Domains Of Staphylococcal Protein A Research Articles

Oxidized pullulan exhibits potent antibacterial activity against S. aureus by disrupting its membrane integrity

The capability of bacteria to withstand the misuse of antibiotics leads to the generation of multi-drug resistant strains, posing a new challenge to curb wound infections. The biological macromolecules, due to their biocompatibility, biodegradability, and antimicrobial properties, have been explored for a variety of antimicrobial and therapeutic purposes. This work reports that a single-step oxidation of pullulan polymer leads to the formation of oxidized pullulan (o-pullulan), which shows striking antibacterial and antibiofilm activities against the Gram-positive bacteria, Staphylococcus aureus, implicated in wound-related infections. Oxidation of pullulan generates 28 % aldehyde groups (3.462 mmol/g) which exerted 97 % bactericidal activity against S. aureus by targeting cell wall-associated membrane protein SpA (Staphylococcal protein A). The molecular docking, gene silencing, and fluorescence quenching studies revealed a direct binding of o-pullulan with the B and C domains of SpA, which alters the membrane potential and inhibits Ca2+-Mg2+-ATPase pumps. O-pullulan also exhibited scavenging activity against intracellular reactive oxygen species (ROS), and non-immunotoxic activity and was found to be non-toxic to mammalian cells. Thus, o-pullulan shows great promise as an antimicrobial polymer against S. aureus for chronic wound management.

Determination of the association constant between the B domain of protein A and the Fc region of IgG

The aim of this work is the numerical modelling of the binding mechanism between the Fc region of human IgG interacting with the B domain of Staphylococcal protein A (SpA). The comprehension of the involved kinetics is of noticeable impact for immunosensor diagnostic applications and consequently contributes to increase the sensitivity and efficiency of such devices based on the immobilization of antibodies on biosensor surface. Brownian dynamics methodology is applied to simulate the Fc–SpA encounter. Then, the association rates kon and koff are estimated from the analysis of the diffusional motion between the Fc region and the B domain of SpA combined with continuum electrostatic calculations. Therefore, the association constant Ka between Fc and SpA is calculated. The behaviour of Ka is analysed taking into account the relative distance between SpA and the Fc fragments. The analyses also include the effects on the binding affinity between SpA and Fc due to the variation of the solvent ionic strength and pH values. The association rates and their analyses are presented and discussed showing that the binding mechanism between the SpA and the Fc fragments is enhanced by the nonpolar interaction, while dissociation is driven by the electrostatic repulsion that occurs at relatively low pH. The numerical estimation of the association constant will support the definition of robust protocols for the detection of antibodies via protein A. Copyright © 2014 John Wiley & Sons, Ltd.

A monoclonal antibody that recognizes the E domain of staphylococcal protein A

Staphylococcal protein A (SpA) binds Fcγ and VH3 clan Fab domains of human and animal immunoglobulin (Ig) with each of its five Ig binding domains (IgBDs), thereby supporting Staphylococcus aureus escape from opsonophagocytic killing and suppressing adaptive B cell responses. The variant SpAKKAA cannot bind Ig yet retains antigenic properties that elicit SpA-neutralizing antibodies and disease protection in mice, whereas S. aureus infection or SpA-immunization cannot elicit neutralizing antibodies. As a test for this model, we analyzed here mAb 358A76, which was isolated from SpA-immunized mice. Unlike SpAKKAA-derived mAbs, mAb 358A76 binds only the first IgBD (E) but not any of the other four IgBDs (D-A-B-C) and its binding can neutralize only the E domain of SpA, which does not provide disease protection in mice. These results are in agreement with a model whereby wild-type SpA-immunization generates a limited antibody response without neutralizing and/or disease protective attributes.

Role of Protein A in the Evasion of Host Adaptive Immune Responses by Staphylococcus aureus

ABSTRACTHeritable defects in human B cell/antibody development are not associated with increased susceptibility to Staphylococcus aureus infection. Protein A (SpA), a surface molecule of S. aureus, binds the Fcγ domain of immunoglobulin (Ig) and cross-links the Fab domain of VH3-type B cell receptors (IgM). Here we generated S. aureus spa variants harboring amino acid substitutions at four key residues in each of the five Ig-binding domains of SpA. Wild-type S. aureus required SpA binding to Ig to resist phagocytosis and SpA-mediated B cell receptor cross-linking to block antibody development in mice. The spaKKAA mutant, which cannot bind Ig or IgM, was phagocytosed and elicited B cell responses to key virulence antigens that protected animals against lethal S. aureus challenge. The immune evasive attributes of S. aureus SpA were abolished in µMT mice lacking mature B cells and antibodies. Thus, while wild-type S. aureus escapes host immune surveillance, the spaKKAA variant elicits adaptive responses that protect against recurrent infection.

Nontoxigenic protein A vaccine for methicillin-resistant Staphylococcus aureus infections in mice.

The current epidemic of hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections has caused significant human morbidity, but a protective vaccine is not yet available. Prior infection with S. aureus is not associated with protective immunity. This phenomenon involves staphylococcal protein A (SpA), an S. aureus surface molecule that binds to Fcγ of immunoglobulin (Ig) and to the Fab portion of VH3-type B cell receptors, thereby interfering with opsonophagocytic clearance of the pathogen and ablating adaptive immune responses. We show that mutation of each of the five Ig-binding domains of SpA with amino acid substitutions abolished the ability of the resulting variant SpAKKAA to bind Fcγ or Fab VH3 and promote B cell apoptosis. Immunization of mice with SpAKKAA raised antibodies that blocked the virulence of staphylococci, promoted opsonophagocytic clearance, and protected mice against challenge with highly virulent MRSA strains. Furthermore, SpAKKAA immunization enabled MRSA-challenged mice to mount antibody responses to many different staphylococcal antigens.

Evaluation of pH-Dependent Staphylococcal Protein A Structural Change Using TOF-SIMS

The evaluation of protein structural change is crucial for the study of protein-protein interactions and protein conformational changes. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used to detect pH dependent structural change in staphylococcal protein A (SpA), which is well known to the bind Fc region of immunoglobulins. TOF-SIMS spectra of SpA-immobilized samples in a pH 7 solution, in an acidic solution, and in a pH 7 solution after having been soaked in an acidic solution, respectively, were compared to select fragment ions specific to normal SpA at pH 7 using spectral analysis techniques such as principal component analysis (PCA) and the mutual information method. A fragment ion depending on change in the B and E domains of SpA were detected with this method. These findings demonstrate that structural change of SpA depending on pH change can be evaluated with TOF-SIMS together with spectrum analysis methods for the multivariate data. [DOI: 10.1380/ejssnt.2009.715]

Validation of helical tilt angles in the solution NMR structure of the Z domain of Staphylococcal protein A by combined analysis of residual dipolar coupling and NOE data.

Staphylococcal protein A (SpA) is a virulence factor from Staphylococcus aureus that is able to bind to immunoglobulins. The 3D structures of its immunoglobulin (Ig) binding domains have been extensively studied by NMR and X-ray crystallography, and are often used as model structures in developing de novo or ab initio strategies for predicting protein structure. These small three-helix-bundle structures, reported in free proteins or Ig-bound complexes, have been determined previously using medium- to high-resolution data. Although the location and relative orientation of the three helices in most of these published 3D domain structures are consistent, there are significant differences among the reported structures regarding the tilt angle of the first helix (helix 1). We have applied residual dipolar coupling data, together with nuclear Overhauser enhancement and scalar coupling data, in refining the NMR solution structure of an engineered IgG-binding domain (Z domain) of SpA. Our results demonstrate that the three helices are almost perfectly antiparallel in orientation, with the first helix tilting slightly away from the other two helices. We propose that this high-accuracy structure of the Z domain of SpA is a more suitable target for theoretical predictions of the free domain structure than previously published lower-accuracy structures of protein A domains.

Staphylococcal protein A as a fusion partner directs secretion of the e1alpha and e1beta subunits of pea mitochondrial pyruvate dehydrogenase by Bacillus subtilis.

Staphylococcal protein A (SPA)-based vectors were constructed to direct secretion of the E1alpha and E1beta subunits of Pisum sativum mitochondrial pyruvate dehydrogenase from Bacillus subtilis. These proteins were not exported when the signal peptide from levansucrase (SacBSP) was fused to their N-termini. Both SacBSP-E1alpha and SacBSP-E1beta fusion proteins were insoluble in the cytoplasm. However, when the SPA open-reading frame was inserted between SacBSP and E1alpha or E1beta, corresponding fusion proteins were secreted from the cells. The first (E) IgG-binding domain of SPA was sufficient to direct low level secretion of both fusion proteins (SacBSP-E-E1alpha and SacBSP-E-E1beta). Adding the second (D) IgG-binding domain improved extracellular protein yields 3- to 4-fold over E alone, but was not as efficient as secretion of the full-length (EDABC) SPA-fusion proteins. All constructs were based on the pUB110-derived multicopy plasmid pWB705. Separate B. subtilis strains transformed with SacBSP-E-E1alpha-His(6) or SacBSP-E1beta were cocultivated in the presence of Ni-NTA agarose. The native pyruvate dehydrogenase alpha2beta2 structure was bound to the affinity matrix, demonstrating assembly after secretion. The use of SPA as a fusion partner during expression of heterologous proteins by B. subtilis provides the basis of a versatile system that can be used to study both secretion and protein:protein interactions.

High-resolution solution NMR structure of the Z domain of staphylococcal protein A

Staphylococcal protein A (SpA) is a cell-wall-bound pathogenicity factor from the bacterium Staphylococcus aureus. Because of their small size and immunoglobulin (IgG)-binding activities, domains of protein A are targets for protein engineering efforts and for the development of computational approaches for de novo protein folding. The NMR solution structure of an engineered IgG-binding domain of SpA, the Z domain (an analog of the B domain of SpA), has been determined by simulated annealing with restrained molecular dynamics on the basis of 671 conformational constraints. The Z domain contains three well-defined α-helices corresponding to polypeptide segments Lys7 to Leu17 (helix 1), Glu24 to Asp36 (helix 2), and Ser41 to Ala54 (helix 3). A family of ten conformers representing the solution structure of the Z domain was computed by simulated annealing of restrained molecular dynamics using the program CONGEN. The average of the root-mean-square deviations (r.m.s.d.) of the individual NMR conformers, relative to the mean coordinates, for the backbone atoms N, C α and C′ of residues Phe5 through Ala56 is 0.69 Å; the corresponding backbone r.m.s.d. for the three-helical core is 0.44 Å. Helices 1, 2 and 3 are antiparallel in orientation (Ω 12 = −170(±4)°, Ω 13 = +16(±3)°, Ω 23 = +173(±7)°). A comparison of backbone amide hydrogen/deuterium exchange rates in free and IgG-bound Z domains demonstrates that the amide protons of helices 1, 2 and 3 are protected from rapid exchange in both states, indicating that all three helices are also intact in the IgG-bound state. These solution NMR results differ from the previously determined X-ray structure of the similar SpA B domain in complex with the F c fragment of a human IgG antibody, where helix 3 is not observed in the electron density map and from the solution NMR structure of the B domain, where helix 3 is observed but helix 1 is tilted by ∼30° with respect to helices 2 and 3. Hydrogen-bonded N-cap and C-cap formation is observed for all three helices of the Z domain; these capping interactions appear to be highly conserved in the five homologous domains of SpA.

Construction and characterization of M13 bacteriophages displaying functional IgG-binding domains of Staphylococcal protein A

Staphylococcal protein A (SPA) is ranked as a versatile probe in immunoassays because of its immunoglobulin G (IgG) -binding capability. However, poor binding of SPA to the IgG of some laboratory animals and its inability to bind human IgG3 restricts its universal utility. In the present study, DNA encoding the four IgG-binding domains of SPA (E, D, A and B) or the B domain alone has been fused, in separate phagemid vectors, to the 5' end of gene III of the phage M13. Upon infection by helper phage M13KO7, phagemid particles encapsulating single-stranded DNA were produced. Dot immunoblot and Western blot analyses showed the presence of fusion proteins on the M13 surface. Binding of rabbit IgG-horseradish peroxidase (IgG-HRP) complex to the phage particles confirmed that the fusion proteins possessed functional IgG-binding domains. The interaction of these phages with immobilised human IgG and its various subclasses was studied by the phage capture immunoassay where the captured phages were detected by a monoclonal antibody to the major coat protein encoded by gene VIII (gVIII). The phages showed maximal binding to IgGlK, followed by IgG2κ, and showed negligible binding to the IgG3κ and IgG3λ. subclasses. The specificity of IgG-binding phages was confirmed in a phage capture and elution assay where the binding of these phages to immobilised human IgG1κ was abolished in the presence of excess of soluble protein A. Moreover, IgG-binding phages could be enriched approx. 1000-fold over non-specific phages in a single round of panning. These IgG-binding phages will be useful in selecting novel protein A molecules with wider specificity and higher affinity.

Nucleotide sequence analysis of the gene for protein A from Staphylococcus aureus Cowan 1 (NCTC8530) and its enhanced expression in Escherichia coli

Nucleotide sequence analysis of the gene ( spa) for staphylococcal protein A (SPA) from Staphylococcus aureus strain Cowan l (NCTC8530) shows that the sequence differs from previously reported SPA nucleotide sequences, especially in the number of repeat units in the cell-wall-binding region of the gene. Dot matrix comparison with streptococcal protein G and the macrophage receptor for the constant fragment (Fc) of immunoglobulins shows a limited but significant homology. The homology to the latter probably identifies the Fc-binding region in the immunoglobulin-binding domains of SPA. Enhanced production of SPA in Escherichia coli was achieved using the lac promoter immediately upstream from the spa gene.