Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46)

Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.     

Differential recognition of surface proteins in Streptococcus pyogenes by two sortase gene homologs

The interaction of Streptococcus pyogenes (group A streptococcus [GAS]) with its human host requires several surface proteins. In this study, we isolated mutations in a gene required for the surface localization of protein F by transposon mutagenesis of the M6 strain JRS4. This gene (srtA) encodes a protein homologous to Staphylococcus aureus sortase, which covalently links proteins containing an LPXTG motif to the cell wall. The GAS srtA mutant was defective in anchoring the LPXTG-containing proteins M6, protein F, ScpA, and GRAB to the cell surface. This phenotype was complemented when a wild-type srtA gene was provided in trans. The surface localization of T6, however, was unaffected by the srtA mutation. The M1 genome sequence contains a second open reading frame with a motif characteristic of sortase proteins. Inactivation of this gene (designated srtB) in strain JRS4 affected the surface localization of T6 but not M6, protein F, ScpA, or GRAB. This phenotype was complemented by srtB in trans. An srtA probe hybridized with DNA from all GAS strains tested (M types 1, 3, 4, 5, 6, 18, 22, and 50 and nontypeable strain 64/14) and from streptococcal groups C and G, while srtB hybridized with DNA from only a few GAS strains. We conclude that srtA and srtB encode sortase enzymes required for anchoring different subsets of proteins to the cell wall. It seems likely that the multiple sortase homologs in the genomes of other gram-positive bacteria have a similar substrate-specific role.     

Group A streptococcal phagocytosis resistance is independent of complement factor H and factor H-like protein 1 binding

Factor H (FH) and factor H-like protein 1 (FHL-1) regulate complement activation through the alternative pathway. Several extracellular bacterial pathogens, prime targets for the complement system, bind FH and FHL-1, thereby acquiring a potential mechanism for minimizing complement deposition on their surface. For group A streptococci (GAS), surface-bound antiphagocytic M proteins mediate the interaction. To study the role of the FH-FHL-1 interaction for complement deposition and opsonophagocytosis of GAS, we first constructed a set of truncated M5 protein variants and expressed them on the surface of a homologous M-negative GAS strain. Binding experiments with the resulting strains demonstrated that the major FH-FHL-1 binding is located in a 42-amino-acid region within the N-terminal third of M5. Measurement of bacteria-bound complement factor C3 after incubation in plasma showed that the presence of this region had little impact upon complement deposition through the alternative pathway. Moreover, streptococci expressing M5 proteins lacking the major FH and FHL-1 binding sequence resisted phagocytosis in human blood as efficiently as bacteria expressing the wild-type protein. Consequently, the data suggest that the binding of the regulators of the alternative pathway is of limited importance for GAS phagocytosis resistance.     

Binding of complement subcomponent C1q to Streptococcus pyogenes: evidence for interactions with the M5 and FcRA76 proteins

Binding of C1q, the first component of the complement system, to some human pathogens has been earlier reported. In the present study, direct binding of C1q to group A streptococci (GAS) of various serotypes as well as some other Gram-positive and Gram-negative species was demonstrated. The interaction between C1q and GAS was investigated more in detail. In hot neutral extracts of a number of GAS strains two components of 64 and 52 kDa, respectively, bound C1q; alkaline and SDS extracts yielded the 52 kDa component as the main C1q-binding substance. Trypsin treatment of the SDS extracts of two GAS strains suggested the C1q-binding component(s) to be of protein nature. C1q-binding material purified from the SDS extract of an avirulent strain, type T27, was separated in 12% SDS-PAGE and probed in Western blot with human C1q and fibrinogen, conjugated to horse radish peroxidase (HRP) as well as rabbit IgG antibodies complexed to HRP (PAP system). The 52 kDa component was non-reactive with fibrinogen or rabbit IgG. However, C1q-binding components purified from the alkaline extracts of two M-positive strains revealed strong binding of either fibrinogen (type M5) or both fibrinogen and rabbit IgG (type M76); the molecular mass of these components, 55 kDa and 43–40 kDa, respectively, was in agreement with the reported molecular mass of the M5 and FcRA76 proteins. Our findings suggest that C1q may interact with GAS through certain M-family proteins as well as by a so far unidentified surface factor of protein nature occurring in most GAS strains. The involvement of M-family proteins, regarded as virulence factors of these organisms, may suggest the interaction of GAS with C1q as biologically important.     

M41型A群链球菌的Ⅰ型胶原样蛋白与人低密度脂蛋白的相互作用

【目的】检测M41型A群链球菌(GAS)ATCC12373中Ⅰ型胶原样蛋白(Scl1)与人低密度脂蛋白(LDL)的相互作用。【方法】克隆了M41型GAS ATCC12373的Ⅰ型胶原样蛋白(Scl1)及其V区(Scl1-V)基因,并表达、纯化重组蛋白rScl1(C176)和rScl1-V(C176V)。通过重组蛋白与人血浆的亲和色谱层析、Western blot及酶联免疫吸附试验(ELISA)检测C176、C176V与LDL的相互作用;通过GAS与LDL的ELISA试验和人血浆与GAS的共孵育试验,检测GAS与LDL的相互作用。【结果】结果证明C176和C176V可以与LDL特异性结合;表达Scl1的M41型GAS可以与LDL相结合。【结论】M41型GAS的Scl1可以与LDL特异性结合。     

The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners.     

Paxillin phosphorylation: bifurcation point downstream of integrin-linked kinase (ILK) in streptococcal invasion

Efficient group A streptococcus (GAS) invasion of mammalian cells requires fibronectin (Fn) binding proteins, such as M1 and PrtF1/SfbI, that bridge bacteria to integrins and activate cellular signalling for ingestion. Previous studies of GAS invasion, mediated by both proteins, suggest a common signalling pathway. However, distinct cellular morphological changes at the port of bacterial entry suggest that different signals are also induced. Here we report that paxillin is phosphorylated in response to Fn-bound GAS that express either M1 or PrtF1/SfbI protein, but is not phosphorylated in response to a mutant deficient in both proteins. Inhibition of paxillin phosphorylation by a tyrosine kinase inhibitor, PP2, or by expression of a dominant negative form of paxillin significantly reduced invasion by M1+ but did not affect ingestion of PrtF1/SfbI+ strains. In contrast, another tyrosine inhibitor, genistein, did not significantly prevent paxillin phosphorylation and had no effect on ingestion of the M1+ strain, but reduced PrtF1/SfbI-mediated entry. This suggests that paxillin phosphorylation is induced by both proteins but only required for M1-mediated invasion. A bifurcation point, downstream of integrin-linked kinase (ILK) and phosphoinositide 3-kinase, likely accounts for the distinct morphological changes. Furthermore, ILK activity is indispensable for M1-induced paxillin recruitment and phosphorylation.     

Comparative studies on the interaction of proteins with a polydimethylsiloxane elastomer. II. The comparative antigenicity of primary and secondarily adsorbed IgG1 and IgG2a and their non-adsorbed counterparts

The antigenicity of bovine IgG1 and IgG2a adsorbed on a polydimethysiloxane (PEP) elastomer, on a widely used polystyrene (Imm 2, Dynatech) or immobilized as biotinylated proteins to streptavidin covalently bound to polystyrene (SA-PS) was compared using various monoclonal (mAbs) and polyclonal antibodies (pAb) to bovine IgG. The IgGs were either adsorbed as native proteins or pre-denatured with 6M Guanidine-HCl (Gu-HCl) or 6 M Gu-HCl/0.1% 2-mercaptoethanol. In special situations, bovine and human IgG was immobilized by secondary adsorption to an albumin monolayer adsorbed on either PEP or Imm 2. Results indicate that pre-denaturation of IgGs with 6 M Gu-HCl/2-mercaptoethanol destroys all antigenicity whereas those IgGs pretreated with 6 M-GuHCl are indistinguishable in their antigenicity from the IgGs adsorbed to either PEP or Imm 2 without such treatment. When immobilized on SA-PS, Gu-HCl-treated IgGs were significantly less detectable, especially when tested using mAbs. In general, IgGs adsorbed on PEP or Imm 2 were less antigenic than when immobilized on SA-PS. However, two monoclonals specific for the IgG2a(A2) allotypic variant, favored the adsorbed protein and one polyclonal best recognized the IgG2a(A1) variant adsorbed on Imm 2 rather than when adsorbed on PEP or immobilized on SA-PS. Both IgG1 and IgG2a, bound by apparent protein-protein interactions to an albumin monolayer, were significantly more detectable than when directly adsorbed on either Imm 2 or PEP. Using ¹²⁵l-antibody or its Fab fragment to reduce steric hindrance in detection, we observed the same differences in detectability as when measured by enzyme-linked immunosorbent assay. Failure to identify a steric hindrance effect and the preference of some antibodies for adsorbed allotypic variants, support the concept of adsorption-induced conformational change (AICC). We conclude that proteins adsorbed as a monolayer on the PEP elastomer used to form the envelope of silicone breast implants are conformationally altered, but not necessarily to the same extent or the same manner as when adsorbed on polystyrene. The significantly great antigenicity of secondarily adsorbed IgG suggests that it may be present in near native conformation. © 1997 John Wiley & Sons, Ltd.     

Comparison of Antibodies Hydrolyzing Myelin Basic Protein from the Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis

It was found that antibodies (Abs) against myelin basic protein (MBP) are the major components of the antibody response in multiple sclerosis (MS) patients. We have recently shown that IgGs from sera of MS patients are active in the hydrolysis of MBP. However, in literature there are no available data concerning possible MBP-hydrolyzing Abs in cerebrospinal fluid (CSF) of MS patients. We have shown that the average content of IgGs in their sera is about 195-fold higher than that in their CSF. Here we have compared, for the first time, the average content of lambda- and kappa-IgGs as well as IgGs of four different subclasses (IgG1-IgG4) in CSF and sera of MS patients. The average relative content of lambda-IgGs and kappa –IgGs in the case of CSFs (8.0 and 92.0%) and sera (12.3 and 87.7%) are comparable, while IgG1, IgG2, IgG3, and IgG4: CSF - 40.4, 49.0, 8.2, and 2.5% of total IgGs, respectively and the sera - 53.6, 36.0, 5.6, and 4.8%, decreased in different order. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We present first evidence showing that IgGs from CSF efficiently hydrolyze MBP and that their average specific catalytic activity is unpredictably ∼54-fold higher than that of Abs from sera of the same MS patients. Some possible reasons of these findings are discussed. We suggest that anti-MBP abzymes of CSF may promote important neuropathologic mechanisms in this chronic inflammatory disorder and in MS pathogenesis development.     

Non-Immune Binding of Human IgG to M-Related Proteins Confers Resistance to Phagocytosis of Group A Streptococci in Blood

The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp) are group A streptococcal (GAS) receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG₁>IgG₄>IgG₂>IgG₃. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.     

A novel sortase, SrtC2, from Streptococcus pyogenes anchors a surface protein containing a QVPTGV motif to the cell wall

The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.     

Complement factor H allotype 402H is associated with increased C3b opsonization and phagocytosis of Streptococcus pyogenes

The main virulence factor of group A streptococcus (GAS), M protein, binds plasma complement regulators factor H (FH) and FH-like protein 1 (FHL-1) leading to decreased opsonization. The M protein binding site on FH is within domain 7 in which also the age-related macular degeneration (AMD)-associated polymorphism Y402H is located. We studied if FH allotypes 402H and 402Y have different binding affinities to GAS. Plasma-derived FH allotype 402H and its recombinant fragment FH5-7(402H) showed decreased binding to several GAS strains. Growth of GAS in human blood taken from FH(402H) homozygous individuals was decreased when compared with blood taken from FH(402Y) homozygous individuals. The effect of the allotype 402H can be explained by combining the previous M protein mutagenesis data and the recently published crystal structure of FH6-8. In conclusion the data indicate that the AMD-associated allotype 402H leads to diminished binding of FH to GAS and increased opsonophagocytosis of the bacteria in blood. These results suggest that the homozygous presence of the allele 402H could be associated with decreased risk for severe GAS infections offering an explanation for the high frequency of the allele despite its association with visual impairment.     

Generation of mutated variants of the human form of the MHC class I-related receptor,FcRn, with increased affinity for mouse immunoglobulin G

Much data support the concept that the MHC class I-related receptor FcRn serves to regulate immunoglobulin G (IgG) concentrations in serum and other diverse body sites in both rodents and humans. Previous studies have indicated that the human ortholog of FcRn is endowed with unexpectedly high stringency in binding specificity for IgGs. In contrast to mouse FcRn, which binds promiscuously to IgGs across species, human FcRn does not bind to mouse IgG1 or IgG2a, and interacts weakly with mouse IgG2b. Here, we investigate the molecular basis for this high-level specificity. We have systematically mutated human FcRn residues to the corresponding mouse FcRn residues in the regions that encompass the FcRn-IgG interaction site. Notably, mutation of the poorly conserved residue Leu137 of human FcRn to glutamic acid (L137E) generates a human FcRn mutant that binds to mouse IgG1 and mouse IgG2a with equilibrium dissociation constants of 13.2 microM and 14.4 microM, respectively. From earlier high-resolution structural analyses of the rat FcRn-rat Fc complex, residue 137 of human FcRn is predicted to contact residue 436 of IgG, which can be either His436 (mouse IgG1, mouse IgG2a) or Tyr436 (human IgG1, mouse IgG2b). The simplest interpretation of our data for the L137E mutant is therefore that replacement of the Leu137-Tyr436 (human) by the Glu137-His436 (mouse) pair generates a receptor that can bind to mouse IgG1 and mouse IgG2a. The L137E mutation reduces the affinity of human FcRn for human IgG1 by about twofold, consistent with the introduction of a less favorable Glu137-Tyr436 interaction. However, the analysis of the effects of other mutations on the binding to different IgGs indicates that the contribution to binding of the interaction of FcRn residue 137 with IgG residue 436 can vary. This suggests the existence of distinct docking topologies that are accompanied by variations in contacts between these two residues for different FcRn-IgG pairs. Our observations are of direct relevance to understanding the molecular nature of the human FcRn-IgG interaction. In turn, understanding human FcRn function has significance for the optimization of the serum half-lives of therapeutic and prophylactic antibodies.     

SipA is required for pilus formation in Streptococcus pyogenes serotype M3

Pili are a major surface feature of the human pathogen Streptococcus pyogenes (group A streptococcus [GAS]). The T3 pilus is composed of a covalently linked polymer of protein T3 (formerly Orf100 or Fct3) with an ancillary protein, Cpa, attached. A putative signal peptidase, SipA (also called LepA), has been identified in several pilus gene clusters of GAS. We demonstrate that the SipA2 allele of a GAS serotype M3 strain is required for synthesis of T3 pili. Heterologous expression in Escherichia coli showed that SipA2, along with the pilus backbone protein T3 and the sortase SrtC2, is required for polymerization of the T3 protein. In addition, we found that SipA2 is also required for linkage of the ancillary pilin protein Cpa to polymerized T3. Despite partial conservation of motifs of the type I signal peptidase family proteins, SipA lacks the highly conserved and catalytically important serine and lysine residues of these enzymes. Substitution of alanine for either of the two serine residues closest to the expected location of an active site serine demonstrated that these serine residues are both dispensable for T3 polymerization. Therefore, it seems unlikely that SipA functions as a signal peptidase. However, a T3 protein mutated at the P-1 position of the signal peptide cleavage site (alanine to arginine) was unstable in the presence of SipA2, suggesting that there is an interaction between SipA and T3. A possible chaperone-like function of SipA2 in T3 pilus formation is discussed.     

Expression of different group A streptococcal M proteins in an isogenic background demonstrates diversity in adherence to and invasion of eukaryotic cells

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M-like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein- and protein F1-deficient GAS strain, SAM2, which also lacks M-like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp-2 and HaCaT cells, a SAM2-derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp-2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.     

Streptococcal DRS (distantly related to SIC) and SIC inhibit antimicrobial peptides, components of mucosal innate immunity: a comparison of their activities

"Streptococcal inhibitor of complement" (SIC) and "distantly related to SIC" (DRS) are related virulence factors secreted by M1 and M12 strains of GAS, respectively. The human mucosal innate immune system, important components of which are beta-defensins, secretory leukocyte proteinase inhibitor (SLPI) and lysozyme, provides the first line of defence against microorganisms. We report the interaction between DRS and these proteins; further investigations into the interaction of SIC with the beta-defensins; and compare the sensitivity of M12 and M1 GAS to SLPI. We show that SLPI, which kills M1 GAS and is inhibited by SIC, cannot kill M12 GAS. DRS cannot inhibit SLPI killing of M1 GAS, although ELISA shows binding of DRS to SLPI. We suggest that the target for SLPI on M1 GAS resembles SIC, and soluble SIC inhibits by acting as a decoy for SLPI. M12 GAS may not have this target and cannot interact with SLPI. DRS inhibits the antibacterial action of hBD-2 and hBD-3. Binding of both SIC and DRS to hBD-2, and DRS to hBD-3, shows small positive enthalpy, suggesting that binding is largely hydrophobic. The data for SIC and hBD-3 indicate that this is not a homogeneous bimolecular interaction. We conclude that DRS shares several of the properties of SIC, and therefore can be considered an important virulence factor of M12 GAS and an aid to colonization of the host mucosae.     

Immune response to superoxide dismutase in group A streptococcal infection

Extracellular localisation of manganese-dependent superoxide dismutase (SodA) by group A streptococcus (GAS) may have a role in protection of this pathogenic bacterium from exogenously produced reactive oxygen species. In this study we show that SodA is found both in surface protein extracts and in culture supernatants of GAS. To investigate whether SodA is a possible vaccine candidate outbred Quackenbush mice were subcutaneously vaccinated with recombinant SodA. Strong antibody responses which were moderately opsonic were elicited. These antibodies were unable to protect mice from intraperitoneal challenge with M1 GAS. We also show that SodA and p145 (a conserved peptide from the M-protein) antibodies are present at significantly higher levels amongst patients with rheumatic heart disease than in control subjects from the same endemic region. The higher SodA antibody levels in patients may be indicative of a role for this protein in pathogenesis of rheumatic heart disease but are more likely to be a marker of recent or recurrent streptococcal infection.     

FbaA- and M protein-based multi-epitope vaccine elicits strong protective immune responses against group A streptococcus in mouse model

We report the construction of a recombinant multivalent vaccine against group A streptococcus (GAS), designated F7M5. It contains seven predominant epitopes of FbaA identified by phage display technology, five non-tissue cross-reactive M protein fragments expressed on four selected serotypes prevalent in China, a Trojan antigen (TA) and a poly-alanine DR epitope (PADRE). BALB/c mice were immunized subcutaneously with F7M5 formulated with Freund's adjuvant, using recombinant FbaA and M protein in parallel as control. Using enzyme-linked immunosorbent assay (ELISA), mouse immune sera were assayed for IgG titers, IgG subclasses, and binding of F7M5 with M1GAS. Results indicated that the multivalent vaccine was highly immunogenic and elicited a balanced IgG1/IgG2a response. We also tested the reactivity of F7M5 to antistreptolysin O (ASO) antibodies in sera of GAS-infected patients and found a 95.8% positive rate, indicating that the epitopes of the vaccine were widely expressed in the prevalent serotypes of GAS. More importantly, the F7M5 vaccine elicited strong protective immune responses against lethal-dose challenge with a survival rate of 90%, but induced no cross-reactions or pathological lesions in mouse model, suggesting that F7M5 can be further developed as an effective and safe anti-GAS vaccine.     

The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.     

Conferring the binding properties of the mouse MHC class I-related receptor, FcRn, onto the human ortholog by sequential rounds of site-directed mutagenesis

The MHC class I-related receptor, FcRn, is involved in binding and transporting immunoglobulin G (IgG) within and across cells. In contrast to mouse FcRn, which binds to IgGs from multiple different species, human FcRn is surprisingly stringent in binding specificity. For example, human FcRn does not bind to mouse IgG1 or IgG2a and interacts only weakly with mouse IgG2b. Here, we have used site-directed mutagenesis in combination with interaction (surface plasmon resonance) studies, with the goal of generating human FcRn variants that more closely resemble mouse FcRn in binding specificity. Our studies show that residues encompassing and extending away from the interaction site on the alpha2 helix of FcRn play a significant and most likely indirect role in FcRn-IgG interactions. Further, by combining mutations in the alpha2 helix with those in a non-conserved region of the alpha1 helix encompassing residues 79-89, we have generated a human FcRn variant that has properties very similar to those of mouse FcRn. These studies define the molecular basis for the marked difference in binding specificity between human and rodent FcRn, and give insight into how human FcRn recognizes IgGs.