RESUMO
In this study a data dependent acquisition label-free based proteomics approach was used to identify pH-dependent proteins that respond in a growth phase independent manner in Campylobacter jejuni reference strain NCTC 11168. NCTC 11168 was grown within its pH physiological normal growth range (pH 5.8, 7.0 and 8.0, µ = â¼0.5 h-1) and exposed to pH 4.0 shock for 2 h. It was discovered that gluconate 2-dehydrogenase GdhAB, NssR-regulated globins Cgb and Ctb, cupin domain protein Cj0761, cytochrome c protein CccC (Cj0037c), and phosphate-binding transporter protein PstB all show acidic pH dependent abundance increases but are not activated by sub-lethal acid shock. Glutamate synthase (GLtBD) and the MfrABC and NapAGL respiratory complexes were induced in cells grown at pH 8.0. The response to pH stress by C. jejuni is to bolster microaerobic respiration and at pH 8.0 this is assisted by accumulation of glutamate the conversion of which could bolster fumarate respiration. The pH dependent proteins linked to growth in C. jejuni NCTC 11168 aids cellular energy conservation maximising growth rate and thus competitiveness and fitness.
Assuntos
Campylobacter jejuni , Campylobacter jejuni/genética , Campylobacter jejuni/química , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteômica , Concentração de Íons de HidrogênioRESUMO
Iron is an essential micronutrient for living organisms as it is involved in a broad variety of important biological processes. However, free iron inside the cell could be potentially toxic, generating hydroxyl radicals through the Fenton reaction. Dps (DNA-binding protein from starved cells) belongs to a subfamily of ferritins and can store iron atoms inside the dodecamer. The presence of a ferroxidase centre, composed of highly conserved residues, is a signature of this protein family. In this study, we analysed the role of two conserved histidine residues (H25 and H37) located at the ferroxidase centre of the Campylobacter jejuni Dps protein by replacing them with glycine residues. The C. jejuni H25G/H37G substituted variant showed reduced iron binding and ferroxidase activities in comparison with wt Dps, while DNA-binding activity remained unaffected. We also found that both CjDps wt and CjDps H25G/H37G were able to bind manganese atoms. These results indicate that the H25 and H37 residues at the ferroxidase centre of C. jejuni Dps are not strictly required for metal binding and oxidation.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/enzimologia , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Histidina/metabolismo , Ferro/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Campylobacter jejuni/química , Campylobacter jejuni/genética , Ceruloplasmina/genética , Sequência Conservada , Histidina/química , Histidina/genética , Cinética , Dados de Sequência Molecular , OxirreduçãoRESUMO
Dps proteins (DNA binding protein from starved cell) form a distinct group within the ferritin superfamily. All Dps members are composed of 12 identical subunits that assemble into a conserved spherical protein shell. Dps oxidize Fe(2+) in a conserved ferroxidase center located at the interface between monomers, the product of the reaction Fe(3+), is then stored inside the protein shell in the form of non-reactive insoluble Fe2O3. The Campylobacter jejuni Dps (CjDps) has been reported to play a plethora of functions, such as DNA binding and protection, iron storage, survival in response to hydrogen peroxide and sulfatide binding. CjDps is also important during biofilm formation and caecal colonization in poultry. In order to facilitate in vitro characterisation of CjDps, it is important to have a simple and reproducible protocol for protein purification. Here we report an observation that CjDps has an unusual high melting temperature. We exploited this property for protein purification by introducing a thermal treatment step which allowed achieving homogeneity by using only two chromatographic steps. Gel filtration chromatography, circular dichroism, mass spectrometry, DNA-binding and iron oxidation analysis confirmed that the CjDps structure and function were unaffected.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Campylobacter jejuni/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Temperatura AltaRESUMO
Oxygen affinity in heme-containing proteins is determined by a number of factors, such as the nature and conformation of the distal residues that stabilize the heme bound-oxygen via hydrogen-bonding interactions. The truncated hemoglobin III from Campylobacter jejuni (Ctb) contains three potential hydrogen-bond donors in the distal site: TyrB10, TrpG8, and HisE7. Previous studies suggested that Ctb exhibits an extremely slow oxygen dissociation rate due to an interlaced hydrogen-bonding network involving the three distal residues. Here we have studied the structural and kinetic properties of the G8(WF) mutant of Ctb and employed state-of-the-art computer simulation methods to investigate the properties of the O(2) adduct of the G8(WF) mutant, with respect to those of the wild-type protein and the previously studied E7(HL) and/or B10(YF) mutants. Our data indicate that the unique oxygen binding properties of Ctb are determined by the interplay of hydrogen-bonding interactions between the heme-bound ligand and the surrounding TyrB10, TrpG8, and HisE7 residues.
Assuntos
Proteínas de Bactérias/química , Campylobacter jejuni/química , Oxigênio/química , Oxigênio/metabolismo , Hemoglobinas Truncadas/química , Proteínas de Bactérias/genética , Campylobacter jejuni/genética , Glicina/genética , Heme/química , Heme/genética , Histidina/química , Histidina/genética , Ligação de Hidrogênio , Ligantes , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Análise Espectral Raman , Hemoglobinas Truncadas/genética , Triptofano/química , Triptofano/genética , Tirosina/química , Tirosina/genéticaRESUMO
Campylobacter jejuni and Campylobacter coli were isolated from aborted pig fetuses which proceeded from different animals and farms between February 2000 and March 2001. Seven Campylobacter jejuni biotype II, three biotype I and one Campylobacter coli biotype I were identified by phenotypic tests and Lior's scheme. To corroborate and compare the phenotypic results, 7.5, 10 and 12.5% polyacrilamide gel electrophoresis (SDS-PAGE) were used under reducing conditions. Characteristic bands of hypervariable dense zone within C. jejuni and C. coli species were observed in all the whole cell protein extracts with differences in mobility. It was possible to establish differences between identical phenotypic Campylobacter isolates and different protein profile from fetuses of the same litter. SDS-PAGE is a stable and reproducible method to establish differences between Campylobacter strains and is considered applicable for the differentiation of the wide variability of Campylobacter species for epidemiologic purposes.
Assuntos
Aborto Animal/microbiologia , Proteínas de Bactérias/análise , Técnicas de Tipagem Bacteriana/métodos , Infecções por Campylobacter/veterinária , Campylobacter coli/isolamento & purificação , Campylobacter jejuni/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Doenças dos Suínos/microbiologia , Animais , Argentina , Infecções por Campylobacter/microbiologia , Campylobacter coli/química , Campylobacter jejuni/química , Idade Gestacional , Fenótipo , Reprodutibilidade dos Testes , Especificidade da EspécieRESUMO
We determined the sensitivity of five strains of Campylobacter jejuni and C. coli isolated from children with diarrhea and from chicken feces to normal human blood serum (undiluted and at concentrations of 10, 30, 50 and 70%), a hypogammaglobulinemic serum and a complement-deficient serum. Both species were highly sensitive to the bactericidal activity of human serum, regardless of their source. The highest bactericidal activity was observed with pooled fresh normal serum, with bacterial survival rates inversely correlated to serum dilutions. Inactivated serum had the least bactericidal activity. When complement was partially restored to inactivated serum, lower survival rates were observed. The hypogammaglobulinemic-normal complement-containing serum had strong bactericidal activity whereas the normal immunoglobulin-containing but complement-deficient serum had little bactericidal activity. These results suggest that Campylobacter may be able to directly activate complement by the alternative pathway.
Assuntos
Atividade Bactericida do Sangue , Campylobacter coli/fisiologia , Campylobacter jejuni/fisiologia , Proteínas do Sistema Complemento/fisiologia , Animais , Campylobacter coli/química , Campylobacter jejuni/química , Galinhas , Criança , Ativação do Complemento , HumanosRESUMO
We determined the sensitivity of five strains Compylobacter jejuni and C. coli isolated from children with diarrhea and from chicken feces to normal human blood serum (undiluted and at concentrationss of 10, 30, 50 and 70 per cent), hypogmmaglobulinemic serum and a complemented-deficient serum. Both species were highly sensitive to the bactecidal activity of human serum, regardless of their source. The highest bactericidal activity was observed with pooled fresh normal serum, with bacterial survival rates inversely correlated to serum dilutions. Inactivated serum had the least bactericidal activity. When complement was partially restored to inactivated serum, lower survival rates were observed. The hypogammaglobulinemic-normal complement-containing serum had strong bactericidal activity whereas the normal immunoglobulin-containing but complement-feficient serum had little bactericidal activity. These results suggest that Campylobacter may be able to directly activate complement by the alternative pathway