Conservation of vaccine antigen sequences encoded by sequenced strains of Streptococcus equi subsp. equi.

BACKGROUND: Streptococcus equi subspecies equi (S equi) is the cause of Strangles, one of the most prevalent diseases of horses worldwide. Variation within the immunodominant SeM protein has been documented, but a new eight-component fusion protein vaccine, Strangvac, does not contain live S equi or SeM and conservation of the antigens it contains have not been reported. OBJECTIVE: To define the diversity of the eight Strangvac antigens across a diverse S equi population. STUDY DESIGN: Genomic description. METHODS: Antigen sequences from the genomes of 759 S equi isolates from 19 countries, recovered between 1955 and 2018, were analysed. Predicted amino acid sequences in the antigen fragments of SEQ0256(Eq5), SEQ0402(Eq8), SEQ0721(EAG), SEQ0855(SclF), SEQ0935(CNE), SEQ0999(IdeE), SEQ1817(SclI) and SEQ2101(SclC) in Strangvac and SeM were extracted from the 759 assembled genomes and compared. RESULTS: The predicted amino acid sequences of SclC, SclI and IdeE were identical across all 759 genomes. CNE was truncated in the genome of five (0.7%) isolates. SclF was absent from one genome and another encoded a single amino acid substitution. EAG was truncated in two genomes. Eq5 was truncated in four genomes and 123 genomes encoded a single amino acid substitution. Eq8 was truncated in three genomes, one genome encoded four amino acid substitutions and 398 genomes encoded a single amino acid substitution at the final amino acid of the Eq8 antigen fragment. Therefore, at least 1579 (99.9%) of 1580 amino acids in Strangvac were identical in 743 (97.9%) genomes, and all genomes encoded identical amino acid sequences for at least six of the eight Strangvac antigens. MAIN LIMITATIONS: Three hundred and seven (40.4%) isolates in this study were recovered from horses in the UK. CONCLUSIONS: The predicted amino acid sequences of antigens in Strangvac were highly conserved across this collection of S equi.

Bacterial composition in Swedish raw drinking water reveals three major interacting ubiquitous metacommunities.

BACKGROUND: Surface raw water used as a source for drinking water production is a critical resource, sensitive to contamination. We conducted a study on Swedish raw water sources, aiming to identify mutually co-occurring metacommunities of bacteria, and environmental factors driving such patterns. METHODS: The water sources were different regarding nutrient composition, water quality, and climate characteristics, and displayed various degrees of anthropogenic impact. Water inlet samples were collected at six drinking water treatment plants over 3 years, totaling 230 samples. The bacterial communities of DNA sequenced samples (n = 175), obtained by 16S metabarcoding, were analyzed using a joint model for taxa abundance. RESULTS: Two major groups of well-defined metacommunities of microorganisms were identified, in addition to a third, less distinct, and taxonomically more diverse group. These three metacommunities showed various associations to the measured environmental data. Predictions for the well-defined metacommunities revealed differing sets of favored metabolic pathways and life strategies. In one community, taxa with methanogenic metabolism were common, while a second community was dominated by taxa with carbohydrate and lipid-focused metabolism. CONCLUSION: The identification of ubiquitous persistent co-occurring bacterial metacommunities in freshwater habitats could potentially facilitate microbial source tracking analysis of contamination issues in freshwater sources.

Accounting for Bacterial Overlap Between Raw Water Communities and Contaminating Sources Improves the Accuracy of Signature-Based Microbial Source Tracking.

Microbial source tracking (MST) analysis is essential to identifying and mitigating the fecal pollution of water resources. The signature-based MST method uses a library of sequences to identify contaminants based on operational taxonomic units (OTUs) that are unique to a certain source. However, no clear guidelines for how to incorporate OTU overlap or natural variation in the raw water bacterial community into MST analyses exist. We investigated how the inclusion of bacterial overlap between sources in the library affects source prediction accuracy. To achieve this, large-scale sampling - including feces from seven species, raw sewage, and raw water samples from water treatment plants - was followed by 16S rRNA amplicon sequencing. The MST library was defined using three settings: (i) no raw water communities represented; (ii) raw water communities selected through clustering analysis; and (iii) local water communities collected across consecutive years. The results suggest that incorporating either the local background or representative bacterial composition improves MST analyses, as the results were positively correlated to measured levels of fecal indicator bacteria and the accuracy at which OTUs were assigned to the correct contamination source increased fourfold. Using the proportion of OTUs with high source origin probability, underpinning a contaminating signal, is a solid foundation in a framework for further deciphering and comparing contaminating signals derived in signature-based MST approaches. In conclusion, incorporating background bacterial composition of water in MST can improve mitigation efforts for minimizing the spread of pathogenic and antibiotic resistant bacteria into essential freshwater resources.

SclC is a member of a novel family of collagen-like proteins in Streptococcus equi subspecies equi that are recognised by antibodies against SclC.

Previously we have reported on a cell surface collagen-like protein, called SclC, from Streptococcus equi subspecies equi. In the present study we show that this protein is a member of a family of seven collagen-like proteins, called SclC-SclI in this subspecies. All proteins contain an N-terminal signal sequence, followed by a unique non-repetitive region called A, a highly repetitive collagen-like region (CL) consisting of Glycine-Xaa-Yaa-triplet repeats. Following the CL-region a C-terminal proline-rich putative wall spanning region (W) preceding an LPXTG-motif and a hydrophobic transmembrane region (M) are found, typical features of cell surface exposed proteins in Gram-positive bacteria. The nucleotide and amino acid sequences, were analysed to investigate the similarities between them, and recombinant proteins encoding different domains (A- and CL-regions) were expressed and purified. Although the novel collagen-like proteins display differences in amino acid sequences, affinity purified antibodies against SclC were found to cross react with the other members of the novel collagen-like proteins. Furthermore, in sera from horses previously diagnosed having strangles, antibodies against these proteins were detected suggesting that these proteins are expressed during the infection.

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.

The von Willebrand factor-binding protein (vWbp) of Staphylococcus aureus is a coagulase.

Staphylococcus aureus encodes a secreted von Willebrand factor-binding protein (vWbp) of 482 amino acids. The N-terminal part of this protein is homologous to staphylocoagulase and therefore we investigated whether vWbp has coagulating activity. Recombinant vWbp was shown to coagulate human and porcine plasma efficiently, but was less active against plasma from other species. The coagulation efficiency was concentration dependent, and could be inhibited by specific antibodies against vWbp. Furthermore, the species-specific coagulation by vWbp depended on the interaction with prothrombin. This interaction also resulted in specific cleavage of vWbp, releasing the C-terminal part from the coagulating domain.

Sorting a Staphylococcus aureus phage display library against ex vivo biomaterial.

A phage display library made from Staphylococcus aureus DNA was sorted against a central venous catheter (CVC) that had been removed from a patient 2 days after insertion. After the first panning, approximately 50% of the clones encoded proteins known to interact with mammalian proteins. After the second and third pannings, fibrinogen-binding and beta2-glycoprotein I (beta2-GPI)-binding phage particles were clearly dominating. Proteins adsorbed to different CVCs were investigated using specific antibodies. Among the proteins probed for, fibrinogen was most abundant, but, interestingly, beta2-GPI was also detected on all tested CVCs.

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.

Identification of a novel collagen-like protein, SclC, in Streptococcus equi using signal sequence phage display.

Strangles is a serious disease in horses caused by Streptococcus equi subspecies equi. In this study, genes encoding putative extracellular proteins in this subspecies have been identified using signal sequence phage display. Among these, one showed similarities to the SclB protein, a member of the collagen-like proteins of Streptococcus pyogenes. The novel gene denoted sclC encodes a protein, SclC, of 302 amino acids, containing typical features found in cell wall-anchored proteins in Gram-positive bacteria. Based on similarities to the S. pyogenes collagen-like proteins the mature SclC protein can be divided into various domains: an N-terminal non-repetitive region (A), a highly repetitive collagen-like region (CL), and a C-terminal proline-rich wall-associated region (W). Using PCR, the sclC gene was detected in all studied strains of S. equi subsp. equi and S. equi subsp. zooepidemicus. Further, antibodies against recombinant SclC were detected in a collection of sera from horses with no history of strangles as well as horses previously infected with S. equi subsp. equi. Interestingly, the sera from convalescence horses were found to have significantly increased antibody titers against the SclC protein indicating that this protein is expressed during infection of S. equi subsp. equi.

Glycerol enhances the antifungal activity of dairy propionibacteria.

Dairy propionibacteria are widely used in starter cultures for Swiss type cheese. These bacteria can ferment glucose, lactic acid, and glycerol into propionic acid, acetic acid, and carbon dioxide. This research examined the antifungal effect of dairy propionibacteria when glycerol was used as carbon source for bacterial growth. Five type strains of propionibacteria were tested against the yeast Rhodotorula mucilaginosa and the molds Penicillium commune and Penicillium roqueforti. The conversion of (13)C glycerol by Propionibacterium jensenii was followed with nuclear magnetic resonance. In a dual culture assay, the degree of inhibition of the molds was strongly enhanced by an increase in glycerol concentrations, while the yeast was less affected. In broth cultures, decreased pH in glycerol medium was probably responsible for the complete inhibition of the indicator fungi. NMR spectra of the glycerol conversion confirmed that propionic acid was the dominant metabolite. Based on the results obtained, the increased antifungal effect seen by glycerol addition to cultures of propionibacteria is due to the production of propionic acid and pH reduction of the medium.

Metabolite profiles of lactic acid bacteria in grass silage.

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 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.

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.

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.

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.

Phage display reveals 52 novel extracellular and transmembrane proteins from Lactobacillus reuteri DSM 20016(T).

Extracellular and transmembrane proteins are important for the binding of bacteria to intestinal surfaces and for their interaction with the host. The aim of this study was to identify genes encoding extracellular and transmembrane proteins from the probiotic bacterium Lactobacillus reuteri by construction and screening of a phage display library. This library was constructed by insertion of randomly fragmented DNA from L. reuteri into the phagemid vector pG3DSS, which was previously developed for screening for extracellular proteins. After affinity selection of the library, the L. reuteri inserts were sequenced and analysed with bioinformatic tools. The screening resulted in the identification of 52 novel genes encoding extracellular and transmembrane proteins. These proteins were classified as: transport proteins; enzymes; sensor-regulator proteins; proteins involved in host/microbial interactions; conserved hypothetical proteins; and unconserved hypothetical proteins. Further characterization of the extracellular and transmembrane proteins identified should contribute to the understanding of the probiotic properties of L. reuteri.

Staphylococcus aureus expresses a cell surface protein that binds both IgG and beta2-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.

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

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 (alpha2-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.