauencinq of the f ibrinoqen reactive clones
By infecting the E. coli host strain simultaneously with a
fl helper phage tR408~ and the~ Zap vector containing the insert,
a single strand DNA containing the pBluescript phagemid with the
insert can be packaged for recirculation, thereby generating
subclones in E. coli cells when plated in LB/ampicillin agar
(Statagene cloning kit instructions). Plasmids for sequencing -
were purified from E. coli by equilibrium centrifugation in
Cesium chloride-ethidium bromide gradients (23). The purity of
the plasmid was confirmed by digestion with restriction enzymes
(New England BioLab, Beverly, MA). By using both T3 and T7
primers flanking the insert, plasmid sequencing of clones 14 and
36 were perfor~ed with the Sequenase Xit (U.S. Biochemicals,
Cleveland, o~) following the mznufacture's instruction (27).
Additional primers were obtained for sequencing from within the
insert .
--8--
~ 1 n ~ oo
Wogs/3~6~s f. 1~ J
Southern blot hybridization
Southern blot hybr;~7~ n ~as perSormed with random primed
samples of gel-purified DNA fragments as probes ~12,23).
Briefly, ~ l~z, -- 1 DNA digested with restriction enzymes was
resolved on 0.7% TBE gel and transferred onto }~ybond-N membrane
Anersham, Arlington }~eights, IL) (14). DNA probes were labeled
with 32p ( _32p deoxycytidine tr;rh~ k~e, AD~ersham) using the
rando2 pri2ed DNA lAh~lin~ kits (Boehringer M~nnhl~;m). The
membrane was then hybridized uith the 32p labelea DNA probe at
65C overnight, washed twice with 2X SSPE with 0.1% SDS at RT for
10 min each followed by lX SSPE with 0.1% SDS at 65C for 15 min.
The membrane was then subject to autoradiography with an
intensifying screen at -70C.
Ex~ression of fibrinoqen-reactive protein of S. aureus in E. coli
one of the fibinogen-binding clones, clone 14 was evaluated
for the expression of the fibrinogen binding protein in E. coli.
E. coli cells containing this clone was grown in 10ml of LB with
50 ug/21 of ampicillin at 37C until the OD 600 reached 1. 0 .
Cells were collected by cetrifugation at 7,000 g for lO min and
resll~p.on~d in 1.25 ml of ice cold TSE buffer (100 _M Tris, pE~
8.0, containing 20% sucrose and 52M EDTA). Lysozy2e was added to
a final ~ onc~ ction of 0.5 mg/ml and the sample was iced for 20
min. For whole cell lysate, 0 . 25 ml of this suspension was
removed and 7 . 5 ul of Triton X-100 and 50 ul of DNase solution
(lO 2M MgCl2 with 100 uglml of DNase) were added. The sa2ple was
frozen (-70C) and thawed twice.
_g_
WO 95134655 2 1 9 2 1 7 ~ , IIU:~YSIU I 100
Magnesiu~ chloride was added to the remaining 1 r. l . cell
suspension (50 m~ final Cu~ LLation) to 51 ~hi 1 i ~o the
spheroplasts which were then pelted at 7,000 g for 15 min. The
supernatant was filtered through a 0.45 um M;llirore membrane to
obtain the periplasmic frâction.
To lyse the spheroplasts, 0 . 25 ml of DNase solution wzs
added to the pellet along wit_ 0 . 75 of water. The spheroplasts
were aspirated vigorously several times with a pasteur pipette,
frozen and thawed twice as described above. The lysate
generated by this treatment was centrifuged for 49, 000 g for 1
hr. The supernatant filtered through a 0.4 um membrane was
designated cytoplasmic fraction.
The transfor~ed E. coli has been deposited at the American
Type Culture Collection under the accession number
SDS-PAGE and ir--ln~lhlt~t analysis
Cellular extracts (lO ul each) were separated on 9% SDS-
polyacryla~ide gel slabs by the method of Laemmli (21).
Prestained molecl~lar standards (BRL, Gaithersburg, ~D) were run
o ù~l~ urr~ntly in adjacent wells. After ele~_LLu,uholesis, the gel
was either stained with silver (Pierce `hPric~l~) or transferred
onto nitro~ llos2 (30). After transfer, the nitrocPlllllose
filters were allowed to react with fihrinogen followed by
affinity purified goat anti-fibrinogen ;~lk~l irP phosphatase
conjugate and reactive suhstrate as described for the screening
of the genomic library . In some experiments, the f ilters were
incubated with 125I fibrinogen (250,000 cpm~, washed and
autoradiographed as descrihe~ above.
--10--
Wo 9s/3~6~ 2 1 9 2 1 ~
To detect other proteins in the cell extracts, the SpPri1';r
antibcdy diluted in blorl-;n~ bu~er (Tris lOmN with O.S M NaC1
and 0.05~6 Tween 20, p~ 8.2) was incubated with the blot for 2
hours at RT. This was followed by incubation with an d"u~uruuLiate
al~:~l;no phosphate Cu~Ju~:t2 for 1 hour and then processed for
band v;c~ll;7ation as previously described (3).
Partial purif ication o~ the f ibrinoqen-reactive protein of S .
aureus
In an attempt to pu~ify the fibrinogen-reactive protein from
clone 14, peripl~-;r extracts fro~ 4 L of culture were ~Lc:~dl~d
as previously described. Briefly, cells were harvested by
centrifugation (7,000 g for 20 ~in) and rpc~lcpondp~ in 7S ml of
TSE buffer containing 37.5 mg of lysozyme. After incubation on
ice for 20 min, MgC12 was added to a final Cu.~cer ~Lc-tion of 50 mM
and spheroplasts segmented at 7,000 g for 30 min. The
periplasmic fraction was aspirated from the supernatant, filtered
through a 0 . 45 um membrane and immediately applied to a
fibrinogen column (2.5 x 20 cm) followed by rotation at 4C
overnight. The fibrinogen column was prepared by mixing 25 mg of
fibrinogen and 5 gm of glutardialdehyde beads as described (4).
After collecting the fall through, the column was washed with lS0
ml. of PBS followed by 150 ml of PBS with 0.5 NaCl. The
f ibrinogen-binding protein was then eluded by rotating the column
with lo ml of 3 M potassium thiocyanate at RT for 20 Min followed
by collection. In prel;m;n~ry studies, a similar elusion
. uce.luL e with o . ~ glycine pE~ 3 . o was not ~ rul. Fractions
from the column were concentrated in a Centricon 10 (A~icon,
Danvers, MA) and analyzed by SDS-PAGE and immunoblots with
~ibrinogen as described.
--11--
~ Wo 95/3~6~ 21~ 2 1 7 ~ JaYalul~ûû
Computer analysis of seS~uenc~ data
DNA protein sPT~Pnre analysis, and se~uence compariscn with
database were rnn~llr od with the Sequence Analysis Software
Package from the GPn~f i ~-q Computer Group (IJniversity of
w;ccnn~:in, Nadison, WI) (9). The deduced amino acid se~uence of
the putative protein was ~ I:d to a se~uence database by the
algorithm of Pearson and Lipman (TFASTA ;mrl~ tation of
GenBank) (25). The fibrinogen reactive protein SP~onre shown in
the figures has been ~c~isnpd to clone 36.
RESULTS
Isolation of f ibrinoqen reactive clones
Using the foregoing ~Luce~uL~s~ a~ Zap library of strain DB,
was screened for clones that were reactive with fibrinogen. Of
100,000 plaques screened, three novel clones, designated 14, 30
and 36, were found to be highly reactive with both l25I
fibrinogen and fibrinogen/antifibrinogen conjugate on
immunoblots. S~hr~ onPc contalning the pBluescript phagemid
together with the insert were subse~uently generated in E. coli
strain Sure. Plasmid DNA from alk~l ;nP lysis minipreps of clones
14, 30 and 36, upon digestion with Eco~ which released the
inserts, revealed DNA LL_, 5 of 4.6, 3.6 and 3.2kb,
respectively. Using the 4 . 6, 3 . 6 and 3 . 2 kb fragments as
separate probes, Southern blot analysis of Eco RI digests of
these clones established that they hybridized each other. These
clones did not hybridize with the EcoRI fragments of pAC8, a
protein A probe of DB, thus eliminating the possibility of a
false positlve reaction between expressed protein A gene product
--12--
1 9 2 1 7 ~ PCr~S95107100
WO 9513'~655 2
and gcat anti-f ibrincgen anti~ody c~...; ,.~te during the screening
pL ~c- duLt:. Further analysis indicated that clone 14 comprises
about 2/3 of the mature molecule, C 36, oYtPn~;ng into the C-
f~n;mlq .
Expression studies of the fibrinoqen reactive protein of S.aureus
Based on restriction analysis, clones 14 and 3 0 were
Oimilar. Although clone 36 contained the co~plete gene as
~et orm;nP-l by SPq-lon--e analysis, expression of the fibrinogen
reactive protein with this clone was found to be difficult.
Notably, a culture of clone 14 when grown to late stationary
phase (i.e. OD600~m 1.5) also resulted in a significantly
decreased yield in f ibrinogen-reactive protein . This result can
be explained either by toxicity of this protein on E. coli or by
increased proteolytic breakdown during stationary phase. For
these reasons, the expression of the partial protein was
evaluated in clone 14 that has been grown to early stationary
phase (OD6001"mm-1. o) .
Expression studies of dif~erent fractions from clone 14 with
Western Blots probed with either 125I fibrinogen or
fibrinogen/anti-fibrinogen conjugate es~hl; ~hod that the
protein, which has a molecular si2e of 34 kD was found in the
whole cell, peri~l~C~;~ and membrane fraction (Fig 1). In
contrast, a crude lysate of an E. coli clone which contained a
llloe:--ript phagemid with a pR~322 insert did not react with
~ibrinogen (Fig. l) . With some fractions (e . g . membrane), there
was also a higher molecular weight band, possibly a dimer, which
reacted with fibrinogen. ~leither of these proteins were fusions
--13--
wo s~/3~6~s 2 1 ~ 2 1 7 ~ P~
proteins as they were not inr;lt~ihl.~ with IPTG. Additionally,
these bands did not react with pclyclonal and ~ 1 anti-
beta-galac~si~l~ce antibody ~1:1000 ~ tion) (Boehringer
Mi~nnh.~;~) cn i ~hlnts. The fibrincgen reactive band alsc did
nct react with a~inity purified chicken anti-prctein A antibcdy
(Accurate C~ c , Westbury , NY), thus prcviding additional
evidence that prctein A was nct cloned which, by binding tc ~gG,
could lead to ~alse positive results.
To conf irm the binding specif icity cf this protein to
fibrinogen, periplasmic extracts which contalned fewer
ccntaminating bands were harvested frcm 4 L cf E. coli cells
expressing the protein of clone 14 and applied to an affinity
column with f ibrinogen linked beads . The cloned proteins of
interest, as analyzed by silver stain and Western blots with 125I
fibrinogen and fibrinogen/anti-fibrinogen conjugate, was found in
precolumn fractions and the 3M potassium thiocyanate eluant.
However, they were not found in the fall-through, P~S with 0.5 N
NaCl eluant, nor in the acid eluant (glycine pH 3.0) Fig. 2).
Although the protein was not purif ied to homogeneity in this one
step pLO~,~dL~ (Fig. 2), these results clearly indicate the
binding specificity of this protein to fibrinogen.
Secuence analysis of the fibrinogen reactive protein
The complete sequence of the f ibrinogen protein expressed by
clone 3 o is shown in Fig . 3 . The sequence revealed an open
reading frame of 1,935 nucleotides. The seguence has a
guanosine-cytosine (GC) content of 34.7%, in contrast to the 30%
GC content in the staphyl~cq~ genome (10). The higher GC
content is attributable to the carboxy terminal half of the
--14--
W0 951346C~ F~ 00
--ler~1P (39.7%). Putative transcription and translation signals
and rih^sr--l binding sites are indicated in Fig. 3. The first
26 a;aino acids have features characteristic of a bacterial signal
peptide (16). Based on the predicted cleavage site, the mature
protein has a predicated size of 69,9gl Da with a deduced pI of
6.5 .
.
Analysis of the deduced amino acid sequence revealed three
distinct domains in this protein. With the exception of residues
27-58, the N-tpr~n;n~l half (residues 58-325) of the protein is
primarily helical as predicted by the Garnier analysis (15).
Two areas (residues 58-lg4 and 264-294) within the helical
portion of the molecule reveal a 7 residue periodicity in which
residue in positions 'a' and d' in a heptad motif 'abcdefg' are
either ~ly~,~hobic or nonpolar (Fig. 4). This finding is
suggestive of a stable coiled-coil conformational structure in
these areas (14.22). The second domain between residues 326 and
505 denotes a proline and glycine rich region (20~). Of the 180
residues present, there are 17 proline and 19 glycine residues.
This contrasts with the N-~Pnm;nAl portion of the molecule in
which only 3~6 of the residues are either proline or glycine while
the r^-~;n;nr carboxyl portion reveals a composition of 14~
proline/glycine residues. The carboxyl-terminal domain (residues
506-645) consists of S tandem, direct repeats of 27 amino acids
each followed by 5 tP~m;nAl amino acids (PRVTK). Div~ ce is
observed mainly in the outside repeats. Conformational analysis
indicated that this repeat region is nrnllP1 ir~1 and contains
mostly beta-sheets. Comparison of the protein sequence with
others in the Gen~ank dataoase revealed significant homology to
three p~ l; ChP~ 5 . aureus coagulases from 5 . aureus strains
8325-4, BB and 213 (18,19,26). With the exception of residue 7
--15--
W0 9513~6~a 2 1 9 2 1 7 ~ P~ on
.
in the primary translaticn product of the fibrinogen reactive
prctein, the N-tP7~inA~ 33 amino acid residue which include the
leader peptides among all four se~uencss were i~pnt;rAl and
therefore are likely to possess j~Pntir~1 sequence cleavage sites
(see Fig. 3). Comparing residues 1 to 422 in the fibrinogen
reactiYe protein to coA~llA~P~ in strains 8325-4, BB and 2~3,
there are 56.2%, 73.2% and 56.2% identity, respectively. The
identity between residues 423 and 645 comprising the five
repeated units to homologous regions in the coagulase sPrll~PnrDc
increased markedly to 93.9~. 95.1% and 96.9% for strains 8325-4,
BB and 213, respectively (Fig. 5). LiXe that of fibrinogen-
reactive protein, the C-termini of coagulase sequences of strains
8325-4, BB and 213 are -s~d of repeating units of 27
homologous, but no irlPn~icAl, amino acids followed by the
tPrm~nAl sequence PRVT~ (Fig. 6). ~Iowever, the number of
repeating units dif f er among strains . Although the f ibrinogen
reactive protein sequence displayed features that are common to
the coagulase sequence, a careful comparison revealed a unique
stretch of 11 amino acids between residues 409 and 419
(S~ITLPSITGES) in the middle of the proline/glycine rich region
(Fig. 5). Of interest is the fact that the motif LPSITGES shares
homology with a cell ~all anchor motif ~r PXTGX) found in other
gram positive surface protein (12,28). ~owever, there is no
complete identity to this heptad motif among se~uences in the
Genbank database.
Based on all of the foregoing, it is clear that a novel
f ibrinogen reactive protein of S . aureus has been cloned. This
protein is both structurally and functionally different from
other apparently similar proteins such as the coagulases. The
protein and the gene which expresses it are illustrated in Fig.
--16--
W0 9s/3~6~ 219 2 ~ 7 5 F~l~u~ oO
3. The ~igure shows the complete clone 36 and the protein it
expresses. Clone 14 runs from nucleotide 6a4 to nucleotide 1935
in Fiq. 3. The protein expressed ~y clone 14 runs from amino
acid residue 229 to 645. Clone 30 is substantially the same as
clone 14 and expresses substantially the same protein.
Sequence analysis clearly indicates that the fibrinogen-
reactive proteins of this invention shares ~ign ~ f; cAnt homology
with staphylococcal coagulases. Recent evidence by Boden and
Flock also suggested that the fibrinogen binding protein of S.
aureus may possess cross-reactivity with anti-coagulase antibody
(2). ~owever, several lines of evidence indicate that clone 36
expresses a unique f ibrinogen-binding protein as do clones 14 and
30. First, eYpression studies of clone 14 which expresses amino
acid residues 229 to 645 show that the ~Y~essed protein is
nPCPe:=Ary for fibrinogen binding. In contrast, ~1 AcsicAl
coagulase has been found to co~pLex with ~Lul~"U~in to from
staphylothrombin which subsequently converts f ibrinogen to f ibrin
(10,17). Secondly, there is a unique stretch of 11 amino acids
in the sequence (residues 409-419) that are not found in any of
coagulase sequence described. Third, this unique amino acid
sequence shares homology with a cell wall anchor motif (LPXTGX)
that is found to be nPcPs~ Ary for anchoring in a variety of gram
positive surface proteins (13,28). Based on these findings, it
would appear that the f ibrinogen reactive protein may belong to a
~amily of coagulase-like proteins, yet it is both structurally
and functionally distinct from any of the cqa~lA~D~ previously
describ~d .
--17--
W0 95~346aa 219 21~ ~ r~ oo
.
The isolation cf fibrinogen binding protein from
staphyl"rocr~1 whcle CPll lysates with conv~n~i~,n~l
chromatographic methcds has been reported in two studies (11,31).
}Iowever, the molecular weight (420 vs 62 kD) and the amino acid
composition differs widely between the two studies. In c~
to the 62 kD protein, methionine and tyrosine, ~ut not cysteine
residues, are present in the protein of this invention. The
fibrinogen reactive protein described here as e~ sced by clone
36 comprises ~)L~ ' jnltely lysine (11.2%), threonine (9.3~s) and
glutamic acid (9%) while glycine (16 . 8%), glutamic acid (15%) and
lysine (13 . 3%) were the abundant amino acids in the 62 XD protein
(31). In addition, the deduced isoelectric point (pI-6.5) of the
fibrinogen reactive protein also differs from the basic pI (about
10 . 2) of the 62XD protein.
Previous studies have revealed that the f ibrinogen binding
-nt of 5. aureus is a cell wall constituerlt because it is
absent in staphylorr,cr 11 ~ form (10) and because bacterial
clumping in the presence of fibrinogen is abolished upon whole
cell digestion with proteinases (4). In reviewing the molecular
architecture of the C-terminal region of other gram positive
surface-anchored proteins, it is evident that they contain
several conserved features (13,14). In the C-tc~m;n~l of these
proteins, a charged tail (4-7 amino acids) is usually preceded by
a highly hydrophobic membrane anchor (about 16-20 amino acids),
the hexamer LPXTGX, a proline-glycine rich domain and a C-
f~-~m;n~l repeat region (14). Clearly, the C-terminal region of
the fibrinogen reactive protein is different from the model
described. In particular, the region preceding the stop codon
lacks a charged tail and a hydrophobic membrane anchor. Instead,
the five t~r~n;n~l amino acids (PRVTK) are preceded by five
--18--
WO 9~3~16aa 21~ 2 i 7 ~ r~ oo
repeats of 27 amino acid each. In addition, the region N-
tP~;n~l to these repeats i5 a broad proline/glycine region
(residues 326-505) in the middle of which is a unique 5Dqn~-n~-e
(IPSITGE) that shares homclogy with a cell wall anchor motif
(LPXTGX) found in other gram positive surfac2 proteins. Notably,
this molecular architecture at the C-terminus is similar to those
described for E -: 1 surface protein A (35). The amino
tPrm;nAl half of E -~ 1 surface protein A, like that of
fibrinogen reactive protein, is ~-helical and is consistent with
an ~-helical coiled protein conformation. The ~ -helical region
is followed by a proline-rich domain and a repeat domain
consisting of ten 20-amino-acid repeats. In addition, it also
lacks a classic membrane anchor and a charyed tail. In contrast
to the f ibrinogen reactive protein, however, there is no LPXTGE
motif in the c-tPrm;n~1 region of 1 :: 1 surface protein A.
The novel proteins of this invention are surf ace proteins of
S. aureus. No such proteins have previously been detected,
isolated and characterized. They are pr;n~;r~lly characterized
by their ability to bind fibrinogen. The ~olecular weight of the
protein ex~,Lessed by clone 36 is about 6g,9gl Da and its
isoelective point 6 . 5 . The gene which expresses this protein
containS about lg35 nucleotides. Other characteristic features
of the protein and of s_ L-. C14 and C30 are described above.
Because of the difficulty in expressing protein from clone 36,
the preferred clone of this invention is clone 36. The preferred
f ibrinogen binding protein is the protein expressed by this
clone .
--19--
Wo gsl3~6~ 2 1 9 2 ~ 7 ~ P~ 00
.
The protein of tbis inventicn, as specifically described
herein, will be re~-o~n i 70~ by the s~cilled artisan as
re ~L~as~:nLltive of a class of protein which may dif~er amongst the
various strains of S. aureus, but will all be ~-hAract~ized as
having substantially the same number of amino acid residues, the
8ame tertiary :~LLU~,LUL~:: and binding activity. They may differ
slightly in the identity of the amino acid residues at 5p-~-; f i r
pOSitions in the protein chain . All such proteins are i n~ d
within the scope of this invention.
The genes which generate the proteins of this invention may
also dif~er slightly amongst various strains, but they all have
the common characteristic of producing a protein of this
invention .
The genes of this invention may be employed, as will be
rPro~Tn; 70~ by the sXilled artisan, to produce plasmids or other
vectors which, in turn are useful for transforming organisms such
as E. coli to produce novel strains of this bacteria which will
express the proteins of the invention.
Inhibition of the binding of S. aureus to endothelial cells
is a major factor in preventing infection. Accordingly
antibodies to proteins ~c~ssed by clone 36, its segments clone
14 and clone 30 and even smaller segments are important factors
in controlling infection. The proteins and protein s~e Ls of
this invention are therefore useful to form vaccines to inhibit
S. aureus infections of mammals, inr~ in~ humans, by
administering an amount of the selected protein which will
stiriulate the production of protective ouantities of antibodies
.
--20--
wo 9~ 16~ 2 ~ g 2 1 7 ~ oo
to limit adhesion cf S. aureus. The proteins may also be used to
produce anti ho~ c in vitro which may be employed for passiYe
; ~ation.
The proteins and polypeptide or peptide s-, L:, of this
invention may be o_tained by any of a number of known process
in~ in~ the re ~;n~nt DNA techniques described above.
Polypeptide and peptides within the scope of the invention
containing, for example from a~out 6 to 20 or more amino acid
Sr~ ~, may be synthesized be standard solid phase p~ uce~uL~:s
with appropriate amino acids using the protection, deprotection
and cleavage techniques and reagents appropriate to each specif ic
amino acid or peptide. A combination of manual and automated
(e.g., Applied Biosystem 430A) solid phase techniques can be used
to synth--ci ~e the novel peptides of this invention. Although
less convenient, classical methods~ of peptide synthesis can also
be employed. For ba~ L-,u.,d on solid phase techniques, reference
is made to Andreu, D., Merrifield, R.B., Steiner, H. and Boman,
H.G., (1983~ Proc. Natl. Acad. sci ~JSA 80, 6475-6479; Andreu, D.
~errifield, R.B., Steiner, H. ~nd Boman, El.G., (1985)
Biochemistry 24, 1683-1688; Fink, J., Boman, A., Boman, H.G., and
~errifield, R.B., tJune 1989) Int. J. Peptide Protein Res. 33,
412-421; Fink, J. Merrifield, R.B., Boman, A. and Boman, H.G.,
(1989) J. Biol. Chem. 264-6260-6267; each of ~-hich being hereby
incorporated herein by ref erence .
The products of the inYentiOn are amphoteric. They can
exist and be utilized as free bases or as pharmaceutically
acceptable metallic or acid addition salts. Suitable metallic
salts include alkali and Alki21 in~ earth metal salts, preferably
--21--
wo ss/346ss 2 19 2 1 7 ~ F~ / I00
sodiuht or potassium salts. Acid addition salts may be p~
from a wide variety of organic and inorganic acids ; n~ ; n~
mineral acids, for example citric, lactic, maleic, tartaric,
phosphoric and hydrochloric acids. These salts can be prepared
by ~ duL~s well 3cnown to those skilled in the art.
Por use as a vaccine, it is presently preferred to
ad~tinister the selected product in a rhA~-~-e~ l ly acceptable
carrier such as a bui~fer. Mice or other Dlam~als, ;nrlur~in~
huhtans, when so ; ; 7Pt?l are protected from colonization and
~-~q-lPnt infection by S. aureus.
Typically, the patient to be protected ~ill be treated with
product of the invention in an amount which is effective to
elicit a protective imhtune response. The sa~ e~-~Pd agent may be
ad~tinistered alone or in a rhA~-^ellti~Ally acceptable liquid or
solid carrier in which it ~say be qispersed, dissolved or
SIlCpPn~P~. If, for example, the patient is to be treated
inL~ clY~ uu5ly~ the peptide may be sllcpPn~p~ as a free base or
dissolved as a ~-~A l l i 1~ salt in isotonic asueous buf f er . Other
htethods of treatment and rhAnr-~ ttically acceptable carriers
will be apparent to the skilled artisan.
The proteins, polypeptides and peptides of this invention
and the genes or oligonucleotides which are employed in their
expression are useful as probes for genes and proteins. They are
also useful to raise antibodies by which specif ic strains of
streptococci can be identif ied . The sequence of nucleotides
which elicit the unique segment from position 409 to position 419
and modification of this sequence are ~CpP~ iAlly useful as
diagnostic probes to identify 5. aureus strains. The procedure
--22--
Wo 9S/3165a 2 1 9 2 t r~ u., 1 ,~ r
ls well known to the s3cilled artisan fcr identifying other
infectious organisms. It involves the prep2ration of labeled
cligonucleotides which aRe used to probe the DNA released fro
the ~ gram positive bacteria by all lysis, either
n i ~ l l y or enzymatically .
Wo gs/3~6ss ~ 1 ~ 2 ~ 7 ~ P~ C
The follcwing citatlons are mentioned in the application.
They are inccrporated by reference.
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