RESUMO
Mature pore-forming OmpF protein from the outer membrane of Yersinia pseudotuberculosis was expressed in Escherichia coli in the form of inclusion bodies (IBs) under different cultivation conditions. The properties and structural organization of the IBs as well as the structure of the recombinant porin (rOmpF) solubilized from the IBs were investigated using electron microscopy, dynamic light scattering, optical spectroscopy, and specific hydrophobic dyes. The size, shape, and stability of the IBs under denaturing solutions were determined. It was found that the IBs were readily soluble in SDS and more resistant to urea. Dissolution of the IBs in both denaturing agents led to formation of a heterogeneous in size population of oligomeric particles. The IBs contained an intermediate form of the rOmpF with native-like secondary structure and elements of tertiary structure, which was able to penetrate a lipid bilayer and adopt a functionally active conformation. There were no significant differences in the properties and structure between the examined IBs formed at different concentrations of the inducer (IPTG). However, the content of amyloids in the IBs increased with increasing concentration of the inducer. These results contribute to the development of new approaches for the production of active proteins from IBs, as well as biologically and functionally active IBs.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Corpos de Inclusão/metabolismo , Porinas/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Microscopia Eletrônica de Varredura , Porinas/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
Recombinant porin OmpF (an integral protein of bacterial outer membrane) from Yersinia pseudotuberculosis was synthesized in Escherichia coli cells as inclusion bodies. By combining the methods of anion-exchange and gel filtration chromatographies, recombinant OmpF (rOmpF) was isolated as an individual protein in its denatured state, and its characteristic properties (molecular mass, N-terminal amino acid sequence, and hydrodynamic radius of the protein in 8 M urea solution) were determined. According to the data of gel filtration, dynamic light scattering, optical spectroscopy, and binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, the rOmpF is fully unfolded in 8 M urea and exists in random coil conformation. In aqueous solutions, rOmpF undergoes conformational changes, reversible self-association, and aggregation. When transferred from 8 M urea into water, PBS (containing 0.15 M NaCl, pH 7.4), or buffer containing 0.8 M urea (pH 8.0), fully unfolded rOmpF forms relatively compact monomeric intermediates prone to self-association with formation of multimers. The oligomeric intermediates have high content of native protein-like secondary structure and pronounced tertiary structure. In acidic media (pH 5.0, close to the protein isoelectric point), rOmpF undergoes rapid irreversible aggregation. Therefore, we found that medium composition significantly affects both porin folding and processes of its self-association and aggregation.
Assuntos
Porinas/química , Yersinia pseudotuberculosis/química , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias , Escherichia coli/genética , Escherichia coli/metabolismo , Corpos de Inclusão , Porinas/biossíntese , Porinas/isolamento & purificação , Conformação Proteica , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Renaturação Proteica/efeitos dos fármacos , Proteínas Recombinantes , Soluções/química , Soluções/farmacologia , ÁguaRESUMO
The OmpF porin gene expression in Yersinia pseudotuberculosis in response to antibiotics of two different classes (kanamycin and nalidixic acid) was analyzed using quantitative PCR and a fluorescence reporter system. Both antibiotics downregulated the expression of the ompF gene. The nalidixic acid significantly reduced ompF expression, while kanamycin, for which porins are considered to be an alternative transport route, only slightly reduced the ompF level.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/biossíntese , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Canamicina/farmacologia , Ácido Nalidíxico/farmacologia , Porinas/biossíntese , Estresse Fisiológico/efeitos dos fármacos , Yersinia pseudotuberculosis/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Regulação Bacteriana da Expressão Gênica/genética , Reação em Cadeia da Polimerase , Porinas/genética , Estresse Fisiológico/genética , Yersinia pseudotuberculosis/genéticaRESUMO
Geographical distribution of individual genotypes of Helicobacter pylori, predominance of virulent types in various regions of Russia, particularly in the Prymorye Territory, remains unclear. We examined 115 children with various gastroduodenal pathology and 33 patients with gastric cancer, of which 57.39 and 60% respectively were infected with H. pylori. All samples positive for H. pylori were further analyzed for gene vacA mosaicism. In all clinical subgroups, variants s1 and m1 predominated; the frequency of genotype s1 was significantly increased (1.3-fold) in the group of cancer patients in comparison with the group of children with gastroduodenal pathology. Three variants of allele combination of signaling and middle regions of the vacA gene (s1m1, s1m2, and s2m2) were revealed; s1m1 was the most frequent in both groups. We suggest that this genotype is a marker of complicated course of gastroduodenitis and a factor of gastric cancer development in local population.
Assuntos
Proteínas de Bactérias/genética , Duodenopatias/microbiologia , Gastroenteropatias/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Neoplasias Gástricas/microbiologia , Adolescente , Adulto , Criança , Duodenopatias/epidemiologia , Feminino , Gastroenteropatias/epidemiologia , Frequência do Gene , Genes Bacterianos , Marcadores Genéticos , Variação Genética , Genótipo , Infecções por Helicobacter/epidemiologia , Humanos , Masculino , Prevalência , Federação Russa/epidemiologia , Neoplasias Gástricas/epidemiologiaRESUMO
To identify the Yersinia genus and pathogenic species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) in a single reaction a multiplex PCR technique was developed. It was optimized by five compounds of PCR buffer and temperature of primers annealing. The multiplex PCR provides an improved and rapid method for detection of the Yersinia genus and identification of pathogenic species.
Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Porinas/genética , Yersinia/classificação , Sequência de Aminoácidos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Yersinia/genética , Yersinia/isolamento & purificação , Yersinia enterocolitica/classificação , Yersinia enterocolitica/genética , Yersinia enterocolitica/isolamento & purificação , Yersinia pestis/classificação , Yersinia pestis/genética , Yersinia pestis/isolamento & purificação , Yersinia pseudotuberculosis/classificação , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/isolamento & purificaçãoRESUMO
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.