C5a peptidase alters clearance and trafficking of group A streptococci by infected mice.

Group A streptococcal C5a peptidase (SCPA) specifically cleaves the human serum chemotaxin C5a at the polymorphonuclear leukocyte (PMNL) binding site. This study tested the proposal that SCPA contributes to virulence by retarding the influx of inflammatory cells and clearance of streptococci during the first few hours after infection. To investigate the specific contribution of SCPA to the virulence of group A streptococci, scpA insertion and deletion mutants were created by directed plasmid insertion into scpA and gene replacement. The precise locations of insertion and deletion mutations were confirmed by PCR and DNA sequence analysis. The impact of mutation on virulence was investigated with a mouse air sac model of inflammation. Experiments evaluated clearance of streptococci from the air sac within 4 h after infection. SCPA- streptococci were cleared more efficiently than wild-type bacteria. Localization of streptococci in lymph nodes and spleens of infected mice revealed a significant difference between mutant and wild-type streptococci. PMNLs and other granulocytes that infiltrated the air sac were quantitated by single-color flow cytometry. The total cellular infiltrate was greater and PMNLs dominated the granulocytic infiltrates of air sacs inoculated with SCPA- mutant bacteria. The data obtained are consistent with the possibility that SCPA- streptococci are initially cleared from the site of infection primarily by PMNLs. Moreover, mutant and wild-type streptococci followed different paths of dissemination. SCPA- bacteria were transported to lymph nodes, whereas wild-type streptococci avoided transport to the lymph nodes and rapidly spread to the spleen.     

Similarity between the group B and A streptococcal C5a peptidase genes.

Group B streptococci (GBS) and group A streptococci (GAS) are known to have surface-associated peptidase activity which specifically cleaves C5a (C5a-ase), the primary chemotaxin for polymorphonuclear leukocytes. Amplification products were obtained from GBS genomic DNA template by using the polymerase chain reaction and primers corresponding to the C5a peptidase gene of GAS (scpA12). A restriction map of the GBS full-length amplified gene (scpB) was developed. The scpB restriction map was found to be highly similar to that of the analogous gene in GAS. A 50-bp deletion was located near the 5' end of scpB in a region where repeat sequences are found in scpA12. Hybridization experiments confirmed that other serotypes of GBS also carry an scpB-like gene. These results show that GBS contain a gene, scpB, which is very similar to that harbored by M12 GAS. The probability that scpB encodes the C5a-ase protein is discussed.     

Intranasal immunization with C5a peptidase prevents nasopharyngeal colonization of mice by the group A Streptococcus.

Early inflammatory events are initiated by phased production of C5a and interleukin-8 in tissue. Most serotypes of group A streptococci express a surface-bound peptidase (SCPA) which specifically cleaves mouse and human C5a chemotaxins. This study investigates the impact of SCPA on colonization of the nasopharyngeal mucosa of mice and evaluates its potential to induce protective immunity. Two strains, serotypes M6 and M49, which contain insertion and deletion mutations in the SCPA gene (scpA) and represent the two major subdivisions of group A streptococci, were characterized and compared in a mouse intranasal infection model. In this model, SCPA mutants were more rapidly cleared from the nasopharynges of inoculated mice compared with wild-type strains. A 2,908-bp fragment of scpA49 gene, obtained by PCR, was ligated to the expression vector pGEX-4T-1 and expressed in Escherichia coli. The affinity-purified deltaSCPA49 protein proved to be highly immunogenic in mice and rabbits. Although the purified deltaSCPA49 immunogen lacked enzymatic activity, it induced high titers of rabbit antibodies which were able to neutralize peptidase activity associated with M1, M6, M12, and M49 streptococci in vitro. This result confirmed that antipeptidase antibodies lack serotype specificity. Intranasal immunization of mice with the deleted form of the SCPA49 protein stimulated significant levels of specific salivary secretory immunoglobulin A (IgA) and serum IgG antibodies and reduced the potential of wild-type M1, M2, M6, M11, and M49 streptococci to colonize. These experiments suggest a new approach to vaccine development for prevention of streptococcal pharyngitis.     

Biological and immunochemical identity of M protein on group G streptococci with M protein on group A streptococci.

Previous evidence for the presence of an M or M-like protein on group G streptococci has been based on the ability of these strains to survive in human blood. In addition, cross-reactions between group A and group G streptococci have been demonstrated, but they have relied either on whole bacterial cell vaccine-induced polyclonal sera or crude protein extracts of these cells. In this study two monoclonal antibodies prepared against the purified, native group A streptococcal M6 protein demonstrated a high degree of cross-reactivity with group G streptococcal clinical isolates (9 and 19 of 22 strains examined, respectively). Ten of these strains exhibited resistance to phagocytosis when rotated in human blood. In addition, immunoblot analysis of crude mutanolysin extracts of group G streptococci with one of the M6 monoclonal antibodies illustrated a remarkable similarity in the protein pattern of these extracts as compared with those of group A streptococcal M protein. The immunoblots further demonstrated a variation in the relative molecular weights of the extracted proteins from strain to strain over a range of 57,000 to 77,000. In addition, a purified, pepsin-derived fragment (Mr, 43,000) from a group G strain was capable of eliciting rabbit antibodies that were opsonic for group G cells in a bactericidal assay. These functional and immunochemical data, in concert with DNA hybridization between group G streptococcal DNA and a group A M6 gene probe (J. R. Scott, W. M. Pulliam, S. K. Hollingshead, and V. A. Fischetti, Proc. Natl. Acad. Sci. USA 82:1822-1826, 1985), provide strong evidence for the presence of an M protein on these organisms and indicate its probable role as a virulence molecule on the surface of group G streptococci.     

Localization of the streptococcal C5a peptidase to the surface of group A streptococci.

Immunofluorescent staining was used to determine that the streptococcal C5a peptidase (SCP) exists as a cell surface antigen on group A streptococci. The ability of hyperimmune serum to neutralize cell-associated SCP activity provided further evidence for the location of SCP. Quantification of SCP during growth in vitro by indirect enzyme-linked immunosorbent assay showed that approximately 90% of the measurable antigen is cell bound.     

Heterogeneity of the streptokinase gene in group A streptococci.

A molecular epidemiological study was conducted to determine the distribution of the streptokinase gene in group A streptococcal strains of different M types and in other streptococcal species. Plasmid pNC1, containing only the internal coding sequence of the streptokinase gene from group C streptococcal strain H46A, was used as a DNA probe in colony and Southern hybridization experiments. Only the pathogenic group A, C, and G streptococci contained a streptokinase gene; 12 other Lancefield group strains did not. A total of 134 group A strains, including 61 M types and 6 T types, were tested. Although only 62% (83 of 134) of the strains tested showed positive streptokinase activity by the casein-plasminogen overlay assay, all strains contained the streptokinase gene as evidenced by strong hybridization with the pNC1 probe. Southern blot DNA hybridizations were carried out with 101 strains of group A streptococci. The restriction enzymes HindIII and HaeIII were used to digest the genomic DNA. Six hybridization patterns were observed after HindIII digestion. Double hybridization bands appeared in all of the patterns, which indicated the existence of a highly conserved HindIII site. More complex hybridization results were obtained after HaeIII digestion. Twelve hybridization patterns were observed; three were characterized by a single hybridization band, and nine were characterized by double bands. Variations in hybridization patterns were observed in strains of both the same and different serotypes. The overall results at the gene level indicate that there is considerable heterogeneity among the streptokinases of group A streptococci, consistent with previous findings of immunological and chemical differences among streptokinases of group A streptococci.     

Bacteriological and serological aspects of group a streptococcal pharyngotonsillitis caused by group a streptococci

Several bacteriological and serological variables were studied in connection with a clinical treatment trial in 212 patients with group A streptococcal pharyngotonsillitis. Anaerobic incubation was not superior to incubation in 5 % CO₂ in air for the detection of group A streptococci. Saliva cultures were inferior to conventional throat cultures in detecting group A streptococci. No strains from patients with recurrences were found to be tolerant to penicillin. In several patients (all asymptomatic), group C and G streptococci were found in follow-up cultures. Group A streptococci serology was more often positive after two months than after one month, also in patients without recurrence.     

Characterization of binding of human alpha 2-macroglobulin to group G streptococci.

An interaction was observed between human alpha 2-macroglobulin (alpha 2M) and streptococci belonging to group A, C, and G. Of 27 group C and 19 group G streptococcal cultures, 13 and 14, respectively, bound 125I-labeled alpha 2M. Some group A streptococci also interacted with alpha 2M. A number of other bacterial species tested did not react with alpha 2M. The binding of 125I-labeled alpha 2M to group G streptococci was time dependent, saturable, and could be inhibited by unlabeled alpha 2M. Inhibition experiments indicated that the streptococcal binding site for alpha 2M differed from the receptors for immunoglobulin G, fibrinogen, aggregated beta 2-microglobulin, albumin, and fibronectin. The alpha 2M binding activity was remarkably sensitive to trypsin and heat treatment indicating its protein nature. Kinetic analysis indicated a homogenous population of binding sites. The number of binding sites per bacterial cell was estimated to be approximately 20,000.     

Binding of human fibronectin to group A, C, and G streptococci

A total of 387 bacterial strains belonging to 35 species were tested in direct binding experiments for the uptake of purified radiolabeled human fibronectin. Positive binding was found in group A, C, and G streptococci and in Staphylococcus aureus. The group C streptococcal species Streptococcus equisimilis, Streptococcus zooepidemicus, Streptococcus equi, and Streptococcus dysgalactiae were uniformly reactive with fibronectin. Beta-hemolytic bovine group G streptococci showed the same degree of reactivity as human group G strains. In contrast, only 4 out of 15 alpha-hemolytic bovine group G strains were able to bind fibronectin. The uptake of fibronectin measured at room temperature with a human group G streptococcus was a slow, time-dependent process with maximum binding after approximately 1 h. Human polyclonal immunoglobulin G and serum albumin tested in inhibition experiments did not affect the fibronectin binding. Fibronectin seems, therefore, to interact with a surface component that is different from the specific binding sites previously described for human immunoglobulin G and serum albumin.     

The gene encoding a new mitogenic factor in a Streptococcus pyogenes strain is distributed only in group A streptococci.

We recently cloned a gene encoding a new mitogenic factor (MF) from Streptococcus pyogenes NY-5. In the present study, we determined the distribution of this MF gene (mf) by PCR based upon its sequence. Of 371 streptococcal group A strains isolated from clinical specimens, 370 (99.7%) were positive for mf. The strain that was negative for the MF gene was also negative for the streptolysin O gene (slo). Some streptococcal strains belonging to groups C and G were negative for mf but positive for slo. Group B strains were negative for both. Furthermore, we examined the presence of mf in 54 strains belonging to 28 families and found mf only in group A streptococci. These results indicate that mf is distributed specifically in group A streptococci and the presence of mf in clinical samples strongly suggests infection with group A streptococci.     

Comparison of albumin receptors expressed on bovine and human group G streptococci.

The albumin receptor expressed by bovine group G streptococci was extracted and affinity purified. The protein was characterized for species reactivity, and monospecific antibodies were prepared to the purified receptor. The bovine group G albumin receptor was compared functionally, antigenically, and for DNA homology with the albumin-binding protein expressed by human group G streptococci. In agreement with previous reports, the albumin-binding activity of human strains was mediated by a unique domain of the type III immunoglobulin G-Fc-binding molecule, protein G. The albumin receptor expressed by bovine group G strains was found to lack any immunoglobulin G-binding potential but displayed a wider profile of species albumin reactivity than protein G. Both albumin receptors could inhibit the binding of the other to immobilized human serum albumin, and each displayed similar binding properties. Antigenic comparison of the two albumin receptors demonstrated a low level of cross-reactivity; however comparison at the DNA level, using an oligonucleotide probe specific for the albumin-binding region of protein G, demonstrated that the two albumin receptors expressed by human and bovine group G streptococcal strains do not display significant homology.     

Secretory antibodies in saliva against group G streptococci (author's transl)

Saliva of 310 persons were examined for their ability to agglutinate 33 streptococcal strains of the groups A-T. It was found that the saliva of persons with blood group O agglutinated significantly more often streptococci of strain G9 than the saliva of persons with blood group A (33.3%, and 18.75% resp., p less than 0.01). A statistical significant difference between persons of blood group B and O, or A and B did not exist. The agglutination reactions were caused probably by unspecific immunoglobulins because this strain G9 binds unspecifically human IgG. It is known, that some streptococcal strains of the groups A, B, C, D, and G contain receptors for the Fc-fragment of IgG and/or IgA. The results demonstrate that persons of the blood group O secrete more often immunoglobulin than those with blood group A.     

Receptor in group C and G streptococci detects albumin structures present in mammalian species.

The presence of albumin structures with the capacity to bind to a surface receptor in group C and G streptococci was studied in serum samples from 45 mammalian species representing 15 different orders, using an inhibition assay. The ability of animal sera to inhibit the uptake of radiolabeled human serum albumin by the streptococci indicated the presence of such albumin structures. Positive reactions were found in species of most orders tested, with Marsupialia as a notable exception. All Carnivora sera tested were strongly positive. In some orders such as Artiodactyla both positive and negative species were identified. Serum samples from 62 bird species representing 15 orders and from 5 fish species were also tested in the inhibition assay. None of these serum samples was capable of inhibiting the uptake of human serum albumin by streptococci. Some differences were also noted in the results obtained with group C and G streptococci from human and bovine sources, respectively, indicating the presence of two types of receptors. The present studies suggest a phylogenetic origin of albumin structures with affinity for the streptococcal receptor to a period after the divergence of Marsupialia from the other mammalian orders.     

Extraction and characterization of IgG Fc receptors from group C and group G streptococci

Immunoglobulin G (IgG) Fc receptors (FcRs) were extracted by proteolytic digestion of four strains each of group C and group G streptococci. The solubilized proteins were analyzed in Western blots and multiple IgG-binding bands were obtained. The banding patterns of some of the strains were very similar, but this property was independent of which streptococcal group the strains belonged to. Highly purified FcRs were prepared from one group C and one group G strain. The 13 N-terminal amino acids were determined, and found to be identical, whereas comparison with the sequence of staphylococcal protein A did not reveal any homology. The isolated streptococcal FcRs also appeared closely related antigenically and functionally. Thus, both molecules were capable of inhibiting each others binding to immobilized IgG, and the radiolabelled group G FcR was completely inhibited from binding to IgG by an antibody to the group C FcR. Finally, in a direct binding assay both proteins were capable of reacting to a similar degree with a wide variety of IgGs, thereby demonstrating the great potential of streptococcal FcRs as tools for binding and detection of IgG antibodies.     

Characterization of haptoglobin-binding properties of streptococci of serological group G

Two group G streptococcal cultures (G 10187, G 11122) with surface antigen T4 possess surface receptors for human haptoglobin (Hp). G 10187 additionally interacted with immunoglobulin G and albumin, G 11122 with fibrinogen and fibronectin. Binding of 125I-Hp 2-1 was time-dependent, saturable, reversible in the presence of unlabelled Hp and could be inhibited by unlabelled human-Hp 2-1, -Hp 2-2, -Hp 1-1, Hp-hemoglobin complexes and by Hp preparations from pigs, horses and rabbits. The Hp binding sites could be destroyed by heat treatment (95 degrees C) and by proteolytic treatment of the bacteria. Hp binding sites were solubilized from group G streptococcal surface by heat treatment of the bacteria at acid pH and subsequently isolated by affinity chromatography on Hp 2-1 sepharose. SDS-PAGE and Western blotting of the Hp binding proteins revealed numerous protein bands with 125I-Hp 2-1 binding activity. Specific antibodies against G streptococcal binding proteins prepared in chickens inhibited binding of 125I-Hp to group G and group A streptococci, but not to Actinomyces pyogenes.     

Isolation and molecular characterization of a novel albumin-binding protein from group G streptococci.

Many streptococcal strains are known to bind the two most abundant plasma proteins, namely, immunoglobulin G and albumin. Protein G isolated from group C and G streptococci has been demonstrated to have separate binding regions for each of these proteins. However, some group G streptococcal strains bind only serum albumin. This report describes the isolation of a 48-kDa albumin-binding protein from such a strain (DG12). The affinity constant of this protein for human serum albumin was determined to be 5 x 10(9) M-1, and the protein interacted strongly also with serum albumin from several other mammalian species. The gene encoding the albumin-binding protein was cloned and expressed in Escherichia coli. DNA sequence analysis of this gene revealed a unique NH2-terminal sequence and three types of repeats in the encoded protein. One of these repeated sequences has significant homology with the albumin-binding domains of protein G, and it was demonstrated that the albumin binding of the DG12 protein was localized within these domains. Another type of repeat is localized in the putative wall-spanning region of the molecule. This repeat sequence, which has the length of only 4 amino acids (LysProGluVal), is repeated 14 times. The relationship of the albumin-binding protein to other cell-wall-associated proteins of pathogenic streptococci is discussed.     

Active and passive intranasal immunizations with streptococcal surface protein C5a peptidase prevent infection of murine nasal mucosa-associated lymphoid tissue, a functional homologue of human tonsils

C5a peptidase, also called SCPA (surface-bound C5a peptidase), is a surface-bound protein on group A streptococci (GAS), etiologic agents for a variety of human diseases including pharyngitis, impetigo, toxic shock, and necrotizing fasciitis, as well as the postinfection sequelae rheumatic fever and rheumatic heart disease. This protein is highly conserved among different serotypes and is also expressed in human isolates of group B, C, and G streptococci. Human tonsils are the primary reservoirs for GAS, maintaining endemic disease across the globe. We recently reported that GAS preferentially target nasal mucosa-associated lymphoid tissue (NALT) in mice, a tissue functionally analogous to human tonsils. Experiments using a C5a peptidase loss-of-function mutant and an intranasal infection model showed that this protease is required for efficient colonization of NALT. An effective vaccine should prevent infection of this secondary lymphoid tissue; therefore, the potential of anti-SCPA antibodies to protect against streptococcal infection of NALT was investigated. Experiments showed that GAS colonization of NALT was significantly reduced following intranasal immunization of mice with recombinant SCPA protein administered alone or with cholera toxin, whereas a high degree of GAS colonization of NALT was observed in control mice immunized with phosphate-buffered saline only. Moreover, administration of anti-SCPA serum by the intranasal route protected mice against streptococcal infection. These results suggest that intranasal immunization with SCPA would prevent colonization and infection of human tonsils, thereby eliminating potential reservoirs that maintain endemic disease.     

Identification of a fibronectin-binding protein (GfbA) in pathogenic group G streptococci.

Attachment to eukaryotic cell surfaces is an essential step in the establishment of colonization and infection by bacterial pathogens. This report examines the adherence capabilities of pathogenic group G streptococci and demonstrates that certain group G streptococcal clinical isolates express a fibronectin-binding protein. This protein, termed GfbA for group G streptococcal fibronectin-binding protein, mediates adherence to human skin fibroblasts (HSF). The gene encoding this protein, gfbA, was isolated, and the complete DNA sequence of gfbA was determined. From this sequence GfbA was predicted to be a 580-amino-acid protein (molecular weight = 64,979) with significant amino acid identity to the group A streptococcal fibronectin-binding proteins SfbI and protein F (PrtF) (76 and 78% identity, respectively). GfbA contains regions with notable identity to the fibronectin-binding repeat domains of PrtF. gfbA(+) strains were able to bind to HSF, and preincubation of the gfbA(+) strains with fibronectin blocked this adherence. In addition, gfbA(+) strains were able to bind radiolabeled fibronectin, and this binding was inhibited with addition of excess unlabeled fibronectin. gfbA-negative strains were not able to bind either the HSF or radiolabeled fibronectin. DNA homologous to gfbA was found in 36% of the group G streptococcal isolates examined. Since not all group G streptococcal strains examined contained gfbA, this suggests there might be other tissue-specific adherence molecules expressed by these pathogenic strains.     

Binding of fibronectin to the surface of group A,C, and G streptococci isolated from human infections

Sixty-nine haemolytic and non-haemolytic streptococcal strains were isolated from various human infections and serogrouped with the coagglutination test. The amount of¹²⁵I-fibronectin bound to bacterial cells in a standard assay was determined. Most of the group A, C, and G strains were able to bind fibronectin. None of the group B or D strains bound significant amounts of fibronectin. Group A, C, and G streptococci appear to preferentially bind the N-terminal region of the fibronectin molecule because the 25K N-terminal fragment of the protein could effectively inhibit the binding of¹²⁵I-fibronectin to cells. Furthermore, the ability of representative strains of group A, C, and G to bind fibronectin was markedly reduced after trypsin treatment of the cells. Fibronectin binding components released from one strain by trypsin treatment inhibited the binding of¹²⁵I-fibronectin to all group A, C, and G streptococci strains. These findings indicate similarities among fibronectin binding proteins of the three groups of streptococci tested. However, the relative susceptibility to trypsin of fibronectin receptors of the three strains differed as did the relative potency of the inhibitory activity of receptors solubilized from different strains. Binding of fibronectin to the cell surface of group A, C, and G streptococci may contribute to virulence, for instance by promoting specific attachment to exposed fibronectin in open wounds and tissue lesions.     

Bacterial infection of wounds: fibronectin-mediated adherence group A and C streptococci to fibrin thrombi in vitro.

Adherence of group A, B, and C streptococci to fibrin thrombi was studied by using a novel fluorochrome microassay carried out in microdilution plates in which fibrin thrombi had been prepared by clotting citrated human, cattle, or horse plasma. Substantial adherence was observed with various strains of group A and C streptococci, whereas little was observed with group B streptococci. Adherence of all group A and C streptococcal strains decreased by up to 40% when fibronectin was depleted from the plasmas used for preparing fibrin thrombi, and fibronectin repletion increased adherence of streptococci in a dose-dependent manner. Addition of the 210-kilodalton C-terminal fragment of fibronectin to fibronectin-depleted plasma restored the adherence of group C but not group A streptococci, whereas addition of the 29-kilodalton N-terminal fragment was without any effect for all tested streptococcal strains. Prior incubation of group A and C streptococcal strains with fibronectin markedly increased their adherence, but treatment with proteases abolished their ability to adhere to fibrin thrombi. Adherence of group B streptococci was not affected by either fibronectin depletion or proteolytic digestion. These results indicate that both fibronectin incorporated into the fibrin matrix of thrombi and soluble fibronectin can mediate adherence of group A and C streptococci to fibrin thrombi and that binding sites for fibronectin located on the bacterial surface mediate this adherence.