Immunochemical characteristics of synthetic peptides with T-cellular and B-cellular epitopes of nonspecific porins of pathogenic <Emphasis Type="Italic">Yersinia</Emphasis>

Multiple antigenic peptides (MAPs) that included the common antigenic epitopes of porins from the outer membranes (OM) of bacteria from the Yersinia genus (Y. pseudotuberculosis, Y. enterocolitica, and Y. pestis that are pathogenic for humans) were synthesized. Mice of the BALB/c line were immunized with these peptides, and antisera to the peptides were obtained. It was demonstrated by EIA that these sera interacted with the porins that were isolated from the OM of pathogenic Yersinia. MAPs were shown to be bound to the antibodies in the blood sera of rabbits immunized with the individual porins and to the antibodies in the blood sera of humans suffering from intestinal yersiniosis and pseudotuberculosis.     

Yersinia pseudotuberculosis mutant OmpF porins with deletions of the external loops: genetic constructions design, expression, isolation and refolding

Yersinia pseudotuberculosis outer membrane (OM) recombinant mutant OmpF porins with deletions of the external loops L1, L6 and L8 were obtained using site-directed mutagenesis of the recombinant plasmid including ompF gene. Heterologeous expression of the mutant proteins was carried out in strain Rosetta of Escherichia coli (Novagen, USA), porins with the deletions were isolated from the inclusion bodies. Mutant proteins in oligomeric form were obtained as result of dialysis and ion-exchange chromatography. Spatial structure of the mutant proteins was demonstrated to have special features in comparison with that of the full-structured OmpF porin on the level of both secondary and tertiary structure. Lacking of the loops L1, L6 and L8 didn't affect the conductivity level of Y pseudotuberculosis porin channel as shown using bilayer lipid membrane (BLM) technique. Lacking of the loops mentioned above has a significant influence on the antigenic structure of the mutant porins as demonstrated with use of immunoblotting technique and ELISA.     

Characterization of Yersinia pseudotuberculosis heat stable serovar specific polypeptides with the use of monoclonal antibodies

The capacity of Y. pseudotuberculosis to express serovar specific polypeptides with different specificity of antigenic determinants was proved with the use of monoclonal antibodies (McAb). For the first time Y. pseudotuberculosis O antigens were found to have heat stable protein components carrying linear epitopes complementary to serovar specific MaAb and ensuring the serological specificity of the infective agent. The possibility of improving intraspecific classification of Y. pseudotuberculosis and their differentiation from other pathogenic Yersinia on the basis of the capacity of these bacteria for synthesizing species and serovar specific proteins is substantiated.     

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.     

Homology models of the Yersinia pseudotuberculosis and Yersinia pestis general porins and comparative analysis of their functional and antigenic regions

The amino acid sequences of the Yersinia pseudotuberculosis porin (YPS) and Y. pestis porin (YPT) have recently deduced but their three-dimensional structures were not known. These sequences were analyzed using the servers 3D-PSSM and PredPort. The YPS and YPT porins were shown to have a high degree of identity (above 50%) in primary and secondary structures. The three-dimensional models of the Yersinia pseudotuberculosis porin (YPS) and Y. pestis porin (YPT) were obtained using the homology modeling approach, SWISS-MODEL Protein Modeling Server and 3-D structure of PhoE porin from E. coli as template. The superposition of the Calpha-atoms of the monomers of the Yersinia porins and PhoE porin gave a root mean square deviations of 0.47 A and 0.43 A for YPS and YPT respectively. Yersinia porins were found to be very similar in their three-dimensional structure to other non-specific enterobacterial porins, having the same features of overall fold and disposition of loop L3. The intrinsic structures of the monomer pores of YPS and YPT were investigated and their conductances were predicted with the program HOLE. The good correspondence between the theoretical and experimental magnitudes of YPS conductance was found. The Yersinia porins were determined to be unusual in containing the substitution, Glu replaced by Val, in a highly conserved pentapeptide (Pro-Glu-Phe-Gly-Gly-Asp), located in the loop L3 tip that disturbs the functionally important cluster of the acidic amino acids in the constriction site. Comparative analysis of structural organization of YPS and E. coli OmpF porin in the regions involved in subunit association and pore lumen was performed. The YPS porin functional properties were predicted. The differences between these porins in polar interactions playing a significant role in stabilization of the porin trimers were found and discussed in term of the variations in trimer stability. The Yersinia porins were shown to have the highest degree of the structural similarity. The differences between the porins were observed in their external loops. Their loops L6 and loops L8 showed 71.4 and 52.9% of sequence identity, respectively. The arrangement of charged residues clustered in the channel external vestibule of these porins was found to be also different suggesting the possible differences in their functional properties. The surface exposed regions of Yersinia porins involved in their potential sequential antigenic determinants were compared. The structural basis of their cross reactivity and antigenic differences is discussed.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. IV. Specificity of antigens

The specificity of the lipopolisacharydes and released proteins (Yop) of Yersinia was tested using the sera of rabbits immunised with pathogenic and non-pathogenic strain of Y. enterocolitica and Y. pseudotuberculosis as well as selected sera of patients. The results of this study showed a cross-reactions between the different serotypes of Y. enterocolitica with the strongest reactions between the pathogenic serotypes O:3 and O:9 and pathogenic serotype O:5,27 and non-pathogenic serotype O:5. Sera positive for B. burgdorferi and from patients with Graves' disease showed a slight cross-reactivity with Yop proteins of Yersinia. However, the higher cross-reactivity was observed between the LPS of Yersinia and Salmonella spp. Due to the evidence of cross-reactivity the results of serological investigations should be interpreted with caution.     

Monoclonal antibodies for studying antigens of protein origin in serotyping of Yersinia pseudotuberculosis

Hybridomas producing monoclonal antibodies (MAb) to Yersinia pseudotuberculosis, serovars I-IV, responsible for serovar appurtenance, were obtained. Virtually all MAbs reacted with protein antigens in immunoblotting. The only exclusion was MAb 3A2 presumably reacting with a glycoprotein epitope of complex structure. Variability of Y. pseudotuberculosis antigenic structure, depending on culturing temperature, was confirmed. Polypeptides with mono- or polydetermined antigenic specificity were determined using MAbs.     

Population structure of the Yersinia pseudotuberculosis complex according to multilocus sequence typing

Multilocus sequence analysis of 417 strains of Yersinia pseudotuberculosis revealed that it is a complex of four populations, three of which have been previously assigned species status [Y.?pseudotuberculosis sensu stricto (s.s.), Yersinia pestis and Yersinia similis] and a fourth population, which we refer to as the Korean group, which may be in the process of speciation. We detected clear signs of recombination within Y.?pseudotuberculosis s.s. as well as imports from Y.?similis and the Korean group. The sources of genetic diversification within Y.?pseudotuberculosis s.s. were approximately equally divided between recombination and mutation, whereas recombination has not yet been demonstrated in Y.?pestis, which is also much more genetically monomorphic than is Y.?pseudotuberculosis s.s. Most Y.?pseudotuberculosis s.s. belong to a diffuse group of sequence types lacking clear population structure, although this species contains a melibiose-negative clade that is present globally in domesticated animals. Yersinia similis corresponds to the previously identified Y.?pseudotuberculosis genetic type G4, which is probably not pathogenic because it lacks the virulence factors that are typical for Y.?pseudotuberculosis s.s. In contrast, Y.?pseudotuberculosis s.s., the Korean group and Y.?pestis can all cause disease in humans.     

The yersiniae--a model genus to study the rapid evolution of bacterial pathogens

Yersinia pestis, the causative agent of plague, seems to have evolved from a gastrointestinal pathogen, Yersinia pseudotuberculosis, in just 1,500-20,000 years--an 'eye blink' in evolutionary time. The third pathogenic Yersinia, Yersinia enterocolitica, also causes gastroenteritis but is distantly related to Y. pestis and Y. pseudotuberculosis. Why do the two closely related species cause remarkably different diseases, whereas the distantly related enteropathogens cause similar symptoms? The recent availability of whole-genome sequences and information on the biology of the pathogenic yersiniae have shed light on this paradox, and revealed ways in which new, highly virulent pathogens can evolve.     

Antigenic relationship and functional properties of Yersinia porins

We have studied the molecular structure and functional properties of major pore-forming proteins isolated as peptidoglycan (PG)-protein complexes from four Yersinia species (Y. intermedia, Y. enterocolitica, Y. kristensenii and Y. frederiksenii) cultured as various temperatures. Despite the close antigenic relationship, Yersinia porins revealed different functional properties. When reconstituted in model membranes, the PG-protein complexes induced conductance which was different for the "cold" (grown at 6-8 degrees C) and "warm" (grown at 37 degrees C) variants of microbial cultures. We conclude that the functional state of Yersinia porins in the outer membrane depends on the cultivation temperature.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions were selected for isolation and refolding of recombinant monomer and porin trimer. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that the recombinant porins are similar in the composition of secondary structure elements to the isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

<Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> mutant OmpF porins with deletions of the external loops: genetic constructions design,expression, isolation and refolding

Yersinia pseudotuberculosis outer membrane (OM) recombinant mutant OmpF porins with deletions of the external loops L1, L6 and L8 were obtained using site-directed mutagenesis of the recombinant plasmid including ompF gene. Heterologeous expression of the mutant proteins was carried out in strain Rosetta of Escherichia coli (Novagen, USA), porins with the deletions were isolated from the inclusion bodies. Oligomers of mutant porins were obtained as result of dialysis and ion-exchange chromatography. Spatial structure of the mutant proteins was found to have special features in comparison with that of the full-structured OmpF porin on the level of both secondary and tertiary structure. As shown using bilayer lipid membrane (BLM) technique the absence of the loops L1, L6 and L8 didn’t affect the conductivity level of Y. pseudotuberculosis porin channel. The absence of the loops mentioned above has a significant influence on the antigenic structure of the mutant porins as demonstrated using immunoblotting technique and ELISA.     

Structure and function of pore-forming proteins from bacteria of the genus Yersinia: I. Isolation and a comparison of physicochemical properties and functional activity of Yersinia porins

The molecular organization and functional activity of porins isolated from the outer membrane (OM) of the Yersinia enterocolitica and three phylogenetically close nonpathogenic Yersinia species (Y. intermedia, Y. kristensenii, and Y. frederiksenii) cultured at 6-8 degrees C were comparatively studied for the first time. The proteins were isolated in two molecular forms (trimeric and monomeric), and their spatial structures were characterized by the methods of optical spectroscopy, CD and intrinsic protein fluorescence. The studied porins were shown to belong to the beta-structural proteins (they have 59-96% total beta structures and 0-17% alpha helices). The spatial structures of the proteins were demonstrated to depend on the nature of the detergent used for solubilization. Unlike the enterobacterial pore-forming proteins, the porin trimers are less stable to sodium dodecyl sulfate (SDS). The spatial structures of the porins become more compact after the substitution of octyl beta-D-glucoside for SDS: the content of beta structures increases and the accessibility of Trp residues to solvent decreases. It was established with the use of the technique of bilayer lipid membranes that the functional properties of the porins are similar to those of the OmpF proteins of Gram-negative bacteria. Trimers are functionally active forms of the porins. Special features of the pore-forming activity of the Yersinia porins were revealed to depend on the microorganism species and the value of the membrane potential.     

Characterization of IS1541-like elements in Yersinia enterocolitica and Yersinia pseudotuberculosis

We characterized Yersinia enterocolitica and Yersinia pseudotuberculosis insertion sequences related to insertion sequence 1541, recently identified in Yersinia pestis. For each of the two species, two insertion sequence copies were cloned and sequenced. Genetic elements from Y. pseudotuberculosis were almost identical to insertion sequence 1541, whereas these from Y. enterocolitica were less related. Phylogenetic analysis of the putative transposases encoded by insertion sequences from the three pathogenic members of the genus Yersinia showed that they clustered with those encoded by Escherichia coli and Salmonella enterica elements belonging to the insertion sequence 200/insertion sequence 605 group. Insertion sequences originating from Y. pestis and Y. pseudotuberculosis constitute a monophyletic lineage distinct from that of Y. enterocolitica.     

Occurrence of yersiniosis and listeriosis in wild boars in Japan

From December 1994 to February 1995, 131 wild boars (Sus scrofa leucomysta) living in a mountainous area in Japan were examined for yersiniosis and listeriosis. Of 131 wild boars, 76 (58%) were males and 55 (42%) were females. Four Yersinia spp. including Y. pseudotuberculosis, Y. enterocolitica, Y. frederiksenii, and Y. aldovei, were isolated from 49 (37%) of 131 wild boars. Yersinia pseudotuberculosis was isolated from five (4%) of 131 wild boars. All Y. pseudotuberculosis isolates were serotype 4b and harbored virulence plasmids. Yersinia pseudotuberculosis was isolated only from boars under 2-yr-old. No human pathogenic Y. enterocolitica was isolated. Listeria monocytogenes was isolated from two (1%) of the wild boars and both isolates were serotype 4b. These findings indicated that wild boar could be a reservoir of Y. pseudotuberculosis and L. monocytogenes in Japan.     

Isolation and characterization of OmpF-like porin from <Emphasis Type="Italic">Yersinia ruckeri</Emphasis>

The polypeptide profile of the porin protein fraction of Yersinia ruckeri, a Gram-negative bacterium causing yersiniosis in fish, has been shown to depend on cultivation temperature. OmpF-like porins are expressed mainly in the outer membrane (OM) of the “cold” variant (4°C) of the microorganism and OmpC-like proteins are expressed in the OM of the “warm” variant (37°C). Both types of porins are present in the OM of Y. ruckeri at room temperature. The OmpF-like porin of the “cold” variant was isolated and characterized. The molecular weight and primary structure of the protein were determined. The methods of optical spectroscopy (circular dichroism and intrinsic protein fluorescence) have shown that the protein has a spatial structure typical of β-structured porins from the OM of Gram-negative bacteria. The functional activity of isolated protein was characterized by the bilayer lipid membrane (BLM) technique. The most probable level of channel conductivity was 320 ± 60 pS, corresponding to the channel conductivity of OmpF porins of the genus Yersinia. The distinctive feature of OmpF porin from Y. ruckeri is high thermostability of its functionally active conformation: the protein forms stable pores in the BLM even after heating to 85°C.     

virF-positive Yersinia pseudotuberculosis and Yersinia enterocolitica found in migratory birds in Sweden

During spring and autumn migrations, 468 fecal samples from 57 different species of migratory birds were collected in Sweden. In total, Yersinia spp. were isolated from 12.8% of collected samples. The most commonly found species was Yersinia enterocolitica, which was isolated from 5.6% of all collected samples, followed by Y. intermedia (3.8%), Y. frederiksenii (3.0%), Y. kristensenii (0.9%), Y. pseudotuberculosis (0.6%), and Y. rohdei (0.4%). The pathogenic, virF-positive Y. pseudotuberculosis strains were recovered from three thrushes. These strains belonged to the same bioserotype, 1/O:2, but had two different profiles as determined by pulsed-field gel electrophoresis with NotI and SpeI enzymes. In addition, 10 Y. enterocolitica strains, all from barnacle geese, belonged to bioserotype 3/O:3, which is associated with human disease. Two of the strains were pathogenic, carrying the virF gene on their plasmids. All pathogenic Y. pseudotuberculosis and Y. enterocolitica strains were recovered during the spring, and as the birds were caught during active migration they likely became infected at an earlier stage of the migration, thus potentially transporting these bacterial pathogens over long geographical distances.     

Molecular Characteristics of OmpF-Like Porins from Pathogenic <Emphasis Type="Italic">Yersinia</Emphasis>

Nonspecific pore-forming proteins (porins) are the major proteins of the outer membrane of Gram-negative bacteria responsible for diffusion of low-molecular-weight compounds. Nucleotide sequences of the OmpF-like porins from the pathogenic bacteria Yersinia pseudotuberculosis (YPS) and Yersinia enterocolitica (YE) were cloned and determined. Values of molecular weights (MW) and isoelectric points (IEP) calculated for these proteins (for OmpF-YPS: MW 37.7 kD, IEP 4.45; for OmpF-YE: MW 39.5 kD, IEP 4.34) are in good agreement with experimental data. The OmpF-like Yersinia porins are highly homologous to each other (83-92%) and also to the OmpF protein from Serratia marcescens (70%); the homology to the OmpF porin from E. coli is significantly lower (52-58%). Multiple alignment of the amino acid sequences of mature OmpF proteins provided the distribution of conservative amino acid residues typical for porins. Moreover, the OmpF-like porins from Yersinia are characterized by the presence of extended regions with high and low homologies, which coincide with the transmembrane domains and "external" loops, respectively, of the topological model of the OmpF porin from E. coli. By predictive methods, the secondary structure of the OmpF-like porins from Yersinia was obtained. This structure is represented by 16 beta-strands connected by short "periplasmic" and longer "external" loops with unordered structure.     

Studies of the biosynthesis of 3,6-dideoxyhexoses: molecular cloning and characterization of the asc (ascarylose) region from Yersinia pseudotuberculosis serogroup VA.

The 3,6-dideoxyhexoses are found in the lipopolysaccharides of gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. Of the five 3,6-dideoxyhexoses known to occur naturally, four have been found in various strains of Salmonella enterica (abequose, tyvelose, paratose, and colitose) and all five, including ascarylose, are present among the serotypes of Yersinia pseudotuberculosis. Although there exists one report of the cloning of the rfb region harboring the abequose biosynthetic genes from Y. pseudotuberculosis serogroup HA, the detailed genetic principles underlying a 3,6-dideoxyhexose polymorphism in Y. pseudotuberculosis have not been addressed. To extend the available information on the genes responsible for 3,6-dideoxyhexose formation in Yersinia spp. and facilitate a comparison with the established rfb (O antigen) cluster of Salmonella spp., we report the production of three overlapping clones containing the entire gene cluster required for CDP-ascarylose biosynthesis. On the basis of a detailed sequence analysis, the implications regarding 3,6-dideoxyhexose polymorphism among Salmonella and Yersinia spp. are discussed. In addition, the functional cloning of this region has allowed the expression of Ep (alpha-D-glucose cytidylyltransferase), Eod (CDP-D-glucose 4,6-dehydratase), E1 (CDP-6-deoxy-L-threo-D-glycero-4- hexulose-3-dehydrase), E3 (CDP-6-deoxy-delta 3,4-glucoseen reductase), Eep (CDP-3,6-dideoxy-D-glycero-D- glycero-4-hexulose-5-epimerase), and Ered (CDP-3,6-dideoxy-L-glycero-D-glycero-4-hexulose-4-reductase), facilitating future mechanistic studies of this intriguing biosynthetic pathway.     

The high-pathogenicity island of Yersinia pseudotuberculosis can be inserted into any of the three chromosomal asn tRNA genes

Pathogenicity islands (PAIs) have been identified in several bacterial species. A PAI called high-pathogenicity island (HPI) and carrying genes involved in iron acquisition (yersiniabactin system) has been previously identified in Yersinia enterocolitica and Yersinia pestis . In this study, the HPI of the third species of Yersinia pathogenic for humans, Y. pseudotuberculosis , has been characterized. We demonstrate that the HPI of strain IP32637 has a physical and genetic map identical to that of Y. pestis . A gene homologous to the bacteriophage P4 integrase gene is located downstream of the asn tRNA locus that borders the HPI of strain IP32637. This int gene is at the same position on the HPI of all three pathogenic Yersinia species. However, in contrast to Y. pestis 6/69, the HPI of Y. pseudotuberculosis IP32637 is not invariably adjacent to the pigmentation segment and can be inserted at a distance ≥ 190 kb from this segment. Also, in contrast to Y. pestis and Y. enterocolitica , the HPI of Y. pseudotuberculosis IP32637 can precisely excise from the chromosome, and, strikingly, it can be found inserted in any of the three asn tRNA loci present on the chromosome of this species, one of which is adjacent to the pigmentation segment. The pigmentation segment, which is present in Y. pestis but not in Y. enterocolitica , is also present and well conserved in all strains of Y. pseudotuberculosis studied. In contrast, the presence and size of the HPIs vary depending on the serotype of the strain: an entire HPI is found in strains of serotypes I only, a HPI with a 9 kb truncation in its left-hand part that carries the IS 100 sequence and the psn and ybtE genes characterizes the strains of serotype III, and no HPI is found in strains of serotypes II, IV and V.