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Originally published In Press as doi:10.1074/jbc.M313613200 on January 28, 2004

J. Biol. Chem., Vol. 279, Issue 16, 15850-15859, April 16, 2004
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Streptococcus pyogenes Fibronectin-binding Protein F2

EXPRESSION PROFILE, BINDING CHARACTERISTICS, AND IMPACT ON EUKARYOTIC CELL INTERACTIONS*

Bernd Kreikemeyer{ddagger}§, Sonja Oehmcke{ddagger}§, Masanobu Nakata{ddagger}, Raimund Hoffrogge¶, and Andreas Podbielski{ddagger}||

From the {ddagger}Department of Medical Microbiology and Hospital Hygiene, Hospital of the Rostock University, Schillingallee 70, 18057 Rostock and the Proteome Center Rostock, Medical Faculty, University of Rostock, Joachim-Jungius-Strasse 9, 18059 Rostock, Germany

Received for publication, December 12, 2003 , and in revised form, January 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Some Streptococcus pyogenes (group A streptococci, GAS) strains have previously been shown to express the fibronectin-binding protein F2 instead of the functionally related but structurally dissimilar protein F1/SfbI. In this study, recombinant N-terminal and C-terminal portions and the two fibronectin-binding domains of protein F2 were used to assess affinity parameters of the interaction with fibronectin and its N-terminal 70-, 30-, and 45-kDa fragments. The association and dissociation equilibrium constants for both binding domains were in the nanomolar range, although the repeat domain of protein F2 exceeded the affinity of the unique domain by up to one order magnitude. Both domains primarily interacted with the 30-kDa fibronectin fragment. Using a prtF2 gene isogenic mutant of a serotype M49 GAS strain that does not harbor the protein F1/SfbI gene, the attachment values of whole bacteria to immobilized fibronectin and to HEp-2 epithelial cells were found to be 6- and 2-fold decreased, respectively. Reduction of prtF2 mutant internalization rates for eukaryotic cells exceeded the reduction of attachment rates, indicating an independent contribution of protein F2 to both processes. The prtF2 transcription and protein F2 expression profiles documented maximum expression at the transition to the stationary phase especially under aerobic growth condition. The protein F2 function as the major fibronectin-binding adhesin in a subset of GAS strains, its expression pattern, and highly specific interaction with fibronectin would be consistent with a status as an indispensable virulence factor for both earlier and later pathogenetic stages of GAS superficial infections.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptococcus pyogenes (group A streptococci, GAS)1 cause a variety of purulent infections ranging from a comparatively benign tonsillopharyngitis to life-threatening necrotizing fasciitis, myositis, and streptococcal toxic shock syndrome. Irrespective of the severity of such diseases, as a necessary initial step the current pathogenetic concept of GAS-associated diseases postulates the specific and firm binding of the GAS to epithelial host cells (1). This attachment relies on the interaction of several types of bacterial and host cell surface molecules. When investigated by in vitro experiments, bacterial fibronectin-binding surface proteins were consistently identified as key players in the process of eukaryotic cell adhesion and internalization.

In the last decade, more than a dozen GAS fibronectin-binding surface proteins have been identified (1–3). Most of them are members of a large family of proteins termed MSCRAMMs (microbial surface components recognizing adhesive matrix molecules (2, 4)). However, only few, i.e. proteins F1/SfbI, Fbp54, and SOF/SfbII, have been characterized concerning important biochemical parameters by determining Km values and binding strength and, simultaneously, their biological role by employing eukaryotic cell interaction assays or animal infection models.

Among the more thoroughly described GAS fibronectin-binding proteins, protein F1/SfbI has received the most extensive attention. The encoding prtF1/sfbI gene is carried by 50–90% of clinical GAS isolates (5–8). The genomic presence of the gene is positively correlated with fibronectin binding of the respective GAS strain. Expression of the gene is more prominent among skin and throat isolates than among blood culture isolates (9). Based on a combination of molecular epidemiological and experimental in vitro data, the prtF1/sfbI gene-positive GAS strains have also been associated with an increased tendency of the bacteria to internalize into eukaryotic cells (10, 11). In turn, this could have some negative impact on the successful applicability of the standard penicillin therapy (11, 12). However, the association of prtF1/sfbI-positive clinical isolates to treatment failures is not unequivocal (13).

In PrtF1/SfbI-positive strains, the absence of susceptibility to penicillin, a drug that predominantly acts outside eukaryotic cells, would be consistent with the well documented role of protein F1/SfbI as a necessary internalization factor during in vitro eukaryotic cell-bacteria interactions. Eukaryotic cell internalization is a multistep process that involves the binding of two C-terminal domains in protein F1/SfbI to two N-terminal domains of the fibronectin molecule (14–18). If in turn fibronectin is bound to integrins of the {alpha}5{beta}1 or {alpha}v{beta}3 varieties, a signal cascade is triggered. This pathway involves the focal adhesion kinase and small GTPases of the Rho family such as Rac and CDC42. Subsequently, the formation of a phagocytic vacuole occurs via actin rearrangements and a membrane engulfment of the bacteria (19–21).

Protein F2 (22), a protein of 1039 amino acid (aa) residues, is another MSCRAMM of the fibronectin-binding subclass. Based on partial sequence homology rates between 0 and 95%, protein F2 could belong to a novel GAS protein family together with the fibronectin-binding proteins PFBP (1161 aa residues (23)), and FbaB (733 aa residues (24)). The encoding prtF2 gene was demonstrated to be present in 36–80% of clinical GAS isolates (25, 26). Its association to specific GAS diseases was examined with contradicting results. Although Delvecchio et al. (27) found that prtF2 was more frequently present in invasive disease strains, Musumeci et al. (28) detected the gene more frequently in GAS isolates from asymptomatic carriers. The picture remains obscure, because Terao et al. (24) observed no difference in fbaB carriage between serotype M3 toxic shock and pharyngitis strains but reported FbaB expression exclusively in the majority of toxic shock-associated isolates. Obviously, the genes are expressed under natural circumstances, because the majority of infected patients harbored antibodies to protein F2 (26).

The prtF1/sfbI and prtF2 genes were simultaneously present in the genomes of 31–100% of the GAS strains tested (25, 26). In genomes with both fibronectin-binding protein genes, prtF1 and prtF2 either resided within a potential pathogenicity island ("fibronectin-binding/collagen-binding/T-antigen-island," serotype M12 (29)) or at separate loci of the genome (serotypes M3, M18 (30–32)), as a potential consequence of genome-scale recombination events. Recombination could also occur on the gene level, because FbaB exhibits medium to high homology to the N terminus of the GAS collagen-binding Cpa protein (24, 33) and protein F2 to the C terminus of Streptococcus dysgalactiae fibronectin-binding FnBA and FnBB proteins (22, 34, 35). Using soluble human matrix proteins, the fibronectin-binding of protein F2 has been associated with two domains within its C terminus. One consisted of three consecutive repeats of 21–37 aa residues ("FbRD"), the other of ~100 non-repeated aa residues ("UFbD" (22)).

Expression of protein F2 was described to primarily depend on the oxygen, but not on the carbon dioxide partial pressure (22). The transcription of the pfbp/prtF2 genes was documented in the exponential growth phase (23) and was found to be negatively controlled by the Nra global regulator (33). Using fbaB-mutant GAS strains, Terao et al. observed 6-fold reduced rates of HEp-2 epithelial cell adhesion and internalization and a similarly reduced mortality rate of mice intraperitoneally infected with such mutants.

In the present study, we use a GAS strain that exclusively carries a prtF2 but not a prtF1/sfbI gene in its genome to (i) perform a thorough analysis of the prtF2 gene expression, (ii) characterize biochemical parameters of the protein F2-fibronectin interaction, (iii) quantitatively describe the protein F2-binding to whole and fragmented immobilized fibronectin, and (iv) document the effects of protein F2 for eukaryotic cell attachment and internalization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Culture Conditions—S. pyogenes (GAS) serotype M49 strain 591 is a skin isolate originally obtained from R. Lütticken, Aachen, Germany. The Escherichia coli strain DH5{alpha} was purchased from Invitrogen (Eggenstein, Germany) and served as a host for plasmids pFW11, pFW11-luc, and pUC-erm (36). An E. coli strain BL21(DE3) was used as the host for plasmid pET28. Both strain and pET28 plasmid were obtained from Invitrogen (Karlsruhe, Germany).

The GAS wild type strain and mutant derivates were cultured in Todd-Hewitt (TH) broth or on TH-agar (Oxoid-Unipath, Wesel, Germany), both supplemented with 0.5% yeast extract (THY) or in chemically defined medium (CDM) (37).

GAS mutants harboring recombinant pFW- or pUC-erm plasmids were maintained in medium containing 60 mg of spectinomycin or 5 mg of erythromycin x liter–1, respectively, except when being used for functional analyses. GAS strains were grown as standing cultures at a temperature of 37 °C under a 5% CO2-20% O2 atmosphere unless otherwise indicated.

E. coli DH5{alpha} isolates transformed with pFW, pUC-erm, or pET derivates were grown on disk susceptibility agar (Oxoid-Unipath) supplemented with 100 mg of spectinomycin, 150 mg of erythromycin, or 30 mg of kanamycin x liter–1, respectively. All E. coli cultures were grown at 37 °C in ambient air except when used for expression of recombinant proteins.

Nucleic Acid Techniques—Chromosomal and plasmid DNA preparations, genetic manipulations, and other conventional DNA techniques, including electroporation of GAS and E. coli strains, were done as described in a previous publication (33).

Construction of Recombinant Vectors and GAS Strains—For the integration of a luciferase reporter box downstream of the prtF2 gene, a central to 3' terminal portion of the prtF2 gene (Fig. 1, aa residue 569, 6 bp downstream of the prtF2 stop codon) was PCR-amplified using the forward/reverse primer pair 5'-AAA AGT TCC AGA CGG CTA CAA G-3'/5'-GCT ATT GTC ACC AAC AG-3' and, utilizing NheI and BamHI 5' primer extensions, was cloned into the multiple cloning site I of plasmid FW11-luc. This plasmid (GenBankTM accession number U41082 [GenBank] ) is an improved version of pFW5-luc (33), which carries a synthetic strong promoter instead of the natural promoter upstream of the aad9 spectinomycin resistance gene. The resulting recombinant pFW-luc plasmid was integrated by a site-specific single-crossover event into the strain 591 genome according to the protocol described by Podbielski et al. (33). The correct insertion site was confirmed using Southern blot hybridizations and appropriate PCR assays on genomic DNA preparations from wild type and mutant strains (data not shown).



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  		FIG. 1.
Schematic presentation of the prtF2 gene and of protein F2 as well as the recombinant constructs used for luciferase reporter gene assays, insertion mutagenesis, and hyperexpression of recombinant protein F2 fragments. The designation of the prtF2 portions used for reporter and insertion constructs is shown underneath the lines. The terms for the protein F2 domains are shown in the respective gray boxes. The numbers of the corresponding nucleotide or amino acid positions are shown under the respective gene or protein fragments used in individual cloning procedures or functional assays. The designation of the two fibronectin-binding domains is according to Jaffe et al. (22). N, N terminus; C, C terminus; aa, amino acids. The schematic presentation is drawn to scale.

 
The nra gene was inactivated following the approach described by Podbielski et al. (33). The 5' terminal nra fragment was amplified by PCR and cloned into the pUC-erm vector using appropriate 5' primer extensions and the BamHI and PstI sites of the plasmid. After confirmation of the insertional mutagenesis, the resulting nra-mutant GAS strain was used for a second insertional mutagenesis with the prtF2-luc reporter construct following the protocol described above.

For the insertional mutagenesis of prtF2, a 5' terminal to central portion of the prtF2 gene (Fig. 1, aa residues 15–432) was PCR-amplified using the forward/reverse primer pair 5'-GGG AAA TCA TCA GTA AAG CAG-3'/5'-GAA TCT CTC CTT GTG TGT TTG-3' and, utilizing SalI and XbaI 5' primer extensions, cloned into the multiple cloning site I of plasmid pFW11. Site-specific single-crossover insertion of the recombinant plasmid and confirmation of the correct insertion were performed as described above.

For generating recombinant plasmids to hyperexpress protein F2 fragments, various sections of the prtF2 gene (Fig. 1) were PCR-amplified using the following primer pairs: aa residues 55–455: 5'-TTC TAC CGA AAT CAT GCC-3'/5'-TCA TAC AGG TCA TAG GTG-3' (F2-NT); aa residues 662–1003: 5'-GGC TCA GGT AAT GAG TGG-3'/5'-GCT GGT AAA CTA GTA TTA CTC-3' (F2-FnB); aa residues 662–798: 5'-GGC TCA GGT AAT GAG TGG-3'/5'-AGT TTT ACC AGA TGA ATC A-3' (F2-UFbD); and aa residues 866–1003: 5'-CCA ACT AAG GGT TCA GGT-3'/5'-GCT GGT AAA CTA GTA TTA CTC-3' (F2-repeat).

Employing the appropriate 5' primer extensions, the resulting PCR products were cloned into the EcoRI and XhoI sites of plasmid pET28. The inserted fragments in each recombinant plasmid were controlled by complete sequencing of the cloned fragments on both DNA strands.

Quantitative Assays for Luciferase Activity—For assessment of the luciferase activity of the prtF2-luciferase reporter fusions, the GAS luc reporter strains were grown in THY or CDM broths without antibiotic supplements as agitated cultures at 200 rpm in ambient air or as standing cultures under a 5% CO2-20% O2 atmosphere or, after prereduction of the media, under a 2% CO2-2% H2-96% N2 atmosphere using the Anoxomat anaerobic culture system (Mart Microbiology, Lichtenvoorde, Holland).

To determine the potential induction of prtF2 transcription by the presence of human matrix proteins in the culture medium, human fibronectin (Sigma) was added to CDM at final concentrations of 0.2, 0.5, 1, and 2 mg/liter. For a potential induction by human cell factors, spent media after 3-day incubation of HEp-2 cells or HEp-2 cells lysed by exposure to distilled water were added at 1/10 of the final assay volume. In these cases, the same amounts of fresh cell culture medium supplements were used for a negative control.

For measurement of luminescence, 1-ml aliquots of the cell suspensions were withdrawn at hourly intervals and processed as described by Podbielski et al. (33). All reported data are representative of at least three independent experiments.

Purification and Characterization of Recombinant Protein F2 Fragments—Due to utilizing pET28 as a vector plasmid, the recombinant internal fragments of protein F2 all carried N-terminal His tags. Hyperexpression of each polypeptide was achieved by growing the recombinant bacteria in LB broth (Invitrogen, Karlsruhe, Germany) as shaking cultures (200 rpm) at 30 °C to mid-exponential phase. Subsequently, 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside was added for induction. After continuing the incubation for another 4 h, the E. coli cells were harvested and processed according to the protocol of the manufacturer of pET28 (Invitrogen).

Purification of the recombinant proteins was achieved by absorption to nickel-nitrilotriacetic acid resin (Qiagen) as outlined in the manufacturer's instructions. The quality of the purified polypeptides was controlled by 12% SDS-PAGE and Coomassie Blue staining (Fig. 3A).



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  		FIG. 3.
Binding characteristics of recombinant protein F2 domains toward whole fibronectin and fibronectin fragments. A, binding of whole fibronectin and the 70-, 45-, and 30-kDa fibronectin fragments to immobilized protein F2 domains as investigated by Western blot techniques. Each protein F2 fragment was used at a concentration of 2.5 µg/lane. M, molecular weight marker; lane 1, F2-FnB; lane 2, F2-NT; lane 3, F2-UFbD; and lane 4, F2-repeat. B, detailed analysis of the interaction of recombinant protein F2 domains (analyte) with immobilized fibronectin and its fragments (ligand) using surface plasmon resonance (SPR) measurements in real-time biospecific interaction analysis. The parts of the figure show representative profiles of the relative SPR responses for the association and dissociation of 200 nM analyte. Note the different scale of the y-axis in the case of the 45kDa fibronectin fragment. Injection of the analyte (A) is marked by an arrow. After reaching the equilibrium phase, the analyte solution was exchanged for washing buffer (W), and the dissociation of the formed complexes was recorded.

 
To detect an interaction with soluble fibronectin, subsequently to SDS-PAGE, the protein F2 fragments were transferred by semidry Western blotting onto polyvinylidene difluoride membranes (Immobilon P, Millipore, Eschborn, Germany). The membranes were blocked for 1 h in PBS containing 10% skimmed milk followed by incubation for 1 h with whole fibronectin and fibronectin fragments at a concentration of 1 µg/ml in PBS. For immunodetection, the blot was first incubated with a rabbit anti-fibronectin antibody (Sigma, Taufkirchen, Germany) diluted 1:1000 in PBS and after washing, with a goat anti-rabbit IgG-HRP-conjugate (Bio-Rad, München, Germany) again diluted 1:1000 in PBS. Finally, visualization was performed with a horseradish peroxidase (HRP) color development solution (Bio-Rad). To generate polyclonal anti-protein F2 rabbit antisera, 500 µg each of the purified recombinant protein F2 N terminus and fibronectin-binding C terminus was used by EuroGentec (Seraing, Belgium) to produce the antisera.

Immunofluorescence Detection of Protein F2 on the Bacterial Surface—Direct immunofluorescence detection of protein F2 was done by reacting whole late exponential phase GAS bacteria with the polyclonal anti-protein F2 fragments antisera or the corresponding pre-immune sera, followed by reaction with a fluorescein isothiocyanate-conjugated anti-rabbit IgG (Alexa Fluor 488® goat anti-rabbit IgG, MoBiTec, Göttingen, Germany). Unlabeled and labeled bacteria were detected by using a BX60 fluorescence microscope and 100 x 1.3 or 60 x 1.25 UplanFl objectives (Olympus, Hamburg, Germany).

Quantitative assays were performed with peroxidase-coupled fibronectin. Labeling of fibronectin was carried out according to the protocol of Schmidt and Wadström (38). For the binding assay, GAS cells were harvested from different points of the growth curve (A600 nm 0.2, 0.5, 1.0, 1.3, and A 1.3 plus 2 h of further incubation and 24 h of total incubation time). To investigate the same number of cfu (colony forming units) in each tube, bacteria were washed in PBS, and all were adjusted to an A600 nm of 1.0. 100 µl of the adjusted GAS suspensions was incubated with 100 µl of HRP-labeled fibronectin (1:1000 dilution of the stock solution recovered after the labeling reaction) for 30 min at 37 °C. After 3-fold repeated washing with PBS, bacteria were transferred to microtiter plates and HRP-developing substrate (Bio-Rad) was added to the wells. The color reaction was stopped with H2SO4 (0.5%) and measured at A405 nm. Binding of HRP to GAS was monitored in control experiments, and A405 nm values were subtracted.

Enzyme-linked Immunosorbent Assay Determination of GAS Binding to Immobilized Fibronectin—For quantifying the binding of GAS strains to immobilized fibronectin, 96-well enzyme-linked immunosorbent assay plates (Greiner Bio-one, Solingen, Germany) were coated overnight at 4 °C with 2.5 µg/well human fibronectin (Sigma, Germany) in PBS. Plates were blocked with 1% BSA in PBS for 1 h at 37 °C and after three washes in 0.05% Tween 20/PBS, late exponential phase GAS strains diluted to an A600 nm of 0.4 in PBS were incubated in the coated wells for 30 min at 37 °C. As determined in control experiments, the number of bacteria used was in the linear range of this bacteria-fibronectin interaction. As a control, GAS strains were incubated in non-coated and BSA-coated wells. After four washes with PBS, a goat antibody to group A Streptococcus-HRP conjugate (Dunn Labortechnik GmbH, Asbach, Germany) diluted 1:5000 in PBS was added, and the plates were further incubated for 1 h at room temperature. After four washes with PBS, bound antibodies were visualized using a TMB Peroxidase EIA Substrate Kit (Bio-Rad). After 10 min, the reactions were stopped with 0.5% H2SO4, and the optical density at 450 nm was monitored. Direct adherence inhibition by soluble protein F2 fragments was tested by blocking the immobilized fibronectin with 1% BSA and adding 10 mg of the protein F2 fragments per liter for 2 h at 37 °C prior to the GAS binding assays.

The results of the adherence assays are reported as the mean of at least three independent experiments. The data were statistically evaluated by means of the Mann-Whitney U test.

Surface Plasmon Resonance Protocols—The interactions between fibronectin (Sigma, Germany), fibronectin fragments (Sigma), and recombinant protein F2 domains were analyzed with a model 3000 BIAcore system (Biosensor, La Jolla, CA) using CM5 and NTA sensor chips. Fibronectin and fibronectin fragments were covalently immobilized on the CM5 carboxymethylated dextran matrix via amine coupling using the amine coupling kit (BIAcore). The amount of immobilized ligand was calculated as follows: resonance units of immobilized ligand = 500 x (molecular mass of immobilized ligand [whole Fn or Fn-fragments]/molecular mass of analyte [recombinant protein F2 portions]) according to the manufacturer's manual. For immobilizing ligands to a pre-set target level and preparing reference surface on the CM5 sensor chip, the software tool "Application Wizard-Surface Preparation" (BIAcore 3000 Instrument Handbook) was used. Binding of analytes was performed in HBS-EP buffer (BIAcore), which was also the running buffer, at 25 °C using a flow rate of 30 µl/min in all experiments. Binding was determined by measuring the resonance unit increment. The affinity surface was regenerated with 100 µl of 0.2% SDS after sample injections and subsequent washing steps.

NTA sensor chips (BIAcore) were used for immobilizing recombinant F2-FNB via the N-terminal His tag. This approach has also the advantage of homogeneously orienting the ligand molecules to make sure that no binding site is blocked after immobilization. Attachment of the ligand was performed following the instructions of the manufacturer (BIAcore). Running and eluent buffer (0.01 M HEPES, 0.15 M NaCl, 50 µM EDTA, 0.005% surfactant P20, pH 7.4) were identical. First, a 1-min pulse of regeneration solution (0.01 M HEPES, 0.15 M NaCl, 0.35 M EDTA, 0.005% surfactant P20, pH 8.3) was injected to remove metal ions potentially present in previously used reagents. After washing the system, a 1-min pulse of nickel solution (500 µM NiCl2 in eluent buffer) at a flow rate of 20 µl/min was injected to saturate the NTA with Ni2+ ions. Then the injection parts of the apparatus were cleaned by applying eluent buffer according to the handbook of the manufacturer (BIAcore). The histidine-tagged ligand (F2-FnB) was dissolved in eluent buffer, injected by a 1-min pulse (flow rate 20 µl/min) and immobilized on the nickel-nitrilotriacetic acid surface. Interaction analysis on the sensor chip NTA essentially followed the procedures described for the CM5 sensor chips.

Kinetic Analysis of BIAcore Sensorgram Data—For calculation of kinetic constants, the recombinant protein F2 portions were injected in increasing concentrations for a contact with the CM5 sensor chips. For an optimization of result reproducibility, the software tool "Application Wizard-Kinetic Analysis" (BIAcore 3000 Instrument handbook) was used, which included the steps "concentration series,""baseline trends," "mass transfer control experiments," and "linked reactions control experiments." The analyte was injected and allowed to interact with the immobilized ligand until the interaction reached equilibrium. At this time the injection of analyte was stopped and buffer was injected to monitor the dissociation phase of the interaction. The data of the BIAcore sensorgrams were first fitted globally and then, locally using different kinetic models of the BIAevaluation software version 3.0, each according to the manufacturer's manual. The results reported here were obtained with locally fitted data using the one step biomolecular association reaction model (1:1 Langmuir model: A + B {leftrightarrow} AB), which resulted in optimum mathematical fits reflected by the lowest Chi values.

Eukaryotic Cell Adherence and Internalization and Determination of Eukaryotic Cell Viability—Assessment of eukaryotic cell adherence and internalization was determined by an antibiotic protection assays and in parallel, by a double immunostaining technique according to the protocols of Molinari et al. (39).

Briefly, for the antibiotic protection assay early stationary phase bacteria were suspended in modified Eagle's medium supplemented with 10% fetal calf serum and were added to HEp-2 cells grown overnight to confluence, establishing a multiplicity of infection of 25. Previous studies indicated this multiplicity of infection to be in the linear range of such experiments. After 2 h, the eukaryotic cells were washed with PBS. One-half of the cells were lysed with distilled water, and the number of bacteria contained in the lysate was assessed by viable counts. The other half of eukaryotic cells was exposed to culture medium supplemented with penicillin and gentamicin for another 2 h. Then these cells were washed and lysed, and the bacterial numbers were counted as above.

Briefly, for the double immunostaining technique, early stationary phase bacteria were added to HEp-2 cells as described above. After 1 h of co-incubation, the eukaryotic cells were washed and fixed with 3% paraformaldehyde for 15 min at 4 °C. Cell monolayers were blocked for 30 min at room temperature with 1% fetal bovine serum/PBS. Subsequently, adherent bacteria were labeled with a 1:5000 diluted rabbit anti-GAS-antibody (Biodesign) and then incubated with an 1:500 diluted goat anti-rabbit-IgG-AlexaFluor 488 conjugate (Molecular Probes, MoBiTec, Göttingen, Germany). Eukaryotic cell membranes were permeabilized by exposure to Triton-X, washed, and again incubated with the anti-GAS-antibody. Then, a goat anti-rabbit IgG-AlexaFluor 647 conjugate (Molecular Probes) was added. For counterstaining of the eukaryotic cell nuclei, the stain 4',6-diamidino-2-phenylindole was used. Finally, the immunostaining results were visualized by fluorescence microscopy at 600- or 1000-fold magnification (see above) and quantitatively assessed by counting extracellular (light gray), cell membrane transmigrating (light and dark gray), and intracellular (dark gray) bacterial chains in at least 10 microscopic fields per assay.

The results are presented as the mean of four independent experiments. The significance of measured differences was determined by the Mann-Whitney U test. For assessment of a specific inhibitory activity, anti-protein F2 antisera were used in 1:5, 1:10, and 1:100 dilutions for pre-incubating the late exponential phase GAS wild type bacteria at room temperature for 1 h and washing the cells before continuing the experiments as outlined for the antibiotic protection assay.

The Live/Dead stain (Molecular Probes, MoBiTec) was used according to the manufacturer's instructions to determine the eukaryotic cell viability in the presence of GAS strains. As in the double immunostaining approach, cells were observed at 600-fold magnification. At least nine microscopic fields containing a minimum of 50 cells were examined in each assay. The results were assembled from three independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Transcription Profile of prtF2 As Determined by Luciferase Reporter Gene Activity—Utilizing Northern blot hybridizations, the transcription rate of the prtF2 gene was found to be very low (33). Thus, based on this method, an assessment of the temporal prtF2 transcription profile was not achievable.

The luciferase reporter box fusions have been successfully employed in GAS for sensitive monitoring of transcriptional up- and down-regulation of regulator genes expressed at very low levels (33). Therefore, the luc reporter box was introduced downstream of the prtF2 gene to generate a transcriptional fusion in the GAS wild type strain (Fig. 1) and the corresponding nra regulator mutant. As in the previous publications, the genomic integration of the luc reporter box did not affect the growth kinetics of the mutant strain under any experimental condition as compared with the corresponding wild type strain (data not shown).

According to the measured luciferase activities in THY medium, which contains a variety of ill defined bovine protein breakdown products, the temporal transcription pattern of prtF2 shows a linear up-regulation during the exponential phase and a maximum at the transition to the stationary growth phase (Fig. 2A). This maximum is followed by a sudden down-regulation in the early stationary growth phase.



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  		FIG. 2.
Transcription profile of the prtF2 gene according to luciferase reporter gene activities. A, prtF2 transcription in bacteria grown in THY broth under different atmospheric conditions; B, prtF2 transcription in wild type and nra mutant bacteria grown in THY broth under an anaerobic atmosphere. For clarity reasons of panel B, the A600 and RLU curves are shown in separate graphs. RLU, relative light units; THY, Todd Hewitt broth; A600, optical density measured at 600-nm wavelength; and h, incubation periods measured in hours. The curves were taken as representative from four individual assays.

 
Exposure to diverse atmospheric conditions revealed an induction of prtF2 transcription by ambient air (Fig. 2A). At generally lower prtF2 transcription levels, little difference was found between CO2-enriched and anaerobic growth conditions (Fig. 2A). Of note, the aerobic incubation imposes exceptional stress on catalase-negative GAS, which results in a delayed growth curve and simultaneously, a delayed up-regulation of prtF2 transcription.

Incubation in CDM broth leads to a slightly reduced prtF2 transcription maximum as compared with incubation in THY (data not shown). Supplementation of CDM with 0.2–2 mg/liter fibronectin appears to be insignificant for the temporal profile and strength of prtF2 transcription (data not shown).

Consistent with previous Northern blot hybridization data (33), prtF2 transcription was about 2- to 3-fold increased in the nra regulator mutant strain, however only when incubated to the transition phase under an anaerobic atmosphere (Fig. 2B). Exposure of the nra mutant to fibronectin-supplemented CDM had no effect on the prtF2 transcription rate. Also, the presence of spent eukaryotic cell culture supernatant and the contents of lysed HEp-2 cells had no consistent effect on prtF2 expression irrespective of the presence or absence of a functional Nra regulator (data not shown).

Binding of Recombinant Protein F2 Fragments to Fibronectin—Without assessing kinetic data, protein F2 fragments have previously been tested for their binding to soluble fibronectin (Fn) and a 29-kDa N-terminal Fn fragment (22). Since then, a cooperative effect of the two C-terminal domains of immobilized protein F1 has been demonstrated to be involved in Fn binding of the protein and in host cell internalization of whole bacteria (14, 16, 17, 40). Therefore, in the present study immobilized recombinant fragments of protein F2 representing the N- and C-terminal portions as well as the previously identified Fn-binding domains (Fig. 1) were quantitatively measured for their specific reactivity with whole and fragmented Fn.

In a first step, the recombinant proteins were subjected to SDS-PAGE and Western blotting, followed by exposure to monomeric whole Fn and its N-terminal 30-, 45-, and 70-kDa fragments. Consistent with the data from Jaffe et al. (22), whole Fn, and its 70- and 30-kDa N-terminal fragments bound to the C-terminal portion and the two Fn-binding domains of protein F2. Unlike protein F1, the 45-kDa sub-N-terminal Fn fragment showed little to no reactivity with the blotted protein F2 fragments (Fig. 3A).

In a second step, the binding strength of the protein F2 fragments to immobilized Fn and its N-terminal fragments was quantified by surface plasmon resonance (SPR) measurements employing the BIAcore system. For this real-time bio-specific interaction analysis, the protein F2 N-terminal (F2-NT) and C-terminal (F2-FnB) portions as well as the unique (F2-UFbD) and repeated (F2-repeat) fibronectin-binding domains were used as soluble analytes and whole fibronectin and several N-terminal fragments as immobilized ligands (Fig. 3B).

Consistent with the Western immunoblot data, the N-terminal protein F2 portion did not bind to any fibronectin fragment (Fig. 3B). Opposed to the non-binding N terminus, the C-terminal protein F2 fragments displayed significant binding to fibronectin and its 70- and 30-kDa N-terminal fragments (Fig. 3B). To calculate Ka- and KD-equilibrium constants, a kinetic analysis was performed. Increasing concentrations of the analyte were allowed to associate with the different fibronectin fragments immobilized as ligands on CM5 chips. After the interaction reached equilibrium, the dissociation phase was monitored by allowing buffer to flow over the chips. The sensorgrams of this interaction were recorded (data not shown), and the data obtained were used for diverse calculations employing kinetic models as suggested by the manufacturer of the BIAcore system. Between the various kinetic models the resulting Ka- and KD-equilibrium constants varied within a maximum range of factor 2. Because the first order kinetic equations corresponded to the observed single site binding in the fibronectin molecule (see below) were in agreement with a former BIAcore-based analysis on protein F1 interaction with fibronectin (41), and led to the most consistent inter-assay results, this type of kinetics was used to calculate the results presented in Table I.


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  		TABLE I
Kinetic data of protein F2-fibronectin interactions as determined by surface plasmon resonance analysis

The association and dissociation data of the interaction were fitted locally using the one step biomolecular reaction model (1:1 Langmuir model: A + B AB), which resulted in optimum mathematical fits reflected by the lowest Chi values. The values for association rate (ka), dissociation rate (kd), association constant (KA), and dissociation constant (KD) were calculated from the binding data with the BIAevaluation software.

 
The association and dissociation constants for the binding of protein F2 fragments to fibronectin were in the range of 108 M–1 and 10–9 M, respectively (Table I), consistent with a high affinity for the binding interaction, which is in the typical range for such a biological function. The rate constant for the protein F2 repeat domain displayed an about 10-fold higher association rate with fibronectin, and a reduced stability of about 10-fold of the formed complex than the unique binding domain of protein F2. Thus, the binding affinity recorded for the F2-FnB portion predominantly relies on the activity of the repeat domain (Table I). In general, the dissociation rate constant (KD), which is in the 10–4 range, suggested a slow dissociation of the protein F2-fibronectin complexes. This is also reflected by the poorly visible dissociation phase in Fig. 3B. When assessing the importance of different fibronectin fragments for protein F2 attachment, the Ka and KD values for the interactions of the F2-FnB, F2-UFbD, and F2-repeat fragments with the N-terminal 70- and 30-kDa fibronectin fragments were in the same range as the values recorded for full sized fibronectin (Table I).

Consistent with the Western immunoblot results, none of the tested protein F2 domains displayed detectable binding to the 45-kDa fibronectin fragment irrespective of using the CM5 sensor chip (45-kDa fibronectin fragment as ligand, Fig. 3B) or the NTA sensor chip (F2-FnB as ligand, data not shown). As for the reactivity toward whole fibronectin, the Ka and KD values for the interaction between the protein F2 repeat domain and the N-terminal fibronectin fragments demonstrated an at least five times higher affinity and lesser dissociation rate than the corresponding data for the unique domain of protein F2 (Table I).

Interaction of GAS Strains with Immobilized Fibronectin—The results from the Western immunoblot and BIAcore analyses clearly showed a reversible attachment of protein F2 to fibronectin, which, concerning its strength and endurance, could well be within a biologically relevant range. In the next set of experiments, we intended to test this prediction by measuring the attachment of whole GAS cells to immobilized fibronectin.

At first, expression and anchoring of protein F2 on the bacterial surface was demonstrated using the anti-protein F2N and C terminus antisera and immunofluorescence microscopy (Fig. 4). Then, a strain with an insertion mutation in its prtF2 gene was created, which should not be able to anchor the C-terminally truncated protein F2 into its cell wall. The growth kinetics and viability of this mutant were identical with the corresponding parameters of the wild type strain (data not shown).



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  		FIG. 4.
Immunofluorescence detection of protein F2 localized on the bacterial surface. Left panel, result of GAS incubation in pre-immune serum. Right panel, result of GAS incubation in anti-F2-FnB antiserum. Visualization was achieved by exposure to a fluorescein isothiocyanate-labeled anti-rabbit-IgG antiserum and fluorescence microscopy at a 600-fold magnification.

 
The effect of the prtF2 mutation on growth-phase dependent protein F2 deposition in the cell wall was then assessed using peroxidase-coupled soluble fibronectin for the quantification of surface-expressed protein F2 and other fibronectin-binding GAS proteins (Fig. 5). In wild type bacteria, a constant basic level of fibronectin-binding was recorded. In addition, a narrow peak of increased fibronectin binding was identified at the transition from exponential to stationary growth phase (A600 nm 1.3, Fig. 5). In the prtF2 mutant, the binding of soluble fibronectin was generally lowered by a factor 2, and no peak of binding activity was observed.



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  		FIG. 5.
Binding of HRP-labeled fibronectin to wild type and prtF2 mutant throughout the growth cycle. Bacteria were investigated from early exponential phase (A600 nm 0.2), mid-exponential phase (A600 nm 0.4), late exponential phase (A600 nm 1.0), early stationary phase (A600 nm 1.3), mid stationary phase (A600 nm 1.3 plus two additional hours of incubation), and late stationary phase (24 h of incubation). Binding of soluble fibronectin was recorded as picograms of fibronectin per 106 colony forming units (cfu) of bacteria. Data represent the means and standard deviation of three independent experiments. Bars marked with asterisks represent results that are significantly different compared with prtF2 mutant binding values.

 
For the following series of experiments, the wild type and prtF2 mutant GAS strains were grown to the early stationary phase, i.e. the growth phase in which the behavior toward fibronectin should show maximum differences between wild type and mutant bacteria (Fig. 5). When testing the binding of both strains to immobilized full-sized fibronectin, the prtF2 mutant displayed statistically significant 6-fold decreased values (Fig. 6). To test the specificity of this attachment, the wells with the immobilized fibronectin were preincubated with recombinant protein F2 fragments. Although the N-terminal fragment showed an insignificant inhibitory effect, the C-terminal fragments interfered with the binding of wild type GAS to fibronectin. The range of attachment reduction was between 40 and 80%, which was statistically significant for each of the three recombinant fragments. Consistent with their Ka and KD values for fibronectin interaction, the inhibitory effect was more prominent for the F2-FnB and F2-repeat fragments than for the F2-UFbD fragment (Fig. 6).



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  		FIG. 6.
Adherence of wt and prtF2 mutant to immobilized fibronectin and competitive inhibition of the wt-fibronectin interaction by recombinant protein F2 domains. Adherent bacteria were quantified employing an enzyme immunoassay format and a peroxidase-conjugated secondary antibody. For details of the assay please refer to "Experimental Procedures." For competitive inhibition experiments 10 µg/well F2-NT, F2-FnB, F2-UFbD, and F2-repeat domains were preincubated with the immobilized fibronectin prior to usage of wild type bacteria for the adherence assay. Data represent the means and standard deviation of three independent experiments. Bars marked with asterisks represent results that are significantly different compared with wild type adherence values.

 
Role of Protein F2 for Interactions between GAS and Eukaryotic Cells—In some GAS serotype strains, the presence of the functionally related protein F1 has been demonstrated to be a prerequisite or at least, in combination with the surface exposed M protein, a major factor for GAS adherence to and internalization into eukaryotic cells (1). These functions were shown to be conclusively associated with the fibronectin-binding activity of protein F1.

The serotype M49 strain of the present investigation does not carry a prtF1/sfbI gene, and its M protein makes little contribution to the capability of the strain to adhere to eukaryotic cells.2 Because the previous results established the fibronectin binding of the serotype M49 strain to significantly rely on its protein F2 expression, testing the impact of protein F2 on the GAS interaction with eukaryotic cells would be a logical consequence.

The serotype M49 wild type strain and the isogenic prtF2 mutant were used for eukaryotic cell attachment and internalization assays. Employing the antibiotic protection assay and subsequent viable counts, the mutant displayed a 17-fold decreased epithelial cell adherence rate and a similarly reduced internalization rate (Fig. 7, A and C). However, this assay at best allows for an initial screen. Because the results of this assay depend on many variables in addition to the activity of adhesins (39), a more reliable alternative approach, i.e. double immunofluorescence staining, was used to confirm this data. Here, compared with the corresponding wt data, the prtF2 mutant showed attachment and internalization rates reduced by the factors 2 and 5, respectively (Fig. 8 and Table II).



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  		FIG. 7.
Protein F2-dependent adherence to and internalization into HEp-2 cells of GAS wild type and prtF2 mutant strains as measured by antibiotic protection assays and viable counts. A, eukaryotic cell adherence rates of the wt and prtF2 mutant bacteria as well as inhibition of wt bacterial adherence to HEp-2 cells using recombinant protein F2 domains. The HEp-2 cell monolayers were preincubated with the indicated amounts of protein F2 domains prior to incubation with bacteria. B, inhibition of wt HEp-2 cell adherence by preincubation of the bacteria with anti-protein F2 antiserum. C and D, bacterial internalization rates with the same experimental setting as shown in A and B. Data represent the means and standard deviation of three independent experiments. The asterisks mark data with statistical significance.

 



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  		FIG. 8.
Representative pictures of GAS wild type and prtF2 mutant strain HEp-2 cell adherence and internalization as detected by double-immunofluorescence microscopy. Adherent GAS chains are presented in light gray, completely internalized chains are presented in dark gray, chains in transition are partially dark gray and partially light gray. Only the nuclei of the eubaryotic cells are visible because of the DAPI stain used in this assay. For the details of the staining procedure please refer to the "Experimental Procedures."

 


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  		TABLE II
Adherence and internalization rates of GAS wild type and prtF2 mutant strains as determined by double immunofluorescence staining

Data represent mean values and standard deviation of three independent assays, with nine microscopic fields counted per experiment.

 
A discrepancy between results obtained by the two methods for measuring GAS-eukaryotic cell interactions could be related to altered host cell viability. When testing this option with the Live/Dead stain and fluorescence microscopy the viability count of HEp-2 cells after 3- and 5-h co-incubation with the wt and the prtF2 mutant bacteria resulted in 10–30% more viable cells after exposure to the prtF2 mutant (data not shown).

The specific influence of protein F2 on these functional parameters was tested by two additional sets of experiments. First, the eukaryotic cells were preincubated with the protein F2 N- and C-terminal fragments at concentrations between 1 and 100 µg/well. The presence of both the C- and N-terminal fragments led to a dose-dependent reduction of attachment and internalization up to 60 and 20% of the reference values, respectively (Fig. 7, A and C).

Second, antiserum directed to the C terminus of protein F2 was added to the GAS bacteria before exposing them to the eukaryotic cells. Again, a significant and dose-dependent reduction of both GAS cell attachment and internalization was recorded, reaching figures as low as 40% of the reference values (Fig. 7, B and D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The specific adhesion of GAS to fibronectin via protein F1/SfbI has been thoroughly characterized. For some GAS strains, this function was shown to be a prerequisite for the development of purulent diseases or intracellular persistence. However, between 10 and 50% of the GAS clinical isolates do not carry the corresponding prtF1/sfbI gene, but instead, several other genes encoding fibronectin-binding proteins such as protein F2, Fbp54, FbaA, SOF, and SfbX.

Using a representative strain from the prtF1/sfbI-negative group, the major objectives of the present investigation were to experimentally assess the affinity constants for the protein F2/fibronectin interaction in detail, to demonstrate the biological impact of this interaction, and to extrapolate the significance of protein F2 in the pathogenetic process by recording its temporal and environmentally dependent expression profile. Although protein F2 was previously shown to be a fibronectin-binding MSCRAMM (22), it was not clear which of its portions predominantly contributed to this activity and whether the binding would support GAS adhesion to eukaryotic target cells or, similar to protein F1/SfbI functions, an additional GAS internalization into host cells.

Utilizing recombinant protein F2 fragments and whole or partial fibronectin molecules for an analysis of their interactions, we could demonstrate by results from Western immunoblots and real-time biospecific interaction analysis (SPR, BIAcore system) that the repeat domain of protein F2 is more important for the fibronectin binding. Its apparent association and dissociation equilibrium constants indicate an affinity for full-sized fibronectin that exceeds that of the unique binding domain by one order of magnitude. For both domains, the fibronectin binding is reversible. Consistent with the higher affinity of the repeat domain, it displays also a lower tendency for dissociation from its ligand. Although decreasing with the size of the fibronectin target fragment, the differences in affinity and dissociation can also be observed for the interaction with the N-terminal 70- and 30-kDa fibronectin portions. Clearly unlike the situation in protein F1/SfbI (14–16, 18), both protein F2 fibronectin-binding domains do not interact with the subterminal 45-kDa fibronectin fragment.

The values of the association and dissociation equilibrium constants of both protein F2 binding domains are in the nanomolar range that has also been described for other relevant MSCRAMMs of Gram-positive cocci and their binding to human matrix proteins (41–43). This suggests a similar importance for the interaction between protein F2 and fibronectin.

After confirming protein F2 surface expression by employing an immunofluorescence assay and assessing the bacterial growth phase in which maximum amounts of protein F2 were deposited on the GAS surface, whole wild type and prtF2 mutant bacteria were measured for their binding to immobilized fibronectin. Consistent with the protein F2/fibronectin equilibrium constants, the attachment of the bacteria to the coated surfaces was shown to predominantly rely on the presence of protein F2. This finding was in complete agreement with the results of Jaffe et al. (22), who measured the binding of their prtF2 mutant to soluble fibronectin, and Terao et al. (24), who tested mutants of the structurally related FbaB protein in serotypes M3 and M18 strains. However, in GAS strains of these two serotypes, genes for other fibronectin-binding proteins such as FbaA (44) or SfbX (45) are not contained in the genomes. Thus, our results indicate a major contribution of protein F2 for fibronectin binding in protein F1/SfbI-negative GAS strains irrespective of the genomic context. Because the kinetic parameters from the two abovementioned fibronectin-binding proteins have not been measured, it is unclear whether protein F2 is the only relevant fibronectin-binding protein in such strains or whether the other proteins display a more important adhesin function under growth conditions that differ from the experimental setting of the present investigation.

Up to this point, the functions of protein F1/SfbI and protein F2 were similar, but not identical. When co-incubating our protein F1/SfbI-negative and protein F2-positive wild type bacteria and the isogenic prtF2 mutant with cultured cells of the epithelial HEp-2 cell line, this pattern of functional similarity but not identity of the two proteins was confirmed. According to the double-immunofluorescence microscopy analysis the protein F2-deficient mutant displayed a decreased attachment to the eukaryotic cells above the range of the diminished fibronectin binding. However, because the numbers of intracellular prtF2 mutant bacteria were about 5-fold lower as compared with the intracellular count of wild type bacteria, protein F2 could additionally and selectively contribute to the internalization of the bacteria into the host cells. Because protein F2 exhibited almost no reactivity toward the subterminal 45-kDa fibronectin portion, the protein F2-dependent internalization pathway obviously differs from the protein F1/SfbI-driven mechanism, which was shown to involve a sequential interaction between the unique and the repeat binding domains of protein F1/SfbI and the 30- and 45-kDa domains of fibronectin (14–16, 46).

The presence of protein F2 could additionally contribute to the intracellular persistence of GAS. Similar to observations with prtF1/sfbI-deficient mutants, eukaryotic cells exposed to prtF2 mutants displayed 10–30% higher numbers of viable cells. In the case of the prtF1/sfbI-related experiments, it was argued that expression of this surface protein led to strong adherence and high internalization rates, consequently, induction of host cell apoptosis occurs (47). Judging from our results, a similar role for protein F2 in apoptosis induction appears to be possible. However, we did not measure apoptosis-related proteins directly.

According to results of the luc reporter gene assays and the quantification of surface-deposited protein F2, the profiles for maximum transcription of the prtF2 gene and peak expression of fibronectin-binding proteins do exactly match. Judged from the different levels for soluble fibronectin in the wild type and prtF2 mutant, and consistent with the attachment experiments to immobilized fibronectin, the binding of this soluble matrix protein predominantly relied on the presence of mature, surface-exposed protein F2. The comparatively narrow peak of maximum fibronectin binding at the transition to stationary phase and the stationary phase decrease back to levels of the early and mid-exponential phase indicate a rapid turnover caused by shedding from the surface or loss of function of surface-deposited protein F2. In most growth phases except the transition to stationary phase, other fibronectin-binding proteins like Fbp54, FbaA, SOF, and SfbX (1, 44, 45) may contribute in a major way to GAS attachment to soluble but not to immobilized fibronectin.

Based on only a few measurements during the growth cycle, a small number of other fibronectin-binding proteins have also been demonstrated to reach their maximum expression in late exponential or early stationary phase (22, 33, 44, 45, 48–50). The biological benefit of this expression profile for the GAS is only partially clear. During a purulent infection, GAS strains show a high multiplication rate most probably because they are exposed to a nutrient-rich environment. Thus, expression of specific adhesins could guarantee such strains to remain in such a favorable neighborhood. The peak expression of the predominant fibronectin-binding protein at the transition phase could indicate that a low multiplication rate is in fact the preferable status for the bacteria to express MSCRAMMs. Once the bacteria have used up their nutrient supplies, the stationary phase begins in which adhesion may not be beneficial any longer. In this phase, secreted proteases like SpeB are produced (51) and cleave some GAS surface proteins potentially to increase the bacterial mobility (52, 53). A processing of protein F2 in the stationary phase as indicated by results of the present investigation, would be consistent with this scenario.

In agreement with the indirect observations of Jaffe et al. (22) on binding of soluble fibronectin to whole bacteria, prtF2 transcription was found to be stimulated by aerobic growth conditions, which in turn would correspond to the postulated function of protein F2 as an adhesin on superficial host tissues. This transcription pattern is not due to the activity of the Nra-negative regulator (33), which was now found to primarily act under anaerobic growth conditions on prtF2 transcription. Because the GAS group in general encodes more than a dozen two-component regulators (31, 54, 55) and especially the serotype M49 GAS strain harbors an additional putative regulator beside Nra within its fibronectin-binding/collagen-binding/T-antigen island (29, 33), the prtF2 gene could be under control of several different regulators, which in turn could be responsible for its increased expression under ambient air.

According to the similar prtF2 transcription results obtained when incubating the bacteria in two different broths or in absence or presence of fibronectin or HEp-2 cell products, prtF2 gene expression does not appear to be influenced by animal tissue-derived factors and, especially, by its cognate receptor on the eukaryotic cell surface. So there are regulatory circuits that affect prtF2 transcription, but the corresponding signals have not been identified under the experimental conditions of this study.

In conclusion, the protein F2 MSCRAMM was shown to be the predominant adhesin and internalin of GAS strains that do not harbor the gene for the protein F1/SfbI adhesin and internalin. Its expression profile indicates a special function for the pathogenesis of superficial GAS-associated diseases potentially during earlier, i.e. adhesion, and later, i.e. internalization, stages of such processes. The specific protein F2-dependent mechanism for eukaryotic cell internalization, which obviously differs from the protein F1/SfbI-dependent pathway, and the molecules of the host environment that stimulate its expression need to be elucidated for a better understanding of pathogenesis of diseases associated with the subset of protein F2-positive and protein F1/SfbI-negative GAS strains.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grants Po391/11-1 and Kr 1765/2-1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

|| To whom correspondence should be addressed. Tel.: 49-381-494-5900; Fax: 49-81-494-5902; E-mail: andreas.podbielski{at}med.unirostock.de.

1 The abbreviations used are: GAS, group A streptococci/streptococcal; CDM, chemically defined medium; cfu, colony forming unit(s); Fn, fibronectin; F2-FnB, protein F2 C-terminal fibronectin-binding portion; F2-Nterm, protein F2 N-terminal portion; F2-UFbD, protein F2 unique fibronectin-binding domain; F2-repeat, protein F2 fibronectin-binding repeat domain; HRP, horseradish peroxidase; NTA, nitrilotriacetic acid; PBS, phosphate-buffered saline; prtF, protein F gene; THY, Todd-Hewitt-Yeast culture medium; aa, amino acid(s); MSCRAMM, microbial surface components recognizing adhesive matrix molecule; wt, wild type. Back

2 A. Podbielski, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank R. Lütticken for providing the GAS strain and H. Kuramitsu for giving the pUC-erm plasmid. We especially acknowledge the expert technical assistance provided by B. Becziczka, Y. Humboldt, and J. Normann. In addition, we thank M. O. Glocker (Proteome Center Rostock) for granting access to the BIAcore system.



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 DISCUSSION
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