Showing papers on "Protein A published in 2009"
TL;DR: A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device.
Abstract: The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.
288 citations
TL;DR: It is demonstrated that Staphylococcus aureus activates type I IFN signaling, which contributes to its virulence as a respiratory pathogen, and mice lacking IFN-alpha/beta receptor 1 (IFNAR-deficient mice), which are incapable of responding totype I IFNs, were substantially protected against lethal S. aUREus pneumonia compared with wild-type control mice.
Abstract: The activation of type I IFN signaling is a major component of host defense against viral infection, but it is not typically associated with immune responses to extracellular bacterial pathogens. Using mouse and human airway epithelial cells, we have demonstrated that Staphylococcus aureus activates type I IFN signaling, which contributes to its virulence as a respiratory pathogen. This response was dependent on the expression of protein A and, more specifically, the Xr domain, a short sequence-repeat region encoded by DNA that consists of repeated 24-bp sequences that are the basis of an internationally used epidemiological typing scheme. Protein A was endocytosed by airway epithelial cells and subsequently induced IFN-beta expression, JAK-STAT signaling, and IL-6 production. Mice lacking IFN-alpha/beta receptor 1 (IFNAR-deficient mice), which are incapable of responding to type I IFNs, were substantially protected against lethal S. aureus pneumonia compared with wild-type control mice. The profound immunological consequences of IFN-beta signaling, particularly in the lung, may help to explain the conservation of multiple copies of the Xr domain of protein A in S. aureus strains and the importance of protein A as a virulence factor in the pathogenesis of staphylococcal pneumonia.
139 citations
TL;DR: It is shown that IsdA binds to involucrin, loricrin, and cytokeratin K10, proteins that are present in the cornified envelope of human desquamated epithelial cells, and is an important adhesin to human ligands, which predominate in its primary ecological niche.
Abstract: The ability of Staphylococcus aureus to colonize the human nares is a crucial prerequisite for disease. IsdA is a major S. aureus surface protein that is expressed during human infection and required for nasal colonization and survival on human skin. In this work, we show that IsdA binds to involucrin, loricrin, and cytokeratin K10, proteins that are present in the cornified envelope of human desquamated epithelial cells. To measure the forces and dynamics of the interaction between IsdA and loricrin (the most abundant protein of the cornified envelope), single-molecule force spectroscopy was used, demonstrating high-specificity binding. IsdA acts as a cellular adhesin to the human ligands, promoting whole-cell binding to immobilized proteins, even in the absence of other S. aureus components (as shown by heterologous expression in Lactococcus lactis). Inhibition experiments revealed the binding of the human ligands to the same IsdA region. This region was mapped to the NEAT domain of IsdA. The NEAT domain also was found to be required for S. aureus whole-cell binding to the ligands as well as to human nasal cells. Thus, IsdA is an important adhesin to human ligands, which predominate in its primary ecological niche.
92 citations
TL;DR: Mutation of a single surface-exposed Phe residue on TbpB within the predicted interface completely abolishes binding to Tf, suggesting that the TBPB N lobe comprises the sole high-affinity binding region for Tf.
85 citations
TL;DR: A comparison of mammalian Cγ gene sequences revealed that IgG diversified into subclasses after speciation, and the effector functions for the IgG subclasses of each species should not be extrapolated from “same name subclasses” in other species.
Abstract: Eleven genomic porcine Cγ gene sequences are described that represent six putative subclasses that appear to have originated by gene duplication and exon shuffle. The genes previously described as encoding porcine IgG1 and IgG3 were shown to be the IgG1a and IgG1b allelic variants of the IGHG1 gene, IgG2a and IgG2b are allelic variants of the IGHG2 gene, while “new” IgG3 is monomorphic, has an extended hinge, is structurally unique, and appears to encode the most evolutionarily conserved porcine IgG. IgG5b differs most from its putative allele, and its CH1 domain shares sequence homology with the CH1 of IgG3. Four animals were identified that lacked either IgG4 or IgG6. Alternative splice variants were also recovered, some lacking the CH1 domain and potentially encoding heavy chain only antibodies. Potentially, swine can transcribe >20 different Cγ chains. A comparison of mammalian Cγ gene sequences revealed that IgG diversified into subclasses after speciation. Thus, the effector functions for the IgG subclasses of each species should not be extrapolated from “same name subclasses” in other species. Sequence analysis identified motifs likely to interact with Fcγ receptors, FcRn, protein A, protein G, and C1q. These revealed IgG3 to be most likely to activate complement and bind FcγRs. All except IgG5a and IgG6a should bind to FcγRs, while all except IgG6a and the putative IgG5 subclass proteins should bind well to porcine FcRn, protein A, and protein G.
82 citations
TL;DR: DRP1nuc is highly expressed in lung adenocarcinomas, and correlates with poor prognosis, and the results suggest that other than the protein level alone, intracellular distribution of the protein is critical for determining the protein function in cells.
70 citations
TL;DR: Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.
70 citations
TL;DR: This work has developed a more stable conjugation strategy utilizing the interaction between avidin and biotin, which is effective in some experimental settings and effective in others.
Abstract: Background
Targeted gene transduction in vivo is the ultimate preferred method for gene delivery. We previously developed targeting lentiviral vectors that specifically recognize cell surface molecules with conjugated antibodies and mediate targeted gene transduction both in vitro and in vivo. Although effective in some experimental settings, the conjugation of virus with antibodies is mediated by the interaction between protein A and the Fc region of antibodies, which is not as stable as covalent conjugation. We have now developed a more stable conjugation strategy utilizing the interaction between avidin and biotin.
57 citations
TL;DR: Results strongly indicated that the proposed strategy, the simultaneous display of two enzymes on the yeast cell surface, was accomplished by quantitatively controlling the display system using affinity binding.
Abstract: We constructed a novel cell surface display system to control the ratio of target proteins on the Saccharomyces cerevisiae cell surface, using two pairs of protein-protein interactions. One protein pair is the Z domain of protein A derived from Staphylococcus aureus and the Fc domain of human immunoglobulin G. The other is the cohesin (Coh) and dockerin (Dock) from the cellulosome of Clostridium cellulovorans. In this proposed displaying system, the scaffolding proteins (fusion proteins of Z and Coh) were displayed on the cell surface by fusing with the 3′ half of α-agglutinin, and the target proteins fused with Fc or Dock were secreted. As a target protein, a recombinant Trichoderma reesei endoglucanase II (EGII) was secreted into the medium and immediately displayed on the yeast cell surface via the Z and Fc domains. Display of EGII on the cell surface was confirmed by hydrolysis of β-glucan as a substrate, and EGII activity was detected in the cell pellet fraction. Finally, two enzymes, EGII and Aspergillus aculeatus β-glucosidase 1, were codisplayed on the cell surface via Z-Fc and Dock-Coh interactions, respectively. As a result, the yeast displaying two enzymes hydrolyzed β-glucan to glucose very well. These results strongly indicated that the proposed strategy, the simultaneous display of two enzymes on the yeast cell surface, was accomplished by quantitatively controlling the display system using affinity binding.
57 citations
TL;DR: The properties of the complex between fragment B of Protein A and the Fc domain of IgG were investigated adopting molecular dynamics with the intent of providing useful insight that might be exploited to design mimetic ligands with properties similar to those of protein A.
48 citations
TL;DR: All 65 mammalian species tested contain gamma globulins reactive with protein A presumably via similar Fc structures of immunoglobulin G with a common evolutionary origin, as well as two mammalian sera not tested previously, which showed marked protein A reactivity.
Abstract: Serum samples from 300 individual birds representing 72 species from 15 of 28 living orders were tested for protein A reactivity in gel diffusion experiments. Only two species, Rhea americana (Greater Rhea) and Pterocnemia pennata (Lesser Rhea), showed positive reactions, whereas serum samples from the remaining 70 species tested were negative. Protein A reactivity in the two Rheiformes species was identified in a slowly migrating serum globulin of unknown function with a molecular weight of 185 000. Absorption of serum from Rhea americana with staphylococci containing protein A almost completely eliminated the slow globulin. In Pterocnemia pennata similar absorption experiments reduced the amount of the corresponding globulin by 40–50 per cent. Two mammalian sera, the wooly opossum and the wallaby, not tested previously were also analyzed. Both species showed marked protein A reactivity. The only negative mammalian serum found earlier, the American opossum, was also reexamined. Concentrated globulin fractions showed a quantitatively small but definite reactivity in tests for protein A reactivity. Thus, all 65 mammalian species tested contain gamma globulins reactive with protein A presumably via similar Fc structures of immunoglobulin G with a common evolutionary origin.
TL;DR: Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports and their effects on the ligand-ligate interaction have been studied in detail.
TL;DR: A novel feasible method to produce Stx2Al in large scale in vitro, which is implicated for the development of multivalent subunit vaccines candidate against EHEC 0157:H7 infections is provided.
Abstract: Shiga toxin 2 (Stx2) is a major virulence factor for enterohemorrhagic Escherichia coli (EHEC), which is encoded by lambda lysogenic phage integrated into EHEC chromosome. Stx2Al, Al subunit of Stx2 toxin has gathered extensive concerns due to its potential of being developed into a vaccine candidate. However, the substantial progress is hampered in part for the lack of a suitable in vitro expression system. Here we report use of the prokaryotic system pET-28a::espA-Stx2Al/BL21 to carry out the fusion expression of Stx2Al which is linked to E. coli secreted protein A (EspA) at its N-terminus. Under the IPTG induction, EspA-Stx2Al fusion protein in the form of inclusion body was obtained successfully, whose expression level can reach about 40% of total bacterial protein at 25 degrees C, much higher than that at 37 degrees C. Western blot test suggested the refolded fusion protein is of excellent immuno-reactivity with both monoclonal antibodies, which are specific to EspA and Stx2Al, respectively. Anti-sera from Balb/c mice immunized with the EspA-Stx2Al fusion protein were found to exhibit strong neutralization activity and protection capability in vitro and in vivo. These data have provided a novel feasible method to produce Stx2Al in large scale in vitro, which is implicated for the development of multivalent subunit vaccines candidate against EHEC 0157:H7 infections.
TL;DR: A vector for soluble protein expression in the cytoplasm of Escherichia coli cells using the cold induced system was constructed and the luminescence properties of recombinant mitrocomin were characterized and compared to other photoproteins including aequorin, clytin-I and cly tin-II.
TL;DR: The complementation assays show that the influences of GdpS on spa and sarS depend on its N-terminal domain, which is predicted to be the sensor of a two-component system, rather than its C- terminal GGDEF domain with conserved GGDEF, suggesting that GDPS functions in S. aureus by an unknown mechanism independent of 3′,5′-cyclic diguanylic acid signaling.
Abstract: Staphylococcus aureus is an important human pathogen that is the principal cause of a variety of diseases, ranging from localized skin infections to life-threatening systemic infections. The success of the organism as a pathogen and its ability to cause such a wide range of infections are due to its extensive virulence factors. In this study, we identified the role of the only GGDEF domain protein (GdpS [GGDEF domain protein from Staphylococcus]) in the virulence of S. aureus NCTC8325. Inactivation of gdpS results in an alteration in the production of a range of virulence factors, such as serine and cysteine proteases, fibrinogen-binding proteins, and, specifically, protein A (Spa), a major surface protein of S. aureus. The transcript level of spa decreases eightfold in the gdpS mutant compared with the parental NCTC8325 strain. Furthermore, the transcript level of sarS, which encodes a direct positive regulator of spa, also decreases in the gdpS mutant compared with the wild type, while the transcript levels of agr, sarA, sarT, and rot display no apparent changes in the gdpS mutant, suggesting that GdpS affects the expression of spa through interaction with SarS by unknown mechanisms. Furthermore, the complementation assays show that the influences of GdpS on spa and sarS depend on its N-terminal domain, which is predicted to be the sensor of a two-component system, rather than its C-terminal GGDEF domain with conserved GGDEF, suggesting that GdpS functions in S. aureus by an unknown mechanism independent of 3',5'-cyclic diguanylic acid signaling.
TL;DR: Sera tested for activity against crude protein A gave results similar to those of normal sera with regard to precipitation of protein A, indirect haemagglutination and bacterial agglutination, with isolated γG-globulins showing activities corresponding to their respective sera.
Abstract: Normal sera from 14 species have been tested for activity against crude protein A. Sera from 6 species produced the protein A line in agar gel, but none of the sera gave the protein B line. Sera from 7 species, including 3 of those precipitating protein A, agglutinated tanned sheep erythrocytes sensitized with crude protein A. With a few individual exceptions, all the sera tested agglutinated Cowan I bacteria. Isolated γG-globulins showed activities corresponding to their respective sera. The active part of the γG-globulins in the precipitation reaction with protein A was located to Fc whereas Fab was the active part both in indirect haemagglutination and bacterial agglutination. Rabbit antiserum to Cowan I bacteria and guinea pig antiserum to crude protein A gave results similar to those of normal sera with regard to precipitation of protein A, indirect haemagglutination and bacterial agglutination. In addition both sera precipitated protein B, this ability being located to the Fab region of the γG-globulins.
TL;DR: Taken together, the results suggest that type II group B streptococci should be divided into two subtypes, II, R+ and II,R-.
Abstract: R-proteins was isolated by affinity chromatography from an alkaline extract of group B streptococci, type III. the identity of the protein was demonstrated by pepsin- and trypsin-treatment and in double diffusion-in-gel with antisera from other laboratories. A monospecific rabbit antiserum against the R-protein was studied in mouse-protection tests, using three type II strains with R-protein and three without, and six type III strains, of which four carried R-protein. the serum protected animals from infection by type II strains carrying R-protein, but not against any of the other strains. The capacity of anti-R-protein serum to opsonize type II strains carrying R-protein was also demonstrated in phagocytosis experiments with mouse peritoneal macrophages. Studies with radio-labelled protein A and anti-R-protein antibodies showed that R-protein was localized on the surface of both type II and type III groups B streptococci. Taken together, the results suggest that type II group B streptococci should be divided into two subtypes, II, R+ and II, R-.
TL;DR: The results suggest that blocking of the binding of complement to IgG is an important virulence mechanism in Fc-receptor-bearing streptococci and staphylococci.
Abstract: Using haemolysis in gel, two bacterial IgG-binding substances, an Fc-receptor isolated from group A streptococci type M15, and protein A from Staphylococcus aureus, were shown to inhibit complement-mediated lysis of sheep erythrocytes sensitized with rabbit IgG. When the crude alkaline extracts of ten types of group A streptococci were tested to see whether streptococcal components other than Fc-binding material might affect lysis, the degree of inhibition was found to be correlated with Fc-binding activity. In no case was the lysis of IgM-coated cells inhibited. Opsonophagocytosis experiments showed that both purified streptococcal Fc-receptor and protein A impaired antibody complement-dependent killing by human polymorphonuclear leukocytes of each of two strains of group B streptococci (lacking IgG Fc-receptors). Furthermore, the impairment was ascribable to interference with the fixation of complement to the antibodies, as demonstrated in pre-opsonization experiments with one of the strains. Our results suggest that blocking of the binding of complement to IgG is an important virulence mechanism in Fc-receptor-bearing streptococci and staphylococci.
TL;DR: In chronic bovine mastitis, the low levels of protein A detected in these strains seem to indicate that other factors play a more important role than host-parasite relationship in staphylococcal infections.
Abstract: Staphylococcus aureus strains from human infections and from bovine cases of acute and of chronic mastitis were studied regarding protein A content. Human strains as well as bovine strains from acute mastitis showed high levels of protein A production in parallel with a high incidence of cell wall associated protein A. In contrast, S. aureus strains from bovine cases of chronic mastitis showed a significantly lower production of protein A. Only about 50 per cent of these strains had detectable cell wall associated protein A. Host-parasite relationship in staphylococcal infections might favour the presence of protein A in strain from acute infections. In chronic bovine mastitis, however, the low levels of protein A detected in these strains seem to indicate that other factors play a more important role.
Patent•
11 Aug 2009TL;DR: In this article, modified immunoglobulin-binding proteins, e.g., Staphylococcus protein A, have been used to improve the specificity of immunoglobalin binding proteins.
Abstract: The present invention relates to modified immunoglobulin-binding proteins, e.g., Staphylococcus protein A, having improved binding specificity for immunoglobulins and methods of making and using the same.
TL;DR: IgG1, IgG2, and IgG3, three subclasses of human IgG, were separated from human serum using the amino acid histidine grafted on to bisoxirane-activated Sepharose, as pseudobiospecific adsorbent, and results compared well with results obtained by use of protein A–Sepharose gel.
Abstract: Adsorption chromatography is increasingly used for protein separations and biomedical applications. Therapeutic molecules such as antibodies, cytokines, therapeutic DNA, and plasma proteins must be purified before characterization and utilization. Use of immunoglobulins as immunodiagnostic and therapeutic tools has initiated many attempts to develop new adsorbents for their separation. Protein A and protein G are the affinity ligands most widely used for separation of immunoglobulins. These proteins are reliable, and have good selectivity and specificity, but are very expensive. Much attention has therefore been devoted to developing alternative methods for separation of immunoglobulins. Pseudobiospecific ligands, for example metal ions and amino acids, can be used for separation of a wide range of biological molecules. In this study, IgG1, IgG2, and IgG3, three subclasses of human IgG, were separated from human serum using the amino acid histidine grafted on to bisoxirane-activated Sepharose, as pseudobiospecific adsorbent. Adsorption of IgG from different animal species on the same chromatographic adsorbent was also tested. The high recovery and purification on histidyl–bisoxirane–Sepharose gel of IgG from all the sources tested compared well with results obtained by use of protein A–Sepharose gel.
TL;DR: Human colostral IgA from three apparently healthy mothers were all divided into protein A reactive and protein A non-reactive fractions on a Sepharose-protein A column.
Abstract: Human colostral IgA from three apparently healthy mothers were all divided into protein A reactive and protein A non-reactive fractions on a Sepharose-protein A column. The reactive fractions, giving no direct precipitation but co-precipitations, were found to interact with protein A through the Fc-region.
TL;DR: It is demonstrated that mammalian immunoglobulins possess F(ab')2- and Fc-binding sites for protein A and for receptors on group C and G streptococci.
Abstract: The distribution among mammalian species of non-immune F(ab')2- and Fc-mediated immunoglobulin interactions with surface proteins of S. aureus (protein A) and of group C and G streptococci was studied. Serum samples from 48 mammalian species representing 15 orders were first tested for their capacity to inhibit streptococcal F(ab')2-mediated binding; 26 of these sera were also tested for streptococcal IgG Fc-mediated binding. Analogous inhibition experiments were then carried out with staphylococci. All mammalian species studied inhibited both types of immunoglobulin binding to streptococci, viz the serum samples contained both F(ab')2- and Fc-reactive immunoglobulins. The reactivity was equal to that of human serum in 26 out of 47 mammalian sera. Seven sera showed a low degree of inhibition compared to human serum. The inhibiting capacities of the two streptococcal non-immune interactions showed a direct correlation (r = 0.91, p less than 0.0001 for the r-value) for individual species. The inhibition patterns observed with S. aureus differed from the profiles recorded with the streptococcal strains, suggesting that these organisms interact with separate sites on the immunoglobulin molecules. Isolated F(ab')2-binding was recorded in 5 out of 24 sera, and Fc-binding alone was noted in 7 sera. Taken together, the present studies demonstrate that mammalian immunoglobulins possess F(ab')2- and Fc-binding sites for protein A and for receptors on group C and G streptococci. The F(ab')2-mediated binding to streptococci is associated with Fc-reactivity, in contrast to protein A which may interact exclusively with a complementary structure in either the F(ab')2- or the Fc-portion of the immunoglobulins.
TL;DR: Results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen, which is specific for a polymorphic lymphocyte antigen.
Abstract: Summary
A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both 125I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20 % of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of 3H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton chain and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen
TL;DR: It was concluded that the elution difference of antibodies with or without oligosaccharides and antibodies with different types of oligosACcharides were due to differential structural changes around the CH2-CH3 domain interface under the low pH conditions used for protein A and protein G chromatography.
TL;DR: The P‐antigen appears to be characteristic of B. catarrhalis and was demonstrated in 69% of normal human sera, and the possibility of a serological identification of the species is introduced.
Abstract: Precipitation patterns of sonicated, acid-extracted and other extracts from Branhamella catarrhalis were examined by double diffusion-in-gel technique, using antiserum to B. catarrhalis. Acid extract gave rise to 4 distinct precipitates. One of these lines was further studied. The bacterial component responsible for this line was trypsin-sensitive, indicating that it was a protein. It was anodally localized by crossed immunoelectrophoresis. By absorption of antiserum with whole bacteria, the precipitating capacity of the serum was diminished, suggesting that the protein antigen (P-antigen) was exposed on the bacterial surface. F(ab')2-fragments of IgG from antiserum, but not from normal rabbit serum, precipitated the P-antigen, indicating that it was a true antigen-antibody reaction. It was possible to make an IgG preparation monospecific for the P-antigen, by absorbing antiserum with trypsinized bacterial extract. 31 strains of B. catarrhalis, 9 strains of N. gonorrhoeae, 10 strains of N. meningitidis, 12 other Neisseria spp. and 2 strains of H. influenzae were investigated for presence of cross-reacting surface antigens, using IgG monospecific for the P-antigen and 125I-labelled protein A from Staphylococcus aureus. After antibody exposure, all 31 strains of B. catarrhalis showed abundant uptake of protein A. No significant uptake was detected on any other investigated strain. Hence, the P-antigen appears to be characteristic of B. catarrhalis. The possibility of a serological identification of the species is introduced. Precipitating antibodies against the P-antigen were demonstrated in 69% of normal human sera.
TL;DR: An increase in the proportion of weak protein A producers during the past decade was found to coincide with a shift in dominant phage types in Denmark.
Abstract: The prevalence of protein A producing strains of S. aureus was found to vary. An increase in the proportion of weak protein A producers during the past decade was found to coincide with a shift in dominant phage types in Denmark. Within phage groups I and II the ability to form large amounts of protein A was shown to be correlated to a deficient haemolysin production. This correlation was also found for group III strains susceptible to phage 75 A. Among group III strains susceptible to phages within the complex 83A, 84, 85, 6557, 89 two subgroups were remarkable. The strains which were resistant to neomycin (antibiogram PSTEN) produced large amounts of protein A, whereas those which were resistant to methicillin (antibiograms PSTEM and PSTM) produced little or no protein A. Analysis of the present material revealed no demonstrable or suspected pathogenic properties of protein A.
TL;DR: Competition ELISA tests suggested that IgG does not compromise Fg binding and vice versa, and FgBP binds Fg avidly through residues located at the extreme N terminus of the molecule, whereas the IgG-binding site is more centrally located between the A and B repeats.
Abstract: Fibrinogen-binding protein (FgBP, also termed SeM) is a cell-wall-associated anti-phagocytic M-like protein of the equine pathogen Streptococcus equi subsp. equi, and binds fibrinogen (Fg) and IgG. FgBP binds Fg avidly through residues located at the extreme N terminus of the molecule, whereas the IgG-binding site is more centrally located between the A and B repeats. FgBP binds equine IgG4 and IgG7 subclasses through interaction with the CH2–CH3 interdomain region of IgG-Fc, and possesses overlapping Fc-binding sites with protein A and protein G. In this study, FgBP truncates containing defined internal deletions were used to identify a stretch of 14 aa (residues 335–348) critical for IgG binding. Protein chimeras consisting of the non-IgG-binding α-helical coiled-coil M5 protein fused to FgBP sequences were used to identify a minimal equine IgG-binding domain consisting of residues 329–360. Competition ELISA tests suggested that IgG does not compromise Fg binding and vice versa.
TL;DR: The protein A content of extracts of staphylococcal cells was estimated by the single radial immunodiffusion technique (Mancini technique) and by a haemagglutination test and the grouping of S. aureus strains according to protein A production using these methods was essentially similar to that based on their different affinity to FITC-labelled immunoglobulins.
Abstract: The protein A content of extracts of staphylococcal cells was estimated by the single radial immunodiffusion technique (Mancini technique) and by a haemagglutination test. The grouping of S. aureus strains according to protein A production using these methods was essentially similar to that based on their different affinity to FITC-labelled immunoglobulins. High yields of protein A were obtained from staphylococcal cells which were harvested after growth on solid medium and extracted in 0.07 M phosphate buffer at pH 5.9 for 10 days at room temperature. The presence of sodium chloride (7.5 per cent) in the medium suppressed the formation of protein A. The immunogenic properties of both protein A producing strains and protein A were dependent on the presence or absence of serum (IgG) in the medium. Due to the complex formation between protein A and IgG, even highly purified protein A induced antibodies which reacted specifically with immunoglobulins when serum or blood had been present in the medium.
01 Jan 2009
TL;DR: The structure of p27 SJ protein of Hypericum perforatum was found to resemble the structure of peroxiredoxin high resolution structure of Ding Protein of Pseudomonas fluorescence.
Abstract: SJ present in a laboratory callus culture of Hypericum perforatum suppresses transcription of the HIV-1 genome in several human cell types including primary culture of microglia and astrocytes. p27 SJ associates with C/EBPβ, a transcription factor that regulates expression of the HIV-1 genome in macrophages and monocytic cells, and the viral transactivator, Tat. It has been found that it can suppress transcription of the HIV-1 genome. Despite its importance, the three dimensional (3D) structure of p27 SJ has not yet been reported. A homology modeling method was used for the prediction of the structure of p27 SJ protein. For the modeling, template protein was obtained by Geno3D server, template protein pdb|2Q9T|chain A having identity 88%, E value 3e-129 and alignment Score 1140. By comparing the template protein a rough model was constructed for the target protein using MODELLER, a program for comparative modelling. The structure of p27 SJ protein of Hypericum perforatum was found to resemble the structure of peroxiredoxin high resolution structure of Ding Protein of Pseudomonas fluorescence . From Ramachandran plot analysis it was found that the portion of residues falling into the most favoured regions was 95.4%. The predicted 3-D model may be further characterized and analyzed using other techniques.