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Article available online at http://www.idealibrary.com on Microbial Pathogenesis 2002; 32: 105–116
PATHOGENESISMICROBIAL
doi:10.1006/mpat.2001.0485
Streptococcus sanguis secretes CD14-bindingproteins that stimulate cytokine synthesis: aclue to the pathogenesis of infective(bacterial) endocarditis?Julia Banksa,b, Stephen Poolec, Sean P. Naira, Jo Lewthwaitea,Peter Tabonaa, Rod McNabb, Michael Wilsonb, Angela Pauld &Brian Hendersona∗aCellular Microbiology Research Group and bDepartment of Microbiology, Eastman Dental Institute,University College London, 256 Gray’s Inn Road, London WC1X 8LD, U.K., cDivision ofEndocrinology, National Institute for Biological Standards and Control, South Mims, Potters Bar,Hertfordshire EN6 3QG, U.K. and dInstitute of Cancer Research, The Centre for Cell and MolecularBiology, Chester Beatty Laboratories, London, U.K.
(Received June 13, 2001; accepted in revised form November 4, 2001)
Streptococcus sanguis is the major causative organism of infective (bacterial) endocarditis but,surprisingly, almost nothing is known about how it induces endocardial inflammation. In earlierstudies we have shown that many bacteria secrete potent cytokine-inducing or -inhibiting proteins.We have therefore isolated the material secreted by S. sanguis grown on blood agar or in brothculture and have tested its ability to induce human peripheral blood monocytes to synthesize pro-inflammatory cytokines. The activation of monocytes by the secreted components of S. sanguiswas almost totally blocked by heat and trypsin treatment but not by the lipopolysaccharide-inactivating antibiotic, polymyxin B, suggesting that activity is due to secreted proteins. The activityof the secreted material was significantly reduced by anti-CD14 monoclonal antibodies suggestingthat the active protein (or proteins) was binding to the CD14/Toll-like receptor (TLR)4 complex.Fractionation of the secreted proteins by high performance liquid chromatography (HPLC) identifiedtwo proteins as being responsible for the majority of the cytokine induction: a manganese-dependent superoxide dismutase and a 190 kDa protein, which could not be sequenced, but whichwas neither CshA nor the PI/II proteins. These proteins, or the receptors to which they bind, maybe therapeutic targets and may allow the development of adjunctive therapies to prevent endocardialdamage during the often prolonged treatment of infective endocarditis with antibiotics. In addition,blocking of CD14 may have some therapeutic benefit. 2002 Elsevier Science Ltd.
Key words: Streptococcus sanguis, infective endocarditis, cytokines, inflammation, CD14.
∗Author for correspondence. E-mail: [email protected]
0882–4010/02/030105+12 $35.00/0 2002 Elsevier Science Ltd.
106 J. Banks et al.
Introduction Results
Isolation and composition of the secretedInfective endocarditis (IE) is a life-threatening materialcondition produced by the colonization of theheart valves by bacteria, particularly streptococci Saline extraction of plate-grown bacteria re-and staphylococci. The most common organism moved macromolecular material representingresponsible for IE is Streptococcus sanguis [1, 2]. 4–5% of the dry weight of the bacteria. ElectronEstimates of the incidence of IE range from 40–70 microscopic examination of thin sections of bac-per million per year [3, 4] and the incidence teria after extraction failed to show evidencecontinues to rise [4]. IE displays two unusual of membrane damage (results not shown). Theaspects. First, the major causative organism, S. protein content of the secreted material wassanguis, is a benign occupant of the mouth with assessed by three different assays. The commer-little evidence of a virulent phenotype. Second, cial Lowry and the Bio-Rad DC assays, whenthe lesion is remarkably resistant to antibiotic compared using the same batch of material, gaveprophylaxis [5]. Attention has focused on the results of 18 and 24% protein respectively. Withadhesion of S. sanguis (and other bacteria) to the Bradford assay this figure was only 2.4%.damaged endocardial tissue, and to platelets, Subsequent use of the Bio-Rad DC assay withto form the characteristic vegetations seen various batches of secreted material found thein IE, and a number of putative adhesins and protein content range from 13 to 33%. The car-platelet aggregation-inducing mechanisms bohydrate content, measured by the method ofhave been described [6]. A major omission Dubois [18] (with glucose as standard) rangedfrom our understanding of this disease is how from 3–4%. The lipid content was only a fewthe bacteria, present in the vegetations, induce percent as was the DNA content. The LPS con-endocardial inflammation and tissue damage. tent was extremely small (<0.00002%) and theUnderstanding how this bacterium causes in- slight response in this assay was probably dueflammation may open novel avenues for treat- to components of S. sanguis. The mass hetero-ment. geneity of the secreted material was assessed by
Cytokines are now recognized to be the prim- one- and two-dimensional SDS-PAGE, whichary mediators of acute and chronic inflammation revealed the presence of more than 50 proteins[7, 8], and circulating levels of pro-inflammatory with molecular masses ranging from less thancytokines are found in patients with Q-fever 14 kDa to greater than 200 kDa (Fig. 1).endocarditis [9]. It is now becoming establishedthat many bacterial components and secretedproducts are capable of stimulating cellular cy- Cytokine-inducing activity of the secretedtokine gene transcription [10, 11]. Most attention materialhas focused on Gram-negative cytokine-in-ducing components (such as LPS), but in the Exposure of human monocytes to the secretedlast decade a number of components from Gram- macromolecular material from plate-grown S.positive organisms, such as lipoteichoic acid sanguis resulted in the reproducible productionand superantigens, have also been discovered (more than eight experiments, each with a dif-to stimulate cytokine synthesis [7, 10]. ferent blood donor) of the four pro-inflammatory
We have previously demonstrated that the cytokines assayed: IL-1�, 1L-6, IL-8 and TNF�.population of secreted macromolecules from a S. sanguis grown in broth culture also secretednumber of oral bacteria implicated in the patho- material with similar cytokine-stimulating ac-logy of periodontitis (a chronic inflammatory tivity (results not shown). Activity was com-disease of the gingivae) contain proteins that pared with the effect of 10 ng/ml of E. coli LPS,possess potent biological activities such as: in- a concentration that gives a maximal response.duction of pro-inflammatory cytokine synthesis Analysis of the kinetics of cytokine synthesis[12, 13]; stimulation of bone destruction [14]; revealed that the secreted material and LPS in-and inhibition of cellular proliferation [15, 16]. duced similar rates of cytokine production thatIn this study we have examined the secreted reached a plateau after 24 h (results not shown).macromolecules produced by S. sanguis for their Cytokine production was stimulated by as littleability to stimulate human monocytes to pro- as 300 ng/ml secreted material and there was a
linear dose response between 0.3 and 300 �g/duce pro-inflammatory cytokines.
Cytokine-inducing bacterial proteins in infective endocarditis 107
kDa
49
18
(a)
kDa
66
49
(b)
1 2
Lane
29
66
14
pH 3 pH 10
29
18
Figure 1. One- and two-dimensional SDS-PAGE of the secreted proteins of S. sanguis stained with Coomassieblue. In (a) lane 1 is the molecular mass markers and lane 2 the surface-associated secreted proteins.
ml except for IL-1�, with which production of cytokine synthesis. Both the LPS- and LTA-peaked at 30 �g/ml of secreted material. The induced IL-1� and IL-6 synthesis were com-secreted material from S. sanguis was very ef- pletely inhibited by 20 �g/ml of polymyxin B.ficacious, particularly in terms of IL-8 synthesis, In contrast, the same concentration of polymyxinwhich was many-fold higher than the maximal B exhibited only a slight inhibitory effect on theLPS response (Fig. 2). Control washes from Wil- IL-6-, IL-8- and TNF�-inducing activity of thekens Chalgren agar plates on which bacteria had exported macromolecular fraction. In a numbernot been grown and TSBY medium itself both of experiments, the polymyxin B produced afailed to stimulate human monocytes to produce small, but significant, enhancement of IL-1� syn-pro-inflammatory cytokines. thesis (results not shown).
Effect of heating and proteolysis of thesecreted material on monocyte cytokine Blocking effect of anti-CD14 monoclonalsynthesis antibodies
Exposure of this fraction to heat (100°C) or to In contrast to the lack of effect of polymyxin Btrypsin had a rapid and profound effect on the on the cytokine-inducing activity of S. sanguiscytokine-inducing activity [Fig. 3(a) and (b)]. For secreted material, two monoclonal antibodiesexample, trypsinization resulted in 90% loss of known to block the binding of LPS to CD14the cytokine-inducing activity. (MY4 and 60bca) were found (at concentrations
of 7.5 or 15 �g/ml) to significantly inhibit thesynthesis of IL-6. Such blockade was not due tothe non-specific effect of murine IgG, as subclass-Effect of polymyxin B on cytokinematched monoclonal antibodies failed to showsynthesisany effect on the activity of the secreted material,although it appeared to stimulate the activity ofThe LPS-binding and inactivating antibiotic,the LPS somewhat. The effects of adding 60bcapolymyxin B, was added to monocytes stimu-on monocyte IL-6 synthesis are shown in Fig.lated with either LPS, LTA or the S. sanguis4. Antibody MY4 gave identical results (notsecreted material. Concentrations of all three
agents were chosen to give similar amounts shown).
108 J. Banks et al.
0
600
[S. sanguis EM] µg/ml
[IL
-8]
ng/
ml
500
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LPS3003030.30.030
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[IL
-1β]
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[TN
Fα]
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LPS3003030.30.030
0
30
[S. sanguis EM] µg/ml
[IL
-6]
ng/
ml
25
20
15
10
5
LPS3003030.30.030
12
14
16
6
7
8
9
Figure 2. Stimulation of human monocytes with the secreted material from S. sanguis [here termed exportedmacromolecules (EM)] and measurement of cytokines produced by two-site ELISA. The black column is theproduction of cytokines by cells stimulated with 10 ng of E. coli LPS. Results are expressed as the mean±SDof three replicate cultures from a representative experiment.
0
3000(a)
[IL
-6]
pg/m
l
Unstim
ulate
d PBM
C
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okin
e] p
g/m
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Untreat
ed E
M
Tryps
in co
ntrol
Tryps
inize
d EM
Heat-t
reat
ed E
M TNFαIL-6IL-1β* *
*
Figure 3. (a) Effect of heat treatment and trypsinization on the IL-6-inducing activity of the secreted fractionfrom S. sanguis. Trypsin by itself had no cytokine-inducing activity nor did it block LPS-induced cytokinesynthesis (results not shown). (b) Comparison of the effect of exposing the secreted fraction to heat on itsability to induce IL-1�, IL-6 or TNF�. The effect of the untreated secreted fraction (Ε), the effect of heat (Φ)and the production of cytokine by unstimulated monocytes (∆) is shown. Results are expressed as themean±SD of three replicate cultures from a representative experiment. ∗ P<0.001.
Cytokine-inducing bacterial proteins in infective endocarditis 109
0
8000
[IL
-6]
pg/m
l
7000
6000
5000
4000
3000
2000
1000
controlmAb
60 bcaLPS +controlmAb
LPS +60 bca
EM +controlmAb
EM +60 bca
**
**
****
**
Figure 4. Effect of adding the neutralizing anti-CD14 monoclonal antibody (mAb), 60bca, or a subclass-matched monoclonal, to cultured human monocytes stimulated by S. sanguis secreted material or by LPS.The release of IL-6 has been assayed in this experiment. Cytokine production induced by EM or LPS (Φ),the effect of adding 7.5 �g/ml antibody (∆) and the effect of adding 15 �g/ml antibody (Ε) is shown.Results are expressed as the mean±SD of three replicate cultures. ∗ P<0.05; ∗∗ P<0.01.
Isolation of cytokine-inducing proteins it was not possible to obtain sequence in-formation. S. sanguis is known to express severalhigh molecular mass surface proteins relatedThe secreted proteins from bacteria grown on
agar or in liquid culture have been chro- to the CshA and PI/II proteins (or SspA) ofStreptococcus gordonii. The latter has been re-matographically fractionated and cytokine-
stimulating molecules of the same apparent mo- ported to have weak cytokine-inducing activity[22]. It was possible that this high molecularlecular mass were found. Most of the analysis of
these proteins has utilized plate-grown bacteria. mass protein was either CshA or the PI/II (SspA)proteins. Using antisera against both proteins,Fractionation of the whole secreted macro-
molecular fraction on an MA7Q anion-exchange we found that the PI/II antiserum recognized aprotein of 178 kDa in a mutanolysin extract of S.column eluted with a gradient of 0–1.0 M NaCl
revealed numerous peaks absorbing at OD280 nm sanguis and a protein of 157 kDa in mutanolysinextracts of S. gordonii. The CshA antiserum de-and four peaks of IL-6-stimulating activity (Fig.
5). The two major peaks of cytokine-inducing tected a protein of 212 kDa in both S. sanguis andS. gordonii extracts (results not shown). However,activity, termed active peak 1 (fractions 15 and
16) and 2 (fractions 19 and 20), were further neither antiserum bound to the 190 kDa proteinpresent in the secreted proteins.fractionated. Active peak 1 was re-fractionated
on a MiniQ anion-exchange column using a Active peak 2 was also re-fractionated using aMiniQ anion-exchange column with a shallowershallower gradient (0–0.5 M NaCl) and three
proteins were identified in the most active frac- salt elution gradient (0–0.5 M). The cytokine-inducing activity co-eluted with a 23 kDa proteintion. There was a protein doublet of molecular
mass 190 kDa, a slightly smaller band running (Fig. 7), which was sequenced by Edman de-gradation. The N-terminal sequence (AILLP-just beneath and a small amount of a 54 kDa
protein. The larger protein doublet was sep- DLPYAYDANEPYI) was compared to sequencesin GenBank, using the NCBI BLAST utility, andarated by gel-filtration chromatography on a
Sephadex 75 column from the 54 kDa protein was found to be 88% identical to a manganese-dependent superoxide dismutase of Strepto-and all fractions were tested for IL-6-inducing
activity. IL-6-stimulating activity was found ex- coccus mutans. The 60 kDa protein, and otherless abundant proteins, which co-eluted in thisclusively in one fraction eluted in the void vol-
ume of the column. The only protein present in fraction was also present in adjacent, inactivefractions (not shown).this fraction was the 190 kDa doublet (Fig. 6). A
number of attempts were made to identify this The late-eluting, weakly bioactive peaks 3and 4 had very low absorbances at OD280 nm andlarge protein by Edman sequencing. However,
110 J. Banks et al.
065
1
Volume (ml)
(a)A
bsor
ban
ce 2
80 n
m
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-6]
pg/m
l
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66
49
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)
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Active peak 1(Fr 15/16)
Active peak 2(Fr 19/20)
Active peak 3
Active peak 4
(b) Fraction
M 10 11 12 13 14 15 16 17 18 198 9 20 21 36 37 38 39 40 41 46 47 48 49 50
Figure 5. Initial purification scheme of the secreted material from S. sanguis on an MA7Q anion-exchangecolumn. The top panel shows the elution profile [OD280 nm ()] and the IL-6 inducing activity (…) of thefractions. The NaCl gradient is shown by h. This reveals four separate peaks of cytokine-inducing activity.The lower panel shows the molecular mass of the proteins in the various 1 ml fractions (beginning at fraction8) that have been collected and assayed for cytokine-inducing activity. The SDS-PAGE gel is stained withsilver. M: represents the column containing the molecular mass markers.
showed up on silver-stained gels as large smears. in IE [4]. There is also growing evidence thatActive peak 3 was associated with material of these organisms are a particular problem inmolecular mass in the range 18–24 kDa while immunocompromised patients who have ra-active peak 4 was associated with material of diation- or chemotherapy-induced neutropaeniamolecular mass greater than 100 kDa. The exact and that they can induce a shock-like state re-nature of these proteins is unclear and their sembling Gram-negative septic shock [23]. Theisolation was not attempted [Fig. 5(b)]. pathology of chronic inflammatory conditions
such as IE and the acute shock-like state is largelydependent on the induction of the synthesisof pro-inflammatory cytokines. However, veryDiscussion little is known about the ability of oral viridansstreptococci to induce cytokine synthesis. It hasbeen shown that a variety of strains of oralThe oral viridans streptococci, of which S. san-viridans streptococci can induce human mono-guis is a member, are part of the commensalcytes to produce cytokines [24] but the onlymicrobiota of the mouth and generally do notidentified components of these organisms re-cause local pathology. These organisms have
been recognized for many years to be important ported to stimulate cytokine synthesis are a
Cytokine-inducing bacterial proteins in infective endocarditis 111
2.5
0.03
Volume (ml)
Abs
orba
nce
280
nm
0
6000
[IL
-6]
pg/m
l
0.025 kDa
49
190 kDadoublet0.02
0.015
0.005
0.01
0
5000
4000
3000
2000
1000
Fraction 7
(b)(a)
29
66
Fraction 7
21.510.5
Figure 6. Isolation of a high molecular mass cytokine-inducing protein. (a) Shows the elution profile of theIL-6-stimulating activity (…) on a Superdex 75 gel filtration column of the most active fraction of activepeak 1 (Fig. 5) after it had been further separated on a MiniQ anion-exchange column with a shallowerNaCl gradient. All the other fractions were inactive. In (b) the protein in fraction 7 has been run on SDS-PAGE and silver-stained and only one high molecular mass (doublet) protein is visible.
9
3
Volume (ml)
Abs
orba
nce
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nm
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-6]
pg/m
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kDa
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60 kDa protein
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66
Fraction 23
876
23 kDa protein(unique to thisactive fraction)
0
0.5
NaC
l (M
)
0.25
05
24
20
45
36
Figure 7. Further fractionation of active peak 2 (see Fig. 5) on a MiniQ anion-exchange column over ashallower salt gradient [0–0.5 M (h)]. The elution profile () and the IL-6-inducing activity (…) have beensuperimposed, with all the IL-6-inducing activity eluting in fraction 23. The composition of this fraction isshown in (b).
partially-purified putative superantigenic frac- are trapped on the bacterium’s surface [12, 13].These consist of the population of secreted pro-tion of Streptococcus mitis [25] and protein I/II
[23]. teins that, because they cannot leach into themedium, concentrate on the bacterium’s surface.We have shown in previous studies that bac-
teria grown on agar secrete a large number of This surface-associated fraction from most of thebacteria we have studied is able to stimulatemolecules and that many, if not most, of them
112 J. Banks et al.
human cells to produce cytokines [12, 13]. We more sensible to compare the activity of thesesecreted proteins to the Gram-positive equi-therefore proposed the hypothesis that the se-
creted macromolecules of S. sanguis contained valent of LPS, such as LTA. When a commercialpreparation of purified LTA from S. sanguis wasproteins with the capacity to induce pro-in-
flammatory cytokine synthesis. compared, it demonstrated maximal cytokine-inducing activity at between 10 and 100 �g/ml,Gentle extraction of S. sanguis grown on blood
agar with saline released a substantial amount of which is in the same range as the activity of thenon-purified secreted material from this bac-material (the secreted macromolecular fraction)
that contained more than 50 proteins or protein terium.Exposure of the S. sanguis secreted macro-subunits with a molecular mass range from 14
to >100 kDa. Bacteria grown in liquid culture in molecular fraction to heat or trypsin causedalmost complete inhibition of activity, stronglyTSBY medium also produced a similar number
of proteins. Curiously, measurement of the pro- suggesting that the active component(s) wasproteinaceous and not peptidoglycan or LTAtein, carbohydrate, lipid and DNA content of
this secreted fraction failed to account for at which, like LPS, are extremely stable to heatingand not affected by trypsin. Assay for LPS con-least half of this material. Measurement of the
protein content by three commonly used protein tamination revealed, as expected, extremely lowlevels and this was substantiated by the findingassays gave markedly different results (ranging
from 2 to 24% w/w) and the maximum result that polymyxin B, while blocking the cytokine-inducing actions of LPS and LTA, failed to inhibitobtained was only 33% protein. The most likely
explanation for this underestimate is differences the activity of the exported macromolecular frac-tion. Indeed, polymyxin B stimulated the IL-in the content of amino acids, such as tyrosine
and tryptophan, in these secreted bacterial pro- 1�-inducing activity of this exported material,suggesting the possibility that it might harbourteins compared to the proteins used as a stand-
ard [13]. The carbohydrate assay used, while a an IL-1� inhibitor, which is blocked by poly-myxin B. It was therefore surprising to findstandard assay, only identifies certain sugars
and thus may underestimate the poly- and oligo- that the activity of the exported macromolecularfraction could be significantly inhibited by anti-saccharide content. The Limulus assay for LPS,
which will also pick up components such as bodies that block the activity of CD14 ligandssuch as LPS and LTA [26]. This was a specificpeptidoglycan, showed little response to this
surface-associated material. effect of the anti-CD14 monoclonal antibodies, asirrelevant class/subclass matched monoclonalsHuman monocytes were exposed to graded
concentrations of this macromolecular secreted had no blocking action. To date, CD14-bindingligands have been almost exclusively non-pro-fraction and the production of four major pro-
inflammatory cytokines was assayed by two-site teinaceous [7]. However, there are a growingnumber of examples of proteins, such as chap-immunosorbent assays. Activity of the Gram-
positive surface fraction was compared with eronin 60 proteins [27], and lipoproteins [28]stimulating cells by binding to CD14.10 ng/ml of a very potent LPS from E. coli. The
exported macromolecular fraction was active Fractionation of the mixture of surface-as-sociated secreted material by anion exchangeat sub-microgram to microgram per millilitre
concentrations and could induce the synthesis chromatography revealed the presence of atleast four physicochemically-distinct cytokine-in-of between 5 and 25 ng of IL-1�, IL-6 or TNF�
per 106 cells in a 16 h period. This was roughly ducing fractions. The two most active peaks werefractionated further. The first peak of activity wassimilar to the maximum activity of LPS. In con-
trast, monocytes exposed to the bacterial surface purified to homogeneity and was found to be aprotein of 190 kDa running as a doublet. Thisfraction were induced to produce around
400–600 ng of the chemokine, IL-8, per 106 cells protein proved to be extremely sticky and majorlosses occurred throughout purification resultingover a 16 h period. In this respect, the exported
macromolecular fraction was very much more in insufficient material being produced for se-quencing by Edman degradation. However, usingefficacious than LPS. The LPS used in these
studies was the hexa-acylated molecule from E. monospecific antibodies recognizing either pro-teins I/II (SspA), a family of structurally-relatedcoli, which is known to be an extremely active
inducer of pro-inflammatory cytokines [7] and proteins [29] with the ability to induce cytokinesynthesis [22], or CshA/CshB [30], it was possiblewas very much more potent than the secreted
macromolecules of S. sanguis. Of course it is to discriminate this S. sanguis cytokine-inducing
Cytokine-inducing bacterial proteins in infective endocarditis 113
protein from these two known families of pro- binding to the cytokine-inducing S. sanguis pro-teins. The antagonism of these receptors shouldteins. Fractionation of the second peak of cy-
tokine-inducing activity revealed that the activity block local inflammation. The anti-CD14 mono-clonal antibody experiments suggest that CD14corresponded to a protein of 23 kDa. It was pos-
sible to obtain enough of this protein for se- may play a key role in the process of cell activationproduced by these S. sanguis proteins. Thus,quencing and the N-terminal sequence revealed
that this protein had very high identity with the blockade of CD14, for example by antibodies,or even by administering exogenous CD14 [33],manganese-dependent superoxide dismutase of
S. sanguis and Streptococcus pneumoniae. may help inhibit inflammation and reducemortality.The nature of the molecules in two minor
cytokine-inducing peaks remain to be identified.As proteolysis destroyed more than 90% of thecytokine-inducing activity of the secreted com-
Materials and Methodsponents it is assumed that these bioactive, lowOD280 nm, peaks are proteins. Their staining pattern
Growth of bacteriaon gels suggest that they have a range of mo-lecular masses and thus they may be bacterial
S. sanguis NCTC 10904 was cultured at 37°Cproteins with substantial degrees of post-trans-under anaerobic conditions on Wilkins Chalgrenlational modification, such as glycosylation. It isagar (Oxoid, Basingstoke, Hampshire, U.K.) sup-possible that it is these proteins, lacking in tyro-plemented with 5% (v/v) defibrinated horsesine and tryptophan, that account for the lowblood (Oxoid). Bacteria were grown for 48 h,values recorded in the protein assays. S. sanguisharvested from the plate with 0.15 M NaCl atproduces a number of pro-inflammatory cy-4°C and pelleted by centrifugation at 8000×gtokine-inducing components, the most active offor 30 min. The pelleted bacteria were lyophil-which appear to be proteins. It was notable thatized and stored at −70°C. Bacteria were alsothese secreted proteins had a particular pro-grown in liquid culture in tryptone soya brothpensity to induce formation of the CXC neutro-(Oxoid) supplemented with 5 g/l yeast extractphil chemokine, IL-8. The ability of these proteins(Oxoid) for 16 h. After this time the bacteriato induce such cytokine synthesis is surprisingwere pelleted by centrifugation at 10 000×g forgiven the fact that this organism is normally a30 min and the culture supernatant was re-cent-harmless oral commensal. Of course such pro-rifuged at 10 000×g and then filtered through ateins may not be produced in the oral cavity and0.45 �m filter and retained.their production in culture may be due to specific
environmental signals. The environmental con-trol of oral viridans streptococcal genes is onlynow being explored [31, 32]. The latter paper Extraction of secreted macromoleculesreports on the genes induced when bacteria areswitched from pH 6.2 (proposed salivary pH) to Secreted macromolecules associated with the
bacteria surface were isolated by a modificationpH 7.3 (blood pH). This resulted in the up-regulation of at least five genes, one of which of the method of Wilson et al. [17]. Briefly, bac-
teria were resuspended in 0.15 M NaCl andwas identified as peptide methionine sulphoxidereductase, an enzyme, like manganese-dependent gently stirred for 3 h at 4°C. Bacteria were cent-
rifuged at 8000×g for 30 min and the super-superoxide dismutase, involved in defenceagainst oxidation [32]. It is conceivable that the natant re-centrifuged to remove any particulate
matter before being vacuum-filtered through aconditions in the mouth inhibit the productionof these cytokine-inducing proteins. However, 0.45 �m polyvinylidene difluoride (PVDF) filter
(Millipore, Watford, U.K.). The supernatant, con-when this organism interacts with the endo-cardium the conditions are different and favour taining secreted macromolecules, was dialyzed
using dialysis tubing (Medicell Internationalthe transcription of the genes encoding cytokine-modulating proteins and thus promote tissue Limited, U.K.) with a molecular mass cut-off of
3.5 kDa against 15 changes of distilled water atpathology.The findings presented in this paper suggest 4°C over 5 days, and was then lyophilized. The
extent of removal of surface-associated exportedtwo additional therapeutic modalities for thetreatment of IE, to be used in conjunction with proteins and the integrity of the bacterial cell
membranes after such removal was assessed byantibiotics. The first is to target the host receptors
114 J. Banks et al.
transmission electron microscopy. Protein in the Cell preparation, culture and activationmedium supporting liquid-grown bacteria was
Human monocytes were prepared from buffyprecipitated by adding ammonium sulphate tocoat blood residue (obtained from the Blooda concentration of 85% (this concentration ofTransfusion Centre, Colindale, London, U.K.)salt produced the maximal yield of protein).by Ficoll Histopaque (Sigma) density gradientPrecipitates were harvested by centrifugationcentrifugation [21]. Cells were seeded at 2×106and dissolved in pyrogen-free water and di-cells/ml in 24 well plates and incubated for 1 halyzed extensively as described above. The pro-at 37°C, and then washed with PBS to removetein concentration of the secreted material wasnon-adherent cells. Medium (1 ml) containingdetermined using the commercial Lowry assayeither no additive or various concentrations of(Bio-Rad, Hemel Hempstead, Hertfordshire,the bacterial secreted material was then addedU.K.), the Bio-Rad detergent-compatible proteinto cells and they were cultured for a further 16 hassay (Bio-Rad) and by Bradford assay. Theat 37°C in an atmosphere of 5% CO2/95% air.carbohydrate content was measured by theAfter this incubation period, the medium sup-method of Dubois et al. [18] using glucose as aporting the cells was removed and stored atstandard. The lipid content was crudely de-−70°C for subsequent assay for cytokines.termined by methanol:chloroform extraction fol-
The LPS-binding and inactivating antibiotic,lowed by drying and weighing of the extractedpolymyxin B (Sigma), was used (at a con-material. DNA contamination was assessed bycentration of 10–20 �g/ml in different ex-measurement of absorption at 260 nm. Theperiments) to determine if there was LPSheterogeneity and molecular mass of the pro-contamination in the secreted material [21]. Toteins isolated were determined by SDS-PAGEaddress the same question, use was made ofusing one-dimensional [19] or two-dimensionalmonoclonal antibodies, which bind to the prim-[20] gels stained using a silver stain kit (Pierce,ary LPS receptor, CD14. Antibody MY4 wasTattenhall, Cheshire, U.K.). The endotoxin con-obtained from Beckman Coulter (U.K.). Anti-tent of the extracted material was measured bybody 60bca was prepared in-house from thecommercial Limulus amoebocyte lysate (LAL)hybridoma cell line HB-247 (ATCC) and purifiedassay (Associates of Cape Cod, Liverpool, U.K.)on a protein A column. Antibodies were usedaccording to the manufacturers instructions. Theat concentrations of either 7.5 or 15 �g/ml inbiological activity of the secreted macro-various experiments. Escherichia coli LPS wasmolecular material was tested by adding it toobtained from Difco (U.K.) and S. sanguis li-cultured human monocytes.poteichoic acid (LTA) was obtained from Sigma.Both the polymyxin B and the antibodies wereadded to cells just prior to addition of the se-creted material.
Heating and proteolysis of secretedmaterial Cytokine ELISA
Two site immunoassays for IL-1�, IL-6, IL-8 andTo determine if the biological activity of the TNF� were conducted as described [21].secreted macromolecules was due to the pres-ence of proteins, it was dissolved in 50 mM TrispH 7.6 (Gibco, Paisley, U.K.) to a concentration Fractionation of secreted materialof 1 mg/ml (w/v) and aliquots were heated ina boiling water bath for 30 min. For trypsin Secreted molecules from plate-grown and brothdigestion, sterile sequencing grade trypsin culture-grown bacteria were fractionated by(Sigma, Dorset, U.K.) was added to give a final anion-exchange and size-exclusion chromato-concentration of 2.5 �g/ml. Samples were di- graphy. Anion-exchange chromatography wasgested for 5 h and the trypsin inactivated by performed in the first instance using a Bio-Radaddition of phenylmethylsulphonyl fluoride HRLC system equipped with an MA7Q column(PMSF) to 1 mM. Control digestion mixtures (5×0.78 cm). The column was equilibrated withcontained no bacterial extract, or contained bac- five column volumes of buffer A (20 mM Tris–terial extract but not trypsin, but were otherwise HCl, pH 8.0) at a flow rate of 1 ml/min. Aliquotstreated in an identical fashion. All samples were (1 ml) of the secreted material, dissolved at
4 mg/ml in buffer A, were loaded onto theassayed for cytokine-inducing activity.
Cytokine-inducing bacterial proteins in infective endocarditis 115
9 Capo C, Zugun F, Stein A, et al. Upregulation of tumorcolumn which was washed with five columnnecrosis factor alpha and interleukin-1 beta in Q fevervolumes of buffer A and eluted with a gradientendocarditis. Infect Immun 1996; 64: 1638–42.of 0–100% buffer B (buffer A+1 M NaCl) 10 Henderson B, Poole S, Wilson M. Bacterial modulins: a
over 60 ml. The absorbance of the eluate novel class of virulence factors which cause host tissuewas monitored at 280 nm and 1 ml fractions col- pathology by inducing cytokine synthesis. Microbiol Rev
1996; 60: 316–41.lected. Separation on a MiniQ anion-exchange11 Wilson M, Seymour R, Henderson B. Bacterial per-chromatography column (0.24 ml bed volume)
turbation of cytokine networks. Infect Immun 1998; 66:was performed on a Pharmacia SMART system 2401–9.(Pharmacia, Peapack, NJ, U.S.A.). Protein was 12 Reddi K, Wilson M, Poole S, Meghji S, Hendersonloaded onto a column pre-equilibrated in buffer B. Relative cytokine-stimulating activities of surface
components of the oral periodontopathic bacteriumA at a flow rate of 250 �l/min. The column wasActinobacillus actinomycetemcomitans. Cytokine 1995; 7:washed with 10 column volumes of buffer A at534–41.a flow rate of 400 �l/min. Protein was eluted
13 Reddi K, Wilson M, Nair S, Poole S, Henderson B.from the column using a gradient of 0–50% Comparison of the pro-inflammatory cytokine-stimu-buffer B over 20 column volumes. The ab- lating activity of the surface-associated proteins of peri-sorbance at 280 nm was monitored and 100 �l odontopathic bacteria. J Periodont Res 1996; 31: 120–30.
14 Kirby AC, Meghji S, Nair SP, et al. The potentfractions collected. Gel-filtration chromato-bone resorbing mediator of Actinobacillus actino-graphy was performed using a Superdex 75mycetemcomitans is homologous to the molecular ch-column attached to a Pharmacia SMART aperone GroEL. J Clin Invest 1995; 96: 1185–94.
system (Pharmacia). The Superdex 75 column 15 White PA, Wilson M, Nair SP, Kirby AC, Reddi K,(2.4 ml bed volume) was pre-equilibrated in PBS. Henderson B. Characterization of an antiproliferative
surface-associated protein from Actinobacillus ac-Fractionation was conducted at a flow rate oftinomycetemcomitans. Infect Immun 1995; 63: 2612–8.40 �l/min and 50 �l fractions collected. The ab-
16 White PA, Patel M, Nair S, et al. Control of human cellsorbance of the eluant was monitored at 280 nm.cycle by a bacterial protein, gapstatin. Eur J Cell Biol1998; 77: 228–38.
17 Wilson M, Kamin S, Harvey W. Bone resorbing activityof purified capsular material from Actinobacillus ac-tinomycetemcomitans. J Periodont Res 1985; 20: 484–91.Acknowledgements
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