RESUMO
GroEL, a prototypical member of the chaperonin class of chaperones, is a large supramocular machine that assists protein folding and plays an important role in proteostasis. GroEL comprises two heptameric rings, each of which encloses a large cavity that provides a folding chamber for protein substrates. Many questions remain regarding the mechanistic details of GroEL facilitated protein folding. Thus, data at atomic resolution of the type provided by NMR and EPR are invaluable. Such studies often require complete deuteration of GroEL, uniform or residue specific 13C and 15N isotope labeling, and the introduction of selective cysteine mutations for site-specific spin labeling. In addition, high purity GroEL is essential for detailed studies of substrate-GroEL interactions as quantitative interpretation is impossible if the cavities are already occupied and blocked by other protein substrates present in the bacterial expression system. Here we present a new purification protocol designed to provide highly pure GroEL devoid of non-specific protein substrate contamination.
Assuntos
Chaperonina 60/isolamento & purificação , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Proteínas de Escherichia coli/isolamento & purificação , Mutação Puntual , Sulfato de Amônio/química , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 60/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Estreptomicina/química , Ureia/químicaRESUMO
Vibrio sp. are autochthonous to marine and estuarine waters. Several species of Vibrio are pathogens. It is of utmost importance to detect and discriminate the Vibrio sp. that are often involved in food and water borne infections. Since 16S rRNA based identification has limited utility in differentiating the closely related pathogenic species from non pathogenic species, we have evaluated the discriminatory power of groEL PCR-RFLP for identification of closely related Vibrio sp. Accordingly, in the current study, the efficiency of groEL PCR- RFLP for detection and accurate differentiation of known pathogens among Vibrio sp. such as V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. fluvialis, V. alginolyticus, V. anguillarum was evaluated. PCR amplified groEL gene fragment of each Vibrio sp. was digested separately using 5 restriction enzymes viz. Hha1, Rsa1, Alu1, Dde1 and Mbo1. The accuracy of the method was further validated by insilico restriction analysis of multiple strains of each species using NEBcutter. The method proved to be efficient for detection and differentiation of Vibrio species under study. Phylogenetic analysis also revealed groEL gene to be a better phylogenetic marker for Vibrio compared to 16S rRNA. Hence, the method can be employed for accurate detection of Vibrio sp. including the closely related species.
Assuntos
Vibrio/classificação , Vibrio/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Proteínas de Bactérias/genética , Sequência de Bases , Chaperonina 60/classificação , Chaperonina 60/genética , Chaperonina 60/isolamento & purificação , Simulação por Computador , Genes Bacterianos , Técnicas Microbiológicas/métodos , Filogenia , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Vibrio/isolamento & purificação , Vibrio/patogenicidadeRESUMO
Aggregatibacter actinomycetemcomitans (Aa) expresses a 64-kDa GroEL protein belonging to the heat shock family of proteins. This protein has been shown to influence human host cells, but the apoptotic capacity of the GroEL protein regarding T cells is not yet known. The purpose of this study was to investigate the ability of A. actinomycetemcomitans GroEL (AaGroEL) protein to induce human peripheral blood T-cell apoptosis. Endogenous, purified AaGroEL protein was used as an antigen. In AaGroEL-treated T cells, the data indicated that phosphatidylserine exposure, an early apoptotic event, was dose- and time-dependent. The AaGroEL-treated T cells were also positive for active caspase-3 in a dose-dependent manner. The rate of AaGroEL-induced apoptosis was suppressed by the addition of the general caspase inhibitor Z-VAD-FMK. Furthermore, cleaved caspase-8 bands (40/36 kDa and 23 kDa) were identified in cells responding to AaGroEL. DNA fragmentation was also detected in the AaGroEL-treated T cells. Overall, we demonstrated that the endogenous GroEL from A. actinomycetemcomitans has the capacity to induce T-cell apoptosis.
Assuntos
Aggregatibacter actinomycetemcomitans/metabolismo , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/toxicidade , Chaperonina 60/toxicidade , Clorometilcetonas de Aminoácidos/farmacologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Inibidores de Caspase/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Chaperonina 60/isolamento & purificação , Chaperonina 60/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Humanos , Microscopia de Fluorescência , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.
Assuntos
Proteínas Arqueais/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Chaperonina 60/isolamento & purificação , Animais , Proteínas Arqueais/química , Proteínas de Bactérias/química , Extratos Celulares/isolamento & purificação , Chaperonina 60/química , Chaperonina 60/metabolismo , Cromatografia de Afinidade , Escherichia coli , Masculino , Mitocôndrias Hepáticas/metabolismo , Estresse Oxidativo , Ligação Proteica , Desnaturação Proteica , Ratos WistarRESUMO
Chaperonins are a class of ubiquitous proteins that assist and accelerate protein folding in the cell. The Escherichia coli groEL is the best known and forms a complex with its co-chaperonin groES in the presence of ATP and assists in the folding of nascent and misfolded substrate proteins. The purification of recombinant groEL results in a nearly homogeneous sample that consistently co-purifies with the major contaminant E. coli ß-galactosidase. Removal of ß-galactosidase using column chromatography alone is exceedingly difficult. This is due to the fact that the overall size, surface charge, isoelectric point and hydrophobicity of groEL and ß-galactosidase are very similar. Therefore purification of groEL chaperonin to homogeneity requires denaturation of the complex into monomers with urea for separating the groEL from contaminating ß-galactosidase followed by reassembly of the chaperonin complex. Here, we present a simple procedure for separating ß-galactosidase along with many other impurities from groEL preparations under non-denaturing conditions. The groEL is first salted out with 50% ammonium sulfate. This step also precipitates ß-galactosidase but this is then salted out by the addition of magnesium chloride which leaves groEL in solution. All remaining contaminants are removed by column chromatography.
Assuntos
Chaperonina 60/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , beta-Galactosidase/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Microscopia Eletrônica de Transmissão , Desnaturação ProteicaRESUMO
HSP60, an intracellular molecular chaperone has been largely described as an alarmin or damage-associated molecular pattern when released outside the cell. HSP60 has been reported as a possible ligand of TLR2 or TLR4 inducing NFκB-dependant signaling pathway leading to cytokine secretion. However, recent publications suggested that HSP60 could not act as an activator of TLR4 by itself. The observed effect could be due to the presence of endotoxin in HSP60 preparation especially LPS. In order to clarify the controversy, we produced recombinant human HSP60 in two different strains of Escherichia coli, standard strain for protein overproduction, BL21(DE3), and the new ClearColi BL21(DE3) strain which lacks LPS-activity through TLR4. Undoubtedly, we have shown that recombinant HSP60 by itself was not able to induce NFκB-dependant signaling pathway in a model of THP1 monocyte cell line. Our data suggest that HSP60 needs either pathogen-associated molecules, specific post-translational modification and/or other host factors to activate immune cells via NFκB activation.
Assuntos
Chaperonina 60/biossíntese , Chaperonina 60/farmacologia , Escherichia coli/metabolismo , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/farmacologia , NF-kappa B/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Chaperonina 60/isolamento & purificação , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/farmacologia , Proteínas Mitocondriais/isolamento & purificação , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Even in asymptomatic cases of Chlamydia trachomatis infection, the aim of the antibiotic strategy is eradication of the pathogen so as to avoid the severe late sequelae, such as pelvic inflammatory disease, ectopic pregnancy, and tubal infertility. Although first-line antimicrobial agents have been demonstrated to be predominantly successful in the treatment of C. trachomatis infection, treatment failures have been observed in some cases. Rich source of antimicrobial peptides was recently discovered in Medicago species, which act in plants as differentiation factors of the endosymbiotic bacterium partner. Several of these symbiotic plant peptides have proved to be potent killers of various bacteria in vitro. We show here that 7 of 11 peptides tested exhibited antimicrobial activity against C. trachomatis D, and that the killing activity of these peptides is most likely due to their interaction with specific bacterial targets.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/metabolismo , Chaperonina 60/metabolismo , Chlamydia trachomatis/efeitos dos fármacos , Medicago truncatula/química , Proteínas de Plantas/farmacologia , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Chaperonina 60/isolamento & purificação , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydia trachomatis/metabolismo , Células HeLa , Humanos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Proteínas de Plantas/síntese química , Proteínas de Plantas/isolamento & purificação , Ligação ProteicaRESUMO
In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50-kDa protein (p50d; α-tubulin), a 25-kDa protein (p25; α-tubulin) and a 52-kDa protein (p52c; ß-tubulin) was enhanced.
Assuntos
Cilióforos/química , Cilióforos/crescimento & desenvolvimento , Regulação da Expressão Gênica , Proteínas de Protozoários/análise , Proteínas de Protozoários/isolamento & purificação , Esporos de Protozoários/química , Esporos de Protozoários/crescimento & desenvolvimento , Actinas/análise , Actinas/química , Actinas/isolamento & purificação , Animais , Chaperonina 60/análise , Chaperonina 60/química , Chaperonina 60/isolamento & purificação , Cromatografia Líquida , Cilióforos/genética , Eletroforese em Gel Bidimensional , Peso Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas de Protozoários/química , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Esporos de Protozoários/genética , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Nontuberculous mycobacteria (NTM) are environmental opportunistic pathogens found in natural and human-engineered waters, including drinking water distribution systems and household plumbing. This pilot study examined the frequency of occurrence of NTM in household potable water samples in Mexico City. Potable water samples were collected from the "main house faucet" and kitchen faucet. The presence of aerobic-mesophilic bacteria (AMB), total coliforms (TC), fecal coliforms (FC) and NTM species were determined. Mycobacteria species were identified by PCR restriction enzyme pattern analysis (PRA) of the 65-kDa heat shock protein gene (hsp65) and sequencing of the hypervariable region 2 (V2) of the 16S rRNA gene and of the rpoB gene. RESULTS: AMB (<100 CFU/ml) were present in 118 out of 120 samples; only two samples were outside guidelines ranges (>100 CFU/ml). TC and FC were detected in four and one samples, respectively. NTM species were recovered from 16% samples (19/120) and included M. mucogenicum (nine), M. porcinum (three), M. avium (three), M. gordonae (one), M. cosmeticum (one), M. fortuitum (one), and Mycobacterium sp (one). All household water samples that contained NTM complied with the standards required to grade the water as "good quality" potable water. CONCLUSION: Household potable water may be a potential source of NTM infection in Mexico City.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Chaperonina 60/isolamento & purificação , Água Potável/microbiologia , Micobactérias não Tuberculosas/isolamento & purificação , RNA Ribossômico 16S/isolamento & purificação , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Chaperonina 60/genética , Farmacorresistência Bacteriana/genética , Características da Família , Humanos , México , Micobactérias não Tuberculosas/genética , Projetos Piloto , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genéticaRESUMO
Current study evaluated the hsp65 Nested PCR Restriction Fragment Length Polymorphism Analysis (hsp65 Nested PCR-PRA) to detect and identify Mycobacterium tuberculosis complex directly in clinical samples for a rapid and specific diagnosis of tuberculosis (TB). hsp65 Nested PCR-PRA was applied directly to 218 clinical samples obtained from 127 patients suspected of TB or another mycobacterial infection from July 2009 to July 2010. The hsp65 Nested PCR-PRA showed 100% sensitivity and 95.0 and 93.1% specificity in comparison with culture and microscopy (acid fast bacillus smear), respectively. hsp65 Nested PCR-PRA was shown to be a fast and reliable assay for diagnosing TB, which may contribute towards a fast diagnosis that could help the selection of appropriate chemotherapeutic and early epidemiological management of the cases which are of paramount importance in a high TB burden country.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Chaperonina 60/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Tuberculose/diagnóstico , Adulto , Efeitos Psicossociais da Doença , Feminino , Humanos , Masculino , Mycobacterium tuberculosis/patogenicidade , Polimorfismo de Fragmento de Restrição , Tuberculose/genética , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
BACKGROUND: Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is widely administered to prevent tuberculosis. Vaccine complications are rare. However, when BCG-related adverse reactions arise there is a need to rapidly and reliably identify BCG from other members of the Mycobacterium tuberculosis complex (TBC). PCR assays based on the detection of the regions of difference (RD), in particular RD1 and RD9, have been invaluable in the identification of BCG. Prior to this study, specimens were identified through HPLC analysis at a local reference laboratory taking up to 2 weeks for a result. We sought to expedite the identification process by validating a RD1, RD9 and hsp65 PCR assay for the identification and differentiation of BCG from TBC. FINDINGS: In last past 3 years, we validated the RD1, RD9 and hsp65 PCR assay for 16 mycobacterial isolates obtained from children who had experienced adverse reactions to BCG vaccination. In these cases, the clinician required a definitive identification of the isolate. The RD1 and RD9 PCR profiles indicated that all 16 isolates were BCG whilst amplification of the hsp65 target functioned as a PCR positive control. When tested against clinical M. tuberculosis (MTB), reference and non-tuberculous mycobacteria the PCR assay demonstrated 100% sensitivity and specificity. CONCLUSIONS: The RD1, RD9 and hsp65 PCR assay is a useful tool for the rapid and reliable identification of BCG. Its ease of use has allowed it to be implemented in our clinical microbiology laboratory.
Assuntos
Proteínas de Bactérias/genética , Chaperonina 60/genética , DNA Bacteriano/genética , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Vacina BCG/administração & dosagem , Vacina BCG/efeitos adversos , Proteínas de Bactérias/isolamento & purificação , Chaperonina 60/isolamento & purificação , Primers do DNA/química , DNA Bacteriano/isolamento & purificação , Feminino , Humanos , Lactente , Masculino , Mycobacterium bovis/genética , Mycobacterium bovis/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , Sensibilidade e Especificidade , Tuberculose Pulmonar/prevenção & controleRESUMO
Avian Pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens of poultry. The lack of suitable vaccines and the emergence of multi-resistant strains have hampered the control of avian colibacillosis. To identify immunogenic proteins of APEC as vaccine candidates, immunoproteomics and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) were applied. Proteins from total cell lysates of APEC DE205B isolated from the brain of a duck with septicemia and neurological symptom in China were separated by two-dimensional electrophoresis (2-DE) and reacted with hyperimmune duck serum against DE205B. Fourteen immunoreactive spots were found, representing 11 distinct proteins. These included two predominant immunogenic components, outer membrane protein A (OmpA) and flagellin (FliC). GroEL, which is a member of the molecular chaperone family and identical structurally to eukaryotic heat shock protein 60 (Hsp60), and the other eight antigens are reported here as immunoreactive proteins of APEC for the first time. Subsequently, nine genes encoding the identified proteins were successfully cloned and expressed in E. coli BL21 (DE3). Seven of the recombinant proteins were able to react with hyperimmune duck serum and three of them, GroEL, OmpA and FliC, showed stronger immunoreactivity. Challenge studies revealed that, just like OmpA and FliC, recombinant GroEL stimulated a strong antibody response and supported protective efficacy against APEC infection in ducks. With high phylogenetic conservation, it is considered that GroEL would be an ideal immunogen of APEC for vaccine development.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Chaperonina 60/genética , Patos/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Doenças das Aves Domésticas/microbiologia , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Encéfalo/metabolismo , Chaperonina 60/imunologia , Chaperonina 60/isolamento & purificação , China , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/isolamento & purificação , Vacinas contra Escherichia coli/imunologia , Flagelina , Imunoproteínas/genética , Imunoproteínas/imunologia , Imunoproteínas/isolamento & purificação , Filogenia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Proteômica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Use of antibodies is a cornerstone of biological studies and it is important to identify the recognized protein with certainty. Generally an antibody is considered specific if it labels a single band of the expected size in the tissue of interest, or has a strong affinity for the antigen produced in a heterologous system. The identity of the antibody target protein is rarely confirmed by purification and sequencing, however in many cases this may be necessary. In this study we sought to characterize the myoplasm, a mitochondria-rich domain present in eggs and segregated into tadpole muscle cells of ascidians (urochordates). The targeted proteins of two antibodies that label the myoplasm were purified using both classic immunoaffinity methods and a novel protein purification scheme based on sequential ion exchange chromatography followed by two-dimensional gel electrophoresis. Surprisingly, mass spectrometry sequencing revealed that in both cases the proteins recognized are unrelated to the original antigens. NN18, a monoclonal antibody which was raised against porcine spinal cord and recognizes the NF-M neurofilament subunit in vertebrates, in fact labels mitochondrial ATP synthase in the ascidian embryo. PMF-C13, an antibody we raised to and purified against PmMRF, which is the MyoD homolog of the ascidian Phallusia mammillata, in fact recognizes mitochondrial HSP60. High resolution immunolabeling on whole embryos and isolated cortices demonstrates localization to the inner mitochondrial membrane for both ATP synthase and HSP60. We discuss the general implications of our results for antibody specificity and the verification methods which can be used to determine unequivocally an antibody's target.
Assuntos
Chaperonina 60/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Óvulo/metabolismo , Urocordados/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Anticorpos Monoclonais/metabolismo , Chaperonina 60/genética , Chaperonina 60/isolamento & purificação , Cromatografia por Troca Iônica , Eletroforese em Gel Bidimensional , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Immunoblotting , Hibridização In Situ , Masculino , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/isolamento & purificação , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/isolamento & purificação , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de Aminoácidos , Urocordados/embriologia , Urocordados/genéticaRESUMO
We report here cloning and expression of full length mitochondrial HSP60 gene of Brugia malayi adult worm (mtHSP60bm), purification of the gene product by affinity chromatography, its in silico 3D structure and the sequence homology of the protein with Escherichia coli GroEL/ES and human HSP60. The ATP binding pocket of human HSP60 and mtHSP60bm were analyzed and compared using in silico models. The distribution of HSP60 in different life-stages of the parasite was determined using antibodies raised against recombinant mtHSP60bm (rmtHSP60bm). mtHSP60bm was present in all life-stages of the parasite except third stage infective larvae, in which it could be induced by heat-shock, and showed high degree of homology with E. coli GroEL/ES. The ATP binding pocket of HSP60 in humans, E. coli and B. malayi were also found structurally conserved. This similarity between human and mtHSP60bm might be useful in understanding the host-parasite interactions. This is the first ever report on distribution, cloning, sequence homology and ATP binding site of mtHSP60bm.
Assuntos
Trifosfato de Adenosina/metabolismo , Brugia Malayi/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Aedes , Animais , Sítios de Ligação , Brugia Malayi/genética , Brugia Malayi/isolamento & purificação , Chaperonina 60/isolamento & purificação , Chaperonina 60/metabolismo , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar/genética , DNA de Helmintos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gerbillinae , Interações Hospedeiro-Parasita , Humanos , Imunização , Masculino , Conformação Molecular , Dados de Sequência Molecular , Murinae , RNA de Helmintos/genética , RNA de Helmintos/isolamento & purificação , Homologia de SequênciaRESUMO
Brucella has a great impact on health and economy in Syria, thus much effort is being placed on the development of diagnostics and vaccines. In this context, a wide Nanobody "immune" library was previously established, from which several Brucella-specific binders were isolated. One of these camel genetically engineered heavy-chain antibody fragments was referred to as NbBruc02. The precise antigen of NbBruc02 was presumed to be, according to proteomic approaches, the Brucella heat shock protein of 60 kDa (HSP-60). HSP-60, or alternatively named GroEL, is an interesting Brucella immunodominant antigen with important roles in the parasite life cycle, mainly adhesion and penetration during the infection of macrophages. In the present work, the capacity of NbBruc02 to filtrate the native GroEL from Brucella total extract was tested by immunochromatography approach. The interaction between NbBruc02 and its antigen was further confirmed using recombinant GroEL from Brucella. Interestingly, NbBruc02 was able to immunodetect the native as well as the denatured forms of the rGroEL in ELISA and immunoblotting, respectively. In agreement with previously reported data, NbBruc02 was able only to detect the denatured Yersinia rGroEL. Using surface plasmon resonance (SPR) biosensor, NbBruc02 showed a strong interaction, with nanomolar affinity (K (D) = ~10(-8) M), with the native rGroEL of Brucella and not of Yersinia. Because the casual conformational changes in the GroEL 3D structure make the base of its function, NbBruc02 by its ability to recognize a "conformational epitope," could open wide perspectives to study the role of GroEL in Brucella physiology.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Brucella/química , Chaperonina 60/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/imunologia , Animais , Antígenos de Bactérias/imunologia , Brucella/imunologia , Camelus/imunologia , Chaperonina 60/imunologia , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Immunoblotting , Epitopos Imunodominantes/imunologia , Conformação Proteica , Proteômica , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Síria , Yersinia/química , Yersinia/imunologiaRESUMO
The bacteria Xenorhabdus spp. are entomopathogenic symbionts that can produce several toxic proteins that interfere the immune system of insects. We purified an insecticidal protein from Xenorhabdus ehlersii, and designated it as XeGroEL with an estimated molecular mass of ~58 kDa. Galleria mellonella larva injected with XeGroEL presented prophenoloxidase activation and hemocyte decrease. XeGroEL can kill G. mellonella larva in 48 h with an LD(50) of 0.76 ± 0.08 µg/larva. Our results demonstrate that X. ehlersii possesses a toxic XeGroEL protein acting as a potential factor to activate proPO in host insect, which also provides a meaningful hypothesis to understand the interaction between nematode-symbiotic bacteria and host.
Assuntos
Catecol Oxidase/metabolismo , Chaperonina 60/metabolismo , Precursores Enzimáticos/metabolismo , Hemócitos/imunologia , Inseticidas/metabolismo , Lepidópteros/microbiologia , Xenorhabdus/patogenicidade , Animais , Chaperonina 60/química , Chaperonina 60/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel Bidimensional , Inseticidas/isolamento & purificação , Larva/imunologia , Larva/microbiologia , Larva/fisiologia , Lepidópteros/imunologia , Lepidópteros/fisiologia , Dose Letal Mediana , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência de DNA , Análise de Sobrevida , Xenorhabdus/químicaRESUMO
Xenorhabdus budapestensis can produce a variety of proteins that help this bacterium and its mutualistic nematode vector kill the host insect. In this report, we purified one protein fraction from the intracellular extract of X. budapestensis D43, which was designated HIP57. By injection, HIP57 caused Galleria mellonella larval bodies to blacken and die with an LD(50) of 206.81 ng/larva. Analyzes of HIP57 by two-dimensional gel electrophoresis showed that this protein was a single spot on the gel with a molecular weight of 57 kDa and a pI of â¼5. Sequencing and bioinformatic analysis suggested that the HIP57 toxin was homologous to GroEL. GroEL has been accepted as molecule chaperon; however, our research revealed that HIP57 (GroEL) possesses another novel function as an insecticide. A GroEL phylogenetic tree defined the relationship among the related species of mutualistic bacteria (Xenorhabdus and Photorhabdus) from the entomopathogenic nematodes and the evolution within the family Enterobacteriaceae. Thus, GroEL could be a complement to 16S rDNA for studying the molecular phylogenies of the family Enterobacteriaceae. Phenoloxidase (PO) activity analysis of G. mellonella larvae injected with HIP57 suggested that the toxin activates the PO cascade, which provides an extensive defense reaction that potentially responsible for G. mellonella larval death.
Assuntos
Catecol Oxidase/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Precursores Enzimáticos/metabolismo , Mariposas/microbiologia , Xenorhabdus/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Chaperonina 60/isolamento & purificação , Eletroforese em Gel Bidimensional , Ativação Enzimática , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Controle Biológico de Vetores , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de Proteína , Xenorhabdus/genéticaRESUMO
The control of strongyloidiasis affecting approximately 100 million people - caused by the gastrointestinal nematode Strongyloides stercoralis - is still based on anti-helminthic treatment. In the current study we analysed the immune response to Strongyloides ratti heat shock protein 60 (srHSP60) as a possible vaccine candidate in the murine system. We show that srHSP60 is a target of both, humoral and cellular response in S. ratti-infected mice. Strikingly, vaccination with srHSP60 without adjuvant or with CFA induced a S. ratti-specific Th1 response in vivo that did not confer protection but slightly increased larval output during challenge infection. Using in vitro T cell stimulation assays we provide further evidence that srHSP60 skewed activated T cells towards a Th1 response that interfered with efficient clearance of S. ratti infection. Vaccination with alum-precipitated srHSP60, in contrast, overruled the Th1-inducing activity intrinsic to srHSP60, induced a Th2 response, and conferred partial protection against a challenge infection. As srHSP60 is actively secreted by S. ratti during all life stages, our findings strongly suggest that srHSP60 induced polarization towards a Th1 response reflects a mechanism of immune evasion by this pathogenic nematode.
Assuntos
Antígenos de Helmintos/imunologia , Chaperonina 60/imunologia , Suscetibilidade a Doenças , Strongyloides ratti/imunologia , Estrongiloidíase/prevenção & controle , Células Th1/imunologia , Vacinação/métodos , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Antígenos de Helmintos/administração & dosagem , Antígenos de Helmintos/isolamento & purificação , Chaperonina 60/administração & dosagem , Chaperonina 60/isolamento & purificação , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Estrongiloidíase/imunologia , Estrongiloidíase/parasitologia , Células Th2/imunologiaRESUMO
AIM: To study the efficacy of heat shock protein 65 kDa (HSP65) of Mybobacterium tuberculosis var. bovis in prevention of autoimmune diabetes by intranasal. METHODS: The HSP65 gene was derived from Mybobacterium tuberculosis var. bovis genome by PCR and successfully expressed as soluble protein in Escherichia coli. The recombinant protein HSP65 was purified by anion exchange column chromatography, then used to immunize prediabetic NOD (non-obese diabetic) mice via three intranasal (i.n.) delivery in absence of adjuvants. Serum samples from the immunized mice were collected at monthly intervals. The anti-HSP65 antibody was detected by enzyme-linked immunosorbent assay (ELISA) and verified by Western blot analysis. The concentration of blood glucose was measured by automatic analyzer. RESULTS: Specific anti-HSP65 antibodies were successfully induced in mice immunized via intranasal routes. Histochemical analysis of mice pancreas tissue showed that HSP65 intranasal vaccination could decrease pathological changes in NOD mice. CONCLUSION: Intranasal vaccination with HSP65 in NOD mice could prevent the development of diabetes. Our results demonstrate that intranasal vaccination with HSP65 reduces significantly the inflammatory process associated with auto-immune diabetes. This approach may offer novel therapeutic avenues for the treatment for of type 1 diabetes mellitus.
Assuntos
Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Chaperonina 60/administração & dosagem , Chaperonina 60/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Pancreatite/prevenção & controle , Vacinação , Administração Intranasal , Animais , Anticorpos/sangue , Anticorpos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Chaperonina 60/genética , Chaperonina 60/isolamento & purificação , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/imunologia , Camundongos , Camundongos Endogâmicos NOD , Pancreatite/tratamento farmacológico , Pancreatite/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
OBJECTIVE: To identify mycobacterial species in the sputum of patients suspected of having pulmonary tuberculosis and to determine the impact that the acquisition of this knowledge has on the therapeutic approach. METHODS: We evaluated 106 patients suspected of having pulmonary tuberculosis and referred to the pulmonology department of a public hospital in the city of Teresina, Brazil. Morning sputum specimens were evaluated for the presence of mycobacteria by sputum smear microscopy and culture. We used PCR and restriction enzyme analysis of the hsp65 gene (PRA-hsp65) to identify the strains of mycobacteria isolated in culture. RESULTS: A total of 206 sputum samples were analyzed. Patient ages ranged from 15 to 87 years, and 67% were male. There was cough in 100% of the cases. The predominant radiographic pattern was moderate disease, observed in 70%. Smear positivity was 76%, and isolation in culture occurred in 91% of the cultures. Traditional tests identified nontuberculous mycobacteria (NTM) in 9% of the isolates. The PRA-hsp65 method confirmed these data, showing seven band patterns that were able to identify the isolated species of NTM: Mycobacterium kansasii; M. abscessus 1; M. abscessus 2; M. smegmatis; M. flavescens 1; M. gordonae 5; and M. gordonae 7. All of the patients with NTM were over 60 years of age, and bronchiectasis was seen in 88% of the X-rays. There were two cases of reinfection, initially attributed to M. abscessus and M. kansasii. CONCLUSIONS: In immunocompetent patients, NTM can infect the lungs. It is important to identify the specific NTM in order to establish the correct diagnosis and choose the most appropriate therapeutic regimen. The PRA-hsp65 method is useful in identifying NTM species and can be implemented in molecular biology laboratories that do not specialize in the identification of mycobacteria.