One-Step Electrochemical Growth of 2D/3D Zn(II)-MOF Hybrid Nanocomposites on an Electrode and Utilization of a PtNPs@2D MOF Nanocatalyst for Electrochemical Immunoassay.

To date, two-dimensional (2D) and three-dimensional (3D) metal organic frameworks (MOFs) have been promising materials for applications in electrocatalysis, separation, and sensing. However, the exploration of a simple method for simultaneous fabrication of 2D/3D MOFs on a surface remains challenging. Herein, a one-step and in situ electrosynthesis strategy for fabrication of 2D Hemin-bridged MOF sheets (Hemin-MOFs) or 2D/3D Zn(II)-MOF hybrid nanocomposites on an electrode is reported. It exhibits varied morphologies at different electrodeposition times and attains a 2D/3D complex morphology by adding 1,3,5-benzenetricarboxylic acid (H3BTC) as an organic ligand. The morphology and size of 2D Hemin-MOFs are important factors that influence their performance. Since Pt nanoparticles (PtNPs) are grown on 2D Hemin-MOF sheets, this composite can serve as the peroxidase mimics and PtNPs can act as an anchor to capture the antibody. Therefore, this hybrid nanosheet-modified electrode is used as an electrochemical sensing platform for ultrasensitive pig immunoglobulin G (IgG) and the surface-protective antigen (Spa) protein of Erysipelothrix rhusiopathiae immunodetection. Moreover, this work provides a new avenue for the electrochemical synthesis of 2D/3D MOF hybrid nanocomposites with a high surface area and biomimetic catalysts.

[Identifying immunogenic proteins of Erysipelothrix rhusiopathiae C43065 by MALDI-TOF mass spectrometry and cloning their encoding genes].

OBJECTIVE: To identify immunogenic proteins of Erysipelothrix rhusiopathiae C43065. METHODS: Antigens were extracted from E. rhusiopathiae C43065 by the alkaline extraction method. Proteins in the NaOH-extracted antigen were separated by SDS-PAGE and transferred to nitrocellulose membranes, and then Western blotting was performed with rabbit antiserum against the NaOH-extracted antigens. The immunogenic protein bands were identified by MALDI-TOF mass spectrometry. The genes encoding 5 major immunogenic proteins was amplified by PCR from the genomic DNA of E. rhusiopathiae C43065, and inserted into the pMD18-T vector and then sequenced. RESULTS: A total of 9 immunogenic surface proteins in the NaOH-extracted antigen from E. rhusiopathiae C43065 were successfully identified by MALDI-TOF mass spectrometry. Four of the proteins were putative virulence-associated proteins: enolase, ATP-binding cassette transporter, glyceraldehyde-3 -phosphate dehydrogenase and fructose-bisphosphate aldolase class-II. The genes encoding the chaperone protein GroEL, enolase, ATP-binding cassette transporter, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase class-II were 1614, 1296, 1260, 1005 and 867 bp in length, and the nucleotide sequences homologies of the genes between the C43065 strain and the previously reported E. rhusiopathiae Fujisawa strain was more than 98%. CONCLUSION: Several putative virulence-associated proteins in the NaOH-extracted antigen of E. rhusiopathiae C43065 will be useful for elucidating the roles of these proteins in the pathogenesis of the organism.

Characterization of Seven New Polystyrene Plates Binding Peptides from a Phage-Displayed Random 12-Peptide Library.

A random 12-peptide library was screened against Erysipelothrix rhusiopthiae and porcine circovirus 2 recombinant Cap protein and the selected peptides were used for detecting the corresponding pathogens quickly and effectively. To our surprise, seven peptides, P1 (WHWNAP WWNGVY), P2 (FHWTWQFPYTST), P3 (GAMHLPWHMGTL), P4 (HWNIWWQHHPSP), P5 (HFFKWHTRTNDQ), P6 (HFFRWHPSAHLG) and P7 (HFAYWWNGVRGP) with the characteristics of polystyrene plate (PS) binding target-unrelated peptides (TUPs), were selected from the library. It has been found that P2 and P4 shared common motif of plastic binding peptide, moreover, P2, P3, P5 and P7 have been isolated repeatedly in other research groups using different targets. Then, the seven peptide phage clones were identified as the PS binding TUP phages by phage-ELISA and elution titration, particularly, P1 and P2 showed strong PS binding affinity which can not be inhibited by usual blocking buffers. In addition, all of the phages were not propagation-related TUP, but P3 showed the similar propagation rate with M13KE (vector phage). We also found that the seven PS-TUPs are rich in W, H, F, P and G, particularly, both W and H are contained in all PS-TUPs. It deduced that they may play a potential role in peptide binding to plastic. Although it is difficult to eliminate the TUP phages in phage display completely, these PS-TUPs can be used to exclude the false positive peptides rapidly and effectively and help us to obtain truly interesting peptides more accurately.

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 .

[Cloning and expression of N-terminal protective domain of spaA gene from Erysipelothrix rhusiopathiae C43311].

The spaA gene was amplified by PCR from the genomic DNA of Erysipelothrix rhusiopathiae C43311 strain, and inserted into the pMD18-T vector and then sequenced. The N-terminal protective domain of the spaA gene was amplified by PCR from the recombinant plasmid pMD18-spaA, then cloned into the prokaryotic expression vector pGEX-6p-2 and expressed in E. coli BL21 (DE3) by IPTG induction. The expressed protein was identified by SDS-PAGE and Western blot. The sequence analyses showed that the coding region of the spaA gene of C43311 strain was 1881bp in length, and the nucleotide sequence homology of the spaA genes between the C43311 strain and the previously reported different serotype strains of E. rhusiopathiae was 93 to 99%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 64kDa, and the Western blot results showed that the GST-SpaA-N fusion protein was recognized specifically by an antiserum against the SpaA protein of C43311 strain, suggesting that the fusion protein of GST-SpaA-N possessed high immunoreactivity.

Adhesive surface proteins of Erysipelothrix rhusiopathiae bind to polystyrene, fibronectin, and type I and IV collagens.

Erysipelothrix rhusiopathiae is a gram-positive bacterium that causes erysipelas in animals and erysipeloid in humans. We found two adhesive surface proteins of E. rhusiopathiae and determined the nucleotide sequences of the genes, which were colocalized and designated rspA and rspB. The two genes were present in all of the serovars of E. rhusiopathiae strains examined. The deduced RspA and RspB proteins contain the C-terminal anchoring motif, LPXTG, which is preceded by repeats of consensus amino acid sequences. The consensus sequences are composed of 78 to 92 amino acids and repeat 16 and 3 times in RspA and RspB, respectively. Adhesive surface proteins of other gram-positive bacteria, including Listeria monocytogenes adhesin-like protein, Streptococcus pyogenes protein F2 and F2-like protein, Streptococcus dysgalactiae FnBB, and Staphylococcus aureus Cna, share the same consensus repeats. Furthermore, the N-terminal regions of RspA and RspB showed characteristics of the collagen-binding domain that was described for Cna. RspA and RspB were expressed in Escherichia coli as histidine-tagged fusion proteins and purified. The recombinant proteins showed a high degree of capacity to bind to polystyrene and inhibited the binding of E. rhusiopathiae onto the abiotic surface in a dose dependent manner. In a solid-phase binding assay, both of the recombinant proteins bound to fibronectin, type I and IV collagens, indicating broad spectrum of their binding ability. It was suggested that both RspA and RspB were exposed on the cell surface of E. rhusiopathiae, as were the bacterial cells agglutinated by the anti-RspA immunoglobulin G (IgG) and anti-RspB IgG. RspA and RspB were present both in surface-antigen extracts and the culture supernatants of E. rhusiopathiae Fujisawa-SmR (serovar 1a) and SE-9 (serovar 2). The recombinant RspA, but not RspB, elicited protection in mice against experimental challenge. These results suggest that RspA and RspB participate in initiation of biofilm formation through their binding abilities to abiotic and biotic surfaces.

Structural investigations on the cell surface of Erysipelothrix rhusiopathiae.

The cell wall of Erysipelothrix rhusiopathiae was investigated. The corrected structure of the murein, which was published earlier, is reported here. The murein belongs to the B1delta type. It has L-alanine in position three of the peptide subunit and is crosslinked via a glycine-L-lysine-L-lysine interpeptide bridge. As expected by the complex serological situation in E. rhusiopathiae the carbohydrate moiety of the cell wall proofed to be very intricate. As accessory polymers polysaccharide(s) and teichoic acid(s) were identified. The teichoic acid(s) possess two novel polyols of which one could not be identified, the other one behaved like 6-O-methylgalactitol in gas liquid chromatography. Glucose and N-acetylgalactosamine were determined as substituents. The polysaccharide(s) is composed of mainly N-acetylfucosamine and smaller amounts of galactose, N-acetylglucosamine, and a further unidentified sugar appearing later than hexaacetylsorbitol in GLC.

Computerized comparison of the protein compositions of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum strains.

Protein profiles of six Erysipelothrix rhusiopathiae strains, five Erysipelothrix tonsillarum strains and three Erysipelothrix strains of uncertain taxonomic position were studied by sodium dodecyl sulphate-polyactylamide gel electrophoresis (SDS-PAGE). In a computerized comparison of the protein patterns of the strains, the level of similarity between the strains was determined. The SDS-PAGE protein bands were divided into 14 groups based on molecular weight. The relative distribution of proteins within these groups was used to characterize the strains. These distribution patterns were analysed by computing Pearson's correlation coefficient between strains, and by cluster analysis based on Euclidean distances and the unweighted pair-group method of arithmetic averages (UPGMA). The geometric mean of the similarities calculated by Pearson's correlation coefficient was 0.980 +/- 0.018 between the E. rhusiopathiae strains and 0.979 +/- 0.013 for E. tonsillarum strains. The value was 0.932 +/- 0.036 between the strains belonging to different species. However, a threshold value applicable for identification of a given strain to a species could not be established. Of the three strains of uncertain taxonomic position, the strains designated Rotzunge and Iszap 4 had a protein composition more similar to that of E. tonsillarum than to that of the E. rhusiopathiae type strain. The strain designated Pécs 56, which may be a member of a new species according to literature data, gave inconsistent results by the two methods used. The computerized evaluation method developed here is suitable for the comparison of the protein composition of the strains and for the construction of the protein similarity tree by cluster analysis.

Computerized evaluation procedure for comparing the electrophoretic protein patterns of bacterial strains.

The advantages and drawbacks of different methods of evaluating electrophoretic (SDS-PAGE) protein patterns were studied by comparing Erysipelothrix rhusiopathiae strains. The computerized evaluation procedure recommended by the authors renders it possible to determine objectively the degree of similarity of the protein composition of the different strains according to molecular mass. The degree of similarity obtained in four parallel analyses of a given strain was 988 +/- 0.55 on a scale on which 1000 equals complete identity. the method can be used without adjustment even in those cases where the two strains to be compared contain unequal amounts of electrophoresed proteins.

Comparison of the protein composition of Erysipelothrix rhusiopathiae strains of subtype 1A isolated from ducks and pigs.

Erysipelothrix rhusiopathiae strains isolated from duck carcasses and pigs were examined by sodium dodecyl sulphate--polyacrylamide gel electrophoresis (SDS-PAGE) and the results were evaluated by a computerised method in order to compare the strains for protein composition according to molecular mass. To characterise the similarity of the strains, Pearson's correlation coefficient was calculated. The degree of similarity between duck strains originating from the same flock was 980-991, while that of strains isolated from ducks and pigs varied between 836 and 991 on a scale of 0 to 1000. The method gives highly reproducible results and can be utilised in epidemiological investigations. Its use may provide new data on E. rhusiopathiae strains, complementing the results of methods recommended for this purpose earlier and increasing the efficiency of epidemiological investigations.

Comparison of the protein patterns of Erysipelothrix rhusiopathiae strains by SDS-PAGE and autoradiography.

The proteins of 12 Erysipelothrix rhusiopathiae strains were radiolabelled with L-[35S] methionine, and the protein fractions were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The strains were found to differ from each other in their protein patterns; thus, it was concluded that the method applied could provide useful data for the identification of strains. The described procedure was used for determining the percentage contents of protein fractions of identical molecular mass within the strains compared. The results show that there is no significant correlation between the type of the strains and the percentage of identical molecular weight fractions.

Cellular fatty acid composition of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum.

The cellular fatty acid compositions in 6 strains of Erysipelothrix rhusiopathiae and 7 strains of Erysipelothrix tonsillarum were determined by gas chromatography. Fatty acids, ranging from C10 to C18, were detected in the test strains. The fatty acid profile was characterized by very high percentages of 18:1 (cis-9) (cis-9-octadecenoic acid; 72.4 to 82.1%) and 16:1 (hexadecenoic acid; 8.7 to 13.7%). The profiles of the E. rhusiopathiae and E. tonsillarum strains resembled each other, indicating that discrimination between E. rhusiopathiae and E. tonsillarum from qualitative or quantitative fatty acid differences is difficult.

Cloning, heterologous expression, and characterization of the Erysipelothrix rhusiopathiae DnaK protein.

The dnaK (hsp70) gene from the facultative intracellular pathogen Erysipelothrix rhusiopathiae was cloned by heterologous DNA hybridization of a genomic library using the Escherichia coli dnaK gene as a probe. A 3.2-kb fragment which encoded an 1,800-bp open reading frame was recovered. The deduced amino acid sequence of this open reading frame shares 56% identity with the E. coli DnaK protein. Expression of the encoded protein in E. coli by using the phage T7 promoter/polymerase system resulted in accumulation of a unique 65-kDa protein. Western blot (immunoblot) analysis of extracts from a recombinant E. coli strain using anti-E. coli DnaK polyclonal antibodies confirmed that the cloned gene encodes a DnaK homolog. The recombinant E. rhusiopathiae DnaK protein was purified to 80% homogeneity by ATP affinity chromatography. The purified material hydrolyzed ATP with a specific activity of 100 nmol min-1 mg of protein-1. Analysis of total protein extracts from E. rhusiopathiae indicates that DnaK is a highly expressed protein in this organism.

Differentiation of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell proteins.

The protein patterns of whole cells of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum were studied by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein patterns of the 16 strains of E. rhusiopathiae and E. tonsillarum studied, including the type strains of these two species, resembled each other, except that there were 71-, 41-, 34-, and 26-kDa proteins in the E. rhusiopathiae pattern and 74-, 44-, 36-, and 25-kDa proteins in the E. tonsillarum pattern. This observation indicates that there is some phenotypic heterogeneity in the genus Erysipelothrix. In addition, the protein patterns of E. rhusiopathiae serotype reference strains representing serotypes 1 through 23 and type N were compared. The protein patterns of serotype 1a, 1b, 2, 4, 5, 6, 8, 9, 11, 12, 15, 16, 19, and 21 and type N strains were similar to the pattern of the type strain of E. rhusiopathiae (strain ATCC 19414). Conversely, the protein patterns of serotype 3, 7, 10, 14, and 20 strains were very similar to the pattern of the type strain of E. tonsillarum (strain ATCC 43339). An atypical pattern was observed in serotype 13, 17, 18, 22, and 23 strains. These results suggest that this method may be used as an aid in studying the taxonomy of these bacteria.

Isolation of a high-molecular mass glycoprotein from culture supernatant of an arthritogenic strain of the bacteria Erysipelothrix rhusiopathiae reacting with "inductive" monoclonal antibodies derived from rats with erysipelas polyarthritis.

A glycoprotein exhibiting a relative molecular mass of about 1000 kDa was purified to homogeneity from culture supernatant of arthritogenic bacteria (Erysipelothrix rhusiopathiae, strain T28) by ultrafiltration, ammonium sulfate precipitation, molecular mass exclusion, and ion exchange chromatography. Fractions obtained were analysed for their antigenic content by an enzyme linked immunosorbent assay (ELISA) using rabbit immune serum raised against this strain of Erysipelothrix rhusiopathiae. Distinct monoclonal antibodies obtained from rats suffering from erysipelas polyarthritis display a unique property by inducing very efficiently protective and regulatory mechanisms while being unable to generate classical "passive immunity". These "inductive" monoclonal antibodies recognize most likely linear epitopes on the purified glycoprotein. This makes it a prime source for analysing the target structure of these in vivo "inductive" antibodies.