Expression of group B protective surface protein (BPS) by invasive and colonizing isolates of group B streptococci.

Group B protective surface protein (BPS) is expressed on the cell surface of some group B streptococcal (GBS) (Streptococcus agalactiae) strains and adds to the identification by capsular polysaccharide (CPS), and c or R proteins. We investigated the prevalence of BPS among GBS clinical isolates (303 invasive, 4122 colonizing) collected over 11 years in four American cities. Hot HCl cell extracts were tested by immunoprecipitation in agarose with rabbit antisera to BPS; the alpha (α) and beta (ß) components of c protein; R1, R3, and R4 species of R protein; and CPS serotypes Ia-VIII. BPS was found in 155 isolates (seven invasive, 148 colonizing). Of these, 87 were Ia, 37 II, 20 V; none were III. BPS was expressed usually with another protein: a species of R by 87 or a component of c by 39. The predominant CPS/protein profiles with BPS were Ia/R1,BPS and II/c(α + ß),BPS. Thus, along with CPS serotype and other surface proteins, BPS can be a valuable marker for precise strain characterization of unique GBS clinical isolates with complex surface protein profiles.

M protein-mediated plasminogen binding is essential for the virulence of an invasive Streptococcus pyogenes isolate.

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.

Trigger for group A streptococcal M1T1 invasive disease.

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.

Genes for the majority of group a streptococcal virulence factors and extracellular surface proteins do not confer an increased propensity to cause invasive disease.

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.

Streptococcal invasion.

The genus Streptococcus consists of large number of species many of which are pathogenic to humans and animals. Although streptococci have long been considered as extracellular pathogens, they are capable of causing serious invasive infections such as necrotizing fasciitis and meningitis. Streptococcal invasion, therefore, has been a focus of many studies in recent years. Streptococci are efficiently internalized by nonprofessional phagocytes and the current research interest has shifted to determine the role of this invasion in the natural infection process. Moreover, characterization of bacterial and eukaryotic components involved in the uptake process might be useful in developing new strategies for combating streptococcal infections.

Changes in erythrocyte permeability due to palytoxin as compared to amphotericin B.

Palytoxin causes within minutes a temperature-dependent K+ loss from human and rat erythrocytes which is followed within hours by haemolysis. It decreases the osmotic resistance in a concentration-dependent manner, so that osmotic influences are negligible for K+ release but considerable in haemolysis. External K+ inhibits the haemoglobin release and Rb+ inhibits the release of K+ and haemoglobin. Ca2+ (over 20 microM) and borate (over 5 microM) enhance the loss of K+ and haemoglobin. With both Ca2+ and borate present, the efficacy of palytoxin is raised about 10 000-fold. Under these conditions, about 15 palytoxin molecules per human cell trigger a 50% K+ loss over a wide range of cell concentrations. The palytoxin effect is reversible. After depletion from K+ by low concentrations of palytoxin, human cells can be refilled with K+ and resealed. The pores formed by palytoxin are small. They allow the entrance of Na+ and choline, whereas inositol is largely excluded and Ca2+, as well as sucrose and inulin, are completely excluded. Amphotericin B resembles palytoxin in its ability to cause a considerable prelytic K+ loss and to form small pores. However, it is about 1000-times weaker than palytoxin, is not inhibited by K+ or Rb+, is not activated by Ca2+ or borate, and has a negative temperature dependence. Thus palytoxin represents a novel type of cytolysin.

Delayed haemolytic action of palytoxin. General characteristics.

1. Palytoxin is a haemolysin. The erythrocytes from various species can be classified into a sensitive and a hardly sensitive group. The former contain potassium as their main inside cation and are arranged according to their sensitivity as hog greater than or equal to rat, mouse greater than rabbit greater than guinea-pig greater than man. The latter, comprising those from sheep and cattle, have sodium as their main inside cation. In addition, chicken erythrocytes are relatively insensitive. 2. Haemolysis of rat erythrocytes is preceded by a lag period of 1--2 h. With increasing temperature the haemolysis proceeds more quickly but reaches the same final range between 25 and 42 degrees C. The pH optimum in Britton-Robinson buffer supplemented with saline is between 7 and 8. Washing off palytoxin during the prelytic period reduces the haemolytic power. 3. The sensitivity of rat erythrocytes decreases with increase of osmolarity between 235 and 415 mosM. Accordingly, their osmotic resistance is lowered by palytoxin in a concentration-dependent manner. 4. With both rat and sheep erythrocytes, potassium loss by far precedes the haemolysis due to palytoxin. Potassium loss is measurable already after 1 min and increases with time. After 2 hours the quotient between the ED50 of haemolysis and that of potassium loss is around 200. Thus palytoxin is an unusually strong but slow haemolysin of the osmotic type. The extreme prelytic potassium loss and the correlation between susceptibility and potassium content of erythrocytes points towards the relevance of ionic fluxes.

Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin.

The culture medium of certain strains of Clostridium botulinum type C contains two separable ADP-ribosyltransferases. Besides the ADP-ribosylation of actin due to botulinum C2 I toxin, a second microbial enzyme causes the mono-ADP-ribosylation of a eukaryotic protein with a molecular mass of about 20 kDa found in platelets, neuroblastoma X glioma hybrid cells, S49 lymphoma cells, chick embryo fibroblasts and sperm. The eukaryotic substrate is inactivated by heating and trypsin treatment. In contrast, the novel ADP-ribosyltransferase, which can be separated by DEAE-Sephadex chromatography, is largely resistant in the short term to trypsin digestion.

Botulinum ADP-ribosyltransferase C3 but not botulinum neurotoxins C1 and D ADP-ribosylates low molecular mass GTP-binding proteins.

Botulinum ADP-ribosyltransferase C3 modified 21-24 kDa proteins in a guanine nucleotide-dependent manner similar to that described for botulinum neurotoxin C1 and D. Whereas GTP and GTP gamma S stimulated C3-catalyzed ADP-ribosylation in the absence of Mg2+, in the presence of added Mg2+ ADP-ribosylation was impaired by GTP gamma S. C3 was about 1000-fold more potent than botulinum C1 neurotoxin in ADP-ribosylation of the 21-24 kDa protein(s) in human platelet membranes. Antibodies raised against C3 blocked ADP-ribosylation of the 21-24 kDa protein by C3 and neurotoxin C1 but neither cross reacted with neurotoxin C1 immunoblots nor neutralized the toxicity of neurotoxin C1 in mice. The data indicate that the ADP-ribosylation of low molecular mass GTP-binding proteins in various eukaryotic cells is not caused by botulinum neurotoxins but is due to the action of botulinum ADP-ribosyltransferase C3. The weak enzymatic activities described for botulinum neurotoxins appear to be due to the contamination of C1 and D preparations with ADP-ribosyltransferase C3.

Identification of novel heparin-binding domains of vitronectin.

Vitronectin is a multifunctional serum protein which provides a unique regulatory link between cell adhesion, humoral defense mechanism and the hemostatic system, and the heparin-binding properties of vitronectin are thought to have participated in various functional aspects. In addition to the carboxy-terminal glycosaminoglycan-binding motif, we report on two novel heparin-binding domains which were identified using phage display technique. One heparin-binding domain is located between amino acids Asp82 and Cys137 at the end of the connector region, while the other is in the second hemopexin-type repeat, between amino acids Lys175 and Asp219 of the vitronectin molecule. Our findings may shed new light to the activities of vitronectin and its binding to cells, which could not be explained solely on the basis of the known heparin-binding domain.

SfbII protein, a fibronectin binding surface protein of group A streptococci, is a serum opacity factor with high serotype-specific apolipoproteinase activity.

Serum opacity factor (SOF) is produced by group A streptococci belonging to certain M types. SOF cleaves the apolipoprotein component of the high density lipoprotein fraction of serum rendering it insoluble which in turn leads to serum opacity. SfbII protein, a fibronectin binding surface protein cloned from group A streptococci, was obtained from a strain of M75. Here we show that this protein has a second functional domain responsible for SOF activity. The fibronectin binding region was located in the C-terminal end of the protein. Deletion analysis showed that the remainder of the protein was required for SOF activity. Sequence analysis of SfbII, when compared with the published sequence of SOF22, showed 99% identity with a difference of only four amino acids. In spite of this high homology, SOF from M75 was type-specific and antibody evoked specifically inhibited only SOF produced by M75. Antibodies found in human serum following natural infection also inhibited the SOF of SfbII in a type-specific manner. The results showed that the SfbII protein from M75 is SOF with a high serotype-specific enzyme activity.

Role of alpha 2-macroglobulin in phagocytosis of group A and C streptococci.

Binding of alpha 2-macroglobulin (alpha 2M) to streptococci and its effects on phagocytosis were investigated. Two types of streptococcal binding sites for alpha 2M were observed: Streptococcus pyogenes from human infections interacted only with native alpha 2M whereas S. dysgalactiae from bovine and S. equi from equine infections bound only a complex of alpha 2M with trypsin (alpha 2M-T). Preincubation of S. pyogenes with native alpha 2M substantially enhanced their phagocytosis by human polymorphonuclear neutrophils (PMN) whereas preincubation with alpha 2M-T was without any effect. On the other hand, incubation of S. dysgalactiae and S. equi with alpha 2M-T markedly reduced their phagocytosis by PMN from the respective host species. Native alpha 2M did not affect the phagocytosis of these streptococci. Digestion of the streptococcal binding sites for alpha 2M and alpha 2M-T pronase abolished the enhancement of phagocytosis of S. pyogenes by native alpha 2M as well as the inhibition of phagocytosis of S. dysgalactiae and S. equi by alpha 2M-T. Thus, binding of alpha 2M or its complexes appeared to play a role in streptococcal pathogenicity.

Characterization of group B streptococcal invasion in HEp-2 epithelial cells.

The invasion of group B streptococci (GBS) in HEp-2 epithelial cells was analyzed by electron microscopy and a quantitative antibiotic survival assay. Invasion of GBS involved intimate attachment of streptococcal chains, engulfment of the adherent bacteria by cellular protrusions, entry of the bacteria in a 'polar' fashion and formation of membrane-bound vacuoles in which most of the intracellular streptococci resided. At later stages of infection bacteria were also found free in the cytoplasm. Efficient uptake of streptococci by HEp-2 cells occurred within 20 min and live intracellular bacteria were detectable up to 48 h post-infection. Invasion of GBS required activation of the eukaryotic actin microfilament system involving, at least partially, protein kinase signal transduction pathways. Invasion was inhibited in a dose-dependent manner by decreasing extracellular Ca2+ levels as well as by substances known to interfere with eukaryotic calcium regulatory systems. These results suggest that GBS invade HEp-2 cells by triggering calcium-dependent phagocytosis-like internalization mechanisms and persist intracellularly both in vacuoles and free in the cytoplasm.

Role of fibronectin in staphylococcal colonisation of fibrin thrombi and plastic surfaces.

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 coagulase-negative 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.

Ouabain inhibits the increase due to palytoxin of cation permeability of erythrocytes.

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.

Inhibitory effects of fibrinogen on phagocytic killing of streptococcal isolates from humans, cattle and horses.

The effects of fibrinogen on phagocytic killing of Streptococcus dysgalactiae from cattle and S. equi from horses were studied in comparison to that of S. pyogenes from humans. Phagocytic killing was determined by a fluorometric microassay using glass adherent polymorphonuclear neutrophils (PMN) from the respective host species, preopsonization with homologous sera led to a dose-dependent increase in phagocytic killing of all streptococcal cultures, preincubation of streptococci with fibrinogen significantly inhibited their phagocytic killing. Fibrinogen had no effect on phagocytic killing of non-fibrinogen binding S. agalactiae cultures. Further characterization studies with S. dysgalactiae and S. pyogenes revealed that a partial inhibition of phagocytic killing could also be achieved by preincubation with monomeric beta-chains of fibrinogen. Digestion of the fibrinogen binding sites on streptococci with proteases resulted in an almost complete loss of the inhibitory effects of fibrinogen on phagocytic killing. It could thus be concluded that by binding fibrinogen animal pathogenic streptococci could evade phagocytic killing in a similar manner as M protein carrying S. pyogenes isolates from human infections.

Heterogeneity of fibronectin reactivity among streptococci as revealed by binding of fibronectin fragments.

Streptococci belonging to serological groups A, B, C, G, L and U were studied for their interaction with 125I-labelled fibronectin and its fragments. Fibronectin purified from humans plasma by affinity chromatography on gelatin-agarose and heparin-agarose was cleaved by thrombin into a 29,000 Dalton and a 210,000 Dalton fragments. Terminal analysis of purified fragments indicated that 29,000 fragment was from amino-terminal and 210,000 fragment from carboxyl-terminal domain of fibronectin. Binding of fibronectin was observed in all streptococci except those of group B. Streptococci of groups A, G, L, U and S. equisimilis reacted only with 29,000 Dalton fragment whereas S. dysgalactiae, S. zooepidermicus and S. equi reacted only with 210,000 Dalton fragment. The streptococcal binding sites for these two fragments were distinct from each other. Fibrinogen blocked the binding of 210,000 Dalton fragment but not of 29,000 Dalton fragment. Trypsinization of streptococci did not affect their binding sites for 210,000 Dalton fragment but destroyed those for 29,000 Dalton fragment. The results indicate that the streptococci of group A and G as well as S. equisimilis which are mainly pathogenic in humans bind amino-terminal fragment of fibronectin. This may facilitate the adherence of these pathogens. On the other hand, the streptococci isolated from animal infections had different binding sites recognized only by carboxyl-terminal part of fibronectin.

Immune responses of a liposome/ISCOM vaccine adjuvant against streptococcal fibronectin binding protein 1 (Sfb1) in mice.

BACKGROUND & OBJECTIVES: 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. METHODS: 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. RESULTS: 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 (P<0.05). 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 (P<0.05) 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%). INTERPRETATION & CONCLUSION: 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.

Adherence of streptococcal isolates from cattle and horses to their respective host epithelial cells.

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.

Role of complement S protein (vitronectin) in adherence of Streptococcus dysgalactiae to bovine epithelial cells.

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.