OmpC-like porin from Yersinia pseudotuberculosis: Molecular characteristics, physico-chemical and functional properties

Pore-forming protein from the outer membrane of Yersinia pseudotuberculosis cultured at 37°C has been isolated and characterized. Comparative analysis of the primary and three-dimensional structures of this protein and of OmpC porin from E. coli was carried out, functional properties of these proteins have been studied using bilayer lipid membranes (BLM) technique. The degree of homology, molecular mass and pore-forming properties of the isolated porin was found to be closer to those of OmpC porin from E. coli than OmpF porin from Y. pseudotuberculosis. The value of the most probable conductivity of OmpC porin from Y. pseudotuberculosis (0.18 pS) in BLM corresponded to the conductivity of the native trimer of this protein. Using CD spectroscopy, the porins investigated were shown to belong to the β-structured proteins. Data of the primary structure and intrinsic protein fluorescence revealed essential differences in localization and microenvironment of tryptophan residues in the porins investigated. Participation of external loops L2 and L6 in the formation of the antigenic structure of OmpC porin from Y. pseudotuberculosis was demonstrated. On the basis of crystal structure of osmoporin from Klebsiella pneumoniae, three-dimensional models of the monomer and trimer of the Y. pseudotuberculosis porin were obtained. Using Web server AGGRESCAN, the localization of protein structure sites with the increased aggregation capability (hot spots) has been deter-mined. It turned out that some of these zones localize in the region of intramonomeric contacts in the porin trimer; however, a large part of them is located on the external surface of the β-barrel. The process of thermal denaturation has been studied and the melting points of the porins were determined. It was found that significant changes in the microenvironment of the indole fluorophores (especially tryptophan residues of spectral class I) took place in the process of the thermodenaturation of the proteins. These changes preceded the irreversible conformational transition observed for the E. coli porin at 77°C and for the Y. pseudotuberculosis porin at 70°C.     

The outer membrane porin from Yersinia enterocolitica in yersiniosis diagnostics

The diagnostic test system based on a species-specific antigen, pore-forming protein from the outer membrane of Yersinia enterocolitica, for yersiniosis verification by the method of ELISA has been developed and approved. The proposed ELISA test system is characterized by high sensitivity (95%) and specificity (89%) and provides a differential diagnostics of yersiniosis from other acute enteric infections with similar clinical manifestations. In comparison with the commercial diagnostics based on indirect hemagglutination reaction, which is conventionally used in clinical practice, the porin-based ELISA provides the high level (90–95%) of yersiniosis identification at early (1st week) and late (2nd–4th week) stages of infection process. It has been found that the ELISA test system reveals antibodies to the Y. enterocolitica porin in patient’s serum irrespective of the serological variant of causative agent.     

Outer membrane permeability and porin proteins of Yersinia enterocolitica

In intact cells of Yersinia enterocolitica, loss of the YompF protein reduced the outer membrane permeability parameters for two beta-lactam compounds, cephaloridine and nitrocefin. Additional loss of the outer membrane YompC protein resulted in further reductions in the permeability parameters. We conclude that the outer membrane proteins YompC and YompF of Y. enterocolitica are porin proteins.     

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 for isolation and refolding of recombinant monomer and porin trimer were selected. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that recombinant porins are similar in the composition of secondary structure elements to 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.     

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.     

The protective properties of a porin from the outer membrane of Yersinia pseudotuberculosis

The protective properties of the species-specific pore-forming polypeptide of Y. pseudotuberculosis outer membrane (porin) were studied. The study showed that 80-90% of mice immunized with porin survived after challenge with 10-30 LD50 of Y. pseudotuberculosis, serovars 1 and 3. The LD50 of the preparation exceeded its ED50 more than 100-fold. Immunization made in two injections was more effective than immunization in one injection. The immunization of the animals with porin led to an increase in the activity of peritoneal exudate macrophages with respect to Y. pseudotuberculosis, serovars 1 and 3.     

Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein

Background

Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains.

Results

The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar.

Conclusions

OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica.     

Influence of cultivation conditions on spatial structure and functional activity of OmpF-like porin from outer membrane of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

The influence of cultivation conditions of pseudotuberculosis bacteria on the spatial structure and the functional activity of nonspecific OmpF-like porin was studied by means of optical spectroscopy, scanning microcalorimetry, and bilayer lipid membrane technique. With this goal, porin samples isolated from microbial masses grown at different temperatures, nutrient medium densities, and growth phases were characterized. According to CD data, the porin samples under investigation represent beta-sheet proteins. It was found that the protein isolated from the colonial culture of pseudotuberculosis bacteria grown at low temperature has the most compact structure. Using intrinsic protein fluorescence, it was shown that different conditions of pseudotuberculosis bacteria cultivation (temperature, medium, growth phase) led to the changes in spectral properties of porin fluorescence due to the redistribution of the contributions of tyrosine and different classes of tryptophan residues to the total protein emission. Heat inactivation of porin samples was studied using CD spectroscopy, intrinsic protein fluorescence, and scanning microcalorimetry. Spatial features of the porin samples were found to affect their functional activities. Considering all these data, it is possible to correlate the spatial structure and functional activity of porin samples isolated under different cultivation conditions of bacteria and the composition of the outer membrane lipid matrix.     

Influence of nutrient-limited growth on pathogenesis-associated outer membrane proteins of Yersinia enterocolitica

From studies based on batch culture, it has been postulated that the expression of the virulence-associated proteins of Yersinia spp. is controlled by temperature and Ca²⁺, such that these proteins are synthesized only at the higher temperature (37°C) and calcium-scarce conditions of the intracellular environment. It was found, however, that in Yersinia enterocolitica one of these proteins (140 kDa) is not synthesized at submaximal growth rates under any of the relevant conditions, and that another of the implicated proteins (34 kDa), is synthesized even at 28°C during nutrient-limited growth. Thus, temperature and Ca²⁺ influence the synthesis of these proteins differently under growth conditions that better approximate the natural environments than do batch cultures.     

Influence of nutrient-limited growth on pathogenesis-associated outer membrane proteins of Yersinia enterocolitica

From studies based on batch culture, it has been postulated that the expression of the virulence-associated proteins of Yersinia spp. is controlled by temperature and Ca2+, such that these proteins are synthesized only at the higher temperature (37 degrees C) and calcium-scarce conditions of the intracellular environment. It was found, however, that in Yersinia enterocolitica one of these proteins (140 kDa) is not synthesized at submaximal growth rates under any of the relevant conditions, and that another of the implicated proteins (34 kDa), is synthesized even at 28 degrees C during nutrient-limited growth. Thus, temperature and Ca2+ influence the synthesis of these proteins differently under growth conditions that better approximate the natural environments than do batch cultures.     

Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis

The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways.     

A heat-modifiable outer membrane protein carries an antigen specific for the species Yersinia enterocolitica and Yersinia pseudotuberculosis

The outer membranes of gram-negative bacteria are considered to be of importance in host-bacteria interaction, in protective immunity, and occasionally in subclassification within a species. In this study, the outer membranes of several strains of Yersinia enterocolitica and Y. pseudotuberculosis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the appearance of the major proteins depended on the temperature at which they were solubilized in SDS. A protein was identified with the use of two-dimensional gels and preparative SDS-PAGE, which was equivalent to the "heat-modifiable protein" (protein II) of other Enterobacteriaceae species. A monoclonal antibody, 4G1, was generated against an isolated preparation of this Y. enterocolitica protein. This antibody was tested with whole cell bacterial antigens of 46 individual bacterial strains. The reactive strains included only Y. enterocolitica and Y. pseudotuberculosis. In addition, the reactivity of the 4G1 monoclonal antibody preparation could be absorbed only with Y. enterocolitica and Y. pseudotuberculosis, and not with other strains of bacteria. The reactivity of this 4G1 monoclonal antibody was also tested by the Western Blot technique. Six individual strains were tested: a Y. enterocolitica serotype 0:3, a Y. enterocolitica serotype 0:9, an Escherichia coli, a Salmonella typhimurium, a Shigella flexneri, and a Klebsiella pneumoniae. The 4G1 antibody reacted with only the proteins of the two Y. enterocolitica strains. In conclusion, the equivalent of the heat-modifiable protein was present in Y. enterocolitica and Y. pseudotuberculosis. Moreover, this protein also carried a species-specific antigenic determinant.     

A novel OmpY porin from Yersinia pseudotuberculosis: structure, channel-forming activity and trimer thermal stability

A novel OmpY porin was predicted based on the Yersinia pseudotuberculosis genome analysis. Whereas it has the different genomic annotation such as "outer membrane protein N" (ABS46310.1) in str. IP 31758 or "outer membrane protein C2, porin" (YP_070481.1) in str. IP32953, it might be warranted to rename the OmpN/OmpC2 to OmpY, "outer membrane protein Y", where letter "Y" pertained to Yersinia. Both phylogenetic analysis and genomic localization clearly support that the OmpY porin belongs to a new group of general bacterial porins. The recombinant OmpY protein with its signal sequence was overexpressed in porin-deficient Escherichia coli strain. The mature rOmpY was shown to insert into outer membrane as a trimer. The OmpY porin, isolated from the outer membrane, was studied employing spectroscopic, electrophoretic and bilayer lipid membranes techniques. The far UV CD spectrum of rOmpY was essentially identical to that of Y. pseudotuberculosis OmpF. The near UV CD spectrum of rOmpY was weaker and smoother than that of OmpF. The rOmpY single-channel conductance was 180 ± 20 pS in 0.1 M NaCl and was lower than that of the OmpF porin. As was shown by electrophoretic and bilayer lipid membrane experiments, the rOmpY trimers were less thermostable than the OmpF trimers. The porins differed in the trimer-monomer transition temperature by about 20°C. The three-dimensional structural models of the Y. pseudotuberculosis OmpY and OmpF trimers were generated and the intra- and intermonomeric interactions stabilizing the porins were investigated. The difference in the thermal stability of OmpY and OmpF trimers was established to correlate with the difference in intermonomeric polar contacts.     

Yersinia enterocolitica outer protein T (YopT)

Pathogenic Yersinia strains evade the innate immune responses of the host by producing effector proteins (Yersinia outer proteins (Yops)), which are directly injected into mammalian cells by a type III secretion system (TTSS). One of these effector proteins (YopT) disrupts the actin cytoskeleton of the host cell. YopT is a cysteine protease which cleaves Rho proteins directly upstream of the post-translationally modified cysteine. Thereby, it releases the GTPases from the membrane leading to their inactivation. Besides a biochemical characterisation of the molecular mechanism and substrate specificity also delivery into host cells with chaperone binding and guidance to the injection apparatus and the patho-physiological role of YopT have been studied and are summarised in this review.     

Molecular epidemiology of Yersinia enterocolitica infections

Yersinia enterocolitica is an important food-borne pathogen that can cause yersiniosis in humans and animals. The epidemiology of Y. enterocolitica infections is complex and remains poorly understood. Most cases of yersiniosis occur sporadically without an apparent source. The main sources of human infection are assumed to be pork and pork products, as pigs are a major reservoir of pathogenic Y. enterocolitica. However, no clear evidence shows that such a transmission route exists. Using PCR, the detection rate of pathogenic Y. enterocolitica in raw pork products is high, which reinforces the assumption that these products are a transmission link between pigs and humans. Several different DNA-based methods have been used to characterize Y. enterocolitica strains. However, the high genetic similarity between strains and the predominating genotypes within the bio- and serotype have limited the benefit of these methods in epidemiological studies. Similar DNA patterns have been obtained among human and pig strains of pathogenic Y. enterocolitica, corroborating the view that pigs are an important source of human yersiniosis. Indistinguishable genotypes have also been found between human strains and dog, cat, sheep and wild rodent strains, indicating that these animals are other possible infection sources for humans.     

Structure-function correlation of outer membrane protein porin from Paracoccus denitrificans

Porins from outer membrane of Gram-negative bacteria have a highly stable structure. Our previous studies on porin from Paracoccus denitrificans showed that the outer membrane protein porin is extremely stable toward heat, pH, and chemical denaturants. The major question we have addressed in this paper is whether the high stability of porin is a consequence of the beta-barrel structure and whether it is required for its function. To explain this we have analyzed two cases: first, we used porin wild-type and mutants and compared their structure and function; second, we compared the activity of porin preheated to different temperatures. Structural changes were monitored by infrared spectroscopy. We observed that the structural stability of porin is not equivalent to functional activity as minor alteration in the structure can result in drastic differences in the activity of porins.     

Effect of natural polymorphism on structure and function of the Yersinia pestis outer membrane porin F (OmpF protein): a computational study

The Yersinia pestis outer membrane porin F (OmpF) is a transmembrane protein located in the outer membrane of this Gram-negative bacterium which is the causative agent of plague, where it plays a significant role in controlling the selective permeability of the membrane. The amino acid sequences of OmpF proteins from 48 Y. pestis strains representing all currently available phylogenetic groups of this Gram-negative bacterium were recently deduced. Comparison of these amino acid sequences revealed that the OmpF can be present in four isoforms, the pestis-pestis type, and the pestis-microtus types I, II, and III. OmpF of the most recent pestis-pestis type has an alanine residue at the position 148, where all the pestis-microtus types have threonine there (T148A polymorphism). The variability of different pestis-microtus types is caused by an additional polymorphism at the 193rd position, where the OmpFs of the pestis-microtus type II and type III have isoleucine-glycine (IG⁺₁₉₃) or isoleucine-glycine-isoleucine-glycine (IGIG⁺₁₉₃) insertions, respectively (IG⁺₁₉₃ and IGIG⁺₁₉₃ polymorphism). To investigate potential effects of these sequence polymorphisms on the structural properties of the OmpF protein, we conducted multi-level computational analysis of its isoforms. Analysis of the I-TASSER-generated 3D-models revealed that the Yersinia OmpF is very similar to other non-specific enterobacterial porins. The T148A polymorphism affected a loop located in the external vestibule of the OmpF channel, whereas IG⁺₁₉₃ and IGIG⁺₁₉₃ polymorphisms affected one of its β-strands. Our analysis also suggested that polymorphism has moderate effect on the predicted local intrinsic disorder predisposition of OmpF, but might have some functional implementations.     

Effects of pH on structural and functional properties of porin from the outer membrane of Yersinia pseudotuberculosis. II. Characterization of pH-induced conformational intermediates of yersinin

pH-Induced intermediates of Omp F-like porin from the outer membrane of Yersinia pseudotuberculosis (yersinin) were characterized by fluorescence and fluorescent probe spectroscopy and circular dichroism. The most dramatic changes in the intrinsic fluorescence of the protein induced by pH titration correlated with different conformational states of the porin molecule. pH-induced conformational transitions of yersinin can be described in terms of a three-state model: (1) disordering of porin associates and formation of porin trimers structurally similar to the native protein; (2) unfolding of individual porin domains followed by cooperative dissociation of trimers into monomers; (3) formation of two loosely structured forms of monomer intermediates. It is assumed that one of these monomeric forms (at pH 3.0) corresponds to the molten-globule state of porin with native secondary structure, while the other one (at 2.0) represents a partly denatured (misfolded) monomer, which retains no more than 50% of the regular secondary structure. The putative mechanism of low pH-induced β-barrel unfolding is discussed in terms of a theoretical model of yersinin spatial structure.     

The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin.

An essential virulence attribute for Yersinia enterocolitica and Yersinia pseudotuberculosis is the ability to invade the intestinal epithelium of mammals. The chromosomal invasin gene (inv) has been cloned from both of these Yersinia species, and the Y. pseudotuberculosis invasin has been well characterized (R. R. Isberg, D. L. Voorhis, and S. Falkow, Cell 50:769-778, 1987). Here we constructed TnphoA translational fusions to the Y. enterocolitica inv gene to identify, characterize, and localize the inv protein product in Escherichia coli. The cloned Y. enterocolitica inv locus encoded a unique protein of ca. 92 kilodaltons when expressed in minicells. A protein of comparable size was detected in immunoblots by using monoclonal antibodies directed against the Y. pseudotuberculosis invasin. This protein, which we also refer to as invasin, promoted both attachment to and invasion of cultured epithelial cells. These two functions were not genetically separable by insertional mutagenesis. We determined that the Y. enterocolitica invasin was localized on the outer membrane and that it was exposed on the bacterial cell surface, which may have implications for how invasin functions to mediate invasion.