scholarly journals Staphylococcus aureus Protein A Recognizes Platelet gC1qR/p33: a Novel Mechanism for Staphylococcal Interactions with Platelets

2000 ◽  
Vol 68 (4) ◽  
pp. 2061-2068 ◽  
Author(s):  
Truc Nguyen ◽  
Berhane Ghebrehiwet ◽  
Ellinor I. B. Peerschke
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Site ◽  
Soluble Protein ◽  
Molecular Mechanisms ◽  
Solid Phase ◽  
Specific Binding ◽  
Protein A ◽  
Alpha Helix ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Terminal

ABSTRACT The adhesion of Staphylococcus aureus to platelets is a major determinant of virulence in the pathogenesis of endocarditis. Molecular mechanisms mediating S. aureus interactions with platelets, however, are incompletely understood. The present study describes the interaction between S. aureus protein A and gC1qR/p33, a multifunctional, ubiquitously distributed cellular protein, initially described as a binding site for the globular heads of C1q. Suspensions of fixed S. aureus or purified protein A, chemically cross-linked to agarose support beads, were found to capture native gC1qR from whole platelets. Moreover, biotinylated protein A bound specifically to fixed, adherent, human platelets. This interaction was inhibited by unlabeled protein A, soluble recombinant gC1qR (rgC1qR), or anti-gC1qR antibody F(ab′)2 fragments. The interaction between protein A and platelet gC1qR was underscored by studies illustrating preferential recognition of the protein A-bearing S. aureus Cowan I strain by gC1qR compared to recognition of the protein A-deficient Wood 46 strain, as well as inhibition of S. aureus Cowan I strain adhesion to immobilized platelets by soluble protein A. Further characterization of the protein A-gC1qR interaction by solid-phase enzyme-linked immunosorbent assay techniques measuring biotinylated gC1qR binding to immobilized protein A revealed specific binding that was inhibited by soluble protein A with a 50% inhibitory concentration of (3.3 ± 0.7) × 10−7 M (mean ± standard deviation; n = 3). Rabbit immunoglobulin G (IgG) also prevented gC1qR-protein A interactions, and inactivation of protein A tyrosil residues by hyperiodination, previously reported to prevent the binding of IgG Fc, but not Fab, domains to protein A, abrogated gC1qR binding. These results suggest similar protein A structural requirements for gC1qR and IgG Fc binding. Further studies of structure and function using a truncated gC1qR mutant lacking amino acids 74 to 95 demonstrated that the protein A binding domain lies outside of the gC1qR amino-terminal alpha helix, which contains binding sites for the globular heads of C1q. In conclusion, the data implicate the platelet gC1qR as a novel cellular binding site for staphylococcal protein A and suggest an additional mechanism for bacterial cell adhesion to sites of vascular injury and thrombosis.

scholarly journals A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

1996 ◽  
Vol 133 (2) ◽  
pp. 431-444 ◽  
Author(s):  
D C Hocking ◽  
R K Smith ◽  
P J McKeown-Longo
Keyword(s):  
Binding Site ◽  
Wound Repair ◽  
Solid Phase ◽  
Focal Adhesions ◽  
Amino Terminus ◽  
Matrix Assembly ◽  
Amino Terminal ◽  
Fibronectin Matrix ◽  
Beta 1 Integrin ◽  
Rgd Site

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

scholarly journals Protein Co-Aggregation Related to Amyloids: Methods of Investigation, Diversity, and Classification

2018 ◽  
Vol 19 (8) ◽  
pp. 2292 ◽  
Author(s):  
Stanislav Bondarev ◽  
Kirill Antonets ◽  
Andrey Kajava ◽  
Anton Nizhnikov ◽  
Galina Zhouravleva
Keyword(s):  
Binding Site ◽  
Molecular Mechanisms ◽  
Amyloid Fibrils ◽  
Specific Binding ◽  
Protein S ◽  
Functional Protein ◽  
Functional Roles ◽  
Protein Fibrils ◽  
Protein Functions ◽  
Functional Amyloids

Amyloids are unbranched protein fibrils with a characteristic spatial structure. Although the amyloids were first described as protein deposits that are associated with the diseases, today it is becoming clear that these protein fibrils play multiple biological roles that are essential for different organisms, from archaea and bacteria to humans. The appearance of amyloid, first of all, causes changes in the intracellular quantity of the corresponding soluble protein(s), and at the same time the aggregate can include other proteins due to different molecular mechanisms. The co-aggregation may have different consequences even though usually this process leads to the depletion of a functional protein that may be associated with different diseases. The protein co-aggregation that is related to functional amyloids may mediate important biological processes and change of protein functions. In this review, we survey the known examples of the amyloid-related co-aggregation of proteins, discuss their pathogenic and functional roles, and analyze methods of their studies from bacteria and yeast to mammals. Such analysis allow for us to propose the following co-aggregation classes: (i) titration: deposition of soluble proteins on the amyloids formed by their functional partners, with such interactions mediated by a specific binding site; (ii) sequestration: interaction of amyloids with certain proteins lacking a specific binding site; (iii) axial co-aggregation of different proteins within the same amyloid fibril; and, (iv) lateral co-aggregation of amyloid fibrils, each formed by different proteins.

Evaluation of Coagulase Activity and Protein A Production for the Identification of Staphylococcus aureus

1995 ◽  
Vol 58 (8) ◽  
pp. 858-862 ◽  
Author(s):  
TSUNG C. CHANG ◽  
SU H. HUANG
Keyword(s):  
Public Health ◽  
Staphylococcus Aureus ◽  
Protein A ◽  
High Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
American Public Health Association ◽  
Bacterial Strains ◽  
Test Sensitivity ◽  
Microtiter Plates ◽  
Health Association

The activities of coagulase and thermostable nuclease (TNase) and the production of protein A were studied in 338 bacterial strains. These included 213 isolates of Staphylococcus aureus to determine which characteristic was most specific for the identification of S. aureus. The evaluation of different protocols for interpretation of coagulase results was also undertaken. Protein A was analyzed by a sandwich enzyme-linked immunosorbent assay using microtiter plates coated with antiprotein A antibodies. Coagulase activities were determined according to the criteria recommended by Association of Official Analytical Chemists (AOAC; any degree of clot formation is a positive reaction), American Public Health Association (APHA; coagulase activities ≥ 3+ are positive reactions), and the Bacteriological Analytical Manual (BAM; only 4+ reaction is positive). It was found that the AOAC protocol, which had a test sensitivity of 97.7% and a specificity of 95.1% and could be completed within six hours, was more practical than the methods used by APHA and BAM. Compared with coagu1ase and TNase, protein A was a better marker of S. aureus; a high sensitivity (100%) and specificity (96.8%) were obtained by using protein A for the identification of S. aureus.

Development of a monoclonal antibody to the rabbit 8.5S uterine progestin receptor

1985 ◽  
Vol 63 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Kiyohide Nakao ◽  
John E. Myers ◽  
Lee E. Faber
Keyword(s):  
Sucrose Gradient ◽  
Solid Phase ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Deae Cellulose ◽  
Enzyme Linked Immunosorbent Assay ◽  
Progestin Receptors ◽  
Sucrose Gradient Centrifugation ◽  
Progestin Receptor ◽  
Solid Phase Radioimmunoassay

Nonactivated (8.5S) rabbit uterine progestin receptor was enriched 10- to 30-fold by chromatography on columns of spheroidal hydroxylapatite and DEAE-cellulose. A total of`~25 μg of receptor (purity ~1%) was injected at multiple sites into a BALB/c mouse. After several injections, splenic lymphocytes were fused with P3x63Ag8.653 mouse myeloma cells. This fusion produced in excess of 240 hybridomas, which were screened by an enzyme-linked immunosorbent assay (ELISA), solid-phase radioimmunoassay, and sucrose gradient centrifugation. One colony (KN 382/EC1) produced a mouse immunoglobulin G1 which bound rabbit 8.5S uterine progestin receptor. The cell line has been repeatedly cloned under conditions of limiting dilution and expanded in mice as ascitic tumors. Antibody was purified by (NH4)2SO4 precipitation, DEAE-cellulose chromatography, and affinity chromatography with protein A – Sepharose CL-4B. Specificity of the antibody was determined by sucrose gradient centrifugation and solid-phase radioimmunoassay. The antibody bound to progestin receptors from rabbit uterus and MCF-7 breast cancer cells. It did not react with progestin receptors from rat uterus, guinea pig uterus, or chick oviduct, nor did it bind to estrogen receptors from any of the tissues we tested.

Solid-phase radioimmunoassay with protein-A-bearing Staphylococcus aureus cells used to assay a protein (ferritin) and a hapten (digoxin).

1980 ◽  
Vol 26 (1) ◽  
pp. 37-40
Author(s):  
J Gauldie ◽  
H K Tang ◽  
A Corsini ◽  
W H Walker
Keyword(s):  
Staphylococcus Aureus ◽  
Matrix Effects ◽  
Solid Phase ◽  
Protein A ◽  
General Applicability ◽  
Rabbit Igg ◽  
Human Sera ◽  
Solid Phase Radioimmunoassay ◽  
Formalin Fixed ◽  
Economical Alternative

Abstract We compared use of protein-A-containing Staphylococcus aureus bacteria with conventional ammonium sulfate precipitation and second-antibody methods of separating bound and free antigen in the radioimmunoassay of a hapten (digoxin) and protein (ferritin) in human sera. In each case, values obtained with the heat-killed, formalin-fixed bacteria correlated well with those found by established methods. No matrix effects were detected in either hapten or protein measurements. Because of the affinity of S. aureus for rabbit IgG, rabbit antisera could be used with a small number of bacteria to detect antigen in the presence of 50-fold excess human IgG. The availability of S. aureus and ease of handling make this reagent a rapid, economical alternative of general applicability in radioimmunoassay.

Solid-phase radioimmunoassay with protein-A-bearing Staphylococcus aureus cells used to assay a protein (ferritin) and a hapten (digoxin).

1980 ◽  
Vol 26 (1) ◽  
pp. 37-40 ◽  
Author(s):  
J Gauldie ◽  
H K Tang ◽  
A Corsini ◽  
W H Walker
Keyword(s):  
Staphylococcus Aureus ◽  
Matrix Effects ◽  
Solid Phase ◽  
Protein A ◽  
General Applicability ◽  
Rabbit Igg ◽  
Human Sera ◽  
Solid Phase Radioimmunoassay ◽  
Formalin Fixed ◽  
Economical Alternative

Abstract We compared use of protein-A-containing Staphylococcus aureus bacteria with conventional ammonium sulfate precipitation and second-antibody methods of separating bound and free antigen in the radioimmunoassay of a hapten (digoxin) and protein (ferritin) in human sera. In each case, values obtained with the heat-killed, formalin-fixed bacteria correlated well with those found by established methods. No matrix effects were detected in either hapten or protein measurements. Because of the affinity of S. aureus for rabbit IgG, rabbit antisera could be used with a small number of bacteria to detect antigen in the presence of 50-fold excess human IgG. The availability of S. aureus and ease of handling make this reagent a rapid, economical alternative of general applicability in radioimmunoassay.

An Enzyme-Linked Immunosorbent Assay for the Rapid Detection of Staphylococcus aureus in Processed Foods

1994 ◽  
Vol 57 (3) ◽  
pp. 184-189 ◽  
Author(s):  
TSUNG C. CHANG ◽  
SU H. HUANG
Keyword(s):  
Staphylococcus Aureus ◽  
Immunosorbent Assay ◽  
Rapid Detection ◽  
Protein A ◽  
Large Degree ◽  
Microtiter Plate ◽  
Specific Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Processed Foods ◽  
Culture Methods

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the rapid detection of Staphylococcus aureus in foods. The assay was based on the detection of protein A which is a specific protein secreted by S. aureus. Following a 24-h incubation in a staphylococcal selective broth containing mannitol as the carbon source, the culture supematant was added to the microtiter plate coated with anti-protein A immunoglobulin G (IgG). After incubation, peroxidase-labeled anti-protein A IgG was used to produce the signal of antigen-antibody reaction. The sensitivity of the assay for protein A was 0.1 ng/ml. For 37 strains of S. aureus studied, all produced protein A, and the amount (13-1,100 ng/ml) of protein A secreted by different strains varied to a large degree. For another 57 strains (including 19 Staphylococcus spp.) of bacteria tested, two strains (5. capilis subsp. capitis CCRC 12161 and S. lentus CCRC 12926) produced very low amounts of protein A (0.6-1 ng/ml) after 24-h incubation. Staphylococcus aureus was detected by the ELISA in all of six samples of precooked foods naturally contaminated with the bacterium. Twenty-two processed foods artificially inoculated with S. aureus at levels of < 2 CFU/g and 10 to 20 CFU/g, respectively, were all positive by the ELISA. As compared to the conventional culture methods which take 5 to 6 days to complete, the ELISA can detect low numbers of S. aureus in processed foods with a total analytical time of only 28 h.

scholarly journals Specific Ligand Binding Attributable to Individual Epitopes of Gonococcal Transferrin Binding Protein A

2002 ◽  
Vol 70 (2) ◽  
pp. 732-740 ◽  
Author(s):  
Heather P. Masri ◽  
Cynthia Nau Cornelissen
Keyword(s):  
Ligand Binding ◽  
Fusion Proteins ◽  
Iron Uptake ◽  
Solid Phase ◽  
Protein A ◽  
Dot Blot ◽  
Affinity Tags ◽  
Amino Terminal ◽  
Dot Blot Assay ◽  
Specific Ligand

ABSTRACT The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.

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