Lars Frykberg

A second IgG-binding protein in Staphylococcus aureus

Most strains of Staphylococcus aureus express IgG-binding activity and this binding has been considered to be solely mediated by protein A. However, the existence of a second gene in S. aureus strain 8325-4 encoding an IgG-binding polypeptide was recently reported. This novel IgG-binding polypeptide was found after panning a shotgun phage display library, made from chromosomal DNA, against immobilized human IgG. The complete gene (sbi) encoding this novel IgG-binding protein, denoted protein Sbi, has now been cloned and sequenced. Analysis of other S. aureus strains showed that this gene is not unique for strain 8325-4. The protein consists of 436 amino acids and exhibits an immunoglobulin-binding specificity similar to protein A. Furthermore, it is shown that Sbi is highly expressed in strain Newman 4, which shows that IgG-binding activity in S. aureus can be mediated by proteins other than protein A.

A novel von Willebrand factor binding protein expressed by Staphylococcus aureus

When a shotgun phage-display library of Staphylococcus aureus Newman was affinity selected (panned) against recombinant von Willebrand factor (vWf), a novel von Willebrand factor binding protein (vWbp) was found. Experimental data indicate that the interaction between vWbp and vWf is very specific and mediated by a region of 26 aa residues in the C-terminal part of vWbp. vWbp has an N-terminal secretory signal sequence but no cell wall anchoring motif, suggesting a soluble extracellular location. Mature vWbp could be purified from the culture supernatant and the identity of the protein was confirmed by N-terminal sequencing. vWbp migrates with an apparent molecular mass of 66 kDa and the deduced protein consists of 482 aa. The gene encoding vWbp, named vwb, was present in all S. aureus strains investigated.

CNE, a collagen-binding protein of Streptococcus equi

Streptococcus equi subspecies equi is an important horse pathogenic bacterium causing a serious disease called strangles. Using bioinformatics we identified a gene denoted cne (gene encoding collagen-binding protein from S. equi) coding for a novel potential virulence factor of this species called protein CNE. The protein is composed of 657 amino acids and has the typical features found in cell surface-anchored proteins in Gram-positive bacteria. CNE displays amino acid sequence similarities to the previously well-studied collagen-binding protein CNA from Staphylococcus aureus, a proven virulence factor in septic arthritis. Based on similarity to CNA the structure of the mature CNE protein can be divided into an N-terminal A domain and a C-terminal B domain. The highest similarity between CNA and CNE is found in the A domains. The A domain in CNA is known to be the collagen-binding domain. Two parts of cne were amplified using polymerase chain reaction (PCR) and ligated into an expression vector, and recombinant CNE proteins were produced in Escherichia coli. The purified CNE proteins were shown to display collagen-binding activity in a Western ligand blot and to inhibit collagen binding to cells of subsp. equi and to CNE-coated microtitre wells. Furthermore, the A domain of CNE was sufficient for binding collagen, and was shown to compete for the same site on collagen as CNA in inhibition studies. Using PCR, the cne gene was detected in all studied strains of subsp. equi and S. equi subsp. zooepidemicus.

Recombinant Streptococcus equi proteins protect mice in challenge experiments and induce immune response in horses

ABSTRACT Horses that have undergone infection caused by Streptococcus equi subspecies equi (strangles) were found to have significantly increased serum antibody titers against three previously characterized proteins, FNZ (cell surface-bound fibronectin binding protein), SFS (secreted fibronectin binding protein), and EAG (α2-macroglobulin, albumin, and immunoglobulin G [IgG] binding protein) from S. equi. To assess the protective efficacy of vaccination with these three proteins, a mouse model of equine strangles was utilized. Parts of the three recombinant proteins were used to immunize mice, either subcutaneously or intranasally, prior to nasal challenge with S. equi subsp. equi. The adjuvant used was EtxB, a recombinant form of the B subunit of Escherichia coli heat-labile enterotoxin. It was shown that nasal colonization of S. equi subsp. equi and weight loss due to infection were significantly reduced after vaccination compared with a mock-vaccinated control group. This effect was more pronounced after intranasal vaccination than after subcutaneous vaccination; nearly complete eradication of nasal colonization was obtained after intranasal vaccination (P < 0.001). When the same antigens were administered both intranasally and subcutaneously to healthy horses, significant mucosal IgA and serum IgG antibody responses against FNZ and EAG were obtained. The antibody response was enhanced when EtxB was used as an adjuvant. No adverse effects of the antigens or EtxB were observed. Thus, FNZ and EAG in conjunction with EtxB are promising candidates for an efficacious and safe vaccine against strangles.

Staphylococcus aureus expresses a cell surface protein that binds both IgG and β2-glycoprotein I

The existence of a second IgG-binding protein, protein Sbi, in Staphylococcus aureus has been reported previously. Later data indicated that protein Sbi also bound another serum component. This component has now been affinity-purified on immobilized protein Sbi and identified as beta2-glycoprotein I (beta2-GPI), also known as apolipoprotein H. The minimal beta2-GPI-binding domain was identified by shotgun phage display and the binding was shown to be mediated by a region of 57 amino acids, clearly separated from the IgG-binding domain. It is also shown that protein Sbi, and thus the beta2-GPI-binding activity, is expressed on the staphylococcal cell surface at levels varying between strains.

M-like proteins of Streptococcus dysgalactiae

ABSTRACT Streptococcus dysgalactiae is one of the most important bacterial species isolated from bovine mastitis. To identify potential virulence factors of this species we prepared chromosomal DNA from strain 8215 and constructed a phage display library. By affinity selection of the library against fibrinogen (Fg), we isolated and characterized a gene, called demA, encoding a protein with the molecular mass of ∼58 kDa, called DemA, displaying both plasma protein binding properties and sequence similarities with the M and M-like proteins of other streptococcal species. Purified recombinant DemA protein was found to completely inhibit Fg-binding to cells ofS. dysgalactiae. A continued sequence analysis revealed that the demA gene was preceded by an open reading frame (dmgA) coding for a putative protein, called DmgA, with high similarities to the Mga proteins of Streptococcus pyogenes. By additional cloning, the correspondingdmgA and demA genes from another strain, called Epi9, were isolated and analyzed. These genes, called dmgBand demB, respectively, revealed a high degree of similarity to the corresponding genes in strain 8215. Increased binding of Fg by cells of strain Epi9, grown in an atmosphere with 10% CO2, was correlated to an enhanced transcription of thedemB gene as shown in a Northern blot. Strain 8215 did not respond to CO2, which could be explained by a nonfunctionaldmgA gene due to insertion of an insertion sequence element. Based on sequence similarities of the described proteins to Mga, M, and M-like proteins and the response to elevated level of CO2, we suggest that the dmg anddem genes are members of a regulon similar to the describedmga regulon in S. pyogenes, which encodes several virulence factors in this species.

Variable Surface Protein Vmm of Mycoplasma mycoides subsp. mycoides Small Colony Type

A variable surface protein, Vmm, of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) has been identified and characterized. Vmm was specific for the SC biotype and was expressed by 68 of 69 analyzed M. mycoides SC strains. The protein was found to undergo reversible phase variation at a frequency of 9 x 10(-4) to 5 x 10(-5) per cell per generation. The vmm gene was present in all of the 69 tested M. mycoides SC strains and encodes a lipoprotein precursor of 59 amino acids (aa), where the mature protein was predicted to be 36 aa and was anchored to the membrane by only the lipid moiety, as no transmembrane region could be identified. DNA sequencing of the vmm gene region from ON and OFF clones showed that the expression of Vmm was regulated at the transcriptional level by dinucleotide insertions or deletions in a repetitive region of the promoter spacer. Vmm-like genes were also found in four closely related mycoplasmas, Mycoplasma capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae, Mycoplasma sp. bovine serogroup 7, and Mycoplasma putrefaciens. However, Vmm could not be detected in whole-cell lysates of these species, suggesting that the proteins encoded by the vmm-like genes lack the binding epitope for the monoclonal antibody used in this study or, alternatively, that the Vmm-like proteins were not expressed.

Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins.

Getting to Grips with Strangles: An Effective Multi-Component Recombinant Vaccine for the Protection of Horses from Streptococcus equi Infection

Streptococcus equi subspecies equi (S. equi) is a clonal, equine host-adapted pathogen of global importance that causes a suppurative lymphodendopathy of the head and neck, more commonly known as Strangles. The disease is highly prevalent, can be severe and is highly contagious. Antibiotic treatment is usually ineffective. Live attenuated vaccine strains of S. equi have shown adverse reactions and they suffer from a short duration of immunity. Thus, a safe and effective vaccine against S. equi is highly desirable. The bacterium shows only limited genetic diversity and an effective vaccine could confer broad protection to horses throughout the world. Welsh mountain ponies (n = 7) vaccinated with a combination of seven recombinant S. equi proteins were significantly protected from experimental infection by S. equi, resembling the spontaneous disease. Vaccinated horses had significantly reduced incidence of lymph node swelling (p = 0.0013) lymph node abscessation (p = 0.00001), fewer days of pyrexia (p = 0.0001), reduced pathology scoring (p = 0.005) and lower bacterial recovery from lymph nodes (p = 0.004) when compared with non-vaccinated horses (n = 7). Six of 7 vaccinated horses were protected whereas all 7 non-vaccinated became infected. The protective antigens consisted of five surface localized proteins and two IgG endopeptidases. A second vaccination trial (n = 7+7), in which the IgG endopeptidases were omitted, demonstrated only partial protection against S. equi, highlighting an important role for these vaccine components in establishing a protective immune response. S. equi shares >80% sequence identity with Streptococcus pyogenes. Several of the components utilized here have counterparts in S. pyogenes, suggesting that our findings have broader implications for the prevention of infection with this important human pathogen. This is one of only a few demonstrations of protection from streptococcal infection conferred by a recombinant multi-component subunit vaccine in a natural host.

MAG, a novel plasma protein receptor from Streptococcus dysgalactiae

The gene encoding a plasma protein receptor from Streptococcus dysgalactiae has been cloned and sequenced. The gene product, with a predicted molecular mass of approx. 44 kDa, binds alpha 2-macroglobulin (alpha 2 M), serum albumin and immunoglobulin G (IgG). By subcloning and expressing various parts of the gene as fusion proteins, we found that the three binding activities reside in discrete domains of the protein. The single IgG-binding domain, localized in the C-terminal part of the molecule, shows high homology to streptococcal type-III Fc receptors. In the middle of the molecule, there is a stretch of 50 amino acids (aa) mediating albumin binding. This region has partial homology with the albumin-binding domains of streptococcal protein G. The alpha 2 M-binding domain is located in the N terminus of the molecule and is composed of a unique aa sequence. We call this trifunctional plasma protein receptor, MAG (binds alpha 2 M, albumin and IgG).