Dermatan sulphate is released by proteinases of common pathogenic bacteria and inactivates antibacterial alpha-defensin.

Defensins represent an evolutionarily conserved group of small peptides with potent antibacterial activities. We report here that extracellular proteinases secreted by the human pathogens Pseudomonas aeruginosa, Enterococcus faecalis and Streptococcus pyogenes release dermatan sulphate by degrading dermatan sulphate-containing proteoglycans, such as decorin. Dermatan sulphate was found to bind to neutrophil-derived alpha-defensin, and this binding completely neutralized its bactericidal activity. During infection, proteoglycan degradation and release of dermatan sulphate may therefore represent a previously unknown virulence mechanism, which could serve as a target for novel antibacterial strategies.

Virulent aggregates of Streptococcus pyogenes are generated by homophilic protein-protein interactions.

Many strains of the important human pathogen Streptococcus pyogenes form aggregates when grown in vitro in liquid medium. The present studies demonstrate that this property is crucial for the adherence, the resistance to phagocytosis and the virulence of S. pyogenes. A conserved sequence of 19 amino acid residues (designated AHP) was identified in surface proteins of common S. pyogenes serotypes. This sequence was found to promote bacterial aggregation through homophilic protein-protein interactions between AHP-containing surface proteins of neighbouring bacteria. A synthetic AHP peptide inhibited S. pyogenes aggregation, reduced the survival of S. pyogenes in human blood and attenuated its virulence in mice. In contrast, mutant bacteria devoid of surface proteins containing AHP-related sequences did not aggregate or adhere to epithelial cells. These bacteria are also rapidly killed in human blood and show reduced virulence in mice, underlining the pathogenic significance of the AHP sequence and S. pyogenes aggregation.

Five homologous repeats of the protein G-related protein MIG cooperate in binding to goat immunoglobulin G.

Protein MIG, from Streptococcus dysgalactiae, binds alpha2-macroglobulin and immunoglobulin G (IgG). MIG-derived fusion proteins with one to five IgG-binding repeats differed up to 72,000- fold in avidity for goat IgG, indicating a considerable cooperativity of the repeats. Significant sequence variation in the IgG-binding repeats was recognized. Protein MIG interacted with goat IgG1 via both the Fc and Fab parts.

Size-selectivity of the glomerular barrier to high molecular weight proteins: upper size limitations of shunt pathways.

To evaluate the large pore radius of the glomerular capillary filter, plasma-to-urine fractional clearances of a number of endogenous proteins were assessed in normal and in nephrotic Wistar rats in which proximal tubular reabsorption had been inhibited using lysine. The proteins studied varied in radius from 16.2 A (Beta 2-microglobulin) to 90 A (alpha 2-macroglobulin). The nephrotic syndrome was induced by puromycin aminonucleoside (PAN). A marked restriction of the transport of large proteins across the glomerular capillary wall was found, indicating that there are no non-discriminatory 'shunt pathways' in the glomerular barrier. Rather, there seems to be large pores of radius 110 to 115 A accounting for the clearance of large proteins into the primary urine. This protein excretion pattern was almost the same for control and nephrotic rats, except that in the latter, the number of large pores was increased 170 times. The ratio between the number of large and small pores was calculated to be approximately equal to 7 x 10(-7) in normal rats and to 1.2 x 10(-4) in PAN nephrotic rats, assuming no classic shunt pathways. If classic shunt pathways had still existed, they would normally contribute to no more than approximately equal to 10(-5) of the total glomerular filtration rate. We postulate that very large macromolecules like IgM will not pass the glomerular filter at all under normal conditions, whereas the urine concentration of alpha2-macroglobulin will normally be extremely low.

Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging.

The bacterium Helicobacter pylori is the causative agent for peptic ulcer disease. Bacterial adherence to the human gastric epithelial lining is mediated by the fucosylated Lewis b (Leb) histo-blood group antigen. The Leb-binding adhesin, BabA, was purified by receptor activity-directed affinity tagging. The bacterial Leb-binding phenotype was associated with the presence of the cag pathogenicity island among clinical isolates of H. pylori. A vaccine strategy based on the BabA adhesin might serve as a means to target the virulent type I strains of H. pylori.

Structure and stability of protein H and the M1 protein from Streptococcus pyogenes. Implications for other surface proteins of gram-positive bacteria.

M proteins and other members of the M protein family, expressed on the surface of Streptococcus pyogenes, bind host proteins such as immunoglobulins, albumin, and fibrinogen. Protein H and the M1 protein are expressed by adjacent genes and both belong to the M protein family. In this work, the structure and stability of these two proteins have been investigated. As judged from sequence analysis and circular dichroism spectroscopy, the proteins are almost entirely in an alpha-helix conformation. The amino acids are arranged in a seven-residue (heptad) repeat pattern along the greater part of the proteins. These observations support the previously accepted model of M proteins as coiled-coil dimers. However, it was also found that the structures of both proteins were thermally unstable; i.e., the content of helix conformation was greatly reduced at 37 degrees C as compared to 25 degrees C or below. Together with previous findings that these proteins appear as monomers at 37 degrees C and dimers at low temperatures, the results suggest that the coiled-coil dimers are unfolded at 37 degrees C. The heptad patterns of protein H and the M1 protein showed a nonoptimal distribution of residues expected for a coiled-coil conformation. This is a possible explanation for the low thermal stability of the proteins. It was also demonstrated that the proteins were stabilized in the presence of the ligands IgG and/or albumin. Protein H and M1 protein show a high degree of sequence similarity in their C-terminal regions, and a fragment from this region displayed a high content of helix conformation, whereas fragments from the nonsimilar N-terminal parts did not adopt any stable folded structure. Thus, the C-terminal parts, which are conserved within the M protein family, may constitute a framework for the formation of the parallel helical coiled-coil structure, and we propose that the less stable N-terminal part may also participate in antiparallel interaction with M proteins on adjacent bacteria. The results suggest that temperature fluctuations in the environment could change the properties of bacterial surface proteins, thereby affecting the molecular interactions between the bacterium and its host.

Protein H--a bacterial surface protein with affinity for both immunoglobulin and fibronectin type III domains.

Several bacterial species express surface proteins with affinity for the constant region (Fc) of immunoglobulin (Ig) G. The biological consequences of the interaction with IgG are poorly understood but it has been demonstrated that genes encoding different IgG Fc-binding proteins have undergone convergent evolution, suggesting that these surface molecules are connected with essential microbial functions. One of the molecules, protein H, is present in some strains of Streptococcus pyogenes, the most significant streptococcal species in clinical medicine. In contrast to other Ig-binding bacterial proteins tested, protein H was found to interact also with the neural cell adhesion molecule (N-CAM), a eukaryotic cell surface glycoprotein mediating homo- and heterophilic cell-cell interactions. The affinity for the interaction between protein H and N-CAM was 1.6 x 10(8)/M and the binding site on protein H was mapped to the NH2-terminal 80 amino acid residues. N-CAM and IgG are both members of the Ig superfamily and analogous to N-CAM, IgG binds to the NH2-terminal part of protein H. However, the binding sites for the two proteins were found to be separate, an unexpected result which was explained by the observation that the fibronectin type III (FNIII) domains and not the Ig-like domains of N-CAM are responsible for the interaction with protein H. Thus, the binding of N-CAM to protein H was blocked with fibronectin but not with IgG. Moreover, apart from fibronectin itself and N-CAM, fragments of fibronectin and the matrix protein cytotactin/tenascin containing FNIII domains also showed affinity for protein H.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein H--a surface protein of Streptococcus pyogenes with separate binding sites for IgG and albumin.

Protein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (IgGFc) region of immunoglobulin (Ig) G. In absorption experiments with human plasma, protein H-sepharose could absorb not only IgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 x 10(9) M-1, which is higher than the affinity between IgG and protein H (Ka = 1.6 x 10(9) M-1). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein-protein interaction assays, the binding of albumin was mapped to three repeats (C1-C3) in the C-terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin- and IgGFc-binding bacterial surface protein. Also IgGFc-binding could be mapped with the protein H fragments and the region was found N-terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized IgG or IgGFc. This sequence was not found in previously described IgGFc-binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify IgGFc- and albumin-binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.

Convergent evolution among immunoglobulin G-binding bacterial proteins.

Protein G, a bacterial cell-wall protein with high affinity for the constant region of IgG (IgGFc) antibodies, contains homologous repeats responsible for the interaction with IgGFc. A synthetic peptide corresponding to an 11-amino acid-long sequence in the COOH-terminal region of the repeats was found to bind to IgGFc and block the interaction with protein G. Moreover, two other IgGFc-binding bacterial proteins (proteins A and H), which do not contain any sequences homologous to the peptide, were also inhibited in their interactions with IgGFc by the peptide. Finally, a decapeptide based on a sequence in IgGFc blocked the binding of all three proteins to IgGFc. This unusually clear example of convergent evolution emphasizes the complexity of protein-protein interactions and suggests that bacterial surface-protein interaction with host protein adds selective advantages to the microorganism.

Immunologically induced pleural and peritoneal mast cell histamine release: difference in responsiveness in relation to secretagogue and rat strain used.

Responsiveness was compared for cell populations harvested from the peritoneal and pleural cavities of rats with respect to histamine release induced by specific antigen, anti-IgE, and Con A. Cell populations were obtained from Fischer, PVG or Sprague-Dawley (SD) rats, which were either untreated or immunized with 10 micrograms ovalbumin together with 100 mg alum intraperitoneally. Mast cell histamine release was examined with crude cell populations. The results show that differences in response capacity to the various secretagogues employed do exist between pleural and peritoneal cells in Fischer and PVG strains but under the present circumstances apparently not in SD rats. These differences vary in magnitude with the secretagogue employed and (for cells from immunized animals) with the time elapsed between immunization and test. In PVG rats, neither pleural nor peritoneal mast cell histamine release induced by antigen paralleled serum OA-IgE antibody levels. Furthermore, an increase in anti-IgE induced release of histamine from serosal mast cells occurred in parallel with a decrease in total serum IgE levels. These data indicate that the functional differences observed with respect to release properties of the two cell populations are due not only to intrinsic differences in mast cell populations but also to differences in reaginic antibodies sensitizing the cells.

Anti-IgE-and ConA-induced histamine release from human basophilic leukocytes: the existence of differences in relative responses within individuals.

Histamine release was induced from human blood leukocytes with Concanavalin A (ConA) and two different preparations of anti-IgE. A dose-response curve was constructed for each cell population and secretagogue. No qualitative difference in response pattern was observed with the two anti-IgEs; however, the relative release-inducing efficacy of ConA and anti-IgE seemed to vary with the individual providing the cells. These findings indicate that release induced by ConA is not completely equivalent to that induced by anti-IgE.

Anti-IgE-induced histamine release from rat tissues passively sensitized in vivo with myeloma IgE differs with strain and mast cell source.

Four different strains of rats, BDII, E3, LE, and OM/N, each with a low basal serum level of IgE, were examined with respect to anti-IgE- and ConA-induced release of histamine from serosal mast cells and chopped lung tissue. Tests were performed before and three days after i.p. injection of a graded dose of myeloma IgE (IR 162)-containing ascitic fluid. There was a clearcut strain difference in response capacity with respect to ConA- and anti-IgE-induced release of histamine from serosal mast cells before myeloma IgE-injection. Response capacity increased in some but not all strains after myeloma IgE injection; increase in response capacity of serosal mast cells did not correlate to that of chopped lung tissue. Analogous findings were observed when two of the strains, LE and E3, were passively sensitized by i.p. injection with serum containing another myeloma IgE (IR2). These results indicate that differences exist between mast cells of different rat strains, and within strain between mast cells of various tissues in capacity to become sensitized to myeloma IgE.

Antigen-induced release of histamine from serosal mast cells, lung, and tracheal tissue: variation with tissue and rat strain in relation to serum IgE-antibody level.

Rats of the Brown Norway (BN)2, Fischer, PVG and Sprague Dawley (SD) strains were immunized intraperitoneally with graded doses of ovalbumin (OA) together with either alum or Silica gel. At specified times after immunization, in vitro histamine release from serosal mast cells and from chopped lung and tracheal tissue was determined after challenge with OA. The development of the capacity to respond in these tests seemed to vary within strain independently for mast cells, lung, and tracheal tissue with immunization dose of antigen and nature of adjuvant. These variations were not closely correlated to observed variations in serum levels of OA-IgE antibody or ratio OA-IgE to total IgE as determined by radioimmunoassay. Furthermore, variations between strains in response capacity did not correlate to inter strain variation in median serum OA-IgE antibody level. These results do not clearly conform to the possibility that one single class of IgE mediates anaphylactic reactivity of various tissues of the rat.

Anti-IgE and Con A-induced histamine release from mast cells of four rat strains: correlation with total serum IgE.

Anti-IgE- and Con A-induced histamine release from serosal mast cells were compared to each other and to total serum levels of IgE in non-immunized, alum-injected, and Silica gel-injected rats of the BN, Fischer, PVG, and SD strains. The results indicate that the degree of anti-IgE- and Con A-induced release is strain-dependent and varies with immunization conditions. Furthermore, there is a gross but not complete correlation between the degree of serosal mast cell histamine release induced by the two secretagogues. However, Con A- or anti-IgE-induced release could significantly be correlated to serum levels of total IgE only in the Fischer strain but not in the BN or the PVG strains. In the SD strain, Con A-induced release correlated to serum IgE levels in Silica gel-injected but not in alum-injected animals.

Anti-IgE-induced histamine release from human basophilic leukocytes: inhibition depends on nature and concentration of inhibitor and secretagogue.

Histamine release from human basophilic leukocytes was induced by increasing concentrations of anti-IgE in the presence of various concentrations of 2-deoxyglucose (2-DOG), the histamine H2-receptor agonist dimaprit, theophylline, or enprofylline (a new antiasthmatic xanthine derivative). The results show that the degree of inhibition produced by each agent differed with the concentration of anti-IgE used and with the nature and concentration of the inhibitor. These data indicate that great care should be used when characterizing an inhibitor of mediator release by simply giving a figure for the per cent inhibition of release observed in its presence.

Antigen-induced release of histamine from rat tissues in vitro: dissociation in development of serosal mast cell, lung tissue, and tracheal tissue response capacity.

We examined the temporal development and the fading in Sprague Dawley rats, actively sensitized to ovalbumin (OA), of the capacity of serosal mast cells, chopped lung tissue, and occasionally chopped tracheal tissue, to respond at antigen challenge in vitro with histamine release. Response capacity of both serosal mast cells and lung tissue developed within 2-3 weeks after injection of 1 microgram OA or more together with 100 mg of alum. Maximum response capacity was observed in cells and tissue from animals injected with 10 micrograms OA, part of the response capacity then remained until 3 months after immunization. Development of serosal mast cell reactivity was occasionally dissociated from that of lung tissue. When low amounts of alum (1 or 10 mg) were employed as adjuvant, lung tissue reactivity could be induced in the virtual absence of serosal mast cell response capacity. Silica gel was less efficient than alum as an adjuvant for induction of a primary response, but 'secondary' tissue responses could be induced when silica gel was used as an adjuvant. Pretreatment of the animals with cyclophosphamide before the booster injection enhanced and prolonged the response capacity of lung tissue. Animals injected with OA together with Freund's complete adjuvant did not provide responding serosal mast cells; response capacity of lung tissue varied with immunization dose of antigen. Antigen-induced histamine release from chopped tracheal tissue did not correlate to response capacity of lung tissue. Thus, the development in the rat of response capacity with respect to antigen-induced histamine release dissociates from serosal mast cells, lung tissue, and tracheal tissue.