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1.
DNA Cell Biol ; 38(8): 773-785, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31339741

RESUMO

Pierisin-5 protein (pie-5) belongs to a family of proteins possessing DNA-dependent ADP-ribosyltransferase activity, which can induce apoptotic cell death. The baculovirus-mediated expression vector system (BEVS) has been commonly used for in vitro expression of heterologous protein subunits for basic scientific research, in addition to the development and production of diagnostics and vaccines. In this study, a new method for the in vitro expression of the cytotoxic protein was established using the baculovirus expression system. The antiproliferative and apoptotic effect of the novel recombinant pierisin-5 protein (rpie-5) was investigated in different human cancer cell lines, such as HeLa, HepG2, and AGS. Cloning, in vitro overexpression, and purification of the rpie-5 protein were performed by using BEVS in Sf21 (Spodoptera frugiperda) insect cell line. The rpie-5 protein exhibits cytotoxicity in all the cell lines, but HeLa (IC50 0.6 µg/mL) was more sensitive when compared with HepG2 (IC50 1.9 µg/mL) and AGS (IC50 3.7 µg/mL) cell lines. The cytotoxic effects of rpie-5 lead to apoptotic cell death in cancer cells and resulted in nuclear fragmentation, enlargement of the nucleus, loss of mitochondrial membrane potential, and finally release of lactose dehydrogenase (LDH) enzyme from the cell membrane. This study reports the molecular mechanism of apoptotic cell death through the upregulation of Bax (Bcl-2 family activating protein-X), Bad, APAF-1 (apoptotic protease activating factor-1), Cyt-c, and caspase-3/9 and the downregulation of Bcl-2 (B-cell lymphoma 2) in rpie-5-treated cancer cells. The study concludes that rpie-5 has p53-independent apoptosis in HepG2 cells and p53-dependent apoptosis in HeLa and AGS cell lines. In the future, this study helps to understand the molecular mechanism of rpie-5 to induction of apoptosis and cell death.


Assuntos
ADP Ribose Transferases/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Proteínas Recombinantes/farmacologia , ADP Ribose Transferases/genética , Animais , Apoptose/fisiologia , Baculoviridae/genética , Linhagem Celular Tumoral , Clonagem Molecular , Humanos , Proteínas de Insetos/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/genética , Células Sf9 , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética
2.
Vet Microbiol ; 235: 63-70, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282380

RESUMO

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid (FT) and substantial economic loss in Korea due to egg drop syndrome and mortality. Despite the extensive use of vaccines, FT still occurs in the field. Therefore, the emergence of more pathogenic SG or the recovered pathogenicity of a vaccine strain has been suspected. SpvB, an ADP-ribosyl transferase, is a major pathogenesis determinant, and the length of the polyproline linker (PPL) of SpvB affects pathogenic potency. SG strains accumulate pseudogenes in their genomes during host adaptation, and pseudogene profiling may provide evolutionary information. In this study, we found that the PPL length of Korean SG isolates varied from 11 to 21 prolines and was longer than that of a live vaccine strain, SG 9R (9 prolines). According to growth competition in chickens, the growth of an SG isolate with a PPL length of 17 prolines exceeded that of an SG isolate with a PPL length of 15 prolines. We investigated the pseudogenes of the field isolates, SG 9R and reference strains in GenBank by resequencing and comparative genomics. The pseudogene profiles of the field isolates were notably different from those of the foreign SG strains, and they were subdivided into 7 pseudogene subgroups. Collectively, the field isolates had gradually evolved by changing PPL length and acquiring additional pseudogenes. Thus, the characterization of PPL length and pseudogene profiling may be useful to understand the molecular evolution of SG and the epidemiology of FT.


Assuntos
Galinhas/microbiologia , Evolução Molecular , Doenças das Aves Domésticas/microbiologia , Salmonelose Animal/microbiologia , Salmonella enterica/genética , ADP Ribose Transferases/genética , Animais , Surtos de Doenças , Ligantes , Peptídeos/genética , Pseudogenes , República da Coreia , Salmonella enterica/isolamento & purificação , Sorogrupo
3.
Int J Oncol ; 55(1): 309-319, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180549

RESUMO

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain­derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA­derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half­life extension, can be used for specific killing of HER2­expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (KD 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (KD) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50­values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA­derivatives are promising agents for targeted cancer therapy.


Assuntos
ADP Ribose Transferases/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Exotoxinas/administração & dosagem , Neoplasias/tratamento farmacológico , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Fatores de Virulência/administração & dosagem , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacocinética , Albuminas/administração & dosagem , Albuminas/química , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Linhagem Celular Tumoral , Exotoxinas/química , Exotoxinas/genética , Exotoxinas/farmacocinética , Feminino , Humanos , Camundongos , Neoplasias/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacocinética , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Ressonância de Plasmônio de Superfície , Distribuição Tecidual , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/farmacocinética
4.
Genome Biol Evol ; 11(1): 1780-1796, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31173069

RESUMO

The diversification of microbial populations may be driven by many factors including adaptation to distinct ecological niches and barriers to recombination. We examined the population structure of the bacterial pathogen Pseudomonas aeruginosa by analyzing whole-genome sequences of 739 isolates from diverse sources. We confirmed that the population structure of P. aeruginosa consists of two major groups (referred to as Groups A and B) and at least two minor groups (Groups C1 and C2). Evidence for frequent intragroup but limited intergroup recombination in the core genome was observed, consistent with sexual isolation of the groups. Likewise, accessory genome analysis demonstrated more gene flow within Groups A and B than between these groups, and a few accessory genomic elements were nearly specific to one or the other group. In particular, the exoS gene was highly overrepresented in Group A compared with Group B isolates (99.4% vs. 1.1%) and the exoU gene was highly overrepresented in Group B compared with Group A isolates (95.2% vs. 1.8%). The exoS and exoU genes encode effector proteins secreted by the P. aeruginosa type III secretion system. Together these results suggest that the major P. aeruginosa groups defined in part by the exoS and exoU genes are divergent from each other, and that these groups are genetically isolated and may be ecologically distinct. Although both groups were globally distributed and caused human infections, certain groups predominated in some clinical contexts.


Assuntos
ADP Ribose Transferases/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Genética Populacional , Pseudomonas aeruginosa/genética , Fluxo Gênico , Genoma Bacteriano , Filogenia , Recombinação Genética
5.
Anaerobe ; 57: 35-38, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30880150

RESUMO

Clostridium (Clostridioides) difficile has been identified in humans and a wide range of animal species, but there has been little study of remote animal populations with limited human contact. The objective of this study was to determine the prevalence and types of C. difficile in wild and captive polar bears (Ursus maritimus). Fecal samples were collected from two populations of wild polar bears in Nunavut Canada; M'Clintock Channel and Hudson Strait (Davis Strait or Foxe Basin), as well as from a facility (PBJ) in Churchill, Manitoba that temporarily houses nuisance polar bears and from captive bears in a zoological park. Enrichment culture was performed and isolates were characterized by ribotyping and toxinotyping. Clostridium difficile was isolated from 24/143 (16.8%) of samples; 18/120 (15%) wild bear samples, 4/7 (57%) from the PBJ and 2/16 (13%) samples from three zoo bears. The prevalence of C. difficile was significantly higher in bears that were housed at the PBJ vs wild bears (P = 0.0042), but there was no difference between wild bears from M'Clintock Channel (14/100, 14%) and those from Hudson Strait (4/20, 20%) (P = 0.50). Fourteen of the 24 (58%) isolates were toxigenic; 13/18 (72%) wild bear isolates, 0/4 PBJ isolate and 1/2 zoo isolates. Four toxigenic ribotypes were identified, with one that possessed tcdB and cdtA predominating. None of the toxigenic isolates were ribotypes that have been identified previously by the authors. There was no overlap in toxigenic ribotypes between the different populations. Clostridium difficile was not uncommonly identified in polar bears, with differences in type distribution amongst the different regions. The presence of strains that have not been identified in humans or domestic animals suggests that polar bears may be a natural reservoir of unique strains of this important bacterium.


Assuntos
Derrame de Bactérias , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/veterinária , Clostridium difficile/isolamento & purificação , Ursidae/microbiologia , ADP Ribose Transferases/genética , Animais , Regiões Árticas/epidemiologia , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridium difficile/classificação , Clostridium difficile/genética , Fezes/microbiologia , Doenças dos Peixes , Manitoba/epidemiologia , Nunavut/epidemiologia , Prevalência , Ribotipagem
6.
J Infect Chemother ; 25(4): 262-266, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30642771

RESUMO

In this study, we investigated all Clostridioides difficile strains isolated from stool samples in Nagasaki University Hospital between January 2012 and December 2014. Toxin genes (tcdA, tcdB and cdtA/cdtB) were analyzed for multiplex PCR in a total of 213 strains. In the toxin gene-positive strain, PCR ribotyping was conducted using capillary gel electrophoresis-based PCR and the Webribo database. Patients' backgrounds were analyzed by departments, disorders, antimicrobials, and clinical dates. The positive rates of tcdA, tcdB, and cdtA/cdtB genes were 62.9%, 63.4%, and 2.8%, respectively. The most frequent PCR ribotype was 047 (14.1%), followed by 014/0 (11.1%) and 002/0 (8.2%). In univariate analysis, the risk factors for the detection of toxin gene-positive strains in patients were older age (p = 0.0036), over ≥ 65 years old (p = 0.0175), the patients hospitalized at Department of Digestive Surgery (P = 0.0059), higher CRP level (P = 0.0395), and lower albumin level (p = 0.0014). In the multivariate analysis, the risk factor for detection of toxin gene-positive strains was the patients hospitalized at Department of Digestive Surgery (OR; 4.62, 95% CI; 1.18-18.0, p = 0.0274). In this study, the percentage of toxin gene-positive and cdtA/cdtB gene-positive strains was almost the same as that reported in previous studies, but the ribotype was different. In addition, we revealed that the risk factor associated with the detection of toxin gene-positive strains was the patients hospitalized at Department of digestive surgery.


Assuntos
Infecções por Clostridium/epidemiologia , Clostridium difficile/genética , Ribotipagem/métodos , ADP Ribose Transferases/genética , ADP Ribose Transferases/isolamento & purificação , Adolescente , Adulto , Fatores Etários , Idoso , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Clostridium difficile/isolamento & purificação , Fezes/microbiologia , Feminino , Hospitais Universitários/estatística & dados numéricos , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Fatores de Risco , Adulto Jovem
7.
BMC Res Notes ; 12(1): 28, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30646938

RESUMO

OBJECTIVE: The present study aimed to determine the prevalence of virulence factors and antimicrobial resistance profile of Pseudomonas aeruginosa strains isolated from Iranian burn patients. RESULTS: This cross-sectional study performed on 100 P. aeruginosa isolates which were recovered from burn wound specimens in 2014-2015. All presumptive isolates were identified by standard microbiologic tests. Antimicrobial susceptibility test was carried out by disk diffusion method. The presence of virulence genes was determined by PCR method. Antibiotic susceptibility results revealed that the isolates were mostly susceptible to amikacin (61%), ceftazidime (60%), and imipenem (55%). Moreover, 59% of the isolates were multi-drug resistance (MDR). The most prevalent MDR pattern was aminoglycosides-penicillins-fluoroquinolones-carbapenems (15%). The presence of exoT, exoY, exoS and exoU genes was detected in 100%, 100%, 59%, and 41% of the tested isolates, respectively. Results points out the pattern of MDR and genetic diversity of type III secretion system among P. aeruginosa strains isolated from the burn population. Overall, the association of MDR and the presence of the specific virulence genes can be a predictive marker for the persistence of these isolates in the hospitals and subsequently a worse clinical condition for the affected patients.


Assuntos
ADP Ribose Transferases/genética , Antibacterianos , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Unidades de Queimados , Queimaduras/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Proteínas Ativadoras de GTPase/genética , Glucosiltransferases/genética , Pseudomonas aeruginosa/genética , Sistemas de Secreção Tipo III , Fatores de Virulência/genética , Estudos Transversais , Humanos , Irã (Geográfico) , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/isolamento & purificação
8.
Nature ; 557(7707): 729-733, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29795346

RESUMO

Ubiquitination is a post-translational modification that regulates many cellular processes in eukaryotes1-4. The conventional ubiquitination cascade culminates in a covalent linkage between the C terminus of ubiquitin (Ub) and a target protein, usually on a lysine side chain1,5. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a ubiquitination method in which a phosphoribosyl ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond6-8. Here we present the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity, and determine the mechanism of this unique post-translational modification. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain and a mono-ADP-ribosyltransferase domain. Biochemical analysis shows that the mono-ADP-ribosyltransferase domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the phosphodiesterase domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous phosphodiesterase domain from the SidE family member SdeD 9 in complexes with Ub and ADPR-Ub. The structures suggest a mechanism for how SdeA processes ADPR-Ub to PR-Ub and AMP, and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-linked ubiquitination and will enable future studies of this unusual type of ubiquitination in eukaryotes.


Assuntos
ADP Ribose Transferases/metabolismo , Legionella pneumophila/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Ubiquitinação , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Difosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Legionella pneumophila/genética , Lisina/metabolismo , Proteínas de Membrana/genética , Modelos Moleculares , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Serina/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo
9.
J Infect Chemother ; 24(8): 641-647, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29685855

RESUMO

We conducted a nationwide molecular epidemiological study of Clostridium difficile infection (CDI) in Japan investigated the correlation between the presence of binary toxin genes and CDI severity. This is the first report on molecular epidemiological analyses for CDI in multiple university hospitals in Japan, to our knowledge. We examined 124,484 hospitalized patients in 25 national and public university hospitals in Japan between December 2013 and March 2014, investigating antimicrobial susceptibilities and toxin-related genes for C. difficile isolates from stools. Epidemiological genetic typing was performed by PCR-ribotyping and repetitive sequence-based (rep)-PCR to examine the genetic similarities. The results detected toxin A-positive, toxin B-positive, binary toxin-negative (A+B+CDT-) detected from 135 isolates (80.8%) and toxin A-negative, toxin B-positive, binary toxin-negative (A- B+CDT-) in 23 (13.8%). Toxin A-positive, toxin B-positive, and binary toxin-positive (A+B+CDT+) were seen in 9 isolates (5.4%). Vancomycin (n = 81, 37.7%) or metronidazole (n = 88, 40.9%) therapies were undertaken in analyzed cases. Ribotypes detected from isolates were 017/subgroup 1, 070, 078, 126, 176, 449, 475/subgroup 1, 499, 451, 566 and newtypes. Rep-PCR classified 167 isolates into 28 cluster groups including 2-15 isolates. In addition, 2 pairs of strains isolated from different institutions belonged to the same clusters. Seven out of 9 (77.8%) of the patients with binary toxin producing strains had "mild to moderate" outcome in evaluated symptoms. In conclusion, we found that binary toxin did not show regional specificity and had no relevance to severity of CDI.


Assuntos
Antibacterianos/uso terapêutico , Infecções por Clostridium/epidemiologia , Clostridium difficile/genética , Hospitais Universitários/estatística & dados numéricos , ADP Ribose Transferases/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/genética , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Clostridium difficile/efeitos dos fármacos , Clostridium difficile/isolamento & purificação , Monitoramento Epidemiológico , Fezes/microbiologia , Feminino , Humanos , Concentração Inibidora 50 , Japão/epidemiologia , Masculino , Metronidazol/uso terapêutico , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Epidemiologia Molecular , Reação em Cadeia da Polimerase , Ribotipagem/métodos , Índice de Gravidade de Doença , Vancomicina/farmacologia , Vancomicina/uso terapêutico , Adulto Jovem
10.
Plant Mol Biol ; 97(1-2): 103-112, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29633168

RESUMO

KEY MESSAGE: This research has shown, for the first time, that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins and the transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. Angiogenesis refers to the formation of new blood vessels, which resulted in the growth, invasion and metastasis of cancer. The vascular endothelial growth factor receptor 2 (VEGFR2) plays a major role in angiogenesis and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins are promising therapeutics for targeted cancer therapy. They consist of an antibody linked to a protein toxin and are designed to specifically kill the tumor cells. In our previous study, VGRNb-PE immunotoxin protein containing anti-VEGFR2 nanobody fused to the truncated form of Pseudomonas exotoxin A has been established. Here, we expressed this immunotoxin in lettuce chloroplasts. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, multigene engineering in a single transformation event and maternal inheritance of the transgenes. Site specific integration of transgene into chloroplast genomes, and homoplasmy were confirmed. Immunotoxin levels reached up to 1.1% of total soluble protein or 33.7 µg per 100 mg of leaf tissue (fresh weight). We demonstrated that transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. These results indicate that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins.


Assuntos
ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Cloroplastos/genética , Exotoxinas/genética , Imunotoxinas/genética , Alface/genética , Fatores de Virulência/genética , ADP Ribose Transferases/farmacologia , Toxinas Bacterianas/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cloroplastos/metabolismo , Clonagem Molecular , Exotoxinas/farmacologia , Células HEK293 , Humanos , Imunotoxinas/farmacologia , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Fatores de Virulência/farmacologia
11.
Spectrochim Acta A Mol Biomol Spectrosc ; 199: 421-429, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29649678

RESUMO

Colorimetric DNA detection is preferred over other methods for clinical molecular diagnosis because it does not require expensive equipment. In the present study, the colorimetric method based on gold nanoparticles (GNPs) and endonuclease enzyme was used for the detection of P. aeruginosa ETA gene. Firstly, the primers and probe for P. aeruginosa exotoxin A (ETA) gene were designed and checked for specificity by the PCR method. Then, GNPs were synthesized using the citrate reduction method and conjugated with the prepared probe to develop the new nano-biosensor. Next, the extracted target DNA of the bacteria was added to GNP-probe complex to check its efficacy for P. aeruginosa ETA gene diagnosis. A decrease in absorbance was seen when GNP-probe-target DNA cleaved into the small fragments of BamHI endonuclease due to the weakened electrostatic interaction between GNPs and the shortened DNA. The right shift of the absorbance peak from 530 to 562nm occurred after adding the endonuclease. It was measured using a UV-VIS absorption spectroscopy that indicates the existence of the P. aeruginosa ETA gene. Sensitivity was determined in the presence of different concentrations of target DNA of P. aeruginosa. The results obtained from the optimized conditions showed that the absorbance value has linear correlation with concentration of target DNA (R: 0.9850) in the range of 10-50ngmL-1 with the limit detection of 9.899ngmL-1. Thus, the specificity of the new method for detection of P. aeruginosa was established in comparison with other bacteria. Additionally, the designed assay was quantitatively applied to detect the P. aeruginosa ETA gene from 103 to 108CFUmL-1 in real samples with a detection limit of 320CFUmL-1.


Assuntos
ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Queimaduras/microbiologia , Sondas de DNA/química , DNA Bacteriano/genética , Endonucleases/metabolismo , Exotoxinas/genética , Ouro/química , Nanopartículas Metálicas/química , Pseudomonas aeruginosa/genética , Fatores de Virulência/genética , ADP Ribose Transferases/análise , Toxinas Bacterianas/análise , Técnicas Biossensoriais/métodos , Calorimetria/métodos , DNA Bacteriano/análise , Exotoxinas/análise , Humanos , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/microbiologia , Espectrofotometria/métodos , Fatores de Virulência/análise
12.
Anaerobe ; 51: 12-16, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29534914

RESUMO

Strains of Clostridium difficile producing only binary toxin (CDT) are found commonly in animals but not humans. However, human diagnostic tests rarely look for CDT. The Cepheid Xpert C. difficile BT assay detects CDT with equal sensitivity (≥92%) in human and animal faecal samples.


Assuntos
ADP Ribose Transferases/genética , Proteínas de Bactérias/genética , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/veterinária , Clostridium difficile/genética , Técnicas de Diagnóstico Molecular/métodos , Animais , Humanos , Sensibilidade e Especificidade
13.
PLoS One ; 13(3): e0194425, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29543870

RESUMO

Scabin is a mono-ADP-ribosyltransferase enzyme and is a putative virulence factor produced by the plant pathogen, Streptomyces scabies. Previously, crystal structures of Scabin were solved in the presence and absence of substrate analogues and inhibitors. Herein, experimental (hydrogen-deuterium exchange), simulated (molecular dynamics), and theoretical (Gaussian Network Modeling) approaches were systematically applied to study the dynamics of apo-Scabin in the context of a Scabin·NAD+·DNA model. MD simulations revealed that the apo-Scabin solution conformation correlates well with the X-ray crystal structure, beyond the conformation of the exposed, mobile regions. In turn, the MD fluctuations correspond with the crystallographic B-factors, with the fluctuations derived from a Gaussian network model, and with the experimental H/D exchange rates. An Essential Dynamics Analysis identified the dynamic aspects of the toxin as a crab-claw-like mechanism of two topological domains, along with coupled deformations of exposed motifs. The "crab-claw" movement resembles the motion of C3-like toxins and emerges as a property of the central ß scaffold of catalytic single domain toxins. The exposure and high mobility of the cis side motifs in the Scabin ß-core suggest involvement in DNA substrate binding. A ternary Scabin·NAD+·DNA model was produced via an independent docking methodology, where the intermolecular interactions correspond to the region of high mobility identified by dynamics analyses and agree with binding and kinetic data reported for wild-type and Scabin variants. Based on data for the Pierisin-like toxin group, the sequence motif Rß1-RLa-NLc-STTß2-WPN-WARTT-(QxE)ARTT emerges as a catalytic signature involved in the enzymatic activity of these DNA-acting toxins. However, these results also show that Scabin possesses a unique DNA-binding motif within the Pierisin-like toxin group.


Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , DNA/metabolismo , Simulação de Dinâmica Molecular , Streptomyces/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sítios de Ligação/genética , Domínio Catalítico , Cristalografia por Raios X , DNA/química , DNA/genética , Cinética , NAD/química , NAD/metabolismo , Ligação Proteica , Streptomyces/genética , Especificidade por Substrato
14.
Anticancer Res ; 38(1): 61-69, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29277757

RESUMO

BACKGROUND: We generated humanized/de-immunized immunotoxins targeting the prostate-specific membrane antigen (PSMA) and tested their cytotoxic activity against prostate cancer cells in vitro. MATERIALS AND METHODS: The humanized/de-immunized version of our murine anti-PSMA single-chain antibody fragment (scFv) D7, termed hD7-1(VL-VH), was ligated to the 40-kDa toxin domain of Pseudomonas aeruginosa exotoxin A (PE40), and to the deimmunized 24-kDa toxin domains PE24 or PE24mut. The immunotoxins designated as hD7-1(VL-VH)-PE40, hD7-1(VL-VH)-PE24 and hD7-1(VL-VH)-PE24mut were bacterially expressed and purified by affinity chromatography. Binding and cytotoxicity were examined by flow cytometry and viability assay, respectively. RESULTS: All immunotoxins revealed strong binding to prostate cancer cells expressing PSMA and specific cytotoxicity, with half-maximal inhibitory concentration values in the picomolar range. CONCLUSION: We successfully created powerful anti-PSMA immunotoxins with reduced immunogenicity for further clinical development and application against advanced prostate cancer.


Assuntos
Antígenos de Superfície/imunologia , Glutamato Carboxipeptidase II/imunologia , Imunotoxinas/farmacologia , Neoplasias da Próstata/imunologia , ADP Ribose Transferases/genética , ADP Ribose Transferases/imunologia , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Linhagem Celular Tumoral , Escherichia coli/genética , Exotoxinas/genética , Exotoxinas/imunologia , Humanos , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Fatores de Virulência/genética , Fatores de Virulência/imunologia
15.
J Microbiol Immunol Infect ; 51(3): 344-351, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28583353

RESUMO

PURPOSE: To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18 bp deletion in the tcdC gene and toxin gene profile (A, B, binary), with eleven RT027 isolates from different Finnish geographical areas and time periods. METHODS: Twenty-eight C. difficile RT027-like isolates and 11 RT027 comparative strains were characterised by capillary-electrophoresis (CE) ribotyping, multi-locus variable tandem-repeats analysis (MLVA), multi-locus sequence typing (MLST), and sequencing of tcdC and gyrA gene fragments. Susceptibility to moxifloxacin was determined by E-test. RESULTS: Of 28 RT027-like isolates, seven RTs (016, 034, 075, 080, 153, 176 and 328), three WEBRIBO types (411, 475, AI-78) and three new profiles (F1-F3) were identified. MLVA revealed six clonal complexes (RTs 016, 027, 176 and F3). MLST showed eleven sequence types (1, 41, 47, 67, 95, 191,192, 223, 229, 264 and new ST). Twenty-two isolates (RTs 016, 080, 176, 328, F1, F2, F3 and WRTAI-78) carried Δ117 in the tcdC gene. Isolates of RTs 016, 027 and 176 were moxifloxacin resistant and harboured Thr82Ile in the GyrA. CONCLUSION: Our results show a high diversity within 28 Finnish RT027-like C. difficile isolates, with twelve CE-ribotyping profiles and eleven STs. MLVA revealed the regional spread of RTs 016, 027, 176 and F3. The presence of Δ117 in the tcdC gene in eight non-027 RTs highlights the importance of careful interpretation of the results from molecular systems targeting this site in the genome of C. difficile and the need of strain typing for epidemiological purposes.


Assuntos
Técnicas de Tipagem Bacteriana , Clostridium difficile/genética , Clostridium difficile/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Ribotipagem/métodos , ADP Ribose Transferases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Toxinas Bacterianas/análise , Toxinas Bacterianas/genética , Sequência de Bases , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Clostridium difficile/classificação , Clostridium difficile/efeitos dos fármacos , DNA Girase/genética , DNA Bacteriano/análise , Farmacorresistência Bacteriana , Enterotoxinas/genética , Feminino , Finlândia , Fluoroquinolonas/farmacologia , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Repetições Minissatélites/genética , Epidemiologia Molecular , Moxifloxacina , Tipagem de Sequências Multilocus/métodos , Proteínas Repressoras/genética , Adulto Jovem
16.
Gene Ther ; 25(1): 47-53, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28937681

RESUMO

Immune cells become increasingly attractive as delivery system for immunotoxins in cancer therapy to reduce the intrinsic toxicity and severe side effects of chimeric protein toxins. In this study, we investigated the potential of human primary T cells to deliver a secreted immunotoxin through transient messenger RNA (mRNA) transfection. The chimeric protein toxin was directed toward the neovasculature of cancer cells by fusing a truncated version of Pseudomonas exotoxin A (PE38) to human vascular endothelial growth factor (VEGF) and to the single chain variable fragment (scFv) of anti-Her2/neu. Protocols for the transient transfection of human embryonic kidney cells (HEK293) as well as activated primary human T cells were established. Transient transfection with mRNA coding for the immunotoxins e23-PE38, VEGF-PE38 and its attenuated variant VEGF-PE38D yielded efficient expression and secretion. Mass spectrometry analysis endorsed that a fraction of VEGF-PE38D was properly translocated into the endoplasmic reticulum. Furthermore, cytotoxic activity of immunotoxin secreting T cells toward cancer cells was confirmed in co-culture with ovarian adenocarcinoma cells in the presence of a bispecific antibody (bsAb), highlighting the potential of primary T cells for mRNA-mediated immunotoxin delivery.


Assuntos
Imunoterapia Adotiva , Imunotoxinas/genética , RNA Mensageiro/genética , Linfócitos T/transplante , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Transporte Biológico , Retículo Endoplasmático/metabolismo , Exotoxinas/genética , Células HEK293 , Humanos , Imunidade Celular , Espectrometria de Massas , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Anticorpos de Cadeia Única/genética , Linfócitos T/imunologia , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Fatores de Virulência/genética
17.
Biochem J ; 475(1): 225-245, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29208763

RESUMO

Scabin was previously identified as a novel DNA-targeting mono-ADP-ribosyltransferase (mART) toxin from the plant pathogen 87.22 strain of Streptomyces scabies Scabin is a member of the Pierisin-like subgroup of mART toxins, since it targets DNA. An in-depth characterization of both the glycohydrolase and transferase enzymatic activities of Scabin was conducted. Several protein variants were developed based on an initial Scabin·DNA molecular model. Consequently, three residues were deemed important for DNA-binding and transferase activity. Trp128 and Trp155 are important for binding the DNA substrate and participate in the reaction mechanism, whereas Tyr129 was shown to be important only for DNA binding, but was not involved in the reaction mechanism. Trp128 and Trp155 are both conserved within the Pierisin-like toxins, whereas Tyr129 is a unique substitution within the group. Scabin showed substrate specificity toward double-stranded DNA containing a single-base overhang, as a model for single-stranded nicked DNA. The crystal structure of Scabin bound to NADH - a competitive inhibitor of Scabin - was determined, providing important insights into the active-site structure and Michaelis-Menten complex of the enzyme. Based on these results, a novel DNA-binding motif is proposed for Scabin with substrate and the key residues that may participate in the Scabin·NAD(+) complex are highlighted.


Assuntos
ADP Ribose Transferases/química , Proteínas de Bactérias/química , Toxinas Bacterianas/química , DNA Bacteriano/química , Glicosídeo Hidrolases/química , Streptomyces/enzimologia , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Ligação Competitiva , Domínio Catalítico , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Cinética , Modelos Moleculares , NAD/química , NAD/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Streptomyces/genética , Streptomyces/patogenicidade , Especificidade por Substrato , Termodinâmica
18.
J Microbiol Biotechnol ; 28(12): 2079-2094, 2018 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-30661346

RESUMO

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.


Assuntos
Quimera , Peptídeos Cíclicos/farmacologia , Inibidores de Proteases/farmacologia , Substâncias Protetoras/farmacologia , Proteólise , Salmonella/genética , Salmonella/metabolismo , ADP Ribose Transferases/genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Toxinas Bacterianas/genética , Transporte Biológico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral/efeitos dos fármacos , Dissulfetos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Exotoxinas/genética , Feminino , Humanos , Neoplasias/tratamento farmacológico , Peptídeos Cíclicos/genética , Inibidores de Proteases/metabolismo , Engenharia de Proteínas , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusão/genética , Tripsina/metabolismo , Fatores de Virulência/genética
19.
J Immunol Res ; 2018: 1090287, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30596104

RESUMO

Previously, we developed a novel EGFR-targeted antibody (denoted as Pan), which has superior antitumor activity against EGFR-overexpressed tumors. However, it shows marginal effect on the growth of esophageal cancers. Therefore, the variable region of Pan was fused to a fragment of Pseudomonas exotoxin A (PE38) to create the immunotoxin, denoted as Ptoxin (PT). Results indicated that PT shows more effective antitumor activity as compared with Pan both on EGFR-overexpressed KYSE-450 and KYSE-150 esophageal cancer cells, especially on KYSE-450 cells. Moreover, treatment of PT induces regression of KYSE-450 tumor xenografts in nude mice. Furthermore, we investigated the potential mechanism involved in the enhanced antitumor effects of PT. Data showed that PT was more potent in reducing the phosphorylation of EGFR and ERK1/2. More importantly, we for the first time found that PT was more effective than Pan in inducing ROS accumulation by suppression of the Nrf2-Keap1 antioxidant pathway, and then induced apoptosis in KYSE-450 esophageal cancer cells, which may partly explain the more sensitive response of KYSE-450 to PT treatment. To conclude, our study provides a promising therapeutic approach for immunotoxin-based esophageal cancer treatment.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Esofágicas/terapia , Imunotoxinas/uso terapêutico , ADP Ribose Transferases/genética , Animais , Anticorpos Monoclonais/genética , Apoptose , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Proliferação de Células , Receptores ErbB/imunologia , Exotoxinas/genética , Humanos , Imunotoxinas/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais , Fatores de Virulência/genética , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Mol Biol (Mosk) ; 51(6): 997-1007, 2017 Nov-Dec.
Artigo em Russo | MEDLINE | ID: mdl-29271963

RESUMO

We have generated and characterized HER2-specific targeted toxin based on the low-immunogenic variant of Pseudomonas exotoxin A (LoPE), in which most of the human immunodominant B-cell epitopes have been inactivated. Nonimmunoglobulin DARPin-based HER2-specific protein was used as a targeting module for toxin delivery to the cellular target. Using confocal microscopy, it has been found that both domains in this hybrid toxin retained their functionality, i.e., the specific interaction with HER2 receptor, as well as the internalization and effective transport to ER typical of the wild-type Pseudomonas exotoxin A. The HER2-dependent cytotoxic effect correlated with receptor expression level at the cell surface, as shown in vitro using cell lines with different levels of HER2 expression. Due to the very high selective cytotoxicity against HER2-positive human tumor cells, as well as expected low immunogenicity, we believe that this new targeted toxin may be promising for future in vivo studies as a therapeutic agent for HER2-positive tumors.


Assuntos
ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Biomarcadores Tumorais/genética , Exotoxinas/genética , Terapia de Alvo Molecular , Proteínas Musculares/genética , Proteínas Nucleares/genética , Receptor ErbB-2/genética , Nanomedicina Teranóstica/métodos , Fatores de Virulência/genética , ADP Ribose Transferases/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Transporte Biológico , Biomarcadores Tumorais/metabolismo , Células CHO , Linhagem Celular Tumoral , Cricetulus , Retículo Endoplasmático/metabolismo , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Exotoxinas/metabolismo , Expressão Gênica , Células HeLa , Humanos , Imunotoxinas/genética , Imunotoxinas/metabolismo , Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Virulência/metabolismo
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