Karin Jacobsson

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.

Metabolite Profiles of Lactic Acid Bacteria in Grass Silage

ABSTRACT The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml−1 for two of the three test organisms).

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.

A unipolarly located, cell-surface-associated agglutinin, RapA, belongs to a family of Rhizobium-adhering proteins (Rap) in Rhizobium leguminosarum bv. trifolii.

The phage-display cloning technique was used to find rhizobial proteins that bind to receptors located on the bacterial cell surface. The aim was to clone the gene(s) encoding rhicadhesin, a universal rhizobial adhesion protein, and/or other cell-surface-binding proteins. Four such Rhizobium-adhering proteins (Rap) were revealed in Rhizobium leguminosarum bv. trifolii strain R200. The binding is mediated by homologous Ra domains in these proteins. One member of the Rap protein family, named RapA1, is a secreted calcium-binding protein, which are also properties expected for rhicadhesin. However, the size of the protein (24 kDa instead of 14 kDa) and its distribution among different rhizobia (present in only Rhizobium leguminosarum biovars and R. etli instead of all members of Rhizobiaceae argue against RapA1 being rhicadhesin. Protein RapA1 consists of two homologous Ra domains and agglutinates R200 cells by binding to specific receptors located at one cell pole during exponential growth. Expression of these cell-surface receptors was detected only in rhizobia that produce the RapA proteins. The authors propose that the homologous Ra domains, found to be present also in other proteins with different structure, represent lectin domains, which confer upon these proteins the ability to recognize their cognate carbohydrate structures.

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.

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.

Phage display as a novel screening method to identify extracellular proteins.

Extracellular proteins are involved in many diverse and essential cell functions and in pathogenic bacteria, and they may also serve as virulence factors. Therefore, there is a need for methods that identify the genes encoding this group of proteins in a bacterial genome. Here, we present such a method based on the phage display technology. A novel gene III-based phagemid vector, pG3DSS, was constructed that lacks the signal sequence which normally orientates the encoded fusion protein to the Escherichia coli cell membrane, where it is assembled into the phage particle. When randomly fragmented DNA is inserted into this vector, only phagemids containing an insert encoding a signal sequence will give rise to phage particles displaying a fusion protein. These phages also display an E-tag epitope in fusion with protein III, which enables isolation of phages displaying a fusion protein, using antibodies against the epitope. From a library constructed from Staphylococcus aureus chromosomal DNA, genes encoding secreted as well as transmembrane proteins were isolated, including adhesins, enzymes and transport proteins.

A novel family of fibrinogen-binding proteins in Streptococcus agalactiae

Streptococcus agalactiae is a contagious pathogen in bovine mastitis. It is also one of the leading causes to neonatal pneumonia, sepsis and meningitis in Europe and North America. Although extracellular bacterial proteins that interact with host structures are putative vaccine components, so far only a few receptins have been identified and characterised from this organism. The aim of the present study was to identify fibrinogen-binding receptins from a shotgun phage display library constructed from the bovine type strain CCUG 4208. A novel extracellular receptin was identified after selecting the library against bovine fibrinogen. This protein is a member of a family of at least three proteins that share the fibrinogen-binding region as well as the N-terminal signal sequence, whereas the intervening region varies in size and has almost no sequence similarity. Proteins of this family are present also in human isolates of S. agalactiae, although binding to human fibrinogen has not been detected.