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Originally published In Press as doi:10.1074/jbc.M706739200 on January 7, 2008

J. Biol. Chem., Vol. 283, Issue 10, 6359-6366, March 7, 2008
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Opacity Factor Activity and Epithelial Cell Binding by the Serum Opacity Factor Protein of Streptococcus pyogenes Are Functionally Discrete*Formula

Christine M. Gillen{ddagger}1, Harry S. Courtney§2, Kai Schulze, Manfred Rohde||, Mark R. Wilson{ddagger}, Anjuli M. Timmer**, Carlos A. Guzman, Victor Nizet**, G. S. Chhatwal||, and Mark J. Walker{ddagger}3

From the {ddagger}School of Biological Sciences, University of Wollongong, New South Wales 2522, Australia, the §Veterans Affairs Medical Center and Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38104, the Department of Vaccinology, Helmholtz Centre for Infection Research, Braunschweig, Germany, the ||Department of Microbial Pathogenicity, Helmholtz Centre for Infection Research, Braunschweig, Germany D-38124, and the **Department of Pediatrics, Division of Pharmacology and Drug Discovery, University of California, San Diego, La Jolla, California 92093-0687

Received for publication, August 14, 2007 , and in revised form, November 26, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Serum opacity factor (SOF) is a unique multifunctional virulence determinant expressed at the surface of Streptococcus pyogenes and has been shown to elicit protective immunity against GAS infection in a murine challenge model. SOF consists of two distinct domains with different binding capacities: an N-terminal domain that binds apolipoprotein AI and a C-terminal repeat domain that binds fibronectin and fibrinogen. The capacity of SOF to opacify serum by disrupting the structure of high density lipoproteins may preclude its use as a vaccine antigen in humans. This study generated mutant forms of recombinant SOF with reduced (100-fold) or abrogated opacity factor (OF) activity, for use as vaccine antigens. However, alterations introduced into the N-terminal SOF peptide (SOF{Delta}Fn) by mutagenesis to abrogate OF activity, abolish the capacity of SOF to protect against lethal systemic S. pyogenes challenge in a murine model. Mutant forms of purified SOF{Delta}Fn peptide were also used to assess the contribution of OF activity to the pathogenic processes of cell adhesion and cell invasion. Using latex beads coated with full-length SOF, SOF{Delta}Fn peptide, or a peptide encompassing the C-terminal repeats (FnBD), we demonstrate that adhesion to HEp-2 cells is mediated by both SOF{Delta}Fn and FnBD. The HEp-2 cell binding displayed by the N-terminal SOF{Delta}Fn peptide is independent of OF activity. We demonstrate that while the N terminus of SOF does not directly mediate intracellular uptake by epithelial cells, this domain enhances epithelial cell uptake mediated by full-length SOF, in comparison to the FnBD alone.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptococcus pyogenes (group A streptococcus, GAS)4 is an important human pathogen responsible for a wide variety of skin and mucosal infections ranging from pharyngitis and impetigo to more severe invasive infections, such as necrotizing fasciitis and streptococcal toxic shock-like syndrome (1-3). The serum opacity factor (SOF) is a large protein of ~110 kDa, which is expressed at the cell surface by approximately half of all clinical isolates (4, 5). Similar to a number of other surface proteins expressed by S. pyogenes, SOF binds fibronectin via a C-terminal-repeated domain (FnBD) (6-8), a function that has been implicated in the adhesion of GAS to epithelial cells (9). In contrast to the conserved C terminus, the N terminus of SOF (SOF{Delta}Fn) is highly variable, exhibiting ~55% identity between different serotypes of S. pyogenes. The N-terminal domain of SOF was originally thought to cleave apolipoprotein A1 (apoAI) in human serum leading to the precipitation of high density lipoproteins (HDLs) (10, 11). However, it has recently been demonstrated that the OF activity of SOF is not enzymatic; rather, the direct binding of apoAI by SOF triggers the release of the HDL lipid cargo of apoAI, initiating the opacity reaction (12). This OF domain of SOF promotes GAS invasion of epithelial cells (13), but it is not known whether the OF activity itself or a discrete domain within the N terminus of SOF contributes to this phenotype. SOF is a virulence determinant of GAS, with insertional inactivation or allelic replacement of sof reducing mortality in an intraperitoneal and subcutaneous murine infection model (13, 14). SOF is also a vaccine candidate, parenteral immunization of mice with SOF protects against lethal intraperitoneal challenge (15).

It is not known what physiological effect that the precipitation of HDL would have upon the human host or how the interaction of SOF with apoAI contributes to the pathogenesis of GAS. ApoAI exerts a potent anti-inflammatory effect by preventing contact between infected T-cells and monocytes, thereby inhibiting cytokine production (namely tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-1) in monocytes (16). In vitro, both HDL and apoAI exert anti-inflammatory effects against the potent bacterial endotoxins, Gram-negative LPS and Gram-positive lipoteichoic acid, binding strongly to both endotoxins and inhibiting the production of TNF-{alpha} (17-19). In vivo, transgenic animal studies of the toxicity of LPS have shown that expression of human apoAI transgenes protected mice from a lethal dose of LPS (20). ApoAI also possesses specific anti-bacterial and anti-viral properties (21-23).

The administration of active SOF protein as a vaccine when the downstream effects of disrupting HDL and its activity in vivo are unknown would not be recommended, as the most effective protection against GAS infection is delivered when the SOF protein is administered parenterally (15), and the localized depletion of HDL may reduce the body's defense against other pathogens or may result in inflammation at the site of immunization. Thus, before SOF could be used as a potential vaccine antigen (alone or as part of a multivalent vaccine formulation) it would be prudent to eliminate the OF activity of the protein. To this end, this study generated mutant forms of recombinant SOF protein with attenuated or eliminated OF activity, for use as vaccine formulations. These mutant SOF proteins have also been used to further delineate OF activity and cell binding activity within the N terminus of SOF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis—A pQE30-based vector encoding a fusion protein of residues 33-872 of SOF from a M75 GAS strain (lacking signal sequence and fibronectin binding repeat region, pSOF75{Delta}Fn) (24), was used as the template for all mutagenesis reactions. Site-directed mutagenesis was performed as previously described (25). Primers for site-directed mutagenesis are given in supplemental Table S1. Two mutagenesis strategies were employed, amino acid residues were substituted with alanine (single residues up to 5 residues) and small deletions were made within rSOF75{Delta}Fn. Deletions within rSOF75{Delta}Fn were generated by using site-directed mutagenesis to introduce two AvrII restriction sites flanking the region to be deleted, followed by digestion with AvrII. The digested fragments were then separated by agarose gel electrophoresis, and the DNA fragment containing the portion of pSOF75{Delta}Fn of interest was extracted from the gel and re-ligated.

Expression and Purification of Wild Type and Mutant Forms of rSOF75 and rSOF75{Delta}Fn Protein—Large scale expression and purification of rSOF75 proteins was conducted essentially according to the manufacturer's instructions (Qiagen), and has been previously described (13, 26). To ensure correct refolding of proteins was achieved, wild type purified proteins were subsequently tested for opacity factor activity using an agarose overlay method (7).

Structural Characterization of Wild Type and Mutant Forms of rSOF75{Delta}Fn Protein—To determine the structural integrity of mutant rSOF75{Delta}Fn proteins in comparison to the wild type, a comparison of the secondary structure of wild type and mutant rSOF75{Delta}Fn proteins was conducted using circular dichroism (CD) spectroscopy. CD spectra were acquired using a Jasco J-810 Spectropolarimeter (Jasco). Experiments were conducted at room temperature with proteins at a concentration of ~0.2 mg/ml in 10 mM sodium phosphate buffer, pH 7.5 containing 50% trifluoroethanol (27, 28). Far UV spectra were recorded from 190-250 nm in a 0.1-cm pathlength cell (Starna) containing 400 µl of protein solution. The data shown represents an average of ten scans, corrected for a buffer baseline. Mean residue ellipticity (MRE; [{theta}]) was calculated using Equation 1 (29).

Formula 1(Eq. 1)
The {alpha}-helical content of wild type and mutant forms of rSOF75{Delta}Fn was calculated from the MRE value at 222 nm using Equation 2 as described by Ref. 30.

Formula 2(Eq. 2)

Opacity Activity Assays—Qualitative opacity factor assays were conducted using the serum agarose overlay method (7). The serum overlay method permits visual confirmation of OF activity, as binding of apoA1 by SOF causes precipitation of apoAI and HDL, which appears as an opaque white band on the solid serum/agarose medium. Data are presented as an inversion of the actual blot with opacity activity appearing as a dark band on a light background. Quantitative opacity factor assays were conducted using purified HDL or human serum using the method of Courtney et al. (12), with opacification measured as absorbance at 405 nm.

ApoAI Binding Capacity—Wells of a microtiter plate were coated with 20 µg/ml rSOF75 protein or gelatin in 0.01 M sodium bicarbonate for 1 h at 37 °C. Plates were washed with PBS and blocked with gelatin (1 mg/ml in PBS) for 1 h at 37 °C, 100 µl of biotinylated (Pierce) apoAI (Calbiochem) was added and incubated at 37 °C for 1 h. Following washing, 100 µl of avidin-peroxidase (0.5 µg/ml) was added to the wells and incubated at 37 °C for 1 h. The plates were then washed and 3,3'-5,5'-tetramethylbenzidine substrate added. Color development was stopped using 1 M phosphoric acid, and the absorbance at 450 nm was recorded. Assays were performed in triplicate.

Interaction of SOF-coated Latex Beads with HEp-2 Cells—Assays of the interaction of SOF-coated latex beads with HEp-2 cells were conducted per previously published methods (13, 31, 32). Preliminary assays were conducted in Dulbecco's modified Eagle's medium HEPES supplemented with either 10% FCS or 1% FCS. However, differential binding was observed between these two assay conditions, the rSOF75{Delta}Fn only mediated binding to HEp-2 cells when incubated in DMEM HEPES 1% FCS, and thus these conditions were used for all further latex bead adherence assays. The efficiency of protein loading onto latex beads was measured by FLUOstar fluorescent plate reader (BMG Labtech) using anti-SOF75 rabbit serum and fluorescent labeling with goat anti-rabbit Alexa 488 (green) (Molecular Probes) (data not shown), protein loading efficiency was found to be comparable for all protein domains. To determine the effect of exogenous addition of the rFNBD domain on the adhesion and internalization of rSOF{Delta}Fn-coated latex beads, purified rFnBD at 1, 5, or 10 µg/ml was preincubated with the HEp-2 cells for 1 h prior to addition of the coated latex beads, and was maintained throughout the subsequent 4-h incubation with the latex beads.

Confocal Microscopy Studies—HEp-2 cells (after incubation with the coated latex beads) were fixed for 30 min on ice in 500 µl/well of prechilled 4% paraformaldehyde in PBS, and then washed twice in PBS. Cells were then blocked by the addition of 200 µl/well of PBS containing 10% fetal calf serum and incubated for 30 min at room temperature. The blocking solution was then removed, and the cells were incubated with either protein-G-purified rabbit polyclonal anti-SOF75 antibodies (26) (30 µg) or rabbit polyclonal anti-FnBD antibodies (1:100 dilution) for 45 min at room temperature. Following washing with PBS, cells were incubated with goat anti-rabbit Alexa 488 diluted 1:400 in PBS containing 10% BSA (Molecular Probes) for 1 h at room temperature and subsequently washed with PBS. Cells were permeabilized with 200 µl/well of 0.1% (v/v) Triton X-100 in PBS for 30 min on ice, washed in PBS, followed by storage at 4 °C overnight. The following day, cells were treated with goat anti-rabbit Alexa 633 diluted 1:400 in PBS containing 10% BSA (Molecular Probes) for 1 h and washed three times in PBS. Cells were then mounted onto a glass slide using Mowiol solution (Calbiochem). Images were recorded using a Leica TCS SP confocal microscope mounted on a Leica DM IRBE inverted microscope with Leica TCS NT software (Version 2.61; Leica Microsystems).

Mouse Immunization and Challenge—To determine the protective efficacy of rSOF75{Delta}Fn proteins challenge studies were performed. BALB/c mice (n = 10) were immunized subcutaneously with 25 µg of wild type or mutant forms of rSOF75{Delta}Fn protein in incomplete Freund's adjuvant. Control mice received a subcutaneous injection of PBS. After 2 weeks, the mice were boosted with an intramuscular injection of another 25 µg of each protein in PBS. Control mice received a PBS injection. Two weeks after the booster injections, all mice were challenged by an intraperitoneal injection of ~1 x 109 CFU of the SOF-positive M49 GAS strain 591 (33). The number of surviving mice was recorded daily. Moribund mice were sacrificed and recorded as dead.

Serum samples were collected on days 0 and 28, and stored at -20 °C prior to determination of rSOF75{Delta}Fn-specific antibodies. In brief, 96-well Nunc-Immuno MaxiSorp assay plates (Nunc) were coated with 2 µg/ml of wild type rSOF75{Delta}Fn in coating buffer (bicarbonate, pH 9.4). After overnight incubation at 4 °C, plates were blocked with 1% BSA in PBS (pH 7.4) for 1 h at 37 °C. Serial 2-fold dilutions of serum in PBS with 1% BSA were added (100 µl/well), and plates were incubated for 1 h at 37 °C. After four washes, secondary biotinylated antibodies were added followed by 1 h of incubation at 37 °C. After six washes, 50 µl/well of peroxidase-conjugated streptavidin (Pharmingen), diluted 1:1000, was added, and plates were further incubated for 45 min at room temperature. After a final six washes, the substrate ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) in 0.1 M citrate-phosphate buffer containing 0.1% H2O2 was added, and plates were incubated for 30-60 min at room temperature. The absorbance was measured at a wavelength of 405 nm.

Statistical Analyses—For apoAI binding experiments, an unpaired Student's t test was used to determine if there was any significant difference in the apoAI binding ability of wild type and mutant SOF proteins. For latex bead experiments, a one way analysis of variance using Bartlett's test for equal variance was used to determine whether there was any significant variation in the median number of beads attached to or taken up by HEp-2 cells, followed by a Tukey's Multiple Comparison Test for individual comparison of adherence and internalization mediated by two different proteins. For immunization and challenge experiments, a Kruskal-Wallis test was used to determine whether there was any significant variation in the median titers of the four groups of antisera. Dunn's Multiple Comparison test was used for individual comparison of two groups of antisera. Difference in survival curves was determined by log rank test. All statistics were performed using GraphPad Prism version 4.02 (Graph-Pad Software Inc., San Diego, CA).


Figure 1
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  		FIGURE 1.
The domain structure, opacity factor activity, and adherence phenotypes of SOF, rSOF75, rSOF75{Delta}FnWT, and the rSOF75{Delta}Fn mutants generated in this study. A, schematic representation of the SOF75 protein. SS, signal sequence; FnBD, fibronectin binding domain; MSD, membrane spanning domain. The domain mediating OF activity is indicated by a bracket. Boxes represent the domains encompassed by the rSOF75 proteins used in this study. B, opacity factor phenotype of wild type and mutant forms of the rSOF75{Delta}Fn peptide as indicated by the serum agarose overlay method.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SOF is a unique multifunctional protein, capable of binding fibronectin via a C-terminal domain designated FnBD (8, 24) and apoAI via an N-terminal domain (12) (Fig. 1A). The N-terminal domain of SOF (SOF{Delta}Fn) has been shown to mediate adhesion to HEp-2 cells and promote HEp-2 cell invasion by whole GAS cells. To assess the contribution of the OF activity of SOF in the processes of HEp-2 epithelial cell adhesion and invasion, deletion mutagenesis was used to eliminate the OF activity of recombinant rSOF75{Delta}Fn. Deletion was undertaken to remove between 22 and 63 amino acid residues of the rSOF75{Delta}Fn protein (between Pro148 and Lys211, Pro210 and Glu232, Lys231 and Asp286, Val285 and Glu315). Each of the rSOF75{Delta}Fn deletion mutants lacked OF activity (Fig. 1B). The rSOF75{Delta}FnDEL[P210-> E232] mutant was also found to lack OF activity when incubated in human serum or human HDL (Fig. 2). To delineate specific amino acid residues that contribute to OF activity, site-directed mutagenesis to alanine was undertaken on 52 amino acids of SOF75{Delta}Fn that are 100% conserved in 16 different SOF sequences (supplemental Table S1). A mutant form of rSOF75{Delta}Fn with attenuated OF activity (100-fold reduction in activity) was constructed by simultaneously substituting 5 amino acids with alanine (Asp218, Ser226, Lys228, Met229, Glu232). The rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A] protein has attenuated OF activity when incubated with either human serum or human HDL (Fig. 2). To determine whether the loss or attenuation of OF activity was due to a decrease in the apoAI binding capacity of SOF, the ability of rSOF75{Delta}FnWT, rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A], and rSOF75{Delta}FnDEL[P210-> E232] to bind biotinylated apoAI was assayed (Fig. 2D). There was no significant decrease in apoAI binding by the mutant proteins, suggesting that the loss of OF activity occurs via an alternative mechanism.


Figure 2
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  		FIGURE 2.
The OF activity and apoAI binding capacity of wild type and mutant forms of rSOF75{Delta}Fn. A, OF activity over 24 h was determined by adding 1 µg/ml of either rSOF75{Delta}FnBDWT, rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A], or rSOF75{Delta}FnDEL[P210-> E232] to human serum and recording the A405 nm at timed intervals. B and C, the opacification of serum (B) or HDL (C) as a function of protein concentration. Human serum or purified human HDL was treated with the indicated concentration of protein for 24 h and the absorbance determined at 405 nm. D, binding of biotinylated apoAI by either rSOF75{Delta}FnBDWT, rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A], rSOF75{Delta}FnDEL[P210-> E232], or gelatin.

 
The impact of the mutations to rSOF75{Delta}Fn on protein structure was analyzed using far-UV CD spectroscopy. rSOF75{Delta}FnWT had a CD emission spectrum typical of proteins containing both {alpha}-helices and β-sheets, with a characteristic minimum at ~220 nm, a second larger minimum at ~207 nm and a maximum at 190 nm (34). Of the rSOF75{Delta}FnDEL mutant proteins generated, rSOF75{Delta}FnDEL[K231-> D286] could not be purified for structural analysis. Far-UV CD spectra obtained for rSOF75{Delta}FnDEL[P148-> K211], rSOF75{Delta}FnDEL[P210-> E232], and rSOF75{Delta}FnDEL[V285-> D315] indicate perturbation to the secondary structure of the proteins. The rSOF75{Delta}FnWT is predicted to contain 27% {alpha}-helicity, with a predicted 24% {alpha}-helical content in rSOF75{Delta}FnDEL[P148-> K211], 21% in rSOF75{Delta}FnDEL[P210-> E232], and 22% in rSOF75{Delta}FnDEL[V285-> D315]. A concomitant shift toward a more disordered structure was observed for each deletion mutant, as indicated by a shift in the 207 nm minima to a shorter wavelength of 204 nm for rSOF75{Delta}FnDEL[P148-> K211] and rSOF75{Delta}FnDEL[P210-> E232] and 205.5 nm for rSOF75{Delta}FnDEL[V285-> D315] (Fig. 3) (34, 35). In contrast to the loss of secondary structural elements in the rSOF75{Delta}FnDEL mutants, the rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A] mutant had increased secondary structure when compared with rSOF75{Delta}FnWT, with an increase in predicted {alpha}-helicity from 27 to 29% (Fig. 3).


Figure 3
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  		FIGURE 3.
rSOF75{Delta}Fn mutants have perturbed secondary structure. The spectra of wild type rSOF75{Delta}Fn (solid line) compared with mutant forms of recombinant SOF75{Delta}Fn (dotted line). A, rSOF75{Delta}FnDEL[P148-> K211]; B, rSOF75{Delta}FnDEL[P210-> E232]; C, rSOF75{Delta}FnDEL[V285-> D315]; D, rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A]. All proteins exhibit CD emission spectra characteristic of proteins containing both {alpha}-helices and β-sheets, displaying a characteristic minimum at ~220 nm, a second minimum at ~207 nm, and a maximum at 190 nm. CD spectra in A, B, and C show a shift in the latter minimum to a shorter wavelength, indicating an increase in the proportion of the protein having disordered structure.

 
To assess the direct contribution of the OF activity of SOF in the processes of HEp-2 epithelial cell adhesion and invasion, the ability of latex beads coated with wild type OF-positive, mutant OF-negative, and OF-attenuated forms of the rSOF75{Delta}Fn protein to bind to the human pharyngeal epithelial cell line HEp-2 was assayed. These studies indicate that the SOF{Delta}Fn domain mediates attachment to HEp-2 cells, with the latex beads coated with rSOF75{Delta}FnWT adhering to HEp-2 cells in numbers equivalent to latex beads coated with the full-length rSOF75 (p > 0.05), and significantly more latex beads coated with rSOF75{Delta}FnWT adhering to HEp-2 cells than latex beads coated with a protein encompassing only the fibronectin binding domain of the SOF protein (rFNBD) (p < 0.01). Furthermore, this HEp-2 adherence mediated by the SOF{Delta}Fn domain is not dependent on OF activity, with latex beads coated with rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A] (attenuated OF activity) and rSOF75{Delta}FnDEL[P148-> K211] (abolished OF activity) mediating adherence at the same level as the rSOF75{Delta}FnWT protein (p > 0.05) (Fig. 4, A and C). Latex beads coated with rSOF75{Delta}FnDEL[P210-> E232] and rSOF75{Delta}FnDEL[V285-> D315] have a significantly reduced capacity to attach to HEp-2 cells when compared with rSOF75{Delta}FnWT (p < 0.01), however, there were significantly more latex beads coated with rSOF75{Delta}FnDEL mutants attached to HEp-2 cells than were observed with the BSA control (p < 0.01). It has been previously demonstrated that the SOF{Delta}Fn domain of SOF possesses pro-invasive properties when expressed on the surface of non-invasive GAS strains or non-invasive Lactococcus lactis (13). However, while the SOF{Delta}Fn promotes epithelial cell invasion in these backgrounds, this study has shown that the SOF{Delta}Fn protein domain is not sufficient per se to mediate intracellular invasion of HEp-2 cells (Fig. 4, B and C). However, while it is apparent that the N terminus of SOF does not directly mediate epithelial cell invasion, it may be concluded that the higher uptake by epithelial cells of latex beads coated with full-length SOF, in comparison to the FnBD domain alone, is attributable to the N-terminal domain of SOF. A significantly greater proportion of latex beads coated with rSOF75 were found to be intracellular (57.1%) than latex beads coated with FnBD alone (31.8% intracellular) (p < 0.01) (Fig. 4B).


Figure 4
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  		FIGURE 4.
SOF{Delta}Fn promotes association of latex beads to HEp-2 cells independent of OF activity. A, total number of SOF-coated latex beads per 10 HEp-2 cells. SOF-coated latex beads were incubated with HEp-2 cells for 4 h in Dulbecco's modified Eagle's medium HEPES supplemented with 1% FCS. Asterisks indicate adherence significantly lower than that mediated by the recombinant full-length SOF, rSOF75 (p < 0.001). B and C, binding and uptake of SOF-coated latex beads by HEp-2 cells. Following incubation with latex beads, internal and external beads were differentiated using double immunofluorescence confocal microscopy. External beads were labeled by incubation with SOF-specific rabbit anti-serum, followed by goat anti-rabbit Alexa-488 conjugated antibody (green). Internal beads were labeled by subsequently permeabilizing the cells using 0.1% Triton X-100, and subsequently incubating the cells with SOF-specific rabbit antiserum, followed by goat anti-rabbit Alexa-633-conjugated antibody (red). C, sample of images obtained using double immunofluorescence microscopy. Binding and uptake mediated by rSOF75, rFnBD, and rSOF75{Delta}Fn peptides is shown. Binding mediated by SOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A] with attenuated OF activity and rSOF75{Delta}FnDEL[P148-> K211] with abrogated OF activity is also shown.

 
While SOF is a protective antigen in murine vaccination studies, the capacity of SOF to opacify serum raises questions about its use in humans. The capacity to knock-out OF activity while retaining the structural and functional integrity of the molecule may be a requirement for further evaluation of SOF as a human vaccine candidate. To this end, the protective efficacy of rSOF75{Delta}FnWT, rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A] and the corresponding deletion mutant rSOF75{Delta}FnDEL[P210-> E232] were assessed in a murine model of lethal GAS challenge. rSOF75{Delta}FnWT, rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A], and rSOF75{Delta}FnDEL[P210->E232] all elicited significantly elevated titers of anti-rSOF75{Delta}FnWT serum IgG (p < 0.001; p < 0.01, p < 0.01) (Fig. 5A). In concordance with the results of Courtney et al. (15), mice immunized with rSOF75{Delta}FnWT showed significantly increased survival when challenged with the SOF-positive M49 GAS strain 591 compared with PBS control mice (p = 0.0069). While mice immunized with rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A], and the corresponding deletion mutant rSOF75{Delta}FnDEL[P210-> E232] showed increased survival when compared with the control mice, this increase was not statistically significant (p > 0.05) (Fig. 5B). While Courtney et al. (15) have previously shown that parenteral immunization with rSOF2{Delta}Fn protects against lethal systemic challenge with an M2 GAS strain, this is the first study to show that immunization with SOF can protect against lethal challenge with a heterologous GAS strain.


Figure 5
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  		FIGURE 5.
The murine response to parenteral immunization with wild type and mutant forms of rSOF75{Delta}Fn and subsequent lethal intraperitoneal challenge. A, antibody titers directed against rSOF75{Delta}FnWT 28-days postintravenous immunization with either rSOF75DFnWT, rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A], or rSOF75{Delta}FnDEL[P210-> E232]. Bars represent specific IgG present in the serum of control and vaccinated mice, with results expressed as geometric means, error bars represent S.E. (n = 10). Asterisks indicate titers significantly greater than the control. B, survival of intravenously immunized mice after lethal challenge with a heterologous S. pyogenes strain. Mice (n = 10) vaccinated with either rSOF75{Delta}FnWT, rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A], or rSOF75{Delta}FnDEL[P210-> E232] were challenged with ~1 x 109 CFU of virulent M49 S. pyogenes strain 591. Mortality was recorded daily. Significance was determined using the log rank test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
SOF is a unique multifunctional protein which may contribute to GAS pathogenesis by virtue of its abilities to interact with a variety of components of plasma and the extracellular matrix. A number of GAS surface proteins have a role in promoting host epithelial cell invasion including SfbI (36, 37), M protein (31, 38, 39), PrtF2 (40, 41), FbaA (42), and SOF (13). M protein and SfbI mediate intracellular invasion via two distinct pathways, both of which are dependant on fibronectin binding. M protein-mediated ingestion of GAS depends on co-engagement of the CD46 receptor and fibronectin via separate domains of the M protein; fibronectin in turn acts as a bridging molecule binding {alpha}5β 1 receptors at the host epithelial cell surface (43-46). The formation of the M protein-fibronectin-{alpha} 5β1 complex and engagement of the CD46 receptor results in intracellular signaling cascades that lead to cytoskeletal rearrangement for ingestion of the bacteria (31, 47). As opposed to M protein, SfbI requires only an interaction with fibronectin to trigger intracellular invasion, because the two fibronectin binding domains on SfbI co-operatively bind fibronectin which in turn binds {alpha}5β1 integrins on the host cell surface (44, 45). The resultant complex triggers the formation and recruitment of caveosomes which may allow GAS to persist within host cells without exposure to the acidic environment of phagosomes or lysosomes (48). While it has been clearly demonstrated through specific gene deletion that SOF, PrtF2 and FbaA are mediators of intracellular invasion of epithelial cells, the mechanism via which these surface protein mediate internalization is not known. The work of Timmer et al. (13), using SOF{Delta}Fn expressed at the surface of SOF-negative GAS and the heterologous species L. lactis, found that the N-terminal OF domain of SOF contributes to epithelial cell invasion independently of the C-terminal fibronectin binding domain. However, using latex beads coated with SOF protein, we demonstrate that the N-terminal OF domain is not intrinsically sufficient to mediate epithelial cell invasion, but will significantly enhance intracellular invasion in the presence of FnBD. Different GAS strains express a wide array of surface anchored proteins that interact with fibronectin, the M49 GAS strain used in the experiments of Timmer et al. (13) expresses both FBP54 and PrtF2 (40, 49). Thus, in this case the SOF{Delta}Fn expressed on the surface of M49 GAS may enhance cellular uptake, as the requirement for fibronectin binding may be achieved by FBP54 and PrtF2 expressed at the surface of the M49 GAS cells. However, the exogenous addition of rFnBD does not induce internalization of rSOF{Delta}Fn (results not shown), suggesting that fibronectin binding must be coupled at the surface of the latex bead or GAS cell for enhanced internalization to occur.

Incubation of cells with exogenous rSOF{Delta}Fn does not enhance uptake of GAS cells but inhibits internalization in a concentration-dependent manner (13), suggesting that the interaction of the N-terminal domain of SOF with the epithelial cell surface occurs via a specific receptor on the surface of HEp-2 cells.

We have previously demonstrated (13) that the N-terminal OF domain of SOF mediates tight adherence to epithelial cells. Thus, SOF{Delta}Fn-mediated adherence to the surface of epithelial cells most likely occurs via a mechanism independent of that required for the opacity reaction. The finding that these mutations clearly diminished the ability of SOF to opacify HDL but did not alter its binding to apoA1 indicated that other functions of SOF related to the opacity reaction were altered. Recent data suggest that SOF is a heterodivalent fusogenic protein that opacifies HDL by binding and cross-linking HDL particles resulting in the displacement of apoA1, fusion of the HDL particles, and the extrusion of a delipidated HDL particle (50). This mechanism produces a very large lipid particle that is enriched in cholesterol esters and essentially depleted of apolipoproteins. Thus, we propose that the described mutations of SOF alter the opacity reaction by interfering with one or more of these processes. The expression of multifunctional macromolecules at the surface of GAS is a common theme. For instance, M protein, SfbI, and the hyaluronic acid capsule each have dual functions in epithelial cell interactions and phagocytosis resistance (31, 36-39, 51-54), while glyceraldehyde-3-phosphate dehydrogenase functions as a glycolytic enzyme and binds multiple serum proteins including plasmin and fibronectin (55, 56). SOF joins an increasing list of multifunctional surface proteins of GAS.

As an immunogenic surface protein of GAS, SOF is a candidate vaccine antigen. While SOF was shown to lack protective efficacy against mucosal challenge when administered intranasally (57), SOF is a promising vaccine against systemic infection as parenteral immunization of mice with SOF2{Delta}Fn protects against lethal intraperitoneal challenge (15). Unlike in Europe and the United States, where the throat is often the primary tissue reservoir, the skin is the major site of infection among a number of populations in which GAS infection is endemic including the Australian aboriginal population (58), populations of India and Trinidad, American Indians, and Polynesians living in New Zealand (59-61). Thus, there is a need for protective antigens for use in areas such as the tropical north of Australia, where the skin is the primary route of GAS entry. Two of the prime vaccine candidates from the surface proteome of GAS, SfbI and the conserved C-terminal epitopes of M protein, while effective at eliciting protection against mucosal colonization, have proven ineffectual at reducing the rate of mortality due to systemic GAS infection in murine models (62, 63). While SOF has proven to be protective against systemic infection, it is not known what physiological effect the interactions between HDL and SOF would have upon the human host following vaccination. It would be prudent to eliminate the OF activity of the SOF protein to ensure undesirable side effects do not occur. To this end the OF-attenuated (rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A]) and corresponding OF-negative (rSOF75{Delta}FnDEL[P210-> E232]) mutants generated in this study were examined to determine the protective efficacy of the mutants against lethal systemic GAS infection. Unfortunately, while the mutant proteins remained immunogenic, the structural alterations introduced upon mutagenesis may have prevented accessibility of protective epitopes or altered the protective epitopes such that mice immunized with rSOF75{Delta}Fn[D218A-S226A-K228A-M229A-E232A] or rSOF75{Delta}FnDEL were not significantly protected. Other protective epitopes in streptococcal antigens such as the Group B streptococcus polysaccharide capsule and alpha C protein (64, 65) will only be protective if presented to the host immune system in their native conformation. In addition, maintenance of the conformational structure may enhance the immune response to epitopes of M proteins of GAS (66). The protective efficacy of the main protective epitope of SOF may have been altered by a loss of conformation associated with mutagenesis performed in this study. This study has shown that SOF can protect against intraperitoneal challenge by a heterologous GAS strain. These data, in conjunction with previous findings that antiserum against one serotype of GAS was bactericidal for multiple GAS serotypes (15), suggest that SOF contains common protective epitopes.

The SOF protein is a virulence determinant in GAS with affinity for multiple serum proteins including fibrinogen (67), fibronectin (8, 24), and apoAI (12). Additionally, SOF plays a role in epithelial cell adhesion and invasion (9, 13) and phagocytosis resistance (68). This study has shown that the N terminus of SOF mediates binding to HEp-2 cells and delineates this binding activity from the ability of the N terminus of SOF to opacify serum. This study is the first to indicate that vaccination with SOF can protect against infection by a heterologous GAS serotype in an animal model.


   FOOTNOTES
 
* 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1. Back

1 Recipient of an Australian Research Council Postgraduate Award. Back

2 Supported by research funds from the Dept. of Veterans Affairs. Back

3 To whom correspondence should be addressed: School of Biological Sciences, University of Wollongong, Wollongong, NSW, 2522, Australia. Tel.: 0061-2-4221-3439; Fax: 0061-2-4221-4135; E-mail: mwalker{at}uow.edu.au.

4 The abbreviations used are: GAS, group A streptococcus; SOF, serum opacity factor; OF, opacity factor; apoAI, apolipoprotein AI; LPS, lipopolysaccharide; SOF{Delta}Fn, N-terminal SOF domain; FnBD, C-terminal fibronectin binding domain; WT, wild type; HDL, high density lipoprotein; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FCS, fetal calf serum. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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