Streptococcal protein G. Gene structure and protein binding properties

Protein G was solubilized from 31 human group C and G streptococcal strains with the muralytic enzyme mutanolysin. As judged by the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the binding patterns of the solubilized protein G molecules in Western blot experiments, the strains could be divided into three groups, represented by the group G streptococcal strains G148 and G43 and the group C streptococcal strain C40. The 65-kDa G148 protein G and the 58-kDa C40 protein G showed affinity for both immunoglobulin G (IgG) and human serum albumin (HSA), whereas the 40-kDa G43 protein G bound only IgG. Despite the different molecular patterns, the three protein G species had identical NH2-terminal amino acid sequences. Apart from the 65-kDa peptide, digestion of G148 streptococci with mutanolysin also produced a 52-kDa IgG- and HSA-binding peptide and a 14-kDa HSA-binding peptide. It was demonstrated that these peptides resulted from cleavage of 65-kDa protein G by proteolytic components in the mutanolysin preparation. The protein G genes of the C40 and G43 strains were cloned and sequenced, and their structure was compared to the previously published sequence of the G148 protein G gene. As compared to G148, both the C40 and G43 genes lacked a 210-base pair fragment in the IgG-binding region, accounting for the 10-fold lower affinity of these proteins for IgG. The G43 gene also lacked a 450-base pair fragment in the 5'-end of the gene, explaining why the G43 protein G did not bind HSA. The differences in protein G structure did not correlate with the clinical origin of the strains used in this study. The IgG-binding region of protein G was further mapped. Thus, a peptide corresponding to a single IgG-binding unit was obtained by the cloning and expression of a 303-base pair polymerase chain reaction-generated DNA fragment. The affinity of this 11.5-kDa peptide for human IgG was 8.0 x 10(7) M-1, as determined by Scatchard plots. Finally, a 55-amino acid-long synthetic peptide, corresponding to one of the three repeated domains in the COOH-terminal half of strain G148 protein G, effectively blocked binding of protein G to IgG.     

Characterization of clinical isolates of group C and G streptococci on the basis of protein G gene

Treatment of human group C and G streptococci with cyanogen bromide results in solubilization of surface protein G molecules. Strain-to-strain variation in the quanity, molar mass and functional activity of protein G extracted from representative group C and G isolates led to the identification of three structurally and functionally distinct forms of the protein. Using different molecular biological approaches it was possible to determine the group of streptococci (C or G), or the quantity of IgG and HSA domains. Presented at theInternational Conference on Recent Problems in Microbiology and Immunology, Košice (Slovakia), 13–15 October 1999.     

Fibrinogen binding inhibits the fixation of the third component of human complement on surface of groups A, B, C, and G streptococci

Effects of fibrinogen binding to M protein-positive and -negative streptococci on fixation of the third component of human complement (C3) were determined. In all test cultures of serological groups A, B, C, and G fixation of C3 was observed in normal human serum as revealed by quantitative fluorescent immunoassay. Fibrinogen binding inhibited the fixation of C3 on streptococci. The degree of inhibition was proportional to the extent of fibrinogen binding. Thus, inhibition of C3 fixation was most pronounced in strongly fibrinogen-positive streptococci of groups A, C, and G and not demonstrable in fibrinogen-negative cultures of groups C and G. Trypsinization of the streptococci destroyed their capacity to bind fibrinogen and consequently the inhibitory effects on C3 fixation. The carboxymethylated alpha and beta chains of fibrinogen moderately inhibited C3 fixation whereas gamma chain had no influence. These studies may indicate that fibrinogen binding structures other than M protein could also be involved in streptococcal pathogenicity.     

Evaluation of the usefulness of selected commercial systems for identification of Streptococcus species

The usefulness was assessed of three commercially available systems for rapid identification of streptococcal strains. The studied material comprised 68 strains of streptococci and enterococci (including 24 standard strains) belonging to serological groups: A (14 strains), B (10), C (11), D (10), F (3) and G (10), as well as 10 S. pneumoniae strains. The strains were isolated from throat, nasal, wound swabs, blood, pus of inpatients and throat and nasal swabs of outpatients. For the identification of streptococci 3 commercially available systems were used: API 20 STREP (bioMerieux, France), rapid ID 32 Strep (bioMerieux, France), Streptoplast PPL 18 (HTL, Poland). The determinations were done according to producer's instructions. The highest percent of correctly identified strains was obtained with the rapid ID 32 Strep--80.9%, with the API 20 STREP--76.4% strains were identified correctly and with the PPL 18--61.8%. The study showed that the API 20 STREP and rapid ID 32 STREP are suitable for the identification of streptococcal strains from groups: A, B, C, D, F and enterococci--group D. The proportions of correctly identified strains from these groups with the Streptoplast PPL 18 were lower than those determined with the bioMerieux systems. Using of three identification systems streptococci from group G and S. pneumoniae strains cannot be identified.     

Novel complex formed between a nonproteolytic cell wall protein of group A streptococci and alpha 2-macroglobulin.

Binding of 125I-labeled alpha 2-macroglobulin (alpha 2M) to streptococci belonging to serological groups A, B, C, and G was studied. Streptococci of groups A and G interacted only with native alpha 2M, and those of group C reacted only with alpha 2M-trypsin complex. Binding of alpha 2M to group A streptococci was saturable and reversible. The dissociation constant was 2.02 X 10(-7) M, and the number of binding sites was calculated to be 18,000 per streptococcus. The alpha 2M-binding protein could be solubilized by treatment of group A streptococci with a murolytic enzyme and subsequently purified by affinity chromatography and high-pressure liquid chromatography. The purified protein was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 78,000. It possessed no proteolytic activity and interacted with native alpha 2M in Western blots (immunoblots). Interaction of purified binding protein with alpha 2M led to a change in the conformation of alpha 2M similar to that obtained by alpha 2M-protease complexes. Reversible binding of a nonproteolytic streptococcal component of alpha 2M is thus a novel feature of alpha 2M reactivity.     

Lysis and Lysogenization of Groups A, C, and G Streptococci by a Transducing Bacteriophage Induced from a Group G Streptococcus

A temperate bacteriophage, designated GT-234, was isolated from a group G Streptococcus after ultraviolet irradiation. After several single-plaque passages in a group G indicator strain, this phage formed plaques in 3 of 14 group A strains, in 3 of 15 group C strains, and in 4 of 13 group G strains-but not in some representatives of several other serogroups. After propagation in each of the sensitive strains, the progeny from each was shown to be the same phage by (i) adsorption and plaque formation in each of the other groups, (ii) lysogenization in each of the other groups, (iii) high titers on infection of each serogroup, regardless of the group of propagating strain, and (iv) neutralization of infection in each of the other groups by antiserum against the phage propagated in group G. Phage GT-234 is serologically related to virulent group A phage A25, from which it is morphologically indistinguishable. Like A25, it is a transducing phage. Other studies showed that A25, as well as a group A temperate transducing phage (AT-298), could also infect strains of group C and G. These results indicate a need for reassessment of group specificity and phage receptors among streptococci of groups A, C, and G and raise possibilities for intergroup transduction.     

Binding of albumin promotes bacterial survival at the epithelial surface

Human serum albumin (HSA) is the dominating protein in human plasma. Many bacterial species, especially streptococci, express surface proteins that bind HSA with high specificity and affinity, but the biological consequences of these protein-protein interactions are poorly understood. Group G streptococci (GGS), carrying the HSA-binding protein G, colonize the skin and the mucosa of the upper respiratory tract, mostly without causing disease. In the case of bacterial invasion, pro-inflammatory cytokines are released that activate the epithelium to produce antibacterial peptides, in particular the chemokine MIG/CXCL9. In addition, the inflammation causes capillary leakage and extravasation of HSA and other plasma proteins, environmental changes at the epithelial surface to which the bacteria need to respond. In this study, we found that GGS adsorbed HSA from both saliva and plasma via binding to protein G and that HSA bound to protein G bound and inactivated the antibacterial MIG/CXCL9 peptide. Another surface protein of GGS, FOG, was found to mediate adherence of the bacteria to pharyngeal epithelial cells through interaction with glycosaminoglycans. This adherence was not affected by activation of the epithelium with a combination of IFN-γ and TNF-α, leading to the production of MIG/CXCL9. However, at the activated epithelial surface, adherent GGS were protected against killing by MIG/CXCL9 through protein G-dependent HSA coating. The findings identify a previously unknown bacterial survival strategy that helps to explain the evolution of HSA-binding proteins among bacterial species of the normal human microbiota.     

Protein G genes: structure and distribution of IgG-binding and albumin-binding domains

Protein G (also designated Fc receptor type III) is the IgG-binding protein of group C and G streptococci. Protein G has also been shown to bind human serum albumin but at a site that is structurally separated from the IgG-binding region. From the known gene sequence of protein G, two synthetic oligonucleotides were constructed for use as probes in DNA-hybridization experiments to study the structure and distribution of the albumin- and IgG-binding regions in bacterial strains belonging to different species. Thus, one of the probes corresponded to repeats within the IgG-binding region (I) and the other corresponded to repeats in the albumin-binding encoding region (II). Probe I showed strong hybridization to DNA isolated from 31 human group C and G strains, whereas hybridization to probe II was variable. With the three restriction endonucleases used, three restriction patterns were found in Southern blot experiments. No fundamental difference could be detected in hybridization experiments, either between strains of group C and G streptococci, or between isolates of different clinical origin. No hybridization to DNA from other bacterial species was found.     

Isolation of non-type-specific protein antigens of Streptococcus group B

Hydrochloride extracts obtained from group B streptococcal strains of different serotypes have proved to be the source of type-nonspecific protein antigens, precipitated with ethanol and studied by gel chromatography and spectrophotometric scanning in ultraviolet rays. Thus, 2 or 3 antigens, one of them found to be common for streptococci of groups A, B and G, as well as the admixture of group-specific polysaccharide, have been detected. In extracts obtained from group B streptococcal strains of different serotypes a common protein antigen, specific only for group B, has been detected. The suitability of gel chromatography with the use Toyopearl gel HW-55F for the preparative isolation of the specific fraction of protein type-nonspecific antigen with a view to the subsequent study of immune response to group B streptococci has been shown.     

Structure of the IgG-binding regions of streptococcal protein G.

The gene encoding the IgG-binding protein G from Streptococcus G148 was isolated by molecular cloning. A subclone containing a 1.5-kb insert gave a functional product in Escherichia coli. Protein analysis of affinity-purified polypeptides revealed two gene products, both smaller than protein G spontaneously released from streptococci, but with identical IgG-binding properties. The complete nucleotide sequence of the insert revealed a repeated structure probably evolved through duplications of fragments of different sizes. The deduced amino acid sequence revealed an open reading frame extending throughout the insert, terminating in a TAA stop codon. Analysis of the two gene products by N-terminal amino acid determination suggests that two different TTG codons are recognized in E. coli for initiation of translation to yield the two products. Based on these results several truncated gene constructions were expressed and analysed. The results suggest that the C-terminal part of streptococcal protein G consists of three IgG-binding domains followed by a region which anchors the protein to the cell surface. Structural and functional comparisons with streptococcal M protein and staphylococcal protein A have been made.     

Specific binding sites for monomeric and aggregated beta 2-microglobulin on surface of groups A, B, C, and G streptococci

Human beta 2-microglobulin (beta 2-m) was isolated from urine samples of patients with tubular dysfunctions and aggregated with glutaraldehyde. Four aggregates with molecular weights of 800,000, 480,000, 260,000, and 60,000 were separated by filtration on Sephacryl S-300. The aggregates and monomeric beta 2-m (11,800 MW) were subsequently labeled with 125I and tested for binding to streptococci. Group A streptococci bound only aggregated beta 2-m with a mean binding of 44.5%. Most of the group G streptococci, on the other hand, bound only monomeric beta 2-m with a mean binding of 58%. Among group B streptococci the serotypes with protein antigens interacted mainly with monomeric beta 2-m and those without protein antigens preferentially with aggregated beta 2-m. Nontypable group B streptococcal serotypes did not bind monomeric or aggregated beta 2-m. Of the streptococci belonging to group C, S. equisimilis reacted with monomeric beta 2-m and S. dysgalactiae with aggregated beta 2-m. S. equi did not interact with monomeric beta 2-m or aggregated beta 2-m. Bindings of monomeric beta 2-m and aggregated beta 2-m were saturable and could be inhibited by the respective unlabeled forms of beta 2-m. Fibrinogen, fibronectin, alpha 2-macroglobulin, haptoglobin, or immunoglobulin G did not inhibit the binding of either form of beta 2-m. The binding sites for monomeric beta 2-m were more susceptible to trypsin than those for aggregated beta 2-m. Treatment of streptococci with pronase destroyed their binding activities for monomeric and aggregated beta 2-m. Both monomeric beta 2-m and aggregated beta 2-m binding sites were sensitive to heat. The Scatchard plots of monomeric beta 2-m and aggregated beta 2-m were linear with Kd of 1.29 X 10(-9) M and 1.9 X 10(-9) M respectively. The number of binding sites per bacterium were estimated to be 81,000 for monomeric beta 2-m and 1,210 for aggregated beta 2-m.     

Peptides as probes for G protein signal transduction

Triggered by agonist binding to cell surface receptors, the heterotrimeric G proteins dissociate into and βγ subunits, each activating distinct second messenger pathways. Peptides from the primary sequences of receptors, G proteins, and effectors have been used to study the molecular interactions between these proteins. Receptor-derived peptides from the second, third and fourth intracellular loops and certain naturally occurring peptides antagonize G protein interactions and can directly activate G protein. These peptides bind to G protein sites that include the N and C terminal regions of the subunit and a yet to be identified region of the β subunit. Peptides have also been useful in characterizing G protein-effector interactions. The identification of the contact sites between proteins involved in G protein signal transduction should aid in the development of non-peptide mimetic therapeutics which could specifically modify G protein-mediated cellular responses.     

Hemagglutination Activity and Localization of Fc Receptor of Group A and G Streptococci

The IgG-Fc binding activity and binding sites on the cell surface of streptococci, strains AR1 (group A) and G148 (group G), and Staphylococcus aureus strain Cowan I were examined by hemagglutination (HA) and immunoelectron microscopic methods. No distinct difference was observed in the HA activity among these three strains. However, the strains differed in the distribution of Fc receptor. Cowan I cells (having protein A) were heavily covered with two layers of ferritin particles, whereas AR1 cells were heavily covered with a single, rough layer of ferritin particles. G148 cells (having protein G) were labeled with a relatively thin, rough ferritin layer. The trypsin susceptibility of the Fc receptors of the AR1 strain was much higher than that of the G148 strain. These results suggest that both streptococcal strains are distinctly different in the arrangement or in the conformation of the Fc receptor from the Cowan I strain. It is also suggested that the Fc receptor molecules of the streptococcal strains differ from each other not only in conformation but also in trypsin susceptibility.     

Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin

Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.     

Comparative studies on surface hydrophobicity of streptococcal strains of groups A, B, C, D and G

Cell surface hydrophobicity of group A, B, C, D and G streptococcal strains has been studied and compared in a new test based on the fact that the degree of bacterial aggregation in ammonium sulphate depends on amphiphilic surface antigens. M-positive group A strains showing good growth in normal human blood aggregated in the standard salt aggregation test at very low concentrations of ammonium sulphate, while M-negative strains, which were killed in normal human blood, usually aggregated at high salt concentrations. Agents such as 2 M-KSCN, 2 M-guanidine. HC1 or 2 M-urea decreased the aggregation of the M-positive strains in the salt aggregation test while non ionic detergents such as Tween 20 (1%, w/v) and ethylene glycol (2 M) did not affect cell aggregation. Binding of fibrinogen and albumin resulted in a decrease of surface hydrophobicity of the group A M-positive strains. Group B strains possess a hydrophilic surface character and did not aggregate, while group C and G strains behaved in the salt aggregation test like M-negative strains of group A streptococci. Group D strains did not aggregate even at high ammonium salt concentrations. The results are discussed in relation to the influence of lipoteichoic acid and other surface antigens on strains of the various groups, and it is suggested that M protein and possibly also other surface proteins contribute to the high surface hydrophobicity of group A strains.     

Nucleic acid hybridization and cell wall composition studies of pyogenic streptococci

Abstract DNA-rRNA and DNA-DNA hybridization studies indicate that the classical pyogenic streptococci can be divided into five homology clusters. Based on these studies the term pyogenic streptococci should be confined to the first cluster consisting of serological groups A, A-variant, C, G ('large' colony, type II) and L.
Streptococci of serological groups B and M form the second cluster. The third cluster is composed of streptococci of serological groups R and S and serological groups U, V and P are found in the fourth cluster. The fifth cluster comprises strains of Streptococcus anginosus S. intermedius, Streptococcus MG and serological groups G ('minute' colony, type I) and F (type I). Most of the test strains contain the peptidoglycan type Lys-Ala_1–3. Only streptococci of serogroups R and S reveal a directly cross-linked peptidoglycan. Rhamnose was found as characteristic component of all cell wall polysaccharides. The impact of our results on the systematics of classical pyogenic streptococci will be discussed.     

G protein beta2 subunit-derived peptides for inhibition and induction of G protein pathways. Examination of voltage-gated Ca2+ and G protein inwardly rectifying K+ channels

Voltage-gated Ca2+ channels of the N-, P/Q-, and R-type and G protein inwardly rectifying K+ channels (GIRK) are modulated via direct binding of G proteins. The modulation is mediated by G protein betagamma subunits. By using electrophysiological recordings and fluorescence resonance energy transfer, we characterized the modulatory domains of the G protein beta subunit on the recombinant P/Q-type channel and GIRK channel expressed in HEK293 cells and on native non-L-type Ca2+ currents of cultured hippocampal neurons. We found that Gbeta2 subunit-derived deletion constructs and synthesized peptides can either induce or inhibit G protein modulation of the examined ion channels. In particular, the 25-amino acid peptide derived from the Gbeta2 N terminus inhibits G protein modulation, whereas a 35-amino acid peptide derived from the Gbeta2 C terminus induced modulation of voltage-gated Ca2+ channels and GIRK channels. Fluorescence resonance energy transfer (FRET) analysis of the live action of these peptides revealed that the 25-amino acid peptide diminished the FRET signal between G protein beta2gamma3 subunits, indicating a reorientation between G protein beta2gamma3 subunits in the presence of the peptide. In contrast, the 35-amino acid peptide increased the FRET signal between GIRK1,2 channel subunits, similarly to the Gbetagamma-mediated FRET increase observed for this GIRK subunit combination. Circular dichroism spectra of the synthesized peptides suggest that the 25-amino acid peptide is structured. These results indicate that individual G protein beta subunit domains can act as independent, separate modulatory domains to either induce or inhibit G protein modulation for several effector proteins.     

Group G streptococcal IgG binding molecules FOG and protein G have different impacts on opsonization by C1q

Recent epidemiological data on diseases caused by beta-hemolytic streptococci belonging to Lancefield group C and G (GCS, GGS) underline that they are an emerging threat to human health. Among various virulence factors expressed by GCS and GGS isolates from human infections, M and M-like proteins are considered important because of their anti-phagocytic activity. In addition, protein G has been implicated in the accumulation of IgG on the bacterial surface through non-immune binding. The function of this interaction, however, is still unknown. Using isogenic mutants lacking protein G or the M-like protein FOG (group G streptococci), respectively, we could show that FOG contributes substantially to IgG binding. A detailed characterization of the interaction between IgG and FOG revealed its ability to bind the Fc region of human IgG and its binding to the subclasses IgG1, IgG2, and IgG4. FOG was also found to bind IgG of several animal species. Surface plasmon resonance measurements indicate a high affinity to human IgG with a dissociation constant of 2.4 pm. The binding site was localized in a central motif of FOG. It has long been speculated about anti-opsonic functions of streptococcal Fc-binding proteins. The presented data for the first time provide evidence and, furthermore, indicate functional differences between protein G and FOG. By obstructing the interaction between IgG and C1q, protein G prevented recognition by the classical pathway of the complement system. In contrast, IgG that was bound to FOG remained capable of binding C1q, an effect that may have important consequences in the pathogenesis of GGS infections.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.     

Distribution and serological specificity of sialidase produced by various groups of streptococci

The occurrence of a streptococcal sialidase (designated St-sialidase) in culture fluids of various streptococci was investigated. St-sialidase was found to occur in strains belonging to groups A, B, C, E, G, H, and L, and the unclassified strains, Streptococcus sanguis and Streptococcus uberis. St-sialidase of group A was confined predominantly to types 4 and 22. St-sialidases, extracted from the culture fluids of some selected strains, were antigenic, eliciting the formation of antibody which effectively neutralized the enzymatic activity of the enzyme. Antisera to the St-sialidases of groups A, B, C, E, G, and L, and Streptococcus sanguis were produced in rabbits. The St-sialidases of groups A, B, and E streptococci were serologically distinct and group-specific. The St-sialidases from groups C, G, and L were serologically homologous, but distinct from St-sialidases of the other groups. Antiserum to the enzyme of strain 10557 (S. sanguis) cross-reacted with the St-sialidase of strain 9927 (S. uberis).