Many group A streptococcal strains express two different immunoglobulin-binding proteins, encoded by closely linked genes: characterization of the proteins expressed by four strains of different M-type

Most group A streptococcal strains are able to bind immunoglobulin (Ig) in a non-immune manner, and the majority of these strains bind both IgA and IgG. Using molecular cloning and immunochemical techniques, we have purified and characterized the Ig Fc-receptors expressed by four such strains. Two of the strains express a novel type of receptor, designated protein Sir, which binds IgA and IgG of all subclasses, and therefore has broader reactivity than any Fc-receptor previously described. The other two strains express protein Arp, a receptor that binds IgA of both subclasses, and also binds polyclonal IgG weakly. Characterization of the weak IgG-binding ability of protein Arp shows that it binds only some monoclonal IgG proteins, in particular those of the IgG3 subclass. The four strains studied here were unexpectedly found to also express a second Ig-receptor, called protein Mrp, encoded by a gene closely linked to the gene for the first receptor. The Mrp protein does not bind IgA, but it binds IgG molecules of the IgG1, IgG2 and IgG4 subclasses, and it also binds fibrinogen. Binding of fibrinogen has been reported to be a characteristic property of streptococcal M proteins, which suggests that the Mrp protein may be an M protein that also binds Ig. Taken together, all available evidence now indicates that most strains of group A streptococci express two different Ig-binding proteins, encoded by closely linked genes.     

Biochemical and biological properties of the binding of human fibrinogen to M protein in group A streptococci.

Fibrinogen is known to bind to group A streptococci and precipitate with extracts containing streptococcal M protein. We have previously shown that the binding of fibrinogen to M-positive streptococci prevents opsonization by complement and protects that organism from phagocytosis in nonimmune blood. In the present study, we used 3H-labeled fibrinogen, a highly purified peptide fragment of type 24 M protein (pep M24), and anti-pep M sera to show that fibrinogen binds to M-positive streptococci with high affinity (dissociation constants, 1 to 5 nM); occupation of the high-affinity binding sites suffices to protect the organism from phagocytosis; proteolytic treatments that remove M protein from streptococcal cells abolish binding; binding is competitively inhibited by anti-pep M sera; pep M24 precipitates fibrinogen; and binding to type 24 cells is inhibited by pep M24. We conclude that M protein is the cell surface structure principally responsible for binding fibrinogen on the surface of M-positive streptococci and that this binding contributes to the known antiopsonic property of M proteins.     

Extensive sequence homology between IgA receptor and M proteins in Streptococcus pyogenes

Many strains of Streptococcus pyogenes are known to express a receptor for IgA. The complete nucleotide sequence of the gene for such a receptor, protein Arp4, has been determined. The deduced amino acid sequence of 386 residues includes a signal sequence of 41 amino acids and a putative membrane anchor region, both of which are homologous to similar regions in other streptococcal surface proteins. The processed form of the IgA receptor has a length of 345 amino acids and a calculated molecular weight of 39544. The N-terminal sequence of the processed form is different from that previously found for a similar IgA receptor isolated from a S. pyogenes strain of type M60. The sequence of protein Arp4 shows extensive homology to the C-terminal half of streptococcal M proteins, but not to the streptococcal IgG receptor protein G or staphlyococcal protein A. Apart from the membrane anchor, this homology includes a sequence of 119 amino acid residues containing three repeated units and a 54-residue sequence without repeats. The protein expressed in Escherichia coli is found in the periplasmic space, in which it constitutes the major protein. Protein Arp4 is the first example of a surface protein that has both immunoglobulin-binding capacity and structural features characteristic of M proteins.     

A role for the fibrinogen-binding regions of streptococcal M proteins in phagocytosis resistance

All virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type-specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen-binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogen-binding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.     

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.     

Class I major histocompatibility proteins are an essential component of the simian virus 40 receptor.

The class I molecules encoded by the major histocompatibility complex (MHC) present endogenously synthesized antigenic peptide fragments to cytotoxic T lymphocytes. We show here that these proteins are an essential component of the cell surface receptor for simian virus 40 (SV40). First, SV40 binding to cells can be blocked by two monoclonal antibodies against class I human lymphocyte antigen (HLA) proteins but not by monoclonal antibodies specific for other cell surface proteins. Second, SV40 does not bind to cells of two different human lymphoblastoid cell lines which do not express surface class I MHC proteins because of genetic defects in the beta 2-microglobulin gene in one line and in the HLA complex in the other. Transfection of these cell lines with cloned genes for beta 2-microglobulin and HLA-B8, respectively, restored expression of their surface class I MHC proteins and resulted in concomitant SV40 binding. Finally, SV40 binds to purified HLA proteins in vitro and selectively binds to class I MHC proteins in a cell surface extract.     

The dipeptide repeat region of the fibrinogen-binding protein (clumping factor) is required for functional expression of the fibrinogen-binding domain on the Staphylococcus aureus cell surface

Clumping factor of Staphylococcus aureus is a fibrinogen-binding protein that is located on the bacterial cell surface. The protein has an unusual repeat domain (region R) comprising mainly the dipeptide aspartate and serine. To determine if region R has a role in the surface display of the fibrinogen-binding region A domain, deletions lacking the region R encoding region of the clfA gene were generated. To determine the minimum length of region R required for wild-type levels of ClfA expression, variants with truncated region R domains were constructed. S. aureus cells expressing mutated clfA genes were tested for (i) proteins released by lysostaphin treatment that reacted with antisera specific for region A, (ii) clumping in soluble fibrinogen, (iii) adherence to immobilized fibrinogen and (iv) expression of the ClfA antigen on the cell surface by fluorescent activated cell sorting analysis. Each construct expressed three major immunoreactive proteins, two of which were putative N-terminal degradation products. Region R residues greater than 40 were required between region A and W (72 residues between region A and the LPDTG sorting signal) for wild-type levels of clumping in fibrinogen. A stepwise decrease in clumping titre was observed as the distance between region A and LPDTG was decreased from 72 to 4 residues. Similarly, a decrease in binding of anti-ClfA serum and in binding to fibrinogen-coated plastic surfaces was observed with cells expressing ClfA with 40 region R residues or less. Nevertheless, low levels of adherence to fibrinogen and binding to anti-ClfA serum occurred with ClfA derivatives that lacked region R altogether. This indicates that a small proportion of the ClfA molecules are linked to peptidoglycan very close to the cell surface but that residues greater than 72 are needed to allow sufficient ClfA molecules to span the entire cell wall and to display the biologically active A domain in a form that can participate fully in fibrinogen binding.     

Identification of the ligand-binding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus

The ability of Staphylococcus aureus to bind to fibrinogen and fibrin is believed to be an important factor in the initiation of foreign-body and wound Infections. Recently, we reported the cloning and sequencing of the gene clfA encoding the fibrinogen receptor (clumping factor, ClfA) of S. aureus strain Newman and showed that the gene product was responsible for the clumping of bacteria in soluble fibrinogen and for the adherence of bacteria to solid-phase fibrinogen. This was confirmed here by showing that antibodies raised against purified Region A inhibited both of these properties. Also, immunofluorescent microscopic analysis of wild-type Newman and a clfA::Tn917 mutant of Newman with anti-ClfA Region A sera confirmed that Region A is exposed on the bacterial cell surface. Furthermore, polystyrene beads coated with the Region A protein formed clumps in soluble fibrinogen showing that the ClfA protein alone is sufficient for the clumping phenotype. Western immunoblotting with anti-ClfA Region A antibodies identified the native ClfA receptor as a 185 kDa protein that was released from the cell wall of S. aureus by lysostaphin treatment. A single extensive ligand-binding site was located within Region A of the ClfA protein. Truncated ClfA proteins were expressed in Escherichia coli. Lysates of E. coli and proteins that had been purified by affinity chromatography were tested for (i) their ability to bind fibrinogen in Western ligand blotting experiments, (ii) for their ability to inhibit clumping of bacteria in fibrinogen solution and adherence of bacteria to solid-phase fibrinogen, and (iii) for their ability to neutralize the blocking activity of anti-ClfA Region A antibody. These tests allowed the ligand-binding domain to be localized to a 218-residue segment (residues 332-550) within Region A.     

The biology of colicin M

Abstract This communication summarizes our present knowledge of colicin M, an unusual member of the colicin group. The gene encoding colicin M, cma , has been sequenced and the protein isolated and purified. With a deduced molecular size of 29 453 Da, colicin M is the smallest of the known colicins. The polypeptide can be divided into functional domains for cell surface receptor binding, uptake into the cell, and killing activity. To kill, the colicin must enter from outside the cell. Colicin M blocks the biosynthesis of both peptidoglycan and O-antigen by inhibiting regeneration of the bactoprenyl-P carrier lipid. Autolysis occurs as a secondary effect following inhibition of peptidoglycan synthesis. Colicin M is the only colicin known to have such a mechanism of action. Immunity to this colicin is mediated by the cmi gene product, a protein of 13 890 Da. This cytoplasmic membrane protein confers immunity by binding to and thus neutralizing the colicin. Cmi shares properties with both immunity proteins of the pore-forming and the cytoplasmically active colicins. Genes for the colicin and immunity protein are found next to each other, but in opposite orientation, on pColM plasmids. The mechanism of colicin M release is not known.     

The biology of colicin M

This communication summarizes our present knowledge of colicin M, an unusual member of the colicin group. The gene encoding colicin M, cma, has been sequenced and the protein isolated and purified. With a deduced molecular size of 29,453 Da, colicin M is the smallest of the known colicins. The polypeptide can be divided into functional domains for cell surface receptor binding, uptake into the cell, and killing activity. To kill, the colicin must enter from outside the cell. Colicin M blocks the biosynthesis of both peptidoglycan and O-antigen by inhibiting regeneration of the bactoprenyl-P carrier lipid. Autolysis occurs as a secondary effect following inhibition of peptidoglycan synthesis. Colicin M is the only colicin known to have such a mechanism of action. Immunity to this colicin is mediated by the cmi gene product, a protein of 13,890 Da. This cytoplasmic membrane protein confers immunity by binding to and thus neutralizing the colicin. Cmi shares properties with both immunity proteins of the pore-forming and the cytoplasmically active colicins. Genes for the colicin and immunity protein are found next to each other, but in opposite orientation, on pColM plasmids. The mechanism of colicin M release is not known.     

Complex formation among murine cytomegalovirus US22 proteins encoded by genes M139, M140, and M141

The murine cytomegalovirus (MCMV) proteins encoded by US22 genes M139, M140, and M141 function, at least in part, to regulate replication of this virus in macrophages. Mutant MCMV having one or more of these genes deleted replicates poorly in macrophages in culture and in the macrophage-dense environment of the spleen. In this report, we demonstrate the existence of stable complexes formed by the products of all three of these US22 genes, as well as a complex composed of the products of M140 and M141. These complexes form in the absence of other viral proteins; however, the pM140/pM141 complex serves as a requisite binding partner for the M139 gene products. Products from all three genes colocalize to a perinuclear region of the cell juxtaposed to or within the cis-Golgi region but excluded from the trans-Golgi region. Interestingly, expression of pM141 redirects pM140 from its predominantly nuclear residence to the perinuclear, cytoplasmic locale where these US22 proteins apparently exist in complex. Thus, complexing of these nonessential, early MCMV proteins likely confers a function(s) independent of each individual protein and important for optimal replication of MCMV in its natural host.     

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 molecules

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.     

Multiple binding of type 3 streptococcal M protein to human fibrinogen, albumin and fibronectin

Abstract M proteins are major virulence factors of group A streptococci which enable the bacteria to resist phagocytic attack. Their binding capacity for different plasma proteins seems to be one reason for the antiphagocytic activity of M protein. In the present study we demonstrate that M3 protein, isolated from the streptococcal culture supernatant of strain 4/55, and the recombinant form (rM3), purified from an E. coli lysate after cloning in phage γ-EMBL3, show a multiple binding to fibrinogen, albumin and fibronectin in Western blot and dot binding assays. Binding of M3 protein to the multifunctional extracellular matrix and plasma protein fibronectin may not only influence phagocytosis but may also contribute to the adherence of these bacteria to endothelial and epithelial cells.     

Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins

Phylogenetic analysis of 42 membrane protein (M) genes of influenza A viruses from a variety of hosts and geographic locations showed that these genes have evolved into at least four major host-related lineages: (i) A/Equine/prague/56, which has the most divergent M gene; (ii) a lineage containing only H13 gull viruses; (iii) a lineage containing both human and classical swine viruses; and (iv) an avian lineage subdivided into North American avian viruses (including recent equine viruses) and Old World avian viruses (including avianlike swine strains). The M gene evolutionary tree differs from those published for other influenza virus genes (e.g., PB1, PB2, PA, and NP) but shows the most similarity to the NP gene phylogeny. Separate analyses of the M1 and M2 genes and their products revealed very different patterns of evolution. Compared with other influenza virus genes (e.g., PB2 and NP), the M1 and M2 genes are evolving relatively slowly, especially the M1 gene. The M1 and M2 gene products, which are encoded in different but partially overlapping reading frames, revealed that the M1 protein is evolving very slowly in all lineages, whereas the M2 protein shows significant evolution in human and swine lineages but virtually none in avian lineages. The evolutionary rates of the M1 proteins were much lower than those of M2 proteins and other internal proteins of influenza viruses (e.g., PB2 and NP), while M2 proteins showed less rapid evolution compared with other surface proteins (e.g., H3HA). Our results also indicate that for influenza A viruses, the evolution of one protein of a bicistronic gene can affect the evolution of the other protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cloning and characterization of a gene for a 19kDa fibrinogen-binding protein from Staphylococcus aureus

Staphylococcus aureus has been shown to interact specifically with fibrinogen. Three different extracellular fibrinogen-binding proteins, two of which have coagulase activity, are produced by S. aureus strain Newman. The role of these fibrinogen-binding proteins during staphylococcal colonization and infection has not yet been fully elucidated. Here we describe the cloning, sequencing and expression of a gene for a 19kDa fibrinogen-binding protein. This gene, called fib, encodes a 165-amino-acid polypeptide, including a 29-amino-acid signal sequence. The recombinant protein, which has an estimated molecular mass of 15.9kDa, bound fibrinogen and was recognized by a polyclonal antiserum against the native Fib protein. Homologies between the Fib protein and the fibrinogen-binding domain of coagulase suggest that amino acids within this domain are involved in the binding to fibrinogen.     

M-like,immunoglobulin-binding protein ofStreptococcus pyogenes type M15

An M-like protein fromStreptococcus pyogenes type M15 strain EF1949 (EMML15) was cloned inEscherichia coli and sequenced. Recombinant EMML15 protein revealed a unique binding pattern for human IgG subclasses not described previously. Comparative analysis of the EMML15 amino acid sequence with those of other M-like proteins of opacity factor positive (OF⁺) serotypes and protein H, an IgG receptor from OF^– serotype M1, showed that IgG-binding proteins with common binding of IgG3 were closely related and distinct from streptococcal IgG receptors not binding IgG3. Thus, the Ig-binding proteins fromS. pyogenes were subdivided into two main categories according to binding pattern, protein structure, and gene location.     

Sodium-dependent myo-inositol transporter 1 is a cellular receptor for Mus cervicolor M813 murine leukemia virus

Retrovirus infection is initiated by binding of the surface (SU) portion of the viral envelope glycoprotein (Env) to specific receptors on cells. This binding triggers conformational changes in the transmembrane portion of Env, leading to membrane fusion and cell entry, and is thus a major determinant of retrovirus tissue and species tropism. The M813 murine leukemia virus (MuLV) is a highly fusogenic gammaretrovirus, isolated from Mus cervicolor, whose host range is limited to mouse cells. To delineate the molecular mechanisms of its restricted host range and its high fusogenic potential, we initiated studies to characterize the cell surface protein that mediates M813 infection. Screening of the T31 mouse-hamster radiation hybrid panel for M813 infectivity localized the receptor gene to the distal end of mouse chromosome 16. Expression of one of the likely candidate genes (slc5a3) within this region in human cells conferred susceptibility to both M813 infection and M813-induced fusogenicity. slc5a3 encodes sodium myo-inositol transporter 1 (SMIT1), thus adding another sodium-dependent transporter to the growing list of proteins used by MuLVs for cell entry. Characterization of SMIT1 orthologues in different species identified several amino acid variations within two extracellular loops that may restrict susceptibility to M813 infection.     

Receptor for IgA in group A streptococci: cloning of the gene and characterization of the protein expressed in Escherichia coli

The gene for an IgA-binding protein from a group A streptococcal strain was cloned and expressed in Escherichia coli. The IgA-binding protein, called protein Arp, was purified on IgA-Sepharose, allowing complete purification in a single step. Analysis of protein Arp by Western immunoblotting demonstrated a major IgA-binding band, with an apparent molecular weight of 42 kD. The purified protein was shown to bind serum IgA and secretory IgA, as well as monoclonal IgA of both subclasses. There was no binding to IgM, IgD or IgE, but a weak binding to IgG. Inhibition experiments with whole bacteria indicated that IgA and IgG bind at separate sites. Experiments with immunoglobulin fragments showed that protein Arp binds to the Fc region of both IgA and IgG. The equilibrium constant of the reaction between protein Arp and polyclonal human IgA was determined to be 5.6 x 10(8) M-1. Amino acid sequencing of protein Arp demonstrated a direct repeat of 7 amino acids in the NH2-terminal region, a feature previously found in several streptococcal M proteins. This suggests that protein Arp, like M proteins, may be a streptococcal virulence factor.     

The modification of the cell wall proteins in group A streptococci type M 29 under the influence of spermidine contained in the culture medium

The addition of spermidine into growth medium used for the cultivation of group A streptococci, type M 29, leads to changes in the amino acid composition of cell walls and surface proteins isolated by the method of E. H. Beachey et al. The separation of surface proteins into fibrinogen-binding proteins and fibrinogen receptors by affinity chromatography techniques on cellulose with covalently bound fibrinogen indicates that the proportion of these proteins in pepsin extracts obtained from different strains varies. Both spermidine and avirulent strains have similar content of fibrinogen-binding proteins, although these proteins are absent in virulent strains. Different amounts of fibrinogen receptors are extracted from all strains. As shown in the enzyme immunoassay, fibrinogen receptors contain no group-specific polysaccharide A, Fc-receptors and interact with total antiserum to group A streptococci, type M 29 [correction of 28]. Fibrinogen receptors isolated from the strains under study have been found to have similar amino acid composition. On the basis of these results we believe that neither receptor capacity to fibrinogen nor amino acid composition is indicative of the protective properties of protein M.     

The A domain of fibronectin-binding protein B of Staphylococcus aureus contains a novel fibronectin binding site

The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μM). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca(2+)-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock-lock-latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with K(D) = 2.5 μM despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, K(D) of 20 μM, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.