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Group A Streptococcus (GAS; Streptococcus pyogenes) is a bacterial pathogen for which a commercial vaccine for humans is not available. Employing the advantages of high-throughput DNA sequencing technology to vaccine design, we have analyzed 2,083 globally sampled GAS genomes. The global GAS population structure reveals extensive genomic heterogeneity driven by homologous recombination and overlaid with high levels of accessory gene plasticity. We identified the existence of more than 290 clinically associated genomic phylogroups across 22 countries, highlighting challenges in designing vaccines of global utility. To determine vaccine candidate coverage, we investigated all of the previously described GAS candidate antigens for gene carriage and gene sequence heterogeneity. Only 15 of 28 vaccine antigen candidates were found to have both low naturally occurring sequence variation and high (>99%) coverage across this diverse GAS population. This technological platform for vaccine coverage determination is equally applicable to prospective GAS vaccine antigens identified in future studies., info:eu-repo/semantics/published

Streptococcus pyogenes (group A Streptococcus (GAS)) causes ∼700 million human infections each year, resulting in over 500,000 deaths. The development of a commercial GAS vaccine is hampered by the occurrence of many unique GAS serotypes, antigenic variation within the same serotype, differences in serotype geographical distribution, and the production of antibodies crossreactive with human tissue that may lead to autoimmune disease. Several independent studies have documented a number of GAS cell wall-associated or secreted metabolic enzymes that contain neither N-terminal leader sequences nor C-terminal cell wall anchors. Here, we applied a proteomic analysis of serotype M1T1 GAS cell wall extracts for the purpose of vaccine development. This approach catalogued several anchorless proteins and identified two protective vaccine candidates, arginine deiminase and trigger factor. These surface-exposed enzymes are expressed across multiple GAS serotypes exhibiting =99% amino acid sequence identity. Vaccine safety concerns are alleviated by the observation that these vaccine candidates lack human homologs, while sera from human populations suffering repeated GAS infections and high levels of autoimmune complications do not recognize these enzymes. Our study demonstrates anchorless cell surface antigens as promising vaccine candidates for the prevention of GAS disease. © Springer-Verlag 2012.

Zusammenfassung Jeweils 6 Kulturen von Streptococcus pyogenes vom Menschen, Sc. dysgalactiae vom Rind, Sc. equisimilis vom Menschen und Sc. equisimilis vom Pferd wurden aufgrund ihrer deutlichen Fc-Rezeptoraktivitat fur Immunglobulin (Ig)G vom Menschen zu Untersuchungen ihrer Bindungsspektren ausgesucht. Die radiale Immundiffusion eignete sich zu quantitativen Bestimmungen der IgG-Fc-Rezeptoraktivitat in den Testseren von Mensch, Pferd, Rind, Schwein, Schaf, Ziege, Kaninchen, Hund und Huhn. Dabei zeigten die Sc.pyogenes-Kulturen die starkste Bindungsaktivitat fur Human-IgG und keine oder nur eine geringe Fc-Rezeptoraktivitat fur IgG von Pferd, Ziege, Hund und Huhn. Sc. dysgalactiae vom Rind band verhaltnismasig viel bovines IgG. Sc. equisimilis vom Menschen hatte eine relativ hohere Fc-Rezeptoraktivitat fur Human-IgG und Sc. equisimilis vom Pferd fur das IgG des Wirtstieres. Die Ergebnisse der radialen lmmundiffusion fur Human-IgG stimmten mit den Meswerten der direkten Bindungen von 125J-markiertem IgG vom Menschen weitgehend uberein. Die Spezifitat der IgG-Fc-Rezeptoraktivitat konnte durch Inhibitionsversuche bestatigt werden. Summary Quantitative Determinations of Immunoglobulin G-Bindings to Streptococci Six cultures of Streptococcus pyogenes from man, Sc. dysgalactiae from cattle, Sc. e-quisimilis from humans and Sc. equisimilis from horses were selected for these studies on the basis of their marked Fc-receptor-activity for human immunoglobulin (Ig)G. Radial immunodiffusion (Fig. 1) proved to be suitable for quantitative determinations of the IgG-Fc-receptor-activity in test-sera from man, horses, cattle, pigs, sheep, goats, rabbits, dogs and chickens. Sc. pyogenes showed the strongest binding-activity with human-IgG and little or now activity with IgG from horses, goats, dogs and chickens. Sc. dysgalactiae from cattle bound more bovine IgG. Sc. equisimilis from man had a somewhat higher Fc-receptor-activity for human IgG and Sc. equisimilis from horses for equine IgG (Fig. 2). The results of the radial immunodiffusion for human IgG corresponded with those of 125I-human-IgG-binding. The specificity of the IgG-Fc-receptor-activity was confirmed by inhibition experiments. Resume Determinations quantitatives des liaisons immunoglobulines G sur des streptocoques Six cultures de Streptococcus pyogenes humaines, de Sc. dysgalactiae bovines, de Sc. equisimilis humaines et de Sc. equisimilis equines ont ete examinees sur la base de leur nette activite Fc-receptrice pour l'immunoglobuline (Ig)G humaine pour rechercher leurs spectres de liaison. L'immunodiffusion radiale convient pour des determinations quantitatives de l'activite receptrice IgG-Fc dans les serums testes humains, equins, bovins, porcines, ovins, caprins, du lapin, du chien et de la poule. Les cultures de Sc. pyogenes ont montrte la plus forte activite de liaison pour l'IgG humaine et aucune ou seulement une faible activite receptrice Fc pour l'IgG du cheval, de la chevre, du chien et de la poule. Sc. dysgalactiae du bovin a lie relativement beaucoup l'IgG bovine. Sc. equisimilis humain avait une activite receptrice Fc relativement elevee pour I'IgG humaine et Sc. equisimilis du cheval pour 1′IgG de l'animal hote. Les resultats de l'immunodiffusion pour l'IgG humaine ont bien correspondu avec les valeurs mesurees des liaisons directes de l'IgG humaine marquee au 125J. La specificite de l'activite receptrice IgG-Fc a pu etre confirmee par des essais d'inhibition. Resumen Determinaciones cuantitativas de la union de la inmunoglobulina G a los streptococcus Se eligieron en cada caso 6 cultivos de Streptococcus pyogenes de la especie humana, Sc. dysgalactiae de vacunos, Sc. equisimilis de la epecie humana y Sc. equisimilis de caballos, en virtud de su actividad manifiesta como receptor para Fc de las inmunoglobulinas (Ig)G de los seres humanos, para estudiar sus especuos de fijacion. La inmunodifusion radial resulto apropiada para valoraciones cuantitativas de la actividad como receptor para Fc de IgG en los sueros de prueba de humanos, caballos, vacunos, cerdos, ovejas, cabras, conejos, perros y gallinas. Se observo que los cultivos de Sc. pyogenes presentaban la maxima actividad de fijacion para las IgG humanas y ninguna o solo escasa actividad de receptor Fc para las IgG de caballo, cabra, perro y gallina. Los Sc. dysgalactiae de vacuno absorbian relativamente mucha IgG bovina. Los Sc. equisimilis humanos tenian una actividad receptora Fc relativamente mas elevada para las IgG humanas y Sc. equisimilis de caballo para las IgG equinas. Los resultados de la inmunodifusion radial para las IgG humanas coincidian en gran parte con los valores de medicion de la union directa de IgG humanas marcadas con 125J. Por medio de ensayos de inhibicion pudo confirmarse la especificidad de la actividad como receptor de Fc de IgG.

The human protease plasmin plays a crucial role in the capacity of the group A streptococcus (GAS; Streptococcus pyogenes) to initiate invasive disease. The GAS strain NS88.2 was isolated from a case of bacteremia from the Northern Territory of Australia, a region with high rates of GAS invasive disease. Mutagenesis of the NS88.2 plasminogen binding M protein Prp was undertaken to examine the contribution of plasminogen binding and cell surface plasmin acquisition to virulence. The isogenic mutant NS88.2prp was engineered whereby four amino acid residues critical for plasminogen binding were converted to alanine codons in the GAS genome sequence. The mutated residues were reverse complemented to the wild-type sequence to construct GAS strain NS88.2prpRC. In comparison to NS88.2 and NS88.2prpRC, the NS88.2prp mutant exhibited significantly reduced ability to bind human plasminogen and accumulate cell surface plasmin activity during growth in human plasma. Utilizing a humanized plasminogen mouse model of invasive infection, we demonstrate that the capacity to bind plasminogen and accumulate surface plasmin activity plays an essential role in GAS virulence.

We have constructed a targeted GAS virulence array containing 228 group A streptococcal genes. Using this array we profiled the virulence gene repertoire of 45 GAS isolates from The Netherlands. Cluster analysis of the array data for these strains successfully grouped strains by emm-type. Statistical analysis of the distribution of genes between emm-types showed that bacteriophage related genes were a significant contributor to the inter-emm-type genetic variation. However we also identified a number of non-phage genes whose distribution varied between emm-types. We hypothesise that these genes may be remnant or uncharacterised phage-related genes.

Fibronectin binding protein F1 (Sfb1) ofStreptococcus pyogenes(group A streptococcus [GAS]) is a well-characterized adhesin that has been shown to induce protection in mice against a lethal intranasal GAS challenge after intranasal immunization with cholera toxin B subunit (CTB) as adjuvant. With a murine skin infection model, we have shown that Sfb1/CTB vaccination neither elicits opsonizing antibodies nor prevents systemic bacterial growth and dissemination to internal organs after a subcutaneous GAS challenge. These results indicate that an Sfb1-based vaccine should be complemented with additional protective antigens in order to be used in areas such as the tropical north of Australia, where the skin is the primary route of entry for invasive streptococcal diseases.

The globally disseminated Streptococcus pyogenes M1T1 clone causes a number of highly invasive human diseases. The transition from local to systemic infection occurs by an unknown mechanism; however invasive M1T1 clinical isolates are known to express significantly less cysteine protease SpeB than M1T1 isolates from local infections. Here, we show that in comparison to the M1T1 strain 5448, the isogenic mutant delta speB accumulated 75-fold more human plasmin activity on the bacterial surface following incubation in human plasma. Human plasminogen was an absolute requirement for M1T1 strain 5448 virulence following subcutaneous (s.c.) infection of humanized plasminogen transgenic mice. S. pyogenes M1T1 isolates from the blood of infected humanized plasminogen transgenic mice expressed reduced levels of SpeB in comparison with the parental 5448 used as inoculum. We propose that the human plasminogen system plays a critical role in group A streptococcal M1T1 systemic disease initiation. SpeB is required for S. pyogenes M1T1 survival at the site of local infection, however, SpeB also disrupts the interaction of S. pyogenes M1T1 with the human plasminogen activation system. Loss of SpeB activity in a subpopulation of S. pyogenes M1T1 at the site of infection results in accumulation of surface plasmin activity thus triggering systemic spread.

Background. The factors behind the reemergence of severe, invasive group A streptococcal (GAS) diseases are unclear, but it could be caused by altered genetic endowment in these organisms. However, data from previous studies assessing the association between single genetic factors and invasive disease are often conflicting, suggesting that other, as-yet unidentified factors are necessary for the development of this class of disease. Methods. In this study, we used a targeted GAS virulence microarray containing 226 GAS genes to determine the virulence gene repertoires of 68 GAS isolates (42 associated with invasive disease and 28 associated with noninvasive disease) collected in a defined geographic location during a contiguous time period. We then employed 3 advanced machine learning methods (genetic algorithm neural network, support vector machines, and classification trees) to identify genes with an increased association with invasive disease. Results. Virulence gene profiles of individual GAS isolates varied extensively among these geographically and temporally related strains. Using genetic algorithm neural network analysis, we identified 3 genes with a marginal overrepresentation in invasive disease isolates. Significantly, 2 of these genes, ssa and mf4, encoded superantigens but were only present in a restricted set of GAS M-types. The third gene, spa, was found in variable distributions in all M-types in the study. Conclusions. Our comprehensive analysis of GAS virulence profiles provides strong evidence for the incongruent relationships among any of the 226 genes represented on the array and the overall propensity of GAS to cause invasive disease, underscoring the pathogenic complexity of these diseases, as well as the importance of multiple bacteria and/ or host factors.

The fibronectin binding protein Sfb1 of Streptococcus pyogenes is a well characterised antigen which induces protection against lethal challenge with group A streptococcus (GAS) when adjuvanted with cholera toxin B-subunit (CTB). As an alternative to CTB adjuvanted intranasal immunisations we investigated the immune responses generated in mice using Sfb1 incorporated in to the skin and mucosal adjuvant SAMA4.Mice (BALB/c) were vaccinated intradermally with 100 microl of either SAMA4 (adjuvant only group) or SAMA4/Sfb1 and were boosted 7 days later. Mice vaccinated with CTB based vaccines were immunised by intranasal inoculation with a mixture containing 30 microg Sfb1 and 10 microg CTB on days 1, 3, 5 and 15. At 14 days after the last booster immunisation the immune response was characterised and mice were challenged with 10(8) CFU of S. pyogenes.Mice vaccinated with SAMA4/Sfb1 elicited a Sfb1-specific IgG response in the sera that was significantly higher than that seen in control mice and mice immunised with the adjuvant only (P0.05). No significant differences were seen for specific IgA antibodies in the sera in all groups examined. Compared with non-immunised and adjuvant only immunised controls, mice immunised with the Sfb1/SAMA4 vaccine exhibited a significant increase (P0.05) in the number of Sfb1 reactive spleen cells in lymphoproliferation assays which were three fold higher than those seen for mice vaccinated with the Sfb1/CTB vaccine. Mice vaccinated with CTB/Sfb1 had the highest level of protection (80%) as where mice vaccinated with SAMA4 and SAMA4/Sfb1 displayed no protection (20% and 40%).These data suggest that the SAMA4 adjuvant used in this study fails to elicit protective immunity in BALB/c mice when used to adjuvant the known protective antigen Sfb1.

Group A streptococci (GAS) specifically attach to and internalize into human epithelial host cells. In some GAS isolates, fibronectin-binding proteins were identified as being responsible for these virulence traits. In the present study, the previously identified global negative regulator Nra was shown to control the binding of soluble fibronectin probably via regulation of protein F2 and/or SfbII expression in the serotype M49 strain 591. According to results from a conventional invasion assay based on the recovery of viable intracellular bacteria, the increased fibronectin binding did not affect bacterial adherence to HEp-2 epithelial cells, but was associated with a reduction in the internalization rates. However, when examined by confocal and electron microscopy techniques, the nra-mutant bacteria were shown to exhibit higher adherence and internalization rates than the corresponding wild type. The mutant bacteria escaped from the phagocytic vacuoles much faster, promoting consistent morphological changes which resulted in severe host cell damage. The apoptotic and lytic processes observed in nra-mutant infected host cells were correlated with an increased expression of the genes encoding superantigen SpeA, the cysteine protease SpeB, and streptolysin S in the nra-mutant bacteria. Adherence and internalization rates of a nra/speB-double mutant at wild-type levels indicated that the altered speB expression in the nra mutant contributed to the observed changes in both processes. The Nra-dependent effects on bacterial virulence were confined to infections carried out with stationary growth phase bacteria. In conclusion, the obtained results demonstrated that the global GAS regulator Nra modulates virulence genes, which are involved in host cell damage. Thus, by helping to achieve a critical balance of virulence factor expression that avoids the injury of target cells, Nra may facilitate GAS persistence in a safe intracellular niche.

The influence of genetic background on the ability to control infection with group A streptococci was investigated in different inbred strains of mice. Whereas BALB/c, C57BL/10, and DBA/2 mice were the most resistant strains, with lower bacteria loads and higher survival times, C3H/HeN and CBA/J mice exhibited substantially higher bacterial growth and 100% mortality. Differences in susceptibility were not dependent on the inoculum size. Resistance was influenced by sex, with males being much more susceptible than females. B cell‐ and T cell‐deficient mice from the resistant background were as resistant to infection as were immunocompetent mice, which suggests that the effector mechanisms are independent of adaptive immunity. These results demonstrate for the first time the influence of genetic background and sex on susceptibility to infection with Streptococcus pyogenes in mice. The use of this mouse model of group A streptococcal infection will allow for a better definition of parameters involved in the outcome of the disease. Group A streptococci (GAS) are among the most common human pathogens, with extremely variable clinical manifestations ranging from self-limited infection on the skin or mucosal surfaces, such as impetigo and pharyngitis, to life-threatening invasive diseases, such as necrotizing fasciitis, cellulitis, and streptococcal toxic shock–like syndrome [1]. Asymptomatic carriage and autoimmune sequelae (rheumatic fever, rheumatic heart disease, and glomerulonephritis) complete the clinical spectrum of streptococcal diseases [2]. Although the molecular basis for the pathogenesis of GAS infection is not clear, differences in the route of infection, among other factors, have been suggested as being responsible for different patterns of clinical presentation. Accordingly, in contrast to impetiginous lesions, the nasopharyngeal mucosa has been suggested as the principal reservoir for GAS strains capable of causing severe invasive diseases [3, 4]. Virulence of the infecting strains also has been associated with varying severity of invasive diseases. Thus, several epidemiological studies indicate that specific M serotypes (M1 and M3) are responsible for the majority of invasive GAS infections [4–8], and the emergence of exceptionally virulent clones of these serotypes in some locations has

Endovascular infection is a highly critical complication of invasive Staphylococcus aureus disease. For colonization, staphylococci must first adhere to adhesive endovascular foci. Von Willebrand factor (vWF) is a large, multimeric glycoprotein mediating platelet adhesion at sites of endothelial damage. Earlier it was demonstrated that vWF binds to and promotes the surface adhesion of S. aureus, prompting this effort to identify the vWF adhesin. In Western ligand assays of S. aureus lysates, staphylococcal protein A (SPA) was recognized by purified vWF. Surface plasmon resonance demonstrated the binding of soluble vWF to immobilized recombinant protein A with a K(d) of 1.49 x 10(-8) mol/L. Using flow cytometry, the binding of fluorescein isothiocyanate-labeled vWF to S. aureus was found to be saturable and inhibitable by unlabeled vWF, antiprotein-A antibodies, or IgG. Isogenic Deltaspa::Tc(r) mutants were constructed by the insertion of a tetracycline resistance cassette into spa using allelic replacement, and it exhibited decreased binding of soluble vWF and decreased adhesion to vWF-adsorbed surfaces. The interaction was restored on complementation of the mutants with spa-containing plasmid pSPA7235. In conclusion, protein A confers interaction of S. aureus with soluble and immobilized vWF in a newly discovered function characterizing protein A as a novel member of the staphylococcal surface protein adhesin superfamily and suggesting its potential role in the pathogenesis of endovascular staphylococcal disease.

SpsA, a pneumococcal surface protein belonging to the family of choline-binding proteins, interacts specifically with secretory immunglobulin A (SIgA) via the secretory component (SC). SIgA and free SC from mouse, rat, rabbit and guinea-pig failed to interact with SpsA indicating species-specific binding to human SIgA and SC. SpsA is the only pneumococcal receptor molecule for SIgA and SC as confirmed by complete loss of SIgA and SC binding to a spsA mutant. Analysis of recombinant SpsA fusion proteins showed that the binding domain is located in the N-terminal region of SpsA. By the use of different truncated N-terminal SpsA fusion proteins, the minimum binding domain was shown to be composed of 112 amino acids (residues 172-283). The sequence of this 112-amino-acids domain was used to spot synthesize 34 overlapping peptides, consisting of 15 amino acids each, with an offset of three amino acids on a cellulose membrane. One of the peptides reacted specifically with both SIgA and SC. By using a second membrane with immobilized synthetic peptides of decreasing length containing parts of the identified 15-amino-acid motif a hexapeptide, YRNYPT was identified as the binding motif for SC and SIgA. SpsA proteins with a size smaller than the assay-positive domain of 112 amino acids were able to inhibit the interaction of SIgA and pneumococci provided they contained the binding motif. The results indicated that the hexapeptide YRNYPT located in SpsA of pneumococcal strain type 1 (ATCC 33400) between amino acids 198 and 203 is involved in SIgA and SC binding. Because synthetic peptides containing only parts of the hexapeptide also assayed positive, these results further suggest that at least the amino acids YPT of the identified hexapeptide are critical for binding to SC and SIgA. Amino acid substitutions in the identified putative binding motif abolished SC-/SIgA-binding activity of the mutated SpsA protein, confirming the functional activity of this hexapeptide and the critical role of the amino acids YPT in SC and SIgA binding. Identification of this motif, which is highly conserved in SpsA protein among different serotypes, might contribute towards a new peptide based vaccine strategy.

A common problem in human vaccinology is the limited availability of efficient and non-toxic adjuvants capable of promoting mucosal responses. The potential usefulness of fibronectin-binding protein I (Sfbl) of Streptococcus pyogenes as immunological adjuvant was assessed using ovalbumin (OVA) as a model antigen. Mice were immunized by intranasal route, either with soluble OVA or OVA covalently coupled to Sfbl. Immunization with OVA-Sfbl resulted in the elicitation of about 100-fold higher titers of anti-OVA serum IgG than using OVA alone. The anti-OVA IgG subclass pattern was dominated in both groups of mice by IgG1, followed by IgG2b, IgG2a, and IgG3. Immunization with OVA-Sfbl also resulted in the elicitation of OVA-specific IgA in lung washes (24% of the total IgA), which was absent in mice immunized with OVA alone. Spleen cells from OVA-Sfbl-immunized mice also gave a much stronger proliferative response to restimulation with soluble OVA in vitro. Phenotypic analysis of proliferating cells showed an enrichment in CD4+ T cells, producing a pattern of cytokines (IL-4, IL-5, IL-6 and IL-10) characteristic of Th2-type cells. In contrast to immunization with soluble OVA alone, OVA-Sfbl induced the generation of CD8+ OVA-specific cytotoxic cells. These results demonstrate that Sfbl represents a promising mucosal adjuvant able to substantially improve cellular, humoral and mucosal responses when coupled to an antigen administered by intranasal route.

Bordetella bronchiseptica is a ureolytic mammalian respiratory pathogen. We have investigated the regulation of urease in B. bronchiseptica and the potential role of this enzyme in eukaryotic invasion and intracellular survival. Our results indicate urease is a bordetella virulence repressed gene. Urease activity in virulent B. bronchiseptica BB7865 is up-regulated from basal levels by 5 gl1 magnesium sulphate at 37 degrees C. At 30 degrees C, urease activity remained at basal levels, even in the presence on magnesium sulphate, suggesting a second temperature dependent mechanism of urease regulation was also operating. Urease was not inducible by 10 mM urea nor up-regulated in nitrogen limiting conditions. To evaluate the role of urease in intracellular invasion and survival urease-negative mutants of B. bronchiseptica BB7865 and B. bronchiseptica BB7866 were created by transposon mutagenesis, and compared to the urease-positive parental strains in a HeLa cell invasion assay. We demonstrate that increasing the concentration of urea in the assay increased survival of the urease-positive but not urease-negative strains after 24 h, suggesting that urease does have a role in intracellular survival. Partial DNA sequence analysis of an 11.0 kb EcoRI DNA fragment encoding urease activity revealed an open reading frame containing 50%, 45%, 45%, and 41% homology to the UreA urease subunit protein of Klebsiella aerogenes, Proteus vulgaris, Helicobacter pylori and Proteus mirabilis respectively. We also show Bordetella pertussis to contain sequences homologous with a DNA probe containing the gene encoding UreA of B. bronchiseptica indicating the possible presence of cryptic urease genes in this species.

The mouse macrophage-like cell line J774 was used to analyze opsonin-independent entry and survival of group B streptococci (GBS). Efficient entry of GBS in J774 cells occurred within 5 min postinfection, and streptococci persisted intracellularly without loss of viability for at least 8 h. At 24 h postinfection, 30% of the total intracellular GBS was recovered from macrophages. Inhibition studies using different biochemical modulators of cellular functions showed that bacterial entry seemed to involve nonglycosylated J774 surface structures different from known receptors such as fibronectin-binding integrins. Internalization of GBS by J774 cells occurred by a microfilament-dependent phagocytosis-like process also involving participation of receptor-mediated endocytosis. Prior opsonization of GBS with human serum containing anti-GBS antibodies did not affect bacterial entry but significantly reduced the intracellular survival of GBS. Transmission electron microscopic analysis confirmed these findings and demonstrated that both opsonized and nonopsonized bacteria were contained within phagosomes during the whole infection period. Transmission electron microscopy further revealed that decreased intracellular survival rates of opsonized GBS appeared to be due to increased lysosomal activities of the macrophages. These results suggest that in the absence of opsonins, GBS are able to enter and persist efficiently in macrophages by evading intracellular antibacterial activities commonly associated with opsonin-mediated uptake.

Summary. The adhesive glycoprotein fibronectin has been proposed as a mediator of adherence of certain gram-positive cocci to host cells and fibrin thrombi. This study compared the role of soluble and immobilised fibronectin in the adherence of coagulasenegative staphylococci (CNS) and Staphylococcus aureus to fibrin thrombi and plastic surfaces. Adherence of S. epidermidis to fibrin thrombi was significantly reduced when fibronectin was removed from the plasma used for thrombus preparation. Adherence was restored through restitution of fibronectin. S. epidermidis also adhered substantially more to plastic surface coated with fibronectin than to non-coated plastic. Increased adherence of CNS to plastic was also observed after coating with the 29-kDa N-terminal fragment of fibronectin. Soluble fibronectin did not affect the adherence of CNS to fibrin thrombi or plastic surfaces. The adherence of S. aureus to fibrin thrombi was significantly increased by the addition of soluble fibronectin, but not by incorporation of fibronectin into the clot. These results indicate that the binding of fibronectin is an important factor in the adherence of staphylococci to fibrin clots and plastic surfaces and, thus, colonisation of these surfaces. However, the two species of staphylococci seem to employ different mechanisms of fibronectin-mediated adherence : S. epidermidis interacts mainly with fibronectin incorporated in fibrin clots or immobilised on implanted synthetic materials, whereas S. aureus adheres to the fibrin matrix through binding of soluble fibronectin present in wound exudates.

The binding of bovine complement S protein (vitronectin) to Streptococcus dysgalactiae isolates from cattle with mastitis and the S protein's role in streptococcal adherence to bovine epithelial cells were investigated. All 25 clinical isolates of S dysgalactiae interacted with bovine S protein. None of the other streptococcal species tested bound to bovine S protein. The S protein-binding sites were saturable and highly sensitive to trypsin. The binding of bovine S protein to S dysgalactiae isolates was specific and could not be inhibited by other plasma proteins, such as fibronectin, albumin, fibrinogen, alpha 2-macroglobulin, or IgG. Similarly, streptococcal binding of bovine S protein was not influenced by the synthetic peptide Gly-Arg-Gly-Asp-Ser, which constituted the host cell attachment sequence of S protein. In adherence experiments, prior binding of bovine S protein to S dysgalactiae enhanced streptococcal adherence to bovine epithelial cells. The enhancing effects by bovine S protein were abolished when the respective binding sites on the streptococci were digested by trypsin. Thus, bovine S protein could be an important mediator of adherence of S dysgalactiae to bovine epithelial cells.

1. Palytoxin in concentrations as low as 1 pM raises the potassium permeability of rat, human and sheep erythrocytes, and the sodium permeability of human erythrocytes. The release of potassium or sodium from human cells also occurs when extracellular sodium is replaced by choline. 2. Ouabain inhibits the release due to palytoxin of potassium ions from human, sheep and rat erythrocytes, and also the release of sodium ions from human cells. The glycoside effect is specific since a) it is already prominent with 5 X 10(-8) M ouabain b) rat erythrocytes are less sensitive than human cells to ouabain c) potassium release due to amphotericin B or the Ca2+ ionophore A 23187 is not influenced by ouabain and d) dog erythrocytes are resistant to palytoxin as well as to ouabain. 3. Palytoxin has no direct influence on the Na+, K+ - ATPase. It inhibits the binding of [3H]ouabain to erythrocyte membranes within the same concentration range as unlabelled ouabain. It partially displaces bound [3H]ouabain, and partially inhibits the inactivation of erythrocyte ATPase by the glycoside. Depletion of ATP or of external Ca2+ renders the cells less sensitive to palytoxin. Nevertheless inhibition by ouabain can be still demonstrated with human cells whose ATP stores had been largely exhausted, and also in the absence of external Ca2+. 4. Palytoxin decreases the surface tension at the air-water interface. We assume that the formation of nonspecific pores by palytoxin is linked with its surface activity. Further experiments should demonstrate whether ouabain prevents the binding of palytoxin to erythrocytes ("receptor hypothesis"), or whether an ouabain-sensitive hydrolysis of trace amounts of ATP ("metabolic hypothesis") promotes the palytoxin effect.

Adherence of Streptococcus dysgalactiae isolates from cattle and S equi isolates from horses to their respective host epithelial cells was compared with the adherence of S pyogenes to human epithelial cells. The adherence was quantitatively determined by use of fluorescein-labeled streptococci. All 3 streptococcal species adhered selectively to their respective host cells. The mechanism of adherence was evaluated by binding studies with adhesive plasma protein, fibronectin. Although all 3 streptococcal species bound fibronectin, S dysgalactiae and S equi interacted preferentially with a 210-kilodalton (kD) C-terminal fragment of fibronectin, whereas S pyogenes bound only a 29-kD N-terminal fragment. A synthetic peptide Gly-Arg-Gly-Asp-Ser, representing the host cell attachment site of fibronectin, partially inhibited the binding of fibronectin and of its 210 kD fragment to S dysgalactiae, but not to S equi. The binding of fibronectin and its 29-kD fragment to S pyogenes was not inhibited by Gly-Arg-Gly-Asp-Ser. These differences in binding activities corresponded to the ability of fibronectin to mediate the adherence of the streptococci to the epithelial cells: fibronectin strongly inhibited the adherence of S pyogenes and S equi to the epithelial cells, but only weakly inhibited that of S dysgalactiae.

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.

Streptococcus pyogenes organisms (group A streptococci) are considered to be highly adhesive extracellular pathogens. However, it has recently been reported that S. pyogenes has the capacity to efficiently invade eukaryotic cells. In this study, we demonstrate that the interaction of S. pyogenes fibronectin-binding protein (SfbI) with fibronectin on nonphagocytic HEp-2 cells triggers bacterial internalization. Blocking of the SfbI adhesin by either antibodies against the whole protein or antibodies against the fibronectin-binding domains of SfbI, as well as pretreatment of HEp-2 cells with purified SfbI protein, prevents both S. pyogenes attachment and internalization. Inert latex beads precoated with the purified SfbI protein are ingested by eukaryotic cells, demonstrating that SfbI is per se enough to trigger the internalization process. Experiments performed with a recombinant SfbI domain encompassing the two fibronectin-binding regions of the SfbI molecule demonstrated that these binding regions are essential and sufficient to activate uptake by HEp-2 cells. These results demonstrate that the fibronectin-binding protein SfbI is involved in both S. pyogenes' attachment to and ingestion by HEp-2 cells and contribute to elucidation of the underlying molecular events leading to eukaryotic cell invasion by S. pyogenes.