Department of Cell and Molecular Biology, Section for Clinical and Experimental Infectious Medicine, BMC, B14, Lund University, S-221 84 Lund, Sweden
Correspondence
Inga-Maria Frick
inga-maria.frick{at}medkem.lu.se
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY600861.
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INTRODUCTION |
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The best-characterized surface protein produced by GCS and GGS is protein G, which has affinity for the constant region of immunoglobulin G (IgGFc) and albumin (Björck et al., 1987; Björck & Kronvall, 1984
; Reis et al., 1984
). Protein G also interacts with
2-macroglobulin (
2-M) and high-molecular-weight (H-) kininogen (Sjöbring et al., 1989
). The role of protein G in virulence is not known, but it has been suggested that the binding of host proteins may aid in evasion of host defence mechanisms. Protein G also acts mitogenically on peripheral blood lymphocytes (Otten & Boyle, 1991
). It has also been proposed that protein G could function as an environmental sensor ensuring an appropriately adapted gene expression (Cleary & Retnoningrum, 1994
). The protein-G-related MIG protein of Streptococcus dysgalactiae, an organism which causes bovine mastitis, was recently shown to prevent phagocytosis by bovine neutrophils, most likely through an interaction with IgG or
2-M (Song et al., 2001
); however, it is not known whether protein G expressed on human isolates of GCS and GGS has a similar function. In addition to protein G, a number of strains of GCS and GGS express M or M-like proteins that are similar in structure and function to those described for GAS (Campo et al., 1995
; Collins et al., 1992
; Schnitzler et al., 1995
).
M proteins are cell wall attached, elongated molecules forming -helical coiled-coil dimers. Their N-terminal parts are highly variable, whereas the C-terminal regions are more conserved. M proteins have affinity for numerous plasma proteins, such as fibrinogen, albumin, IgG and complement factors (for references, see Fischetti, 1989
). The M protein is traditionally regarded as a major virulence factor, primarily through its ability to promote bacterial survival in human blood. Specific interactions with fibrinogen and regulatory proteins of complement have been reported as important mechanisms by which M protein of GAS exerts its antiphagocytic effect (Horstmann et al., 1988
; Perez-Casal et al., 1995
; Ringdahl et al., 2000
; Thern et al., 1995
; Whitnack & Beachey, 1982
). Possible proposed mechanisms are the circumvention of opsonization by C3 at the bacterial surface, or the hindering of binding to granulocytes (Podbielski et al., 1996
). Although M proteins of GCS and GGS have also been reported to confer resistance of these bacteria to phagocytosis, it is not known whether these bacteria utilize mechanisms identical to those described for GAS to achieve survival in human blood.
In the present work, the contribution of protein G to survival of GGS in human blood was investigated. Surprisingly, a clinical isolate expressing protein G was found to be rapidly killed. Instead, the experiments performed identified a self-associating M-like protein, denoted FOG for fibrinogen binding protein of G streptococci, as crucial for bacterial survival.
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METHODS |
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CNBr extraction and papain digestion.
CNBr chemically cleaves proteins on the carboxyl side of methionine residues. Bacterial surface proteins were extracted as described by Otten & Boyle (1991). In brief, bacterial cells were collected by centrifugation at 1400 g for 10 min, and resuspended in 2 ml PBS (g bacteria)1. An equal volume of CNBr solution (30 mg per ml of 0·2 M HCl) was added, and the sample was rotated overnight at room temperature, followed by centrifugation at 12 900 g for 15 min. The supernatant was filter-sterilized to remove remaining bacteria, dialysed against 0·1 M HCl for removal of excess CNBr, and, finally, the pH was raised to 7·5 by the addition of 1 M Tris. Released material was then subjected to SDS-PAGE and Western blotting. G148 bacteria were digested with papain, as described by Björck & Kronvall (1984)
, and analysed for binding of IgG.
SDS-PAGE, ligand blotting and N-terminal amino acid sequencing.
SDS-PAGE was performed using the buffer system described by Laemmli (1970). Separated proteins were stained with Coomassie brilliant blue, or transferred onto PVDF membranes (Amersham Biosciences) using the Bio-Rad TransBlot SemiDry Transfer Cell System. Membranes were washed with blocking buffer (PBS containing 0·25 % Tween 20, 0·25 % gelatin) four times for 20 min, and then incubated with radiolabelled protein for 3 h at room temperature, or overnight at 4 °C. The membranes were subsequently washed four times with blocking buffer. Bound ligand was measured using the Fuji Imaging System, after an exposure of 3 h minimum. For detection of protein G in CNBr-released material, membranes were blocked in PBS containing 0·05 % Tween 20 (PBST) and 5 % dry milk powder (blocking buffer), incubated with antibodies against protein G (see next section; 1 : 1000) in blocking buffer for 30 min at 37 °C, washed with PBST, and incubated with horseradish-peroxidase-conjugated protein A (1 : 5000; Sigma) for 30 min at 37 °C. The membranes were washed, and bound antibodies were detected by the chemiluminescence method (Nesbitt & Horton, 1992
). Proteins were also directly applied onto PVDF membranes using a Milliblot-D System. Membranes were incubated with radiolabelled probe as described above. Samples subjected to N-terminal amino acid sequencing were separated by 10 % SDS-PAGE, and transferred onto a PVDF membrane as described by Matsudaira (1987)
. Membranes were stained with 0·1 % Coomassie blue R-250 in 50 % methanol, bands of interest were excised, and sequence analysis was performed at Eurosequence (Meditech Center, Groningen, The Netherlands).
Reagents, labelling and binding assays.
Human serum albumin (HSA), human fibrinogen and polyclonal human IgG were purchased from Sigma. Human factor H was kindly provided by Dr L. Truedsson, Lund University, Sweden. Protein PAB from Finegoldia magna was purified as described by de Château & Björck (1994), and human
2-M was kindly provided by Dr H.-P. Müller, Lund University, Sweden. GAS M1 protein was purified as described by Åkesson et al. (1994)
. Recombinant protein G containing the IgGFc-binding regions was obtained from Sigma, while protein G containing both the IgGFc- and the albumin-binding regions was purified as described by Björck et al. (1987)
. Antiserum against the N-terminal albumin-binding region of protein G was raised in rabbits. HSA was labelled with 125I using the Bolton and Hunter reagent (Amersham). Fibrinogen, IgG and factor H were radiolabelled using the Chloramine T method as described by Greenwood et al. (1963)
, and purified protein FOG fragments were labelled using Iodobeads (Pierce). Overnight cultures, and bacteria subjected to CNBr extraction or papain digestion, were harvested by centrifugation at 1400 g for 10 min, and washed with PBST. 125I-labelled proteins were bound to bacterial cells, as described by Björck & Kronvall (1984)
.
Bactericidal assay, analysis of blood aggregates and blood exchange experiments.
Bacteria were grown to early mid-exponential phase (OD620 0·15) and diluted to approximately 1·5x103 cells ml1 in TH. A 100 µl bacterial suspension was then added to 1 ml human heparinized whole blood or 1 ml human plasma, and incubated with gentle rotation at 37 °C. Aliquots were taken at different time points, mixed with 2·5 ml 0·5 % agar, plated onto THA, and incubated at 37 °C overnight. The number of c.f.u. was counted, and the multiplication factor was calculated by dividing the number of c.f.u. by the initial number of c.f.u. In protection assays, 100 µl G148 bacterial suspension was mixed with soluble proteins, prior to incubation with blood. Samples were taken and plated as above. For cocultivation experiments, G41 was heat inactivated at 80 °C for 10 min, and then 2·5x104 cells were mixed with 100 µl G148 (1·5x103 cells ml1), and the mixture was added to 1 ml whole blood. Samples were taken and plated directly on THA. From the 5 h sample, single colonies were further cultivated in TH for binding analysis and PCR. Heparinized blood was used in all experiments. After 1 h incubation of whole blood and protein FOG, when aggregates were seen, the blood was removed by pipetting, and the aggregates were carefully washed four times in isotonic PBS (0·1 M NaCl, 1·7 mM KH2PO4, 4·9 mM Na2HPO4, pH 7·2). A 1 ml volume of fresh blood from the same donor was then added together with a fresh aliquot of diluted G148 bacteria. Samples were incubated at 37 °C, and bacterial survival was monitored as described above. For analyses by light microscopy, the aggregates were resuspended in 100 µl isotonic PBS, diluted 1 : 15, and applied onto glass slides by means of cytospin, using 300 r.p.m. for 4 min. Specimens were fixed in 2 % paraformaldehyde on ice for 30 min in a humidified chamber, washed twice with cold PBS, stained with haematoxylin and eosin, and analysed by light microscopy or subjected to immunostaining. For immunostaining, samples were incubated with rabbit anti-human fibrinogen (1 : 3000; Dako) for 1 h at 37 °C, washed extensively with PBST, and then incubated with Alexa-Fluor-488-conjugated goat anti-rabbit Fab'2-fragments as secondary antibody (1 : 2000; Molecular Probes). After washing with PBST, samples were analysed using a Nikon Eclipse TE300 inverted fluorescence microscope equipped with a Hamamatsu C4742-95 cooled CCD camera, using a Plan Apochromat x100 objective and a high numerical aperture oil-condenser.
Electron microscopy.
Human polymorphonuclear neutrophils were isolated from heparinized blood using Polymorphprep (Nycomen Pharma), according to the manufacturer's instructions. For scanning electron microscopy, the cells were resuspended in RPMI (Difco), and diluted to a final concentration of 1x107 cells ml1. An aliquot (1x106 cells) was incubated with 5 µg protein FOG, or fragments 1-C or 1-B, at 37 °C for 30 min. Aliquots (30 µl) were then applied onto poly-L-lysine-coated coverslips, and subsequently fixed in 2·5 % glutaraldehyde in 0·15 M sodium cacodylate, pH 7·4, (cacodylate buffer) for 30 min at room temperature. For analyses of G41 and G148 bacteria, 100 µl overnight culture, containing 2x107 c.f.u., was applied onto poly-L-lysine-coated coverslips. After 30 min incubation, the samples were fixed as described above. Specimens were washed with cacodylate buffer, and dehydrated with an ascending ethanol series from 50 % (v/v) to absolute ethanol (10 min per step). The specimens were then subjected to critical-point drying in carbon dioxide, with absolute ethanol as intermediate solvent, mounted on aluminium holders, sputtered with 50 nm palladium/gold, and examined in a JEOL JSM-350 scanning electron microscope.
For transmission electron microscopy, bacterial cells were pelleted, fixed for 1 h at room temperature, and then overnight at 4 °C in 2·5 % glutaraldehyde in cacodylate buffer. Samples were washed with cacodylate buffer, postfixed for 1 h at room temperature in 1 % osmium tetroxide in cacodylate buffer, dehydrated in a graded series of ethanol, and then embedded in Epon 812 using acetone as an intermediate solvent. Specimens were sectioned into 50-nm-thick ultrathin sections with a diamond knife on an LKB ultramicrotome. The ultrathin sections were stained with uranyl acetate and lead citrate. Specimens were observed in a JEOL JEM 1230 electron microscope operated at 80 kV accelerating voltage, and images were recorded with a Gatan Multiscan 791 CCD camera.
Sequencing, cloning procedures, PCR and computational analysis.
Genomic DNA was isolated according to Pitcher et al. (1989), with modifications described by Rasmussen et al. (1999)
. Primers were based on the signal sequence (5'-AGAAAATTAAAAAAAGGTACTGCATC-3') of an M-like protein of GGS (Schnitzler et al., 1995
), and on a conserved area (5'-TGCCATAACAGCAAGGGC-3') in emm genes of GCS, GGS and GAS, located C-terminally of the cell wall anchor motif LPXTG. PCR was performed using 200 ng DNA, 25 pmol of each primer, 0·2 mM dNTP mix (Amersham Pharmacia Biotech), 2 mM MgCl2, 1x PCR buffer, 5 U Taq polymerase (all from Sigma), and sterile H2O to a final volume of 100 µl. Primers were from DNA Technologies, and all reactions were performed with an Eppendorf Mastercycler Personal. Amplification was initiated at 94 °C for 10 min, and terminated at 72 °C for 10 min. Thirty cycles of amplification were run, each cycle consisting of 94 °C for 1 min, 56 °C for 1·5 min, and 72 °C for 1·5 min. PCR products were analysed by agarose (1 %) gel electrophoresis, and purified by using the High Pure PCR product purification kit (Roche). For sequencing of the fog gene, reactions were carried out by primer walking, using BigDye sequencer version 3.0 (Applied Biosystems). Each sequencing reaction consisted of 2 µl BigDye terminator, 40 ng PCR-product, 5 pmol primer, and sterile H2O to a final volume of 10 µl. Sequencing PCR was performed in accordance with the manufacturer's protocol. Samples were precipitated with 29 µl 95 % ethanol and 1 µl 3 M NaAc, pH 5·2, and subsequently centrifuged at 16 100 g for 30 min at room temperature. Samples were vacuum centrifuged, and the sequencing reactions were performed on an ABI 3100 at the BM-unit, Lund University, Sweden. New primers were based on the sequences obtained. Sequences were aligned using MacVector version 7.0, with a minimum overlap of 100 bp, and each base pair was sequenced at least twice with independently purified templates. Computational prediction of signal sequence cleavage was performed using the web-based program SignalP V1.1 (http://www.cbs.dtu.dk/services/SignalP/). Prediction of dimerization and coil formation was done using services available at http://www.ch.embnet.org and http://multicoil.lcs.mit.edu/cgi-bin/multicoil.
Fragments of protein FOG were cloned and expressed in Escherichia coli using the GST Gene Fusion System (Amersham Biosciences). The mature protein FOG (aa 1557) was amplified by PCR using the 5' primer 5'-GCGGATCCGCGGAGAATACATACGATAGATGG-3', containing a BamHI site, and the 3' reverse primer 5'-GCTGAATTCTTATTAACCTGTTGATGGTAACTGTCTCTT-3', containing an EcoRI site. For the generation of fragment 1-B (aa 1278), the 3' reverse primer 5'-GTTGAATTCTTATTAAGCTGTTAGACTGTCAACAATGCC-3', containing an EcoRI site, and for fragment 1-C (aa 1493), the 3' reverse primer 5'-GATGAATTCTTATTAGTTAAGTTTTTCAAGAGCAGCTAATTT-3', containing an EcoRI site, were used in combination with the 5' primer used for the mature protein FOG. After digestion with the indicated restriction enzymes (New England Biolabs), the fragments were cloned into the pGEX-6P-1 vector. Following standard ligation (Invitrogen), the plasmid was transformed into competent E. coli JM109 cells. Transformants were grown on LuriaBertani agar containing 100 µg ampicillin ml1, and screened for the correct fragment insert. Plasmid DNA was then isolated, and transformed into competent E. coli BL21. Induction and procedures of purification followed the instructions of the manufacturer.
PCR amplification of the protein G gene was performed using the 5' primer 5'-TTGGTCGACTGATGATAGGAGATTTATTTG-3', containing a SalI restriction site, and the 3' reverse primer 5'-CGGGGATCCCATATTGAAAAGGCCTCAATG-3', containing a BamHI site. Template DNA was prepared by boiling G41 or G148, grown on THA overnight, in sterile water for 5 min. Following centrifugation at 10 000 g, the supernatant was recovered and used as a template.
Surface plasmon resonance spectroscopy and flow cytometry analyses.
The association and dissociation rate constants for the interactions between protein FOG and fibrinogen, HSA or IgG were determined by surface plasmon resonance spectroscopy, using a Biacore-X system. Protein FOG, HSA and fibrinogen were immobilized on research-grade CM5 sensor chips in 10 mM sodium acetate at pH 3·5, using the amine coupling kit supplied by the manufacturer. Analyses were performed in PBST at 25 °C and at a flow rate of 10 µl min1. Analyte (35 µl) was applied in serial dilutions starting at a concentration of 1 mg ml1. Surfaces were regenerated with 35 µl 0·1 M glycine/HCl, pH 2·0, at a flow rate of 10 µl min1. The kinetic data were analysed by the BIAEVALUATION 2.2 program (Biacore).
Complement deposition on the cell surface of G41 and G148 was analysed by measuring the amount of C3 deposition, as described by Kotarsky et al. (2001). For detection of fibrinogen on the neutrophil surface, purified neutrophils (1x106) were incubated for 30 min at 37 °C with rabbit anti-human fibrinogen diluted 1 : 3000 (Dako) in MEM (Life Technologies), washed twice with PBS, and subsequently incubated for another 15 min in darkness at room temperature with Alexa-Fluor-488-conjugated Fab'2-fragments from goat (1 : 800) (Molecular Probes) directed towards rabbit IgG. Neutrophils were washed twice in PBS for 5 min, resuspended, and analysed by flow cytometry using a FACS-Calibur flow cytometer (Becton-Dickinson) equipped with a 15 mW argon laser tuned at 488 nm. The FL1 fluorescence channel (
em 530 nm) was used to record the emitted fluorescence of Alexa 488.
Heparin-binding protein (HBP) release and precipitation experiments.
Varying amounts of protein FOG, or fragments thereof, were incubated for 30 min at 37 °C in 1 ml human whole blood diluted 1 : 10 in Dulbecco's PBS (Difco). For maximum release of HBP, 5 % Triton X-100 was used, and for background level, one tube was put directly on ice, and one was included in the series incubated at 37 °C. Following incubation, samples were centrifuged at 10 400 g for 15 s, and the resulting supernatants were recovered. HBP was quantified by means of ELISA as described by Tapper et al. (2002). Precipitation of protein FOG in PBS containing 10 % plasma or fibrinogen (300 µg ml1) was performed as described by Herwald et al. (2004)
. Fibrinogen-deficient human plasma was purchased from Enzyme Research Laboratories.
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RESULTS |
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Sequencing of the gene encoding protein FOG
In order to sequence the fog gene, primers were constructed based on a region in the signal sequence of an M-like protein from GGS (Schnitzler et al., 1995), and on a region in the homologous C-terminal membrane-spanning part of M proteins from GCS, GGS and GAS, which was found in database searches (Altschul et al., 1990
). Using these primers, a PCR product of 1·8 kb was produced, with chromosomal DNA from G41 used as a template. This PCR product was then used as a template in sequencing reactions (see Methods). Analysis of the protein sequence deduced from the nucleotide sequence (GenBank/EMBL accession number AY600861) demonstrated that the N-terminal amino acid sequence obtained from the 66 kDa protein band (see Fig. 1d
) was consistent with the N-terminal of the mature protein FOG. This was also in agreement with a putative cleavage of the signal sequence predicted by the web-based server SignalP V1.1. Protein FOG is structurally similar to other M-like proteins of GAS, GCS and GGS species, and it has a signal sequence typical of surface proteins from Gram-positive bacteria. Hence, we follow the structural nomenclature used by Åkesson et al. (1994)
. A schematic representation of protein FOG is shown in Fig. 2
(a). The A domain contains two short repeated sequences (aa 8488 and 108112), and is followed by two identical B domains (B1, aa 133172; B2, aa 193232). The S region is 108 aa long, and is followed by three highly homologous repeats (C1C3, aa 340368, 375406 and 424448, respectively). C1 and C2 are 100 % identical on the amino acid level, and they are encoded by nucleotides that are identical to a level of 92 %. C3, being the shortest of the three repeats, shows 100 % identity on the amino acid level, and 93 % identity on the genetic level. The D domain shows sequence identity with M proteins of class I (Bessen & Fischetti, 1992
), and it includes an LPXTG anchor motif. The size of the fibrinogen-binding fragments generated by CNBr cleavage corresponded to the positions of the methionine residues 38, 284 and 546 (Fig. 2a
). Computer-aided analysis showed that protein FOG has a high probability of dimerization and
-helical coil formation. Amino acids 227245, 336355, 371389 and 406424 of protein FOG show similarity to a region of other M and M-like proteins described to be responsible for self-association (Frick et al., 2000
). Analogous to the M-like protein FAI of GCS (Talay et al., 1996
), there are unique segments in protein FOG, such as the B-repeats, showing no homology to M proteins or any other protein available in the database.
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Survival of GGS in human blood is mediated by protein FOG
As shown above, the G41 strain survives and multiplies 100-fold in human whole blood, while the G148 strain, which lacks protein FOG, is killed (Table 1). In contrast, both strains multiply equally well in human plasma (not shown). To confirm the absence of the fog gene in G148 bacteria, PCR was performed with the primers used for the sequencing of fog, and chromosomal DNA from G148 as the template. No product was obtained with these primers or others used in primer walking the fog sequence, confirming that the G148 strain lacks the fog gene (data not shown). A PCR product corresponding to the 1·8 kbp product was generated in another 30 clinical isolates of GGS, all of which bound fibrinogen (not shown).
To analyse the contribution of protein FOG to the survival of G41 bacteria in blood, the G148 strain was used in a series of experiments. Soluble protein FOG, and fragments 1-C or 1-B, at a concentration of 5 µg ml1, were added to G148, and growth in human whole blood was monitored for a period of 5 h. The amount of protein FOG added corresponds well with the amount that is secreted into the growth medium of G41 bacteria (2·510 µg ml1) in late-exponential growth phase. As shown in Table 2, the addition of FOG resulted in a 500-fold multiplication of G148. FOG also mediated survival of BMJ71, an mga mutant of the AP1 strain of GAS (Kihlberg et al., 1995
) lacking M protein. The M1 protein of the wild-type strain AP1 was also found to protect G148 from being killed (Table 2
). Addition of fragment 1-C gave an intermediate response, resulting in a maintained but not increased number of c.f.u. In contrast, the addition of fragment 1-B, the GST-tag alone or the buffer control failed to restore growth (Table 2
). Neither protein PAB, an albumin-binding surface protein of F. magna (de Château & Björk, 1994
) nor protein G could protect G148 bacteria from phagocytosis (data not shown).
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Protein FOG promotes survival of GGS through aggregation of neutrophils
Survival of GAS in human blood has been attributed to M protein binding of fibrinogen, as well as proteins regulating complement activity (Horstmann et al., 1988; Perez-Casal et al., 1995
; Ringdahl et al., 2000
; Thern et al., 1995
; Whitnack & Beachey, 1982
). The ability of protein FOG to interact with factor H was tested in the slot-binding assay. As shown in Fig. 3
(a), radiolabelled factor H bound to protein FOG, suggesting that this interaction might be of some benefit for FOG-expressing bacteria. The binding was retained in fragment 1-C, but not fragment 1-B. However, protein G was also found to interact with factor H (Fig. 3b
), and no difference in complement deposition on the surfaces of G41 and G148 by the alternative pathway was detected in flow cytometry analyses (not shown).
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DISCUSSION |
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Protein FOG is a self-associating surface protein of GGS. Self-association among GAS has been ascribed to M proteins (Frick et al., 2000); however, other bacterial species, such as Staphylococcus aureus, Mycobacterium tuberculosis and Bordetella pertussis, also show similar self-associating characteristics (McDevitt et al., 1994
; Menozzi et al., 1994
, 1996
). Bacterial aggregation may be of benefit for the bacteria, since larger particles are less at risk of being engulfed by host phagocytes. Also, aggregation may mediate a firmer attachment to host cells, facilitating invasion, and supporting a sustained infection. Protein FOG contains several regions that show high similarity to the AHP sequence involved in self-association of GAS M proteins (Frick et al., 2000
). These regions are located in the middle and C-terminal parts of protein FOG, which might help to explain the antiphagocytic effect of the intact FOG molecule.
A multitude of studies have demonstrated that M protein interactions with regulating proteins of complement, as well as with fibrinogen, play important roles in the survival of GAS in human blood. Factor H was shown early on to bind to the C-repeated region of M proteins, suggesting that an activation of the alternative pathway of complement would thus be inhibited (Horstmann et al., 1988). Protein FOG interacts with factor H in a similar mode, but the fact that protein G also has affinity for factor H, and still has no protective effect, further excludes factor H-binding as an important mechanism for GGS survival in blood. This is in agreement with Kotarsky et al. (2001)
, who demonstrated that resistance to phagocytosis of GAS was independent of factor H.
Fibrinogen binding to M-protein-expressing GAS has for some strains been suggested to modulate bacteriaphagocyte interactions, resulting in inefficient killing of bacteria (Ringdahl et al., 2000; Whitnack & Beachey, 1982
). The mechanism is unclear, but the finding that proteins M1 and M5 both contribute to streptococcal survival, although they interact with different regions of fibrinogen (Ringdahl et al., 2000
), underlines the importance of fibrinogen binding. Neutrophils interact with human fibrinogen through the CD18 family of integrins, where the N-terminal part of the A
chain of fibrinogen binds to CD11c/CD18 (Loike et al., 1991
). This has consequences such as cell spreading, respiratory burst and degranulation, which all are relevant in the bacteriahost interplay. Furthermore, the C-terminal part of the
chain has been reported to bind to CD11b/CD18 (Wright et al., 1988
). In addition, both receptors mediate attachment of unopsonized bacteria to neutrophils (Ross et al., 1992
). Thus, it is conceivable that, by binding fibrinogen, M and M-like proteins, such as protein FOG, could interfere with receptor attachment to activated C3 and C4 on the bacterial surface. Recently, it was also demonstrated that M proteinfibrinogen complexes aggregate and activate neutrophils by cross-linking of the CD18 integrins (Herwald et al., 2004
). Activation resulted in release of HBP and frustrated phagocytes, which, as in our case, seem to be unable to function.
Although protein FOG is structurally related to GAS M proteins, differences in terms of function appear. The binding site for fibrinogen on protein FOG is located in the N-terminal region, but, surprisingly, only the mature molecule exerted a protective effect on killing of non-protein FOG-expressing bacteria (Table 2). Moreover, to achieve an efficient precipitation of fibrinogen and neutrophil clumping, a FOG fragment equivalent to that presented on the bacterial surface is needed. This is in contrast to the GAS M1 protein, in which a fibrinogen-binding N-terminal fragment still forms active complexes with fibrinogen (Herwald et al., 2004
). The affinity constant for the interaction between M1 and fibrinogen is 2·5x108 (Ringdahl et al., 2000
), which is about 100-fold more than the affinity between protein FOG and fibrinogen. Thus, it is tempting to speculate that, due to lower affinity, the complex formation between protein FOG and fibrinogen depends on a conformationally stable
-helical dimer. In this context, it is notable that the C-terminal parts of M proteins, which are well conserved within members of this family, were found to adopt a more stable folded structure than the N-terminal regions (Nilson et al., 1995
). This was later found to be the case also with the fibrinogen-binding protein (FgBp) of Streptococcus equi subsp. equi, in which the C-terminal part contributes to thermal stability of the molecule (Meehan et al., 2002
). The same group also showed that neither A nor B repeats are important for binding of FgBp to fibrinogen (Meehan et al., 2000
). These domains, however, seem important for conformation and multimerization. The results of Meehan and co-workers support our findings that emphasize the difference between having the ability to bind fibrinogen (fragment 1-B) and actually reaching the effect of the binding, i.e. inhibition of neutrophil function. Our results clearly show that an intact protein FOG molecule is a structural prerequisite for the functions investigated in this study, and from which the bacterium may benefit.
Although GGS most often cause skin or mucosal infections, and are less frequently found in blood, neutrophils will be recruited to the site of infection. A possible cross-linking of the CD18 integrins, triggered by protein FOG, would result in HBP release and exhausted neutrophils. This could contribute to streptococcal survival, and, in addition, the HBP release will cause vascular leakage, providing the bacteria with nutrients and a route of bacterial dissemination. During cultivation of FOG-expressing strains, the protein is found in the growth medium at late-exponential growth phase. Most likely, protein FOG is also released from the bacterial surface in vivo; thereby an inhibition of neutrophil function could take place at sites distant from the bacterial surface. In GAS, M proteins are cleaved from the bacterial surface by the bacterial cysteine protease SpeB (Berge & Björck, 1995). Such enzymic activity has not yet been reported for GGS, but the possibility of enzymic surface protein release cannot be ruled out. Streptococcal resistance to neutrophilic killing is a complex mechanism. Here we demonstrate that an M-like molecule of GGS, protein FOG, triggers aggregation of neutrophils in human whole blood, at least in part through an interaction with fibrinogen. However, other properties of protein FOG might affect the function of neutrophils in later stages of encounter. Studies to further understand the molecular interactions between M-like proteins and phagocytic cells are of great interest and importance.
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ACKNOWLEDGEMENTS |
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Received 22 April 2004;
revised 6 August 2004;
accepted 9 September 2004.
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