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1.
Nat Commun ; 12(1): 423, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33462232

RESUMO

Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca2+- and Mg2+-dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear.


Assuntos
Toxinas Bacterianas/metabolismo , Desoxirribonucleases/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Infecções por Yersinia pseudotuberculosis/patologia , Yersinia pseudotuberculosis/patogenicidade , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/toxicidade , Desoxirribonucleases/genética , Desoxirribonucleases/isolamento & purificação , Desoxirribonucleases/toxicidade , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Mutagênese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Yersinia pseudotuberculosis/metabolismo , Infecções por Yersinia pseudotuberculosis/microbiologia
2.
Sci Rep ; 10(1): 2781, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066776

RESUMO

Harmful algal blooms formed by colony-forming cyanobacteria deteriorate water resources by producing cyanotoxins, which frequently occur at high intracellular concentrations. We aimed to localize toxic microcystins (MCs) and bioactive anabaenopeptins (APs) at the subcellular level under noninvasive conditions. Since both metabolites are synthesized nonribosomally, the relaxed specificity of key enzymes catalyzing substrate activation allowed chemical labeling through a standard copper-catalyzed click chemistry reaction. The genera Planktothrix and Microcystis specifically incorporated unnatural amino acids such as N-propargyloxy-carbonyl-L-lysine or O-propargyl-L-tyrosine, resulting in modified AP or MC peptides carrying the incorporated alkyne moiety. The labeled cells were quantitatively differentiated from the unlabeled control cells. MCs and APs occurred intracellularly as distinct entities showing a cell-wide distribution but a lowered spatial overlap with natural autofluorescence. Using the immunofluorescence technique, colocalization with markers of individual organelles was utilized to relate the distribution of labeled MCs to cellular compartments, e.g., using RbcL and FtsZ (cytosol) and PsbA (thylakoids). The colocalization correlation coefficients calculated pairwise between organelles and autofluorescence were highly positive as opposed to the relatively low positive indices derived from labeled MCs. The lower correlation coefficients imply that only a portion of the labeled MC molecules were related spatially to the organelles in the cell.


Assuntos
Toxinas Bacterianas/isolamento & purificação , Cianobactérias/química , Microcistinas/isolamento & purificação , Peptídeos Cíclicos/isolamento & purificação , Aminoácidos/química , Aminoácidos/metabolismo , Toxinas Bacterianas/química , Química Click , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/metabolismo , Água Doce/química , Proliferação Nociva de Algas , Microcistinas/química , Peptídeos Cíclicos/química
3.
J Vet Diagn Invest ; 32(2): 222-225, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31328697

RESUMO

Storage procedures are known to affect the detectability of Clostridium difficile toxins in equine and human feces. We assessed the impact of different storage conditions on the detectability of C. difficile toxins in swine feces. Specimens were inoculated with toxins, 112 ng/g of toxin A (TcdA) and 16 ng/g of toxin B (TcdB) and subjected to the following 3 storage treatments: 4°C, -30°C, repetitive freezing at -30°C and thawing. Toxin determination was assessed at 1, 2, 7, 14, and 21 d with ELISA. A decrease in concentrations of TcdA with time was observed for samples stored at 4°C and repetitive freezing-thawing (p ≤0.05). On day 14, storage at 4°C resulted in decreased TcdA concentration as opposed to storage at -30°C and repetitive freezing-thawing (p ≤0.05). On day 21, storage at 4°C resulted in decreased TcdA detectability compared with storage at -30°C (p ≤0.05). The TcdB concentration was unaffected. These results on toxin detectability in swine feces should be carefully considered in in vitro studies on toxigenic C. difficile. Our results also offer valuable information for microbiologists and veterinarians monitoring the presence of virulent C. difficile in pigs.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas/isolamento & purificação , Infecções por Clostridium/veterinária , Enterotoxinas/isolamento & purificação , Fezes/microbiologia , Manejo de Espécimes/métodos , Doenças dos Suínos/diagnóstico , Animais , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/microbiologia , Feminino , Suínos , Doenças dos Suínos/microbiologia , Fatores de Tempo
4.
J Vet Diagn Invest ; 32(2): 252-258, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31650911

RESUMO

Clostridium perfringens type G is one of the pathogens involved in enteric diseases in poultry. NetB, a pore-forming toxin, is considered the main virulence factor responsible for necrotic enteritis during C. perfringens infection. We carried out a field study involving 14 farms to evaluate the occurrence of netB-positive C. perfringens and the impact of infection in Italian poultry flocks. Environmental samples (n = 117) and 50 carcasses were screened by microbiologic and molecular methods. Microbiologic investigations yielded 82 C. perfringens isolates. DNA was extracted from all samples and screened for α-toxin and NetB encoding genes by real-time PCR. The C. perfringens α-toxin gene was detected in 151 of 167 extracts (90.4%), and 31 of 151 (20.5%) were netB gene positive also. Sixteen isolates from a turkey flock with mild enteric disorders were also netB positive, demonstrating their occurrence not only in broiler but also in turkey flocks. A pulsed-field gel electrophoresis protocol was optimized to evaluate the diversity among isolates and revealed high genetic heterogeneity. The complete NetB toxin-coding gene of 2 C. perfringens isolates from turkey and broiler flocks were analyzed and showed very high relatedness with analogous sequences worldwide.


Assuntos
Galinhas , Infecções por Clostridium/veterinária , Clostridium perfringens/isolamento & purificação , Doenças das Aves Domésticas/epidemiologia , Perus , Animais , Toxinas Bacterianas/isolamento & purificação , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Clostridium perfringens/genética , Enterotoxinas/isolamento & purificação , Itália/epidemiologia , Doenças das Aves Domésticas/microbiologia , Prevalência
5.
J Fish Dis ; 43(2): 207-214, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31752048

RESUMO

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VPAHPND ), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb-protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of goat anti-mouse immunoglobulin G antibody was used to capture the free colloidal gold conjugate MAb. The sample in the application buffer could be applied directly to the application well, and the test result was obtained within 15 min. The sensitivity of the kit is approximately 6.25 µg/ml of toxin, which was equivalent to the toxin produced by approximately 107  cfu/ml of bacteria. This kit is convenient and easy to use since it can be used to identify VPAHPND directly using a single colony of bacteria grown on agar culture plates. Because of its high specificity and simplicity, as well as not being reliant on sophisticated equipment or specialized skills, this strip test could be used by farmers for surveillance for ToxB-producing bacteria.


Assuntos
Toxinas Bacterianas/isolamento & purificação , Cromatografia de Afinidade/veterinária , Hepatopâncreas/microbiologia , Imunoensaio/veterinária , Penaeidae/microbiologia , Vibrio parahaemolyticus/isolamento & purificação , Animais , Anticorpos Antibacterianos , Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Imunoensaio/métodos
6.
Proc Natl Acad Sci U S A ; 116(49): 24808-24818, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31744876

RESUMO

Myxobacteria are an example of how single-cell individuals can transition into multicellular life by an aggregation strategy. For these and all organisms that consist of social groups of cells, discrimination against, and exclusion of, nonself is critical. In myxobacteria, TraA is a polymorphic cell surface receptor that identifies kin by homotypic binding, and in so doing exchanges outer membrane (OM) proteins and lipids between cells with compatible receptors. However, TraA variability alone is not sufficient to discriminate against all cells, as traA allele diversity is not necessarily high among local strains. To increase discrimination ability, myxobacteria include polymorphic OM lipoprotein toxins called SitA in their delivered cargo, which poison recipient cells that lack the cognate, allele-specific SitI immunity protein. We previously characterized 3 SitAI toxin/immunity pairs that belong to 2 families. Here, we discover 4 additional SitA families. Each family is unique in sequence, but share the characteristic features of SitA: OM-associated toxins delivered by TraA. We demonstrate that, within a SitA family, C-terminal nuclease domains are polymorphic and often modular. Remarkably, sitA loci are strikingly numerous and diverse, with most genomes possessing >30 and up to 83 distinct sitAI loci. Interestingly, all SitA protein families are serially transferred between cells, allowing a SitA inhibitor cell to poison multiple targets, including cells that never made direct contact. The expansive suites of sitAI loci thus serve as identify barcodes to exquisitely discriminate against nonself to ensure populations are genetically homogenous to conduct cooperative behaviors.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Myxococcales/genética , Myxococcales/metabolismo , Receptores de Superfície Celular/metabolismo , Alelos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Toxinas Bacterianas/classificação , Toxinas Bacterianas/imunologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Lipoproteínas , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Filogenia , Análise de Sequência
7.
Rev Neurol (Paris) ; 175(10): 644-649, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31629544

RESUMO

Intranasal administration delivers molecules directly to the brain bypassing the blood-brain barrier. Three distinct routes of access have been identified; olfactory, trigeminal and via the paranasal sub-mucosa of the posterior sinuses. Consequently, environmental toxins may access the CNS directly to induce inflammatory and degenerative disease. They may also activate bacterial species of the nasal mucosal microbiome to release both immune-deviating cell wall antigens and transportable neurotoxins with local direct access to the CNS. Evidence is reviewed that toxins of the nasal bacterial microbiota may be directly implicated in the inflammatory and degenerative pathogenesis of multiple sclerosis.


Assuntos
Toxinas Bacterianas/isolamento & purificação , Encéfalo/efeitos dos fármacos , Microbiota/fisiologia , Esclerose Múltipla/etiologia , Nariz/microbiologia , Administração Intranasal , Animais , Toxinas Bacterianas/efeitos adversos , Toxinas Bacterianas/toxicidade , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/microbiologia , Humanos , Esclerose Múltipla/microbiologia , Fatores de Risco
8.
Ann Agric Environ Med ; 26(3): 392-395, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559791

RESUMO

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/microbiologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Animais , Antraz/imunologia , Antraz/prevenção & controle , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Vacinas contra Antraz/isolamento & purificação , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/química , Bacillus anthracis/genética , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios Proteicos
9.
N Z Vet J ; 67(6): 329-332, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31378159

RESUMO

Aims: To determine if presence of the Bacteroides fragilis toxin (bft) gene, a molecular marker of colonic carriage of entertoxigenic Bacteroides fragilis (ETBF) in humans, was associated with a finding of small intestinal adenocarcinomas (SIA) in sheep in New Zealand. Methods: Samples of jejunal tissue were collected from the site of tumours and from grossly normal adjacent tissue in 20 sheep, in different consignments, diagnosed with SIA based on gross examination of viscera following slaughter. Two jejunal samples were also collected from a control sheep in the same consignment that had no gross evidence of SIA. A PCR assay was used to detect the presence of the bft gene in the samples. Results: Of the sheep with SIA, the bft gene was amplified from one or both samples from 7/20 (35%) sheep, and in sheep that had no gross evidence of SIA the bft gene was amplified from at least one sample in 11/20 (55%) sheep (RR 0.61; 95% CI = 0.30-1.25; p = 0.34). Of 11 positive samples analysed, ETBF subtype bft-1 was detected in one, bft-2 was detected in 10, and none were bft-3. Conclusions and Clinical Relevance: There was a high prevalence of detection of the bft gene in both SIA-affected and non-affected sheep, but there was no apparent association between carriage of ETBF, evidenced by detection of the bft gene, and the presence of SIA. ETBF are increasingly implicated in the aetiology of human colorectal cancer, raising the possibility that sheep may provide a zoonotic reservoir of this potentially carcinogenic bacterium. Abbreviation: Bft: Bacteroides fragilis toxin; ETBF: Enterotoxigenic Bacteroides fragilis; SIA: Small intestinal adenocarcinoma.


Assuntos
Adenocarcinoma/veterinária , Toxinas Bacterianas/genética , Neoplasias Intestinais/veterinária , Metaloendopeptidases/genética , Doenças dos Ovinos/diagnóstico , Adenocarcinoma/diagnóstico , Adenocarcinoma/microbiologia , Animais , Toxinas Bacterianas/isolamento & purificação , DNA Bacteriano/análise , Genes Bacterianos , Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/microbiologia , Metaloendopeptidases/isolamento & purificação , Ovinos , Doenças dos Ovinos/microbiologia
10.
J Pharm Biomed Anal ; 174: 650-654, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31279895

RESUMO

Recombinant ExoProtein A (EPA), a detoxified form of Pseudomonas aeruginosa Exotoxin A, is used as a protein carrier in the vaccine field. A scaled manufacturing process, in which EPA was expressed in Escherichia coli, yielded a product that approached or exceeded our upper limit of E. coli host cell protein (HCP) content per human dose. The purification process was redeveloped to reduce HCP levels in the bulk product and HCP content was evaluated by orthogonal methods. Using a platform specific immunoassay, the HCP level from the original purification method was 1,830 ppm (0.18% w/w) while the revised purification process yielded the HCP below the detection limits of the assay. With a 2D/LC-MSE methodology the reference sample from the original process was found to contain 57 unique HCPs at a total level of 37,811 ppm (3.78% w/w). Two lots were tested after purification with the revised process and contained 730 and 598 ppm (0.07% and 0.06% w/w), respectively. To develop a high-throughput MS method, the samples were tested on a 1D/LC-MS/MS. The data sets from the two mass spectrometers correlated well. These improved HCP profiles support implementing the revised purification process for manufacturing the EPA protein carrier and 1D/LC-MS/MS for HCP analysis.


Assuntos
ADP Ribose Transferases/isolamento & purificação , Toxinas Bacterianas/isolamento & purificação , Cromatografia Líquida/métodos , Exotoxinas/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Fatores de Virulência/isolamento & purificação , Algoritmos , Anticorpos Monoclonais/química , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Reações Falso-Positivas , Immunoblotting , Proteólise , Proteínas Recombinantes/isolamento & purificação , Reprodutibilidade dos Testes
11.
J Surg Res ; 244: 111-116, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31279995

RESUMO

BACKGROUND: Clinical practice guidelines define Clostridium difficile infections (CDI) as diarrhea (≥3 unformed stools in 24 h) with either a positive C difficile stool test or detection of pseudomembranous colitis. Diagnostic modalities such as toxigenic culture and nucleic acid amplification testing can identify the presence of toxigenic C difficile in stools. But these tests are confounded by the presence of asymptomatic colonization of toxigenic C difficile and lead to overdiagnosis of CDI. The presence of two large toxins, toxin A and B (TcdA and TcdB) is necessary for pathogenicity. Detection of toxins using toxin enzyme immunoassay is difficult as it has low sensitivity and moderate specificity. Raman spectroscopy (RS) is a novel technology that is used to detect bacteria and their toxins. RS does not require any reagents for detection such as antibodies, enzymes, primers, or stains. We hypothesize that RS is a sensitive method to detect C difficile toxins in stool and will solve the problem of overdiagnosis of CDI. MATERIALS AND METHODS: CDI negative stool samples were spiked with concentrations (1 ng/mL, 100 pg/mL, 1 pg/mL, and 0.1 pg/mL) of TcdA and TcdB. RS was performed on air-dried smeared samples of stool supernatant on a mirror-polished stainless-steel slide. As RS of feces is difficult because of confounding background material and autofluorescence, samples were photo-bleached before spectral acquisition to reduce autofluorescence. Raman spectra were obtained, background corrected, and vector normalized. The data were split into training (70%) and test (30%) datasets. The machine learning methods used on the training data set were Support Vector Machine with Linear and Radial Kernels, Random Forest, Stochastic Gradient Boosting Machine, and Principle Component Analysis-Linear Discriminant Analysis. Results were validated using a test data set. The best model was chosen, and its accuracy, sensitivity, and specificity were determined. RESULTS: In our preliminary results, at all concentrations (1 ng/mL, 100 pg/mL, 1 pg/mL, and 0.1 pg/mL), TcdA or TcdB spiked stool was distinguished from unspiked stool by all models with accuracies ranging from 64% to 77%. Gradient Boosting Machine, Principle Component Analysis-Linear Discriminant Analysis, and Support Vector Machine Linear Kernel performed best with sensitivities ranging from 69% to 90% and specificities ranging from 43% to 78%. CONCLUSIONS: Using RS, we successfully detected TcdA and TcdB in stool samples albeit with moderate-to-high sensitivity and low-to-moderate specificity. Sensitivity and specificity could be further increased with the implementation of deep learning methods, which require large sample sizes. In terms of sensitivity, RS performs better than toxin enzyme immunoassay and has the potential to rapidly detect C difficile toxins in stool at clinically relevant concentrations and thereby help mitigate overdiagnosis of CDI.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas/isolamento & purificação , Enterocolite Pseudomembranosa/diagnóstico , Enterotoxinas/isolamento & purificação , Fezes/química , Análise Espectral Raman , Enterocolite Pseudomembranosa/microbiologia , Estudos de Viabilidade , Fezes/microbiologia , Humanos , Técnicas Imunoenzimáticas , Sensibilidade e Especificidade , Fatores de Tempo
12.
Toxins (Basel) ; 11(6)2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31234410

RESUMO

Cyanobacteria have been shown to produce a number of bioactive compounds, including toxins. Some bioactive compounds obtained from a marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) have been recognized as drug leads; one of these compounds is aplysiatoxin. We have isolated various aplysiatoxin derivatives from a M. producens sample obtained from the Okinawan coastal area. The frozen sample was extracted with organic solvents. The ethyl acetate layer was obtained from the crude extracts via liquid-liquid partitioning, then separated by HPLC using a reversed-phase column. Finally, 1.1 mg of the compound was isolated. The chemical structure of the isolated compound was elucidated with spectroscopic methods, using HR-MS and 1D and 2D NMR techniques, and was revealed to be oscillatoxin I, a new member of the aplysiatoxin family. Oscillatoxin I showed cytotoxicity against the L1210 mouse lymphoma cell line and diatom growth-inhibition activity against the marine diatom Nitzschia amabilis.


Assuntos
Toxinas Bacterianas/isolamento & purificação , Cianobactérias , Toxinas da Lyngbya/isolamento & purificação , Animais , Toxinas Bacterianas/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Diatomáceas/efeitos dos fármacos , Diatomáceas/crescimento & desenvolvimento , Toxinas da Lyngbya/toxicidade , Camundongos
13.
Microb Pathog ; 134: 103593, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31195111

RESUMO

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA+ clinical strains. The aim of this study was to evaluate UPEC tosA+ strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA+ strains and two phylogenetic groups (B2 and D) were identified, including resistance to ß-lactam and non-ß-lactam antibiotics. The UPEC tosA+ clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA+ belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule.


Assuntos
Toxinas Bacterianas/genética , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/genética , Escherichia coli Uropatogênica/genética , Fatores de Virulência/genética , Adesinas de Escherichia coli/genética , Animais , Toxinas Bacterianas/classificação , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/isolamento & purificação , Linhagem Celular , Clonagem Molecular , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/isolamento & purificação , Feminino , Regulação Bacteriana da Expressão Gênica , Aptidão Genética , Variação Genética , Humanos , Testes de Sensibilidade Microbiana , Fenótipo , Filogenia , Coelhos , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Virulência/genética
14.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
15.
Microb Pathog ; 134: 103600, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31202906

RESUMO

INTRODUCTION: Severe intestinal infections caused by V. cholerae, ETEC and EHEC have contributed to the mortality rate in developing countries. Vibrio Cholera, ETEC and EHEC bacterium with the production of CT, LT and Stx2 toxins respectively lead to severe watery and bloody diarrhea. This study aimed to investigate a trimeric vaccine candidate containing recombinant chimeric protein, encapsulate the protein in chitosan nanoparticles and assess its immunogenicity. METHODS: The LSC recombinant gene was used. It is composed of LTB (L), STXB (S) and CTXB (C) subunits respectively. The LSC recombinant protein was expressed and purified and confirmed by western blotting. The purified protein was encapsulated in chitosan nanoparticles, and its size was measured. BalB/c mice were immunized in four groups through oral and injection methods by LSC protein. The antibody titer was then evaluated by ELISA, and finally, the challenge test of the toxins from all three bacteria was done on the immunized mouse. RESULTS: After expression and purification LSC protein size of nanoparticles containing protein was measured at 104.6 nm. Nanoparticles were able to induce systemic and mucosal immune responses by generating a useful titer of IgG and IgA. The challenge results with LT, CT and Stx toxins showed that the LSC protein might partially neutralize the effect of toxins. CONCLUSION: LSC chimeric protein with the simultaneous three essential antigens have a protective effect against the toxins produced by ETEC, EHEC and Vibrio cholera bacteria and it can be used in vaccines to prevent Diarrhea caused by these three bacteria.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Quitosana/farmacologia , Imunização , Nanopartículas/química , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Diarreia/microbiologia , Diarreia/prevenção & controle , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/imunologia , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Regulação Bacteriana da Expressão Gênica , Imunidade nas Mucosas , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Proteínas Recombinantes de Fusão/genética , Toxinas Shiga/genética , Toxinas Shiga/imunologia , Análise de Sobrevida , Vibrio cholerae/genética , Vibrio cholerae/imunologia
16.
Int J Biol Macromol ; 134: 1120-1131, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31129209

RESUMO

Immunotoxins are a class of recombinant proteins which consist of an antibody and a part of a bacterial or herbal toxin. Immunotoxins containing Pseudomonas aeruginosa exotoxin A (PEA) have been found to be very applicable in clinical trials. Many obstacles such as solubility and absorbency reduce their usability in solid tumors. The current study aims to overcome the mentioned barriers by addition and removal of functional and non-functional domains with a structural approach. In the experimental section, we took advantage of molecular dynamics simulations to predict the functionality of candidate immunotoxins which target human HER2 receptors and confirmed our findings with in vitro experiments. We found out when no changes were made to domain II of PEA, addition of solubilizing domains to immunotoxins would not reduce their targeting and anti-tumor activity, while increasing the yield of expression and stability. On the other side, when we replaced domain II with eleven amino acids of furin cleavage site (FCS), the activity of the immunotoxin was mainly affected by the FCS neighboring domains and linkers. A combination of seven beneficial point mutations in domain III was also assessed and reconfirmed that the toxicity of the immunotoxin would be reduced dramatically. The obtained results indicate that the addition or removal of domains cannot depict the activity of immunotoxins and the matter should be assessed structurally in advance.


Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Imunotoxinas/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Fatores de Virulência/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/isolamento & purificação , Toxinas Bacterianas/química , Toxinas Bacterianas/isolamento & purificação , Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Exotoxinas/química , Exotoxinas/isolamento & purificação , Humanos , Imunotoxinas/química , Imunotoxinas/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão , Solubilidade , Relação Estrutura-Atividade , Sumoilação , Fatores de Virulência/química , Fatores de Virulência/isolamento & purificação
17.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1118-1119: 194-202, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31059926

RESUMO

A rapid and sensitive liquid chromatography-mass spectrometry assay was developed and used to quantify emetic cereulide peptide exotoxin, which can be related to possible Bacillus cereus contamination in monoclonal antibody (mAb) bioprocess feeds. The assay limit of detection was 0.05 ng/mL (~1 fmol injected) and limit of quantification 0.16 ng/mL (~3 fmol injected) over a standard curve with >3 orders of magnitude linear dynamic range. The assay allowed quantification of toxin removal in an established two-step mAb purification process consisting of Protein A affinity chromatography followed by multi-modal anion exchange chromatography. Toxin content was ascertained in process stream sample fractions as well as on the Protein A affinity column. An optimized analytical method allowed separation of cereulide toxin from other mAb cell culture components within 6 min. Spiking experiments showed that samples should be collected in high (80% v/v) content acetonitrile to reduce nonspecific losses of the cereulide. The majority of mAb purification process-associated cereulide was detected in the Protein A flow through fraction, whereas only residual amounts were found in wash, strip, and elution fractions. Column cleaning-in-place (CIP) procedures were evaluated to prevent carryover between affinity capture cycles. No carryover was detected between cycles, however trace amounts of cereulide were extracted from the Protein A resin. Increasing the CIP NaOH concentration from 0.1 M to 0.5 M, and contact time from 15 min to 1 h, improved removal of residual cereulide from the resin. Applicability of CIP clearance of cereulide during Protein A chromatography was confirmed with three different mAb feeds. Post Protein A polishing, via target flow through on a multi-modal anion exchange chromatography column, resulted in a product pool with no detectable cereulide. Approximately 5 logs of reduction in cereulide concentration was obtained over the two-step chromatography process. Cereulide contamination is well known and of concern in food processing, however this research may be the first LC-MS quantification of cereulide contamination, and its clearance, in biopharmaceutical mAb processing. The analytical method may also be used to rapidly screen for cereulide contamination in upstream cell culture process streams, prior to downstream product purification. This will allow appropriate measures to be taken to reduce toxin exposure to downstream bioprocess raw materials, consumables and equipment.


Assuntos
Anticorpos Monoclonais/química , Toxinas Bacterianas/isolamento & purificação , Cromatografia de Afinidade/métodos , Depsipeptídeos/isolamento & purificação , Proteína Estafilocócica A/metabolismo , Animais , Bacillus cereus , Células CHO , Cricetinae , Cricetulus , Depsipeptídeos/metabolismo , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
18.
Toxins (Basel) ; 11(4)2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987300

RESUMO

Mycolactone, the amphiphilic macrolide toxin secreted by Mycobacterium ulcerans, plays a significant role in the pathology and manifestations of Buruli ulcer (BU). Consequently, it follows that the toxin is a suitable target for the development of diagnostics and therapeutics for this disease. Yet, several challenges have deterred such development. For one, the lipophilic nature of the toxin makes it difficult to handle and store and contributes to variability associated with laboratory experimentation and purification yields. In this manuscript, we have attempted to incorporate our understanding of the lipophilicity of mycolactone in order to define the optimal methods for the storage, handling, and purification of this toxin. We present a systematic correlation of variability associated with measurement techniques (thin-layer chromatography (TLC), mass spectrometry (MS), and UV-Vis spectrometry), storage conditions, choice of solvents, as well as the impact of each of these on toxin function as assessed by cellular cytotoxicity. We also compared natural mycolactone extracted from bacterial culture with synthesized toxins in laboratory (solvents, buffers) and physiologically relevant (serum) matrices. Our results point to the greater stability of mycolactone in organic, as well as detergent-containing, solvents, regardless of the container material (plastic, glass, or silanized tubes). They also highlight the presence of toxin in samples that may be undetectable by any one technique, suggesting that each detection approach captures different configurations of the molecule with varying specificity and sensitivity. Most importantly, our results demonstrate for the very first time that amphiphilic mycolactone associates with host lipoproteins in serum, and that this association will likely impact our ability to study, diagnose, and treat Buruli ulcers in patients.


Assuntos
Toxinas Bacterianas , Macrolídeos , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Camada Delgada , Humanos , Lipoproteínas HDL/química , Lipoproteínas LDL/química , Macrolídeos/química , Macrolídeos/isolamento & purificação , Macrolídeos/toxicidade , Camundongos , Mycobacterium ulcerans , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta
19.
Bull Exp Biol Med ; 166(6): 759-765, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31028588

RESUMO

Campylobacter genus bacteria causing campylobacteriasis are difficult to culture. This fact necessitates creation of special approaches to studies of the behavior of these pathogens during the manufacture and storage of foodstuffs. The regularities of Campylobacter jejuni transition into an uncultivable state are studied under conditions simulating the process of immersion cooling of fresh poultry products. The proportion of viable colony-forming (CFU) cells to the total count of planktonic and uncultivable cells in the population was calculated by the level of genomic DNA in the samples evaluated by quantitative real-time PCR with intercalating dyes. PCR was carried out with primers detecting the cytolethal toxin subunit B gene cdtB and invasion gene ciaB in C. jejuni strains. The count of detected cells was 5-10-fold higher than the count of CFU; the cultural method failed to detect the agent in 40% analyzed samples of superficially infected products, while the level of uncultivable cells detected by PCR was significantly higher. The relationship between culturing conditions and formation of C. jejuni biofilms was studied. The most intensive formation of film exomatrix was observed under unfavorable conditions for this microorganism at 25oC. In microaerophilic gaseous medium, weak formation of films and intensive growth of C. jejuni populations were observed. Culturing at higher temperatures (37-42oC) was in fact inessential for the film formation process.


Assuntos
Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Campylobacter jejuni/crescimento & desenvolvimento , Produtos Avícolas/microbiologia , Animais , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/metabolismo , Carga Bacteriana , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Galinhas , Contagem de Colônia Microbiana , Contaminação de Alimentos/análise , Expressão Gênica , Humanos , Reação em Cadeia da Polimerase , Temperatura
20.
J Microbiol ; 57(6): 450-460, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31012060

RESUMO

Next-generation DNA sequencing technology was applied to generate molecular data from semiarid reservoirs during well-defined seasons. Target sequences of 16S-23S rRNA ITS and cpcBA-IGS were used to reveal the taxonomic groups of cyanobacteria present in the samples, and genes coding for cyanotoxins such as microcystins (mcyE), saxitoxins (sxtA), and cylindrospermopsins (cyrJ) were investigated. The presence of saxitoxins in the environmental samples was evaluated using ELISA kit. Taxonomic analyses of high-throughput DNA sequencing data showed the dominance of the genus Microcystis in Mundaú reservoir. Furthermore, it was the most abundant genus in the dry season in Ingazeira reservoir. In the rainy season, 16S-23S rRNA ITS analysis revealed that Cylindrospermopsis raciborskii comprised 46.8% of the cyanobacterial community in Ingazeira reservoir, while the cpcBAIGS region revealed that C. raciborskii (31.8%) was the most abundant taxon followed by Sphaerospermopsis aphanizomenoides (17.3%) and Planktothrix zahidii (16.6%). Despite the presence of other potential toxin-producing genera, the detected sxtA gene belonged to C. raciborskii, while the mcyE gene belonged to Microcystis in both reservoirs. The detected mcyE gene had good correlation with MC content, while the amplification of the sxtA gene was related to the presence of STX. The cyrJ gene was not detected in these samples. Using DNA analyses, our results showed that the cyanobacterial composition of Mundaú reservoir was similar in successive dry seasons, and it varied between seasons in Ingazeira reservoir. In addition, our data suggest that some biases of analysis influenced the cyanobacterial communities seen in the NGS output of Ingazeira reservoir.


Assuntos
Biodiversidade , Cianobactérias/classificação , Cianobactérias/isolamento & purificação , Água Potável/microbiologia , Análise de Sequência de DNA/métodos , Microbiologia da Água , Abastecimento de Água , Alcaloides , Toxinas Bacterianas/análise , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Brasil , Cianobactérias/genética , DNA Bacteriano/análise , Monitoramento Ambiental/métodos , Genes Bacterianos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Microcystis/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Saxitoxina/genética , Estações do Ano , Uracila/análogos & derivados
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