Role of Lipopolysaccharide and Nonspecific Porins of Yersinia pseudotuberculosis in the Reception of Pseudotuberculous Diagnostic Bacteriophage

Applied Biochemistry and Microbiology - The role of the outer membrane components of Yersinia pseudotuberculosis and Yersinia pestis in the adsorption of pseudotuberculous diagnostic bacteriophage...     

A comparative analysis of the spatial structure of nonspecific porins from <Emphasis Type="Italic">Yersinia ruckeri</Emphasis> using optical spectroscopy and molecular modeling

The spatial organization of outer-membrane porins is studied by optical spectroscopy and molecular modeling. It was found that the OmpF and OmpC porins from Yеrsiniа ruckeri are β-structured membrane proteins typical of the pore-forming proteins of other Gram-negative bacteria. The spatial structures of monomers and trimers of the OmpC and OmpF porins from Y. ruckeri are simulated using methods of structural bioinformatics. It was found that the structural stability of the more thermostable OmpF trimer is sustained by a greater number of hydrogen bonds and hydrophobic interactions. The main differences of the spatial structures of the test porins are observed in the structure of their outer loops. There are three tryptophan residues in the molecules of the OmpC and OmpF porins of Y. ruckeri. It is demonstrated by moleculardynamics methods that after thermal denaturation the solvent accessibility of the Trp212 residue in OmpF porin increased by two times, while the solvent accessibility of a Trp184 residue in OmpC porin was not increased. It is hypothesized that the red-shifted tryptophan fluorescence spectrum of OmpF porin during thermal denaturation is due to the behavior of the Trp212 residue.     

Mutant OmpF porins of Yersinia pseudotuberculosis with deletions of external loops: Structure–functional and immunochemical properties

Recombinant mutant OmpF porins from Yersinia pseudotuberculosis outer membrane were obtained using site-directed mutagenesis. Here we used four OmpF mutants where single extracellular loops L1, L4, L6, and L8 were deleted one at a time. The proteins were expressed in Escherichia coli at levels comparable to full-sized recombinant OmpF porin and isolated from the inclusion bodies. Purified trimers of the mutant porins were obtained after dialysis and consequent ion-exchange chromatography. Changes in molecular and spatial structure of the mutants obtained were studied using SDS–PAGE and optical spectroscopy (circular dichroism and intrinsic protein fluorescence). Secondary and tertiary structure of the mutant proteins was found to have some features in comparison with that of the full-sized recombinant OmpF. As shown by bilayer lipid membrane technique, the pore-forming activity of purified mutant porins was identical to OmpF porin isolated from the bacterial outer membrane. Lacking of the external loops mentioned above influenced significantly upon the antigenic structure of the porin as demonstrated using ELISA.     

Study of effect of substitution of the penultimate amino acid residue on expression,structure, and functional properties of Yersinia pseudotuberculosis OmpY porin

The purpose of the study was to compare the expression of two Yersinia pseudotuberculosis proteins, wild-type porin OmpY and the mutant porin OmpY designated as OmpY-Q having the uncharged amino acid residue Gln instead of positively charged Arg at the penultimate position in the same heterologous host. According to the literature, a similar substitution (Lys to Gln) of the penultimate amino acid residue in Neisseria meningitidis porin PorA drastically improved the assembly of the protein in the E. coli outer membrane in vivo. Site-directed mutagenesis was used to replace Arg by Gln (R338Q) in OmpY, and the conditions for optimal expression and maturation of OmpY-Q were selected. It was found that the growth rates of E. coli strains producing OmpY and OmpY-Q and the expression levels of the porins were approximately equal. Comparative analysis of recombinant OmpY and OmpY-Q did not show significant differences in structure, antigenic, and functional properties of the porins, or any noticeable effect of the R338Q substitution in OmpY on its assembly in the E. coli outer membrane in vivo. The probable causes of discrepancies between our results and the previous data on porin PorA are discussed considering the known mechanisms of biogenesis of porins at the periplasmic stage.     

Biogenesis of β-barrel integral proteins of bacterial outer membrane

Gram-negative bacteria are enveloped by two membranes, the inner (cytoplasmic) (CM) and the outer (OM). The majority of integral outer membrane proteins are arranged in β-barrels of cylindrical shape composed of amphipathic antiparallel β-strands. In bacteria, β-barrel proteins function as water-filled pores, active transporters, enzymes, receptors, and structural proteins. Proteins of bacterial OM are synthesized in the cytoplasm as unfolded polypeptides with an N-terminal sequence that marks them for transport across the CM. Precursors of membrane proteins move through the aqueous medium of the cytosol and periplasm under the protection of chaperones (SecB, Skp, SurA, and DegP), then cross the CM via the Sec system composed of a polypeptide-conducting channel (SecYEG) and ATPase (SecA), the latter providing the energy for the translocation of the pre-protein. Pre-protein folding and incorporation in the OM require the participation of the Bam-complex, probably without the use of energy. This review summarizes current data on the biogenesis of the β-barrel proteins of bacterial OM. Data on the structure of the proteins included in the multicomponent system for delivery of the OM proteins to their destination in the cell and on their complexes with partners, including pre-proteins, are pre-sented. Molecular models constructed on the basis of structural, genetic, and biochemical studies that describe the mechanisms of β-barrel protein assembly by this molecular transport machinery are also considered.     

Protein composition of the cell envelope of the bacterium <Emphasis Type="Italic">Shewanella frigidimarina</Emphasis> Pi 2-35 (Gammaproteobacteria: Shewanellaceae)

The protein composition of the outer (OM) and cytoplasmic (CM) membranes of the cell envelope of the psychrotolerant marine bacterium Shewanella frigidimarina Pi 2-35, which was isolated from a sample of sea ice from the Amur Bay (Sea of Japan), was analyzed using SDS-PAGE. The cell envelope of this microorganism has been found to contain high molecular weight proteins, cytochromes, which were extracted with detergents of different types. It is shown that the composition of the CM of S. frigidimarina Pi 2-35 does not depend on the cultivation temperature. Proteins with a molecular weight (MW) within the range of 30–50 kDa, which is characteristic for nonspecific porins of terrestrial Gram-negative bacteria, were detected only in the OM of bacteria that were cultivated at a low temperature (6–8°C). Two porin-like proteins, P_ZW (MW 40 kDa) and P_LYS (MW 34.5 kDa), were isolated from the OM by extraction with the 0.5% non-ionic detergent Zwittergent 3-14 and by digestion of the peptidoglycan layer of the cell envelope with lysozyme, respectively. The use of the bilayer lipid membrane (BLM) technique showed that both proteins exhibit a pore-forming activity. The efficiency of channel formation and the pattern of fluctuations of current in the BLM indicate that the isolated porins are not identical in their functional properties.     

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.     

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.     

Physicochemical and Immune Properties of Glycoglycerolipids from Laminaria japonicain Immunostimulating Complexes (ISCOMs)

Certain physicochemical properties of glycoglycerolipids from marine alga Laminaria japonica (monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol) and their ability to be incorporated into immunostimulating complexes (ISCOMs) used for delivery of microbial and tumor antigens in vesicular form were comparatively described. These glycolipids proved to considerably differ by fatty acid composition, degree of unsaturation, and phase transition temperatures. Production of modified ISCOMs through incorporation of these glycolipids into the vesicle instead of the glycolipid component was demonstrated. Preliminary data demonstrated no significant increase in immune response to Yersinia pseudotuberculosis porin in the modified (with monogalactosyldiacylglycerol) and classical (with phosphatidylcholine) ISCOMs as compared to pure porin.