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Timothy J Foster - One of the best experts on this subject based on the ideXlab platform.
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The role of Staphylococcus aureus surface protein SasG in adherence and biofilm formation.
Microbiology (Reading England), 2020Co-Authors: Rebecca M Corrigan, David Rigby, Pauline Handley, Timothy J FosterAbstract:Staphylococcus aureus colonizes the moist squamous epithelium of the anterior nares. One of the adhesins likely to be responsible is the S. aureus surface protein G (SasG), which has sequence similarity with the proteins Pls (plasmin sensitive) of S. aureus and Aap (accumulation associated protein) of Staphylococcus epidermidis. Expression of SasG by a laboratory strain of S. aureus could not be detected by Western immunoblotting. To enable investigation of SasG, the gene was cloned into two expression vectors, the IPTG-inducible pMUTIN4 and the tetracycline-inducible pALC2073, and introduced into S. aureus. Expression of SasG masked the ability of exponentially grown S. aureus cells expressing protein A (Spa), clumping factor B (ClfB) and the fibronectin binding proteins A and B (FnBPA and FnBPB) to bind to IgG, Cytokeratin 10 and fibronectin, respectively. SasG also masked binding to fibrinogen mediated by both ClfB and the FnBPs. Western immunoblotting showed no reduction in expression of the blocked adhesins following induction of SasG. SasG size variants with eight, six or five B repeats masked binding to the ligands, whereas variants with four, two or one repeats had no effect. SasG-expressing strains formed peritrichous fibrils (53.47+/-2.51 nm long) of varying density on the cell wall, which were labelled by immunogold negative staining with anti-SasG antibodies. SasG-expressing strains of S. aureus also formed biofilm independently of the polysaccharide intercellular adhesin (PIA). SasG variants with eight, six and five repeats formed biofilm, whereas variants with four, two or one repeats did not. It was concluded that the fibrillar nature of SasG explains its ability to mask binding of S. aureus microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) to their ligands and to promote formation of biofilm. In addition, the strong adhesion of SasG to desquamated nasal epithelial cells likely compensates for its blocking of the binding of S. aureus ClfB to Cytokeratin 10, which is important in adhesion to squames by cells lacking SasG. Several clinical isolates expressed SasG at levels similar to those of SH1000 sasG : : pMUTIN4, indicating that the properties described in the laboratory strain SH1000 may be relevant in vivo.
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Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the alphaC-domain of human fibrinogen.
Microbiology (Reading England), 2020Co-Authors: Evelyn J Walsh, Helen Miajlovic, Oleg V. Gorkun, Timothy J FosterAbstract:Clumping factor B (ClfB) of Staphylococcus aureus binds to Cytokeratin 10 and to fibrinogen. In this study the binding site in human fibrinogen was localized to a short region within the C terminus of the Aalpha-chain. ClfB only bound to the Aalpha-chain of fibrinogen in a ligand-affinity blot and in solid-phase assays with purified recombinant fibrinogen chains. A variant of fibrinogen with wild-type Bbeta- and gamma-chains but with a deletion that lacked the C-terminal residues from 252-610 of the Aalpha-chain did not support adherence of S. aureus Newman expressing ClfB. A series of truncated mutants of the recombinant Aalpha-chain were tested for their ability to support adherence of S. aureus Newman ClfB(+), which allowed the binding site to be localized to a short segment of the unfolded flexible repeated sequence within the C terminus of the Aalpha-chain. This was confirmed by two amino acid substititions within repeat 5 of the recombinant Aalpha-chain which did not support adherence of Newman ClfB(+). Lactococcus lactis expressing ClfB mutants with amino acid substitutions (N256 and Q235) located in the putative ligand-binding trench between domains N2 and N3 of the A-domain were defective in adherence to immobilized fibrinogen and Cytokeratin 10, suggesting that both ligands bind to the same or overlapping regions.
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Molecular Characterization of the Multiple Interactions of SpsD, a Surface Protein from Staphylococcus pseudintermedius, with Host Extracellular Matrix Proteins
PLOS ONE, 2013Co-Authors: Giampiero Pietrocola, Timothy J Foster, Joan A Geoghegan, Simonetta Rindi, Antonella Di Poto, Antonino Missineo, Valerio Consalvi, Pietro SpezialeAbstract:Staphylococcus pseudintermedius, a commensal and pathogen of dogs and occasionally of humans, expresses surface proteins potentially involved in host colonization and pathogenesis. Here, we describe the cloning and characterization of SpsD, a surface protein of S. pseudintermedius reported as interacting with extracellular matrix proteins and corneocytes. A ligand screen and Western immunoblotting revealed that the N-terminal A domain of SpsD bound fibrinogen, fibronectin, elastin and Cytokeratin 10. SpsD also interfered with thrombin-induced fibrinogen coagulation and blocked ADP-induced platelet aggregation. The binding site for SpsD was mapped to residues 395–411 in the fibrinogen γ-chain, while binding sites in fibronectin were localized to the N- and C-terminal regions. SpsD also bound to glycine- and serine-rich omega loops within the C-terminal tail region of Cytokeratin 10. Ligand binding studies using SpsD variants lacking the C-terminal segment or containing an amino-acid substitution in the putative ligand binding site provided insights into interaction mechanism of SpsD with the different ligands. Together these data demonstrate the multi-ligand binding properties of SpsD and illustrate some interesting differences in the variety of ligands bound by SpsD and related proteins from S. aureus.
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Staphylococcus pseudintermedius expresses surface proteins that closely resemble those from Staphylococcus aureus.
Veterinary microbiology, 2009Co-Authors: Joan A Geoghegan, Emma J Smith, Pietro Speziale, Timothy J FosterAbstract:Staphylococcus pseudintermedius is a commensal of dogs that is implicated in the pathogenesis of canine pyoderma. This study aimed to determine if S. pseudintermedius expresses surface proteins resembling those from Staphylococcus aureus and to characterise them. S. pseudintermedius strain 326 was shown to adhere strongly to purified fibrinogen, fibronectin and Cytokeratin 10. It adhered to the alpha-chain of fibrinogen which, along with binding to Cytokeratin 10, is the hallmark of clumping factor B of S. aureus, a surface protein that is in part responsible for colonisation of the human nares. Ligand-affinity blotting with cell-wall extracts demonstrated that S. pseudintermedius 326 expressed a cell-wall anchored fibronectin binding protein which recognised the N-terminal 29kDa fragment. The ability to bind fibronectin is an important attribute of pathogenic S. aureus and is associated with the ability of S. aureus to colonise skin of human atopic dermatitis patients. S. pseudintermedius genomic DNA was probed with labelled DNA amplified from the serine-aspartate repeat encoding region of clfA of S. aureus. This probe hybridised to a single SpeI fragment of S. pseudintermedius DNA. In the cell-wall extract of S. pseudintermedius 326, a 180kDa protein was discovered which bound to fibrinogen by ligand-affinity blotting and reacted in a Western blot with antibodies raised against the serine-aspartate repeat region of ClfA and the B-repeats of SdrD of S. aureus. It is proposed that this is an Sdr protein with B-repeats that has an A domain that binds to fibrinogen. Whether it is the same protein that binds Cytokeratin 10 is not clear.
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Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the αC-domain of human fibrinogen
Microbiology, 2008Co-Authors: Evelyn J Walsh, Helen Miajlovic, Oleg V. Gorkun, Timothy J FosterAbstract:Clumping factor B (ClfB) of Staphylococcus aureus binds to Cytokeratin 10 and to fibrinogen. In this study the binding site in human fibrinogen was localized to a short region within the C terminus of the Aα-chain. ClfB only bound to the Aα-chain of fibrinogen in a ligand-affinity blot and in solid-phase assays with purified recombinant fibrinogen chains. A variant of fibrinogen with wild-type Bβ- and γ-chains but with a deletion that lacked the C-terminal residues from 252–610 of the Aα-chain did not support adherence of S. aureus Newman expressing ClfB. A series of truncated mutants of the recombinant Aα-chain were tested for their ability to support adherence of S. aureus Newman ClfB+, which allowed the binding site to be localized to a short segment of the unfolded flexible repeated sequence within the C terminus of the Aα-chain. This was confirmed by two amino acid substititions within repeat 5 of the recombinant Aα-chain which did not support adherence of Newman ClfB+. Lactococcus lactis expressing ClfB mutants with amino acid substitutions (N256 and Q235) located in the putative ligand-binding trench between domains N2 and N3 of the A-domain were defective in adherence to immobilized fibrinogen and Cytokeratin 10, suggesting that both ligands bind to the same or overlapping regions.
Donizeti W. Santos - One of the best experts on this subject based on the ideXlab platform.
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A novel highly reactive Fab antibody for breast cancer tissue diagnostics and staging also discriminates a subset of good prognostic triple-negative breast cancers
Cancer Letters, 2014Co-Authors: Thaise G. Araújo, Carlos E. Paiva, Rafael M. Rocha, Yara C.p. Maia, Angela A.s. Sena, Carlos Ueira-vieira, Juliana F. Almeida, Paulo R. De Faria, Ana Paula Carneiro, Donizeti W. SantosAbstract:The discovery of novel markers for breast cancer (BC) has been recently relied on antibody combinatorial libraries and selection through phage display. We constructed a recombinant Fab library, and after selections against BC tissues, the FabC4 clone was thoroughly investigated by immunohistochemistry in 232 patients with long-term follow-up. The FabC4 ligand was determined by mass spectrometry. The FabC4 expression was associated with younger age, lack of progesterone receptor, higher histological grades and non-luminal subtypes, and it also identified a subset of good prognostic triple-negative BCs, possibly targeting a conformational epitope of Cytokeratin-10 (CK10). This new CK10-epitope specific antibody may open new possibilities in diagnostic and therapeutic strategies. © 2013 Elsevier Ireland Ltd.
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A novel highly reactive Fab antibody for breast cancer tissue diagnostics and staging also discriminates a subset of good prognostic triple-negative breast cancers
Cancer Letters, 2013Co-Authors: Thaise G. Araújo, Carlos E. Paiva, Rafael M. Rocha, Angela A.s. Sena, Carlos Ueira-vieira, Juliana F. Almeida, Paulo R. De Faria, Ana Paula Carneiro, Yara Cristina De Paiva Maia, Donizeti W. SantosAbstract:Abstract The discovery of novel markers for breast cancer (BC) has been recently relied on antibody combinatorial libraries and selection through phage display. We constructed a recombinant Fab library, and after selections against BC tissues, the FabC4 clone was thoroughly investigated by immunohistochemistry in 232 patients with long-term follow-up. The FabC4 ligand was determined by mass spectrometry. The FabC4 expression was associated with younger age, lack of progesterone receptor, higher histological grades and non-luminal subtypes, and it also identified a subset of good prognostic triple-negative BCs, possibly targeting a conformational epitope of Cytokeratin-10 (CK10). This new CK10-epitope specific antibody may open new possibilities in diagnostic and therapeutic strategies.
Evelyn J Walsh - One of the best experts on this subject based on the ideXlab platform.
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Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the alphaC-domain of human fibrinogen.
Microbiology (Reading England), 2020Co-Authors: Evelyn J Walsh, Helen Miajlovic, Oleg V. Gorkun, Timothy J FosterAbstract:Clumping factor B (ClfB) of Staphylococcus aureus binds to Cytokeratin 10 and to fibrinogen. In this study the binding site in human fibrinogen was localized to a short region within the C terminus of the Aalpha-chain. ClfB only bound to the Aalpha-chain of fibrinogen in a ligand-affinity blot and in solid-phase assays with purified recombinant fibrinogen chains. A variant of fibrinogen with wild-type Bbeta- and gamma-chains but with a deletion that lacked the C-terminal residues from 252-610 of the Aalpha-chain did not support adherence of S. aureus Newman expressing ClfB. A series of truncated mutants of the recombinant Aalpha-chain were tested for their ability to support adherence of S. aureus Newman ClfB(+), which allowed the binding site to be localized to a short segment of the unfolded flexible repeated sequence within the C terminus of the Aalpha-chain. This was confirmed by two amino acid substititions within repeat 5 of the recombinant Aalpha-chain which did not support adherence of Newman ClfB(+). Lactococcus lactis expressing ClfB mutants with amino acid substitutions (N256 and Q235) located in the putative ligand-binding trench between domains N2 and N3 of the A-domain were defective in adherence to immobilized fibrinogen and Cytokeratin 10, suggesting that both ligands bind to the same or overlapping regions.
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Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the αC-domain of human fibrinogen
Microbiology, 2008Co-Authors: Evelyn J Walsh, Helen Miajlovic, Oleg V. Gorkun, Timothy J FosterAbstract:Clumping factor B (ClfB) of Staphylococcus aureus binds to Cytokeratin 10 and to fibrinogen. In this study the binding site in human fibrinogen was localized to a short region within the C terminus of the Aα-chain. ClfB only bound to the Aα-chain of fibrinogen in a ligand-affinity blot and in solid-phase assays with purified recombinant fibrinogen chains. A variant of fibrinogen with wild-type Bβ- and γ-chains but with a deletion that lacked the C-terminal residues from 252–610 of the Aα-chain did not support adherence of S. aureus Newman expressing ClfB. A series of truncated mutants of the recombinant Aα-chain were tested for their ability to support adherence of S. aureus Newman ClfB+, which allowed the binding site to be localized to a short segment of the unfolded flexible repeated sequence within the C terminus of the Aα-chain. This was confirmed by two amino acid substititions within repeat 5 of the recombinant Aα-chain which did not support adherence of Newman ClfB+. Lactococcus lactis expressing ClfB mutants with amino acid substitutions (N256 and Q235) located in the putative ligand-binding trench between domains N2 and N3 of the A-domain were defective in adherence to immobilized fibrinogen and Cytokeratin 10, suggesting that both ligands bind to the same or overlapping regions.
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clumping factor b a fibrinogen binding mscramm microbial surface components recognizing adhesive matrix molecules adhesin of staphylococcus aureus also binds to the tail region of type i Cytokeratin 10
Journal of Biological Chemistry, 2004Co-Authors: Evelyn J Walsh, Louise Obrien, Xiaowen Liang, Magnus Hook, Timothy J FosterAbstract:Abstract The primary habitat of Staphylococcus aureus in humans is the moist squamous epithelium of the anterior nares. We showed previously that S. aureus adheres to desquamated epithelial cells and that clumping factor B (ClfB), a surface-located MSCRAMM (microbial surface components recognizing adhesive matrix molecules) known for its ability to bind to the α-chain of fibrinogen, is partly responsible (O'Brien, L. M., Walsh, E. J., Massey, R. C., Peacock, S. J., and Foster, T. J. (2002) Cell. Microbiol. 4, 759–770). We identified Cytokeratin 10 (K10) as the ligand recognized by ClfB. Here we have shown that purified recombinant human and murine K10 immobilized on a plastic surface supports adherence of S. aureus in a ClfB-dependent manner. Furthermore, the recombinant A domain of ClfB (rClfB 45–542) bound to immobilized K10 dose-dependently and saturably. Subdomains of human and murine K10 were expressed and purified. The N-terminal head domain (residues 1–145) did not support the binding of rClfB or adherence of S. aureus ClfB+. In contrast, the C-terminal tail domains (human rHK10 452–593, mouse rMK10 454–570) promoted avid binding and adherence. Isothermal titration microcalorimetry and intrinsic tryptophan fluorescence experiments gave dissociation constants for rClfB 45–542 binding to rMK10 454–570 of 1.4 and 1.7 μm, respectively. The tail region of K10 is composed largely of quasi-repeats of Tyr-(Gly/Ser)n. A synthetic peptide corresponding to a typical glycine loop (YGGGSSGGGSSGGY; Y-Y loop peptide) inhibited the adherence of S. aureus ClfB+ to immobilized MK10 to a level of 80%, whereas control peptides had no effect. The KD of rClfB 45–542 for the Y-Y loop peptide was 5.3 μm by intrinsic tryptophan fluorescence. Thus ClfB binds to the glycine loop region of the tail domain of keratin 10 where there are probably multiple binding sites. Binding is discussed in the context of the dock-lock-latch model for MSCRAMM-ligand interactions. We provide an explanation for the molecular basis for S. aureus adherence to the squamous epithelium and suggest that nasal colonization might be prevented by reagents that inhibit this interaction.
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staphylococcus aureus clumping factor b clfb promotes adherence to human type i Cytokeratin 10 implications for nasal colonization
Cellular Microbiology, 2002Co-Authors: Louise Obrien, Evelyn J Walsh, Ruth C Massey, Sharon J Peacock, Timothy J FosterAbstract:Summary Staphylococcus aureus is an important cause of sepsis in both community and hospital settings, a major risk factor for which is nasal carriage of the bacterium. Eradication of carriage by topical antibiotics reduces sepsis rates in high-risk individuals, an important strategy for the reduction of nosocomial infection in targeted patient populations. Understanding the mechanisms by which S. aureus adheres to nasal epithelial cells in vivo may lead to alternative methods of decolonization that do not rely on sustained antimicrobial susceptibility. Here, we demonstrate for the first time that the S. aureus surface-expressed protein, clumping factor B (ClfB), promotes adherence to immobilized epidermal Cytokeratins in vitro. By expressing a range of S. aureus adhesins on the surface of the heterologous host Lactococcus lactis, we demonstrated that adherence to epidermal Cytokeratins was conferred by ClfB. Adherence of wild-type S. aureus was inhibited by recombinant ClfB protein or anti-ClfB antibodies, and S. aureus mutants defective in ClfB adhered poorly to epidermal Cytokeratins. Expression of ClfB promoted adherence of L. lactis to human desquamated nasal epithelial cells, and a mutant of S. aureus defective in ClfB had reduced adherence compared with wild type. ClfB also promoted adherence of L. lactis cells to a human keratinocyte cell line. Cytokeratin 10 molecules were shown by flow cytometry to be exposed on the surface of both desquamated nasal epithelial cells and keratinocytes. Cytokeratin 10 was also detected on the surface of desquamated human nasal cells using immunofluorescence, and recombinant ClfB protein was shown to bind to Cytokeratin K10 extracted from these cells. We also showed that ClfB is transcribed by S. aureus in the human nares. We propose that ClfB is a major determinant in S. aureus nasal colonization.
Thaise G. Araújo - One of the best experts on this subject based on the ideXlab platform.
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A novel highly reactive Fab antibody for breast cancer tissue diagnostics and staging also discriminates a subset of good prognostic triple-negative breast cancers
Cancer Letters, 2014Co-Authors: Thaise G. Araújo, Carlos E. Paiva, Rafael M. Rocha, Yara C.p. Maia, Angela A.s. Sena, Carlos Ueira-vieira, Juliana F. Almeida, Paulo R. De Faria, Ana Paula Carneiro, Donizeti W. SantosAbstract:The discovery of novel markers for breast cancer (BC) has been recently relied on antibody combinatorial libraries and selection through phage display. We constructed a recombinant Fab library, and after selections against BC tissues, the FabC4 clone was thoroughly investigated by immunohistochemistry in 232 patients with long-term follow-up. The FabC4 ligand was determined by mass spectrometry. The FabC4 expression was associated with younger age, lack of progesterone receptor, higher histological grades and non-luminal subtypes, and it also identified a subset of good prognostic triple-negative BCs, possibly targeting a conformational epitope of Cytokeratin-10 (CK10). This new CK10-epitope specific antibody may open new possibilities in diagnostic and therapeutic strategies. © 2013 Elsevier Ireland Ltd.
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A novel highly reactive Fab antibody for breast cancer tissue diagnostics and staging also discriminates a subset of good prognostic triple-negative breast cancers
Cancer Letters, 2013Co-Authors: Thaise G. Araújo, Carlos E. Paiva, Rafael M. Rocha, Angela A.s. Sena, Carlos Ueira-vieira, Juliana F. Almeida, Paulo R. De Faria, Ana Paula Carneiro, Yara Cristina De Paiva Maia, Donizeti W. SantosAbstract:Abstract The discovery of novel markers for breast cancer (BC) has been recently relied on antibody combinatorial libraries and selection through phage display. We constructed a recombinant Fab library, and after selections against BC tissues, the FabC4 clone was thoroughly investigated by immunohistochemistry in 232 patients with long-term follow-up. The FabC4 ligand was determined by mass spectrometry. The FabC4 expression was associated with younger age, lack of progesterone receptor, higher histological grades and non-luminal subtypes, and it also identified a subset of good prognostic triple-negative BCs, possibly targeting a conformational epitope of Cytokeratin-10 (CK10). This new CK10-epitope specific antibody may open new possibilities in diagnostic and therapeutic strategies.
Paulo R. De Faria - One of the best experts on this subject based on the ideXlab platform.
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A novel highly reactive Fab antibody for breast cancer tissue diagnostics and staging also discriminates a subset of good prognostic triple-negative breast cancers
Cancer Letters, 2014Co-Authors: Thaise G. Araújo, Carlos E. Paiva, Rafael M. Rocha, Yara C.p. Maia, Angela A.s. Sena, Carlos Ueira-vieira, Juliana F. Almeida, Paulo R. De Faria, Ana Paula Carneiro, Donizeti W. SantosAbstract:The discovery of novel markers for breast cancer (BC) has been recently relied on antibody combinatorial libraries and selection through phage display. We constructed a recombinant Fab library, and after selections against BC tissues, the FabC4 clone was thoroughly investigated by immunohistochemistry in 232 patients with long-term follow-up. The FabC4 ligand was determined by mass spectrometry. The FabC4 expression was associated with younger age, lack of progesterone receptor, higher histological grades and non-luminal subtypes, and it also identified a subset of good prognostic triple-negative BCs, possibly targeting a conformational epitope of Cytokeratin-10 (CK10). This new CK10-epitope specific antibody may open new possibilities in diagnostic and therapeutic strategies. © 2013 Elsevier Ireland Ltd.
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A novel highly reactive Fab antibody for breast cancer tissue diagnostics and staging also discriminates a subset of good prognostic triple-negative breast cancers
Cancer Letters, 2013Co-Authors: Thaise G. Araújo, Carlos E. Paiva, Rafael M. Rocha, Angela A.s. Sena, Carlos Ueira-vieira, Juliana F. Almeida, Paulo R. De Faria, Ana Paula Carneiro, Yara Cristina De Paiva Maia, Donizeti W. SantosAbstract:Abstract The discovery of novel markers for breast cancer (BC) has been recently relied on antibody combinatorial libraries and selection through phage display. We constructed a recombinant Fab library, and after selections against BC tissues, the FabC4 clone was thoroughly investigated by immunohistochemistry in 232 patients with long-term follow-up. The FabC4 ligand was determined by mass spectrometry. The FabC4 expression was associated with younger age, lack of progesterone receptor, higher histological grades and non-luminal subtypes, and it also identified a subset of good prognostic triple-negative BCs, possibly targeting a conformational epitope of Cytokeratin-10 (CK10). This new CK10-epitope specific antibody may open new possibilities in diagnostic and therapeutic strategies.
