Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi.
Abstract: Cell-wall-associated proteins from Streptococcus equi subsp. equi, the causative agent of strangles, were analysed with a view to identifying a potential protective antigen. Preparations of these proteins, isolated from mutanolysin extracts of cell walls, were shown to contain one major high-M(r) protein species (apparent M(r) 220,000 and 550,000 when analysed by SDS-PAGE and gel-filtration chromatography, respectively). The high-M(r) protein bound horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. The fibrinogen-binding protein (FgBP) reacted with serum taken from horses recovering from strangles and protected mice against lethal challenge from S. equi subsp. equi. The sequence of the corresponding gene (fbp) was determined and shown to encode a mature protein (M(r) 54,597) with predicted coiled-coil structure. An FgBP truncate, lacking the C-terminal cell wall/membrane anchor domain, was overexpressed in and purified from Escherichia coli and was shown to behave in an analogous fashion to the wild-type product in terms of M(r) estimation, fibrinogen binding and seroreactivity.
Publication Date: 1998-05-14 PubMed ID: 9579073DOI: 10.1099/00221287-144-4-993Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research paper is about a study of proteins found in Streptococcus equi subsp. equi, with the aim of identifying a potential protective antigen. The protein, which bonds with horse fibrinogen, was found to react with serum from horses recovering from strangles and also shielded mice from lethal exposure to S. equi.
Identification of Potential Protective Antigen
- Researchers sought to identify potential protective antigens in Streptococcus equi subsp. equi, the bacteria causing strangles, a severe infection in horses.
- The team isolated proteins from mutanolysin extracts of cell walls of the bacteria.
- Among these proteins, they discovered a major high-M(r) protein species, which showed different molecular masses when analyzed through SDS-PAGE and gel-filtration chromatography.
Purification and Characterization of Fibrinogen-binding Protein
- The high-M(r) protein showed an affinity towards horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. This protein was named the Fibrinogen-binding protein (FgBP).
- Interestingly, FgBP reacted with serum obtained from horses who were recovering from strangles, and provided protection to mice against a lethal dose of S. equi.
- The scientists mapped the sequence of the gene (fbp) linked with FgBP, showing that it encoded a protein with a predicted coiled-coil structure.
Further Investigation and Findings
- Diving further into their investigation, the researchers engineered an FgBP truncate, which did not have the C-terminal cell wall/membrane anchor domain. This was accomplished by overexpressing FgBP truncate in Escherichia coli and purifying it from the same.
- The FgBP truncate was found to be analogous to its wild-type counterpart in terms of molecular weight estimation, fibrinogen binding, and seroreactivity, indicating that the bioactive properties of the protein were not lost due to the truncation.
Cite This Article
APA
Meehan M, Nowlan P, Owen P.
(1998).
Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi.
Microbiology (Reading), 144 ( Pt 4), 993-1003.
https://doi.org/10.1099/00221287-144-4-993 Publication
Researcher Affiliations
- Department of Microbiology, Moyne Institute of Preventive MedicineTrinity College, Dublin 2, Ireland.
- National Pharmaceutical Biotechnology Centre, BioResearch IrelandTrinity College, Dublin 2, Ireland.
- Bioresources UnitTrinity College, Dublin 2, Ireland.
- Department of Microbiology, Moyne Institute of Preventive MedicineTrinity College, Dublin 2, Ireland.
MeSH Terms
- Amino Acid Sequence
- Animals
- Bacterial Outer Membrane Proteins / chemistry
- Bacterial Outer Membrane Proteins / immunology
- Bacterial Outer Membrane Proteins / isolation & purification
- Bacterial Proteins
- Blotting, Western
- Carrier Proteins / chemistry
- Carrier Proteins / immunology
- Carrier Proteins / isolation & purification
- Electrophoresis, Polyacrylamide Gel
- Enzyme-Linked Immunosorbent Assay
- Horses
- Mice
- Molecular Sequence Data
- Sequence Analysis
- Streptococcus equi / chemistry
Citations
This article has been cited 13 times.- Frosth S, Morris ERA, Wilson H, Frykberg L, Jacobsson K, Parkhill J, Flock JI, Wood T, Guss B, Aanensen DM, Boyle AG, Riihimäki M, Cohen ND, Waller AS. Conservation of vaccine antigen sequences encoded by sequenced strains of Streptococcus equi subsp. equi.. Equine Vet J 2023 Jan;55(1):92-101.
- Pickering AC, Fitzgerald JR. The Role of Gram-Positive Surface Proteins in Bacterial Niche- and Host-Specialization.. Front Microbiol 2020;11:594737.
- D'Gama JD, Ma Z, Zhang H, Liu X, Fan H, Morris ERA, Cohen ND, Cywes-Bentley C, Pier GB, Waldor MK. A Conserved Streptococcal Virulence Regulator Controls the Expression of a Distinct Class of M-Like Proteins.. mBio 2019 Oct 22;10(5).
- Baiano JC, Tumbol RA, Umapathy A, Barnes AC. Identification and molecular characterisation of a fibrinogen binding protein from Streptococcus iniae.. BMC Microbiol 2008 Apr 23;8:67.
- Lewis MJ, Meehan M, Owen P, Woof JM. A common theme in interaction of bacterial immunoglobulin-binding proteins with immunoglobulins illustrated in the equine system.. J Biol Chem 2008 Jun 20;283(25):17615-23.
- Kelly C, Bugg M, Robinson C, Mitchell Z, Davis-Poynter N, Newton JR, Jolley KA, Maiden MC, Waller AS. Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks.. J Clin Microbiol 2006 Feb;44(2):480-6.
- Lannergård J, Flock M, Johansson S, Flock JI, Guss B. Studies of fibronectin-binding proteins of Streptococcus equi.. Infect Immun 2005 Nov;73(11):7243-51.
- Barocchi MA, Masignani V, Rappuoli R. Opinion: Cell entry machines: a common theme in nature?. Nat Rev Microbiol 2005 Apr;3(4):349-58.
- Dubreuil JD, Giudice GD, Rappuoli R. Helicobacter pylori interactions with host serum and extracellular matrix proteins: potential role in the infectious process.. Microbiol Mol Biol Rev 2002 Dec;66(4):617-29, table of contents.
- Barnett TC, Scott JR. Differential recognition of surface proteins in Streptococcus pyogenes by two sortase gene homologs.. J Bacteriol 2002 Apr;184(8):2181-91.
- Harrington DJ, Greated JS, Chanter N, Sutcliffe IC. Identification of lipoprotein homologues of pneumococcal PsaA in the equine pathogens Streptococcus equi and Streptococcus zooepidemicus.. Infect Immun 2000 Oct;68(10):6048-51.
- Vasi J, Frykberg L, Carlsson LE, Lindberg M, Guss B. M-like proteins of Streptococcus dysgalactiae.. Infect Immun 2000 Jan;68(1):294-302.
- Cope LD, Lafontaine ER, Slaughter CA, Hasemann CA Jr, Aebi C, Henderson FW, McCracken GH Jr, Hansen EJ. Characterization of the Moraxella catarrhalis uspA1 and uspA2 genes and their encoded products.. J Bacteriol 1999 Jul;181(13):4026-34.
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