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Interaction between Complement Regulators and Streptococcus pyogenes: Binding of C4b-Binding Protein and Factor H/Factor H-Like Protein 1 to M18 Strains Involves Two Different Cell Surface Molecules¹

David Pérez-Caballero^*, Isabel García-Laorden, Guadalupe Cortés, Michael R. Wessels, Santiago Rodríguez de Córdoba^2,* and Sebastián Albertí^2,3,

^* Departamento de Inmunología, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; Institut Universitari d'Investigacions en Ciències de la Salut and Departmento de Biología, Universidad de las Islas Baleares, Palma de Mallorca, Spain; and Channing Laboratory, Brigham and Women’s Hospital, and Division of Infectious Diseases, Children’s Hospital, Harvard Medical School, Boston, MA 02115

	Abstract

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Streptococcus pyogenes, or group A Streptococcus, is one of the most frequent causes of pharyngitis and skin infections in humans. Many virulence mechanisms have been suggested to be involved in the infectious process. Among them is the binding to the bacterial cell surface of the complement regulatory proteins factor H, factor H-like protein 1 (FHL-1), and C4b-binding protein. Previous studies indicate that binding of these three regulators to the streptococcal cell involves the M protein encoded by the emm gene. M-type 18 strains are prevalent among clinical isolates and have been shown to interact with all three complement regulators simultaneously. Using isogenic strains lacking expression of the Emm18 or the Enn18 proteins, we demonstrate in this study that, in contradistinction to previously described S. pyogenes strains, M18 strains bind the complement regulators factor H, FHL-1, and C4b-binding protein through two distinct cell surface proteins. Factor H and FHL-1 bind to the Emm18 protein, while C4BP binds to the Enn18 protein. We propose that expression of two distinct surface structures that bind complement regulatory proteins represents a unique adaptation of M18 strains that enhances their resistance to opsonization by human plasma and increases survival of this particular S. pyogenes strain in the human host. These new findings illustrate that S. pyogenes has evolved diverse mechanisms for recruitment of complement regulatory proteins to the bacterial surface to evade immune clearance in the human host.

	Introduction

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Group A Streptococcus, or Streptococcus pyogenes, is a Gram-positive bacterium that frequently produces suppurative infections in humans (1, 2, 3), including pharyngitis and skin infections. These localized infections can be complicated by spread to deeper tissues and by the postinfectious sequelae of acute rheumatic fever and glomerulonephritis (4, 5). Recently, there has been an increase in severe, highly invasive streptococcal infections of soft tissues with high mortality (4, 6, 7, 8). S. pyogenes cells express one or more surface-associated fibrillar proteins, called M proteins, which are major virulence factors because they confer to the bacteria resistance to phagocytosis (9, 10). Among the different S. pyogenes strains, there are >100 different antigenically distinguishable M proteins (9, 11). The M proteins belong to a family of structurally related proteins that are encoded by genes within the mga regulon in the streptococcal chromosome. For most S. pyogenes strains, this gene cluster includes not only the emm gene encoding M (Emm) protein, but also one or two additional genes encoding structurally related proteins designated Enn and Mrp (12, 13). The Emm proteins have affinity for several plasma proteins, including fibrinogen, IgG, IgA, and the complement regulatory proteins, factor H, factor H-like protein 1 (FHL-1),⁴ and C4b-binding protein (C4BP) (12, 14, 15). It has been suggested that bacterial binding of these plasma proteins contributes to the antiphagocytic properties conferred by most Emm proteins.

The interaction between a complement regulatory protein and S. pyogenes was first reported by Horstmann et al. (16). They demonstrated that M6 and other M serotypes (M5, M19, M24, and M28) bind factor H through the conserved regions (B or C repeats) in the Emm protein. The binding of this complement regulatory protein resulted in reduced activation by the alternative pathway on the bacterial surface, which could decrease phagocytosis by macrophages and polymorphonuclear leukocytes. Binding of factor H was, therefore, proposed as the mechanism by which Emm proteins exert their antiphagocytic effect (16). Later studies in an M4 strain demonstrated that C4BP bound to the N-terminal region of the Emm4 protein (17) and that binding of C4BP could confer bacterial protection against classical pathway activation (14). More recently, FHL-1, another complement regulator that results from the alternative splicing of the factor H gene, has been shown to bind to streptococcal cells throughout the N-terminal region of the Emm protein (15).

The present studies were stimulated by the observation that strains of the M18 serotype vary significantly in their capacity to bind C4BP, but not in the binding of FHL-1 (18), despite both complement regulators having been reported to interact with the same N-terminal hypervariable region of the Emm proteins studied previously. To investigate the molecular basis of the interaction between S. pyogenes surface proteins and C4BP or FHL-1, we have characterized the emm gene in different M18 strains and performed binding assays in a wild-type M18 strain and isogenic mutants deficient in expression of specific surface molecules. Our data argue against a role of the Emm18 protein in the interaction between C4BP and M18 S. pyogenes strains and support the conclusion that C4BP binds to a different molecule on the M18 streptococcal surface, Enn18. The results provide evidence that closely related complement regulatory proteins recognize distinct molecular targets on the surface of this important human pathogen.

	Materials and Methods

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Bacterial strains and culture conditions

The S. pyogenes M18 strains 87–282, 346, SS77, 9003, and SS344 have been described previously (18). The capsule and M18 mutants derived from 87 to 282 strain (TX72, 282KZ, and TX74) were also previously described (19, 20). S. pyogenes M1 strain 8763 was previously described and does not bind C4BP (18). Streptococci were grown in Todd-Hewitt broth supplemented with 0.2% yeast extract or on solid medium containing Todd-Hewitt broth supplemented with 0.2% yeast extract supplemented with 1.8% Bacto Agar and 5% (v/v) defibrinated sheep blood. Escherichia coli was grown in Luria-Bertani broth. Erythromycin was used in the cloning experiments at 100 µg/ml for E. coli and 1 µg/ml for S. pyogenes. Cell concentration was determined using a Petroff-Hausser counting chamber (Thomas Scientific).

Proteins and Abs

Human C4BP and factor H were purified from EDTA-plasma, as described (21, 22). FHL-1 was partially purified free of factor H from EDTA-plasma using a heparin-Sepharose column and an anionic exchange chromatography using a Q-Sepharose Fast Flow column (Amersham Biosciences). C4BP was radiolabeled with ¹²⁵I using Iodogen (Pierce, Rockford, IL). mAbs and polyclonal Abs against human C4BP have been previously described (21, 22, 23). The 35H9 anti-human factor H mAb (IgM isotype) recognizes both factor H and FHL-1 proteins (18).

DNA and RNA manipulations

Plasmid DNA was isolated using the Wizard Miniprep Kit (Promega, Madison, WI), according to the manufacturer’s instructions. S. pyogenes genomic DNA was prepared, as described previously (24). Restriction endonuclease digestions, DNA ligations, transformation of CaCl₂ competent E. coli, agarose gel electrophoresis, and Southern hybridizations (ECL kit; Amersham Biosciences) were performed using standard techniques (25). S. pyogenes competent cells were prepared and transformed, as described (24). Total cellular RNA was isolated from S. pyogenes grown to A₆₀₀ = 0.2 using the Qiagen miniRNA prep (Promega, Madison, WI), according to the recommended procedure.

The DNA sequence of the emm18 and the enn18 genes in M18 strains was determined by automated sequence analysis (ABIPRISM 3700) of a PCR product generated using the primers GASM1/GASM2 (26) and GASN1/GASN2, respectively.

Polymerase chain reactions

Primers used in PCR and RT-PCR experiments are listed in Table I. PCRs were performed using a 5-min hot start and the following reaction conditions: 94°C for 1 min, 48°C for 1 min, and 68°C for 1 min for 35 cycles, and final extension step at 68°C for 10 min (GeneAmp PCR 2400 System; PerkinElmer, Wellesley, MA). RT-PCR was performed using the Acces RT-PCR kit (Promega) with a 45-min reaction at 48°C using 15 ng of total cellular RNA as template, followed by PCR for 35 cycles under the conditions described above.

To derive an Enn18 mutant from S. pyogenes acapsular strain TX72, a 771-bp internal fragment of the enn18 was amplified by PCR from S. pyogenes genomic DNA using primers Enn18FB and Enn18RB. The PCR product was cloned into the temperature-sensitive E. coli-streptococcus shuttle vector pJRS233 (24) digested with BamHI to yield plasmid pJENN18. This plasmid was introduced into strain TX72 by electroporation, and erythromycin-resistant transformants were isolated at the nonpermissive temperature (37°C), as described previously (24). Growth of transformants at the nonpermissive temperature resulted in the integration of plasmid through the enn18 homologous DNA sequences found on both the plasmid and the chromosomal DNA and in formation of inactive enn18 alleles.

Binding of C4BP, factor H, and FHL-1 to streptococcal M18 strains in EDTA-plasma

Binding of the three regulators to S. pyogenes in EDTA-plasma was performed essentially as described by Kotarsky et al. (27). Briefly, 2 x 10⁸ bacteria were washed in PBS, pelleted, and incubated with 50 µl of EDTA-plasma diluted four times in PBS for 2 h at room temperature with agitation. After incubation, samples were centrifuged and the pellet was washed twice with 1.5 ml of PBS containing 0.05% Tween 20. Bound proteins were eluted with 50 µl of SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromphenol blue) and subjected to Western blot analysis using the mAb 35H9 to identify factor H and FHL-1, or a rabbit polyclonal Ab to identify C4BP.

Binding of C4BP, factor H, and FHL-1 to 87–282 strain and its isogenic mutants

The binding was performed, as described above, incubating different number of cells of 87–282 (5 x 10⁷), TX72 (5 x 10⁷), 282KZ (2.5 x 10⁹), and TX74 (10¹⁰) strains with 10 µg of factor H or 50 ng of FHL-1. The amount of cells from the different strains for the factor H and FHL-1 binding was adjusted to have approximately equal quantities of total surface proteins. The binding of C4BP was performed incubating 5 x 10⁷ cells of 87–282, TX72, 282KZ, and TX74 with 1 µg of C4BP.

C4BP-binding assays on wild-type and mutants of 87–282 strain

To determine the C4BP association constant and the number of binding sites in the different strains, binding of ¹²⁵I-labeled C4BP (¹²⁵I-C4BP) to S. pyogenes cells was performed, as described above, using 2.5 x 10⁶ bacteria of 87–282, TX72, 282KZ, and TX74 strains and incubating with increasing amounts of ¹²⁵I-C4BP. Nonspecific binding (<5%) was determined using cells of 8763 strain. Free nonincorporated C4BP was determined by subtracting the bound ¹²⁵I-C4BP from the initially added ¹²⁵I-C4BP.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
S. pyogenes strains of the M18 serotype are frequently found in clinical isolates from patients with pharyngitis, invasive infections, and acute rheumatic fever (3, 6, 28). Although some M18 strains bind all three complement regulators, factor H, FHL-1, and C4BP, we have identified a number of M18 strains with reduced capacity to bind C4BP. These differences in the capacity to bind C4BP are in contrast to the apparently identical capacity of all M18 strains to bind factor H and FHL-1. Fig. 1 illustrates the binding of C4BP, factor H, and FHL-1 to five selected M18 strains (87–282, 346, 9003, SS77, and SS344). Although all five M18 strains show similar capacity to bind factor H and FHL-1, the capacity to bind C4BP varies dramatically among these M18 strains. Strain 346 binds C4BP very efficiently; 87–282, SS77, and 9003 show intermediate binding; and SS344 shows no capacity to bind C4BP.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Binding of factor H, FHL-1, and C4BP to different M18 strains. a, Cells of the strains 87–282, 346, SS77, 9003, and SS344 were incubated with EDTA-plasma. The proteins bound to the streptococcal surface were eluted, separated by SDS-PAGE, and subjected to a Western blot using a mouse mAb (35H9) that recognizes both factor H and FHL-1, or a rabbit polyclonal antiserum against human C4BP. b, Alignment of Emm18 amino acid sequences. The complete amino acid sequences of Emm18 protein of all five M18 strains are shown. The 87–282 strain was used as the reference sequence. A gap in the sequences of 346, SS77, 9003, and SS344 strains was introduced at position 204 to accommodate an insertion of 42 aa present in the 87–282 strain.

Our binding studies suggest that the C4BP-binding differences among the M18 strains are not a consequence of different levels of expression of the emm18 gene because all M18 strains show identical capacity to bind factor H or FHL-1 (Fig. 1).

To verify that binding of C4BP to M18 strains was not mediated by the Emm18 protein, we performed binding assays using the 87–282 strain and its isogenic mutants TX72, 282KZ, and TX74. The TX72 strain is a mutant lacking the capsule, the 282KZ strain is an Emm18 protein-deficient mutant, and TX74 is a double mutant lacking both capsule and M protein (19, 20).

The two mutants that lack Emm18 protein expression, 282KZ and TX74, failed to bind factor H or FHL-1 (Fig. 2). These results are consistent with previous data indicating that both proteins have binding sites in the Emm18 protein (15, 29). Factor H binds to the B or C repeat region, depending on the M serotype (15, 29), and FHL-1 to the N-terminal hypervariable region (15). Therefore, lack of binding to factor H and FHL-1 is consistent with lack of expression of the Emm18 protein on the cell surface of the 282KZ and TX74 strains. In marked contrast to these results, both Emm18-deficient mutant strains bound C4BP (Fig. 2), a finding that implies that the lack of Emm18 protein does not impair the binding of C4BP to the streptococcal surface. Whereas data from other S. pyogenes serotypes indicate that both C4BP and FHL-1 interact with the same N-terminal hypervariable region of the Emm proteins (15, 17), results of the present study strongly suggest that FHL-1 and C4BP bind to different molecules on the streptococcal surface in M18 strains. Moreover, because mutants lacking Emm18 protein are able to bind C4BP, we also conclude that in M18 strains the binding site for C4BP on the streptococcal surface is located in a molecule different from the Emm18 protein. As expected, lack of the capsule had no effect on the capacity to bind C4BP, FHL-1, or factor H by the 87–282 streptococcal strain.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Binding of complement regulators to wild-type strain 87–282 and its isogenic mutants. Wild-type or mutant strains were incubated with factor H, FHL-1, or C4BP. After elution of bound proteins, Western blot was performed using mAb 35H9 against both factor H and FHL-1, and a rabbit polyclonal antiserum against C4BP.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. Binding assays on wild-type and 87–282 mutant strains. Wild-type or mutant strains were incubated with increasing amounts of 125I-C4BP. Nonspecific binding (<5%) was determined using 108 cells of the non-C4BP-binding strain 8763. The number of C4BP molecules/cell at saturation and the dissociation constant are shown.

Results of these experiments suggest that other molecules on the streptococcal cell surface, different from the Emm18 protein, are responsible for the interaction between C4BP and M18 strains. Therefore, we considered whether other members of the M protein family might be binding partners for C4BP. As is shown in Fig. 4, in the M18 strains included in this study the mga regulon contains only two genes encoding for two emm-like proteins, Emm18 and Enn18. M18 strains have no mrp18 gene, as it was previously reported (13). Enn18 has been reported to bind human IgG3, but its role in interaction with complement regulatory proteins has not been studied (31).

View larger version (6K): [in this window] [in a new window]   FIGURE 4. Schematic diagram of the mga regulon in M18 strains. Subdivisions of emm-like genes are described by the generic terms emm18, encoding for Emm18 protein, and enn18, encoding for Enn18 protein. The scpA gene, encoding for the C5a peptidase, is located downstream of the emm-like genes.

To investigate the role of the Enn protein in the binding of C4BP to M18 strains, we interrupted by insertion-duplication mutagenesis the enn18 gene required for the synthesis of the Enn18 protein. Because the lack of capsule had no effect on the binding of C4BP to S. pyogenes, we used the acapsular mutant TX72, in which the efficiency of transformation is higher than in the encapsulated strain 87–282, to generate an Enn18 mutant. A schematic representation of the chromosome of parent strain TX72 and its Enn18-deficient mutant TX76 is shown in Fig. 5a. Southern blot analysis of the TX76 chromosome, using as probe an enn18 internal fragment, confirmed that two incomplete copies of the enn18 gene were generated by the integration of the plasmid (Fig. 5b). To further demonstrate that interruption of enn18 gene abolished its expression, we used RT-PCR to estimate the levels of enn18 gene transcription in 87–282 and the four isogenic mutants (282KZ, TX72, TX74, and TX76). We detected enn18 gene transcripts in all strains, including the Emm18 mutant strains 282KZ and TX74. However, inactivation of enn18 resulted in the interruption of the gene transcription in TX76 (Fig. 5c, lower panel). As expected, emm18 gene transcription was only interrupted in the Emm18 mutant strains 282KZ and TX74, but not in the wild-type strain 87–282, in the capsule-deficient mutant TX72, or in the Enn18-deficient mutant TX76 (Fig. 5c, upper panel).

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Inactivation of enn18 in S. pyogenes strain TX72. a, Diagram of insertion-duplication mutagenesis showing homologous recombination between the S. pyogenes chromosome of the TX72 strain and the pJENN18 disruption construct to generate the isogenic enn18 mutant TX76. DNA fragment size in the diagram is not to scale. The continuous line in the mutant genome represents the plasmid DNA integrated into the chromosome. The thicker line of the plasmid indicates the BamHI enn18 internal fragment cloned in pJENN18 and used as probe in the Southern blot analysis. Expected sizes of the fragments that hybridize with the probe are indicated in kb. b, Southern blot analysis of S. pyogenes strain TX72 and Enn18 mutant chromosomes digested with EcoRV and probed with an enn18 internal fragment. Molecular size markers (in kb) are shown on the left. c, Emm18 and enn18 gene transcription by wild-type strain 87–282 and its derived mutants. For each strain, the figure shows the product of RT-PCR using primers (GASM1 and GASM2) specific for emm18, upper panel, and primers (NF and NR2) specific for enn18, lower panel.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Binding of complement regulators to strain TX72 and its isogenic mutants. S. pyogenes strain TX72 or Emm18 and Enn18 mutant strains TX74 and TX76, respectively, were incubated with EDTA-plasma. The proteins bound to the streptococcal surface were eluted, separated by SDS-PAGE, and subjected to a Western blot using a mouse mAb (35H9) that recognizes both factor H and FHL-1, or a rabbit polyclonal antiserum against human C4BP.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our findings that FHL-1 and C4BP bind to different molecules on the surface of the M18 streptococcal cell, and that binding of C4BP does not require expression of the Emm18 protein contrast with data previously reported for other streptococcal serotypes. Elegant studies performed in an M22 strain showed that C4BP binds to the N-terminal hypervariable region of the Emm22 protein and not to the Enn22 protein (32). Results of the present investigation imply that the molecular mechanisms involved in the binding of complement regulators by the different M serotypes are not necessarily conserved, and that different M serotypes may use distinct surface proteins to bind C4BP. In this regard, it has been reported that while factor H binds to the surface of M5 strains through a binding site in the B repeats of the Emm5 protein, the same factor H uses a binding site in the C repeats of the Emm6 protein to bind to M6 strains (15, 29). Moreover, Pandiripally et al. (33) have recently shown that Fba, a protein unrelated to the M protein family, is the molecule responsible for the factor H and FHL-1 binding in an M1 strain. In addition, factor H and C4BP bind to different molecules in Neisseria gonorrhoeae: factor H binds to sialic acids or to loop 5 of porin protein 1A (34, 35), whereas C4BP binds to this pathogen by type IV Pili, Por1A (loop 1), or to Por1B (loops 5 and 7) (36, 37).

In conclusion, we have demonstrated that the complement regulators FHL-1 and C4BP bind to M18 S. pyogenes strains through two different cell surface proteins. Factor H and FHL-1 bind to the Emm18 protein, while C4BP binds to the Enn18 protein. Expression of two distinct binding sites for complement regulatory proteins may represent a unique adaptation of M18 strains that enhances their resistance to complement-mediated opsonophagocytosis. This virulence mechanism further illustrates the diverse and sophisticated strategies used by S. pyogenes for survival in the human host.

	Acknowledgments

 
We thank S. Vara de Rey for her contribution to this work.

	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.

¹ This research was supported by the Spanish Ministerio de Ciencia y Tecnologia (SAF2002-01083). D.P.-C. and I.G.-L. were supported by a fellowship from the Spanish Council for Scientific Research and a fellowship from Red Respira, respectively.

² S.R.d.C. and S.A. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Sebastián Albertí, Unidad de Investigación, Edificio D, 1^a planta, Hospital Universitario Son Dureta, Andrea Doria, 55, Palma de Mallorca 07014, Spain. E-mail address: salberti{at}hsd.es

⁴ Abbreviations used in this paper: FHL-1, factor H-like protein 1; C4BP, C4b-binding protein; ¹²⁵I-C4BP, ¹²⁵I-labeled C4BP.

Received for publication June 4, 2004. Accepted for publication September 26, 2004.

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