Immunogenicity and Protection by DnaK and SpaA Recombinant Proteins Against Erysipelothrix Rhusiopathiae in a Murine Model.

BACKGROUND/AIMS: Swine erysipelas is a disease caused by Erysipelothrix rhusiopathiae, a Gram-positive bacillus, which has great economic importance because it leads to the loss of the swine herd. To control this disease, animals are immunized with a cellular vaccine of killed or attenuated E. rhusiopathiae, but even with herd vaccination, cases of swine erysipelas outbreaks have been reported in the United States, China and Japan, leading to the search for other antigenic components of the bacteria that may promote greater protection against E. rhusiopathiae. The surface protein SpaA from E. rhusiopathiae has been shown to be a candidate to constitute a subunit vaccine, since it has already been reported to induce a host immune response against the bacterium. DnaK, a hsp70 molecular chaperone, also seems to be a good candidate in the composition of a vaccine, as it has been demonstrated to be an antigenic protein of the bacteria. METHODS: This work evaluated the immunogenicity and protection induced by the E. rhusiopathiaee SpaA and DnaK recombinant proteins in a murine model, by intramuscular administration to mice with two doses of 100 µg at 21-day interval between them. The candidate proteins were tested either separately and together, compared with the commercial vaccine and the non-vaccination condition, and mice were challenged with a virulent strain of E. rhusiopathiae. Serum was collected to assess the produced antibodies and peripheral blood cells, whereas spleen and kidney tissues were assayed for E. rhusiopathiae presence by colony counting. RESULTS: A survival curve of the animals was performed, which confirmed the protection induced by the proteins. IgG antibodies increased in the animal serum inoculated with the proteins when compared to the control, and a significant delay in disease symptoms was observed. CONCLUSION: These results suggest that E. rhusiopathiae DnaK and SpaA are immunogenic in mice and interfere with the disease development.

An invasive infection with an unusual spaB-possessing Erysipelothrix rhusiopathiae in a human.

Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a variety of animals. In humans, in contrast to the cutaneous form called erysipeloid, which is an occupational disease and common in individuals who handle raw meat and fish, invasive systemic infections are unusual. E. rhusiopathiae expresses an immunogenic surface protein, Spa (surface protective antigen), which is involved in virulence. Among the antigenically different Spa proteins (SpaA, B and C), which are mostly associated with serovars, SpaA is by far the most prevalent in E. rhusiopathiae isolates from diseased animals. However, the Spa type has not been examined for human isolates, and it is unknown whether SpaB- or SpaC-possessing isolates can cause disease in humans. A Gram-positive, rod-shaped bacterium isolated from a case of human pyogenic spondylitis was analysed. The bacterium was identified as E. rhusiopathiae by a routine biochemical test and MS, and ultimately confirmed by an E. rhusiopathiae-specific PCR assay. Spa typing by sequencing revealed the SpaB type, and the serovar of the strain was identified as untypeable by a conventional agar gel precipitation test, but determined to be serovar 6 by a serotyping PCR assay. Sequence analysis of the serovar-defining chromosomal region revealed that the isolate displayed the same gene organization as the serovar 6 reference strain, but the region was disrupted by an insertion sequence element, suggesting that the isolate originated from a serovar 6 strain. These results highlight that unusual, spaB-possessing E. rhusiopathiae strains can potentially pose serious risks to humans.

Metabolic Fate of 13C-Labeled Polydextrose and Impact on the Gut Microbiome: A Triple-Phase Study in a Colon Simulator.

The present study introduces a novel triple-phase (liquids, solids, and gases) approach, which employed uniformly labeled [U-13C] polydextrose (PDX) for the selective profiling of metabolites generated from dietary fiber fermentation in an in vitro colon simulator using human fecal inocula. Employing 13C NMR spectroscopy, [U-13C] PDX metabolism was observed from colonic digest samples. The major 13C-labeled metabolites generated were acetate, butyrate, propionate, and valerate. In addition to these short-chain fatty acids (SCFAs), 13C-labeled lactate, formate, succinate, and ethanol were detected in the colon simulator samples. Metabolite formation and PDX substrate degradation were examined comprehensively over time (24 and 48 h). Correlation analysis between 13C NMR spectra and gas production confirmed the anaerobic fermentation of PDX to SCFAs. In addition, 16S rRNA gene analysis showed that the level of Erysipelotrichaceae was influenced by PDX supplementation and Erysipelotrichaceae level was statistically correlated with SCFA formation. Overall, our study demonstrates a novel approach to link substrate fermentation and microbial function directly in a simulated colonic environment.

Characterization of pathogenic roles of two Erysipelothrix rhusiopathiae surface proteins.

Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and human erysipeloid. E. rhusiopathiae HP0728 and HP1472 have been reported to be down regulated in low-virulence or avirulent strains, but their pathogenic roles are not known. In this study, it was found that E. rhusiopathiae HP0728 and HP1472 were displayed on the surface of E. rhusiopathiae. Moreover, recombinant HP1472 could adhere to pig vascular endothelial cells. Recombinant HP0728 could bind host plasminogen but could not bind fibronectin. In conclusion, our work suggested that HP0728 and HP1472 are virulence factors of E. rhusiopathiae.

Erysipelothrix rhusiopathiae recruits host plasminogen via the major protective antigen SpaA.

Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and human erysipeloid. Some pathogenic bacteria are able to recruit host plasminogen and then use the plasminogen system for migration across tissue barriers or for nutritional demands during infection. However, there is no study on E. rhusiopathiae recruitment of plasminogen. SpaA has long been known to be a major protective antigen of E. rhusiopathiae, but its roles in virulence have not yet been well clarified. The aim of this study was to detect the activity of E. rhusiopathiae to recruit host plasminogen and evaluate the ability of SpaA to act as a receptor in the recruitment process. It was found that E. rhusiopathiae could recruit host plasminogen. SpaA could specifically bind host plasminogen. Anti-SpaA serum could significantly decrease the activity of E. rhusiopathiae to recruit plasminogen. In addition, this binding activity was lysine dependent. In conclusion, E. rhusiopathiae was able to recruit host plasminogen via SpaA. To our knowledge, this is the first report on E. rhusiopathiae recruitment of host plasminogen and the receptor in the process.

Proteomic and Transcriptomic Analyses of Swine Pathogen Erysipelothrix rhusiopathiae Reveal Virulence Repertoire.

E. rhusiopathiae is the causative agent of erysipelas in animals and erysipeloid in humans, but its pathogenicity is poorly understood. To identify virulence factors associated with E. rhusiopathiae and screen engineered vaccine candidates, we used proteomics and transcriptomics to compare the highly virulent strain HX130709 with an isogenic avirulent derivative, HX130709a. 1,299 proteins and 1,673 transcribed genes were identified and 1,292 of the proteins could be associated with genes. In a comparison between HX130907 and HX130709a, 168 proteins and 475 genes exhibited differences in regulation level. Among these, levels for 61 proteins and transcripts were positively or negatively correlated. Gene Ontology (GO) analysis suggests that many of the down-regulated proteins in the attenuated strain have catalytic or binding functions. Potential protein-protein interactions suggest that some of the down-regulated proteins may regulate PTS, GMP synthase and ribosomal proteins. Morphological results showed that HX130709 and HX130709a have similar colony and capsule morphology. Growth curves and pyruvate measurements suggest that TCA cycle and saccharide phosphorylation levels were decreased and gluconeogenesis was increased in HX130709a. Our study confirms that SpaA and neuraminidase, but not hyaluronidase and capsule, are associated with virulence in E. rhusiopathiae. We conclude that the virulence of E. rhusiopathiae may be associated with slow reactions of the TCA cycle and down-regulation of selected proteins.

A combinational approach of multilocus sequence typing and other molecular typing methods in unravelling the epidemiology of Erysipelothrix rhusiopathiae strains from poultry and mammals.

Erysipelothrix rhusiopathiae infections re-emerged as a matter of great concern particularly in the poultry industry. In contrast to porcine isolates, molecular epidemiological traits of avian E. rhusiopathiae isolates are less well known. Thus, we aimed to (i) develop a multilocus sequence typing (MLST) scheme for E. rhusiopathiae, (ii) study the congruence of strain grouping based on pulsed-field gel electrophoresis (PFGE) and MLST, (iii) determine the diversity of the dominant immunogenic protein SpaA, and (iv) examine the distribution of genes putatively linked with virulence among field isolates from poultry (120), swine (24) and other hosts (21), including humans (3). Using seven housekeeping genes for MLST analysis we determined 72 sequence types (STs) among 165 isolates. This indicated an overall high diversity, though 34.5% of all isolates belonged to a single predominant ST-complex, STC9, which grouped strains from birds and mammals, including humans, together. PFGE revealed 58 different clusters and congruence with the sequence-based MLST-method was not common. Based on polymorphisms in the N-terminal hyper-variable region of SpaA the isolates were classified into five groups, which followed the phylogenetic background of the strains. More than 90% of the isolates harboured all 16 putative virulence genes tested and only intI, encoding an internalin-like protein, showed infrequent distribution. MLST data determined E. rhusiopathiae as weakly clonal species with limited host specificity. A common evolutionary origin of isolates as well as shared SpaA variants and virulence genotypes obtained from avian and mammalian hosts indicates common reservoirs, pathogenic pathways and immunogenic properties of the pathogen.

Phosphorylcholine and SpaA, a choline-binding protein, are involved in the adherence of Erysipelothrix rhusiopathiae to porcine endothelial cells, but this adherence is not mediated by the PAF receptor.

A crucial event in the initiation of many bacterial infections is the adherence of the bacteria to host cells, and bacterial surface structures and their interactions with host cell receptors play an important role in this process. Erysipelothrix rhusiopathiae is the causative agent of swine erysipelas, which may cause acute septicemia or chronic endocarditis and polyarthritis. To study the pathogenic mechanism of the widespread vascular disease observed in the acute form of swine erysipelas, we investigated the role of phosphorylcholine (PCho), a component of the E. rhusiopathiae capsule, in bacterial adherence to porcine endothelial cells (PECs) in vitro. We found that adherence of E. rhusiopathiae strain Fujisawa to PECs was twice that of adherence to control COS-7 cells and that the adherence rates of PCho-defective mutants were approximately 30-50% lower than those of the Fujisawa strain. The adherence of the Fujisawa strain to COS-7 cells transfected with the porcine platelet-activating factor receptor (PAFR) gene, which encodes a G protein-coupled receptor that has been shown to directly bind to Streptococcus pneumoniae via PCho in the bacterial cell wall, was not enhanced. Treatment with a PAFR antagonist (WEB-2086) did not inhibit bacterial adherence to PECs. Incubation of the bacterial cells with an antibody against PCho or SpaA, a choline-binding protein anchored to PCho of the Fujisawa strain, reduced the adherence of the strain to PECs. This effect was not observed when PCho-defective mutants were used. These results suggest that E. rhusiopathiae adheres to PECs via PCho and SpaA and that the PCho-mediated adherence is independent of PAFR.

Collagen adhesin-nanoparticle interaction impairs adhesin's ligand binding mechanism.

BACKGROUND: Pathogenic bacteria specifically recognize extracellular matrix (ECM) molecules of the host (e.g. collagen, fibrinogen and fibronectin) through their surface proteins known as MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) and initiate colonization. On implantation, biomaterials easily get coated with these ECM molecules and the MSCRAMMs mediate bacterial adherence to biomaterials. With the rapid rise in antibiotic resistance, designing alternative strategies to reduce/eliminate bacterial colonization is absolutely essential. METHODS: The Rhusiopathiae surface protein B (RspB) is a collagen-binding MSCRAMM of Erysipelothrix rhusiopathiae. It also binds to abiotic surfaces. The crystal structure of the collagen-binding region of RspB (rRspB31-348) reported here revealed that RspB also binds collagen by a unique ligand binding mechanism called "Collagen Hug" which is a common theme for collagen-binding MSCRAMMs of many Gram-positive bacteria. Here, we report the interaction studies between rRspB31-348 and silver nanoparticles using methods like gel shift assay, gel permeation chromatography and circular dichroism spectroscopy. RESULTS: The "Collagen Hug" mechanism was inhibited in the presence of silver nanoparticles as rRspB31-348 was unable to bind to collagen. The total loss of binding was likely because of rRspB31-348 and silver nanoparticle protein corona formation and not due to the loss of the structural integrity of rRspB31-348 on binding with nanoparticles as observed from circular dichroism experiments. GENERAL SIGNIFICANCE: Interaction of rRspB31-348 with silver nanoparticle impaired its ligand binding mechanism. Details of this inhibition mechanism may be useful for the development of antimicrobial materials and antiadhesion drugs.

The genome of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, reveals new insights into the evolution of firmicutes and the organism's intracellular adaptations.

Erysipelothrix rhusiopathiae is a Gram-positive bacterium that represents a new class, Erysipelotrichia, in the phylum Firmicutes. The organism is a facultative intracellular pathogen that causes swine erysipelas, as well as a variety of diseases in many animals. Here, we report the first complete genome sequence analysis of a member of the class Erysipelotrichia. The E. rhusiopathiae genome (1,787,941 bp) is one of the smallest genomes in the phylum Firmicutes. Phylogenetic analyses based on the 16S rRNA gene and 31 universal protein families suggest that E. rhusiopathiae is phylogenetically close to Mollicutes, which comprises Mycoplasma species. Genome analyses show that the overall features of the E. rhusiopathiae genome are similar to those of other Gram-positive bacteria; it possesses a complete set of peptidoglycan biosynthesis genes, two-component regulatory systems, and various cell wall-associated virulence factors, including a capsule and adhesins. However, it lacks many orthologous genes for the biosynthesis of wall teichoic acids (WTA) and lipoteichoic acids (LTA) and the dltABCD operon, which is responsible for d-alanine incorporation into WTA and LTA, suggesting that the organism has an atypical cell wall. In addition, like Mollicutes, its genome shows a complete loss of fatty acid biosynthesis pathways and lacks the genes for the biosynthesis of many amino acids, cofactors, and vitamins, indicating reductive genome evolution. The genome encodes nine antioxidant factors and nine phospholipases, which facilitate intracellular survival in phagocytes. Thus, the E. rhusiopathiae genome represents evolutionary traits of both Firmicutes and Mollicutes and provides new insights into its evolutionary adaptations for intracellular survival.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the collagen-binding region of RspB from Erysipelothrix rhusiopathiae.

RspB is a surface adhesin of Erysipelothrix rhusiopathiae. A recombinant form of the collagen-binding region of this protein, RspB((31-348)), has been overexpressed in Escherichia coli in native and selenomethionine-derivative forms and purified using affinity and gel-permeation chromatography. Thin plate-like crystals were obtained by the hanging-drop vapour-diffusion method using the same condition for both forms. The native crystals diffracted to a resolution of 2.5 A using an in-house X-ray source, while the selenomethionine-derivative crystals diffracted to a resolution of 2.2 A using synchrotron radiation. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 46.19, b = 66.65, c = 101.72 A, beta = 94.11 degrees .

Bioreactor aeration conditions modulate growth and antigen expression during Erysipelothrix rhusiopathiae cultivation.

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, was cultivated in a 5-L stirred and aerated bioreactor under different dissolved oxygen tensions (0%, 5%, and 30% of saturation) for evaluation of the influence of oxygen on cell growth as well as on the production of the main antigenic component of the vaccine against erysipelas, a 64-69 kDa protein (SpaA). The microorganism presented different growth profiles for different aeration conditions. However, at the end of the batch cultivations, similar cell concentrations were obtained under the studied conditions. In order to maximize biomass titers and antigen production, the microorganism was cultivated in fed-batch operation mode under aerobic conditions. Under this condition, there was a fivefold increase in biomass production in comparison to the results attained in batch cultivations. To follow up antigen expression, samples collected during batch cultivations were concentrated and treated with choline for antigen extraction. Antigen expression was then assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by murine immunization tests. It was observed a direct influence of oxygen availability upon antigen expression, which is favored in the presence of oxygen. Analysis of the samples collected throughout the fed-batch process also revealed that antigen production is growth associated.

Neuraminidase production by Erysipelothrix rhusiopathiae.

In order to characterise neuraminidase activity by Erysipelothrix, 85 isolates of Erysipelothrix spp. from a variety of sources including human clinical, marine and terrestrial animals, and the environment were investigated for neuraminidase production. Neuraminidase activity was detected by a peanut lectin haemagglutination method. The effects of media, incubation conditions and pH on the production and activity of neuraminidase were also investigated. Enzyme activity was detected only in the supernatants of the isolates of Erysipelothrix rhusiopathiae which had been incubated in cooked meat broth and Todd Hewitt broth supplemented with horse serum after 16 and 36 h incubation at 37 degrees C. The maximum titres were reached at 40 h in cooked meat broth and 56 h in Todd Hewitt broth supplemented with horse serum. All 58 isolates and the type strain (ATCC 19414) of E. rhusiopathiae produced detectable neuraminidase activity with titres between 10 and 320. The optimal pH for the enzyme activity varied among the isolates with a pH between 6.0 and 7.0 covering the highest enzyme activity of the most. There was no statistically significant difference in the level of neuraminidase activity between isolates from different sources (p > 0.05). Neuraminidase activity was not detected in the non-pathogenic Erysipelothrix spp. such as E. tonsillarum. Neuraminidase was detected only in E. rhusiopathiae suggesting its possible role as a virulence factor. Enzyme production and activity were medium and pH dependent. The peanut lectin haemagglutination assay is a simple, rapid and sensitive method and is particularly useful for the analysis of multiple samples.

A novel protein of Erysipelothrix rhusiopathiae that confers haemolytic activity on Escherichia coli.

Erysipelothrix rhusiopathiae, the cause of swine erysipelas and human erysipeloid, produces a haemolysin. A recombinant plasmid, pHLY, conferring haemolytic activity on Escherichia coli was isolated from a genomic library of Ery. rhusiopathiae strain Tama-96. This plasmid was stable in RecA- E. coli, but unstable in a RecA+ strain. A spontaneous deletion plasmid, pMini-HLY, also conferring haemolytic activity was derived from pHLY. Two ORFs were detected in pHLY. Analysis of pMini-HLY and other deletion clones established that ORF2 was associated with haemolytic activity. The sequence of ORF1 was highly homologous to those of transposases in the IS30 family. The deletion which generated pMini-HLY was between two short direct repeat (DR) sequences flanking the ORF1 sequence, and there were inverted repeat sequences inside the two DR sequences, suggesting an insertion element. No sequence homology to the deduced amino acid sequence of ORF2 was detected in the databases, but its sequence was characteristic of a surface lipoprotein. Western blot analysis, using antiserum against the 16 kDa protein produced from ORF2, found the protein to be commonly distributed in all Erysipelothrix species.

Correlation between adherence of Erysipelothrix rhusiopathiae strains of serovar 1a to tissue culture cells originated from porcine kidney and their pathogenicity in mice and swine.

Adherence of four virulent and four avirulent strains of Erysipelothrix rhusiopathiae, serovar 1a, to porcine kidney cell lines, PK-15 and ESK cells, was examined in an in vitro system. The virulent strains adhered well to the cells (range of means, 9.95 +/- 0.87-36.01 +/- 1.10 per cell). In contrast, the avirulent strains showed negligible adherence to the cells (range of means, 0.11 +/- 0.04-1.41 +/- 0.13 per cell). Pretreatment of bacteria with heat, trypsin, or antiserum resulted in a marked decrease in adherence. Scanning electron microscopic examination revealed that the bacteria attached directly to the microvilli of cells.

Effect of C3 depletion on the genesis of thrombocytopenia induced in rats by Erysipelothrix rhusiopathiae.

The role of complement in the pathogenesis of marked thrombocytopenia induced in rats by Erysipelothrix rhusiopathiae (E. rhusiopathiae) was examined. In cobra venom factor (CoF)-treated rats thrombocytopenia was not induced by the bacterium. The content of 5-hydroxytryptamine (5-HT) in platelets was decreased significantly after inoculation only in untreated rats. E. rhusiopathiae could bind C3 and generate platelet-bacteria aggregation when incubated in the plasma diluted with veronal-buffered saline containing calcium and magnesium (VBS++) or gelatin-VBS containing magnesium and ethyleneglycol tetra-acetic acid (GVB-Mg-EGTA), but not when incubated in GVB-ethylenediamine tetra-acetic acid (GVB-EDTA) diluted plasma or in CoF-treated or anti rat C3-treated plasma. When platelets were preincubated with activated zymosan, no bacteria could bind to platelets. From the results, we speculate that the alternative complement pathway, activated by E. rhusiopathiae, appears to mediate the formation of platelet-bacterial aggregations that may accelerate the removal of platelets from circulating blood.

Esculin hydrolysis by Gram positive bacteria. A rapid test and it's comparison with other methods.

A number of bacteria hydrolyze esculin enzymatically to esculetin. This characteristic is used by taxonomists and clinical microbiologists in the differentiation and identification of bacteria, especially to distinguish Lance-field group D streptococci from non-group D organisms and Listeria monocytogenes from morphologically similar Erysipelothrix rhusipoathiae and diphtheroids. Conventional methods used for esculin hydrolysis require 4--48 h for completion. We developed and evaluated a medium which gives positive results more rapidly. The 2,330 isolates used in this study consisted of 1,680 esculin positive and 650 esculin negative organisms. The sensitivity and specificity of this method were compared with the PathoTec esculin hydrolysis strip and the procedure of Vaughn and Levine (VL). Of the 1,680 esculin positive organisms, 97% gave positive reactions within 30 minutes with the rapid test whereas PathoTec required 3--4 h incubation for the same number of organisms to yield a positive reaction.

Use of the rapid fermentation test in determining carbohydrate reactions of fastidious bacteria in clinical laboratories.

The rapid fermentation test was used to determine the carbohydrate reactions of some of the fastidious bacteria encountered in clinical laboratories, such as: Haemophilus species, including Haemophilus vaginalis; Actinobacillus actinomycetemcomitans; Cardiobacterium hominis; Kingella species; Corynebacterium species; Propionibacterium species; and Erysipelothrix rhusiopathiae. Results were usually obtained within 4 h by using inocula from 24- or 48-h blood or chocolate agar media.