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1.
Gene ; 759: 144999, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32717305

RESUMO

Clostridium perfringens beta2 (CPB2), a key virulence factor, is produced by C. perfringens type C that is the main pathogenic microorganism causing diarrhea in piglets. However, little is known concerning the toxic damage effect of CPB2 on intestinal cells of piglets. In present study, CPB2 toxin obtained by genetic recombination technology was evaluated for its cytotoxicity property using the intestinal porcine epithelial (IPEC-J2) cells, which aims to attempt to understand and explain its mechanism of action in porcine small intestinal epithelial cells. IPEC-J2 cells were treated with different concentrations of CPB2 toxin (5, 10, 20, 30, 40, and 50 µg/mL), and MTT assay results showed that the cell viability of CPB2-treated IPEC-J2 cells decreased in a dose-dependent manner. Lactate dehydrogenase (LDH) assay results revealed that CPB2 significantly increased the LDH release, relative to the control. The expression of tumor necrosis factor α (TNF-α) and interleukin 8 (IL-8) gradually increased, while the expression of interleukin 10 (IL-10) gradually decreased in IPEC-J2 cells with increasing concentration of CPB2 (10-30 µg/mL), as analyzed by quantitative real-time PCR (RT-qPCR). Also, CPB2 increased the content of intracellular reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) of IPEC-J2 cells. Western blot and immunofluorescence results demonstrate that CPB2 decreased the expression of zonula occludens (ZO-1), claudin12 (CLDN12) and occludin (OCLN) in IPEC-J2 cells. In addition, CPB2 increased Bax expression, and inhibited Bcl-2 and Bcl-xL expression, as measured by Western blot. Considering all of these findings, it was concluded that CPB2 toxin shows significant cytotoxicity, cell growth inhibition and increase in cell permeability in IPEC-J2 cells in a concentration-dependent manner, thus leading to abnormal cell apoptosis and functions in porcine small intestinal epithelial cells.


Assuntos
Toxinas Bacterianas/toxicidade , Células Epiteliais/efeitos dos fármacos , Estresse Oxidativo , Animais , Apoptose , Linhagem Celular , Claudinas/genética , Claudinas/metabolismo , Células Epiteliais/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Potencial da Membrana Mitocondrial , Ocludina/genética , Ocludina/metabolismo , Suínos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
2.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G121-G132, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32567324

RESUMO

Nongenomic glucocorticoid (GC) and serum- and glucocorticoid-inducible kinase 1 (SGK1) signaling regulate ion transport, but CFTR has not been investigated in the intestine. We examined GC, SGK1, and phosphatidylinositol 3-kinase (PI3K) kinase signaling of CFTR ion transport in native intestine and the role of GCs on mRNA, protein, surface expression, and cyclic guanosine monophosphate (cGMP)-elicited diarrhea. Rats were treated with dexamethasone (DEXA; 2 mg/kg ip) or DMSO for 1, 4, and 24 h. Cyclic adenosine monophosphate (cAMP)-activated ion transport was examined in the presence or absence of SGK1 and PI3K inhibitors. Phosphorylation of SGK1, phosphoinositide-dependent kinase 1, and Akt kinases was confirmed by immunoblots using phosphor-specific antibodies. Tissue lysates were analyzed by mass spectrometry. CFTR and SGK1 mRNA were measured by quantitative PCR. Changes in total and surface CFTR protein were determined. The role of GC in cGMP-activated CFTR ion transport was examined. GC synergistically increased CFTR ion transport by SGK1 and PI3K signaling and increased CFTR protein without altering SGK1 or CFTR mRNA. GC induced highest levels of CFTR protein at 4 h that were associated with marked increase in surface CFTR, phosphorylation of the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-like (Nedd4-2), and 14-3-3ε, supporting their roles in surface retention and stability. Coimmunoprecipitation of CFTR, Nedd4-2, and 14-3-3ε indicated that assembly of this complex is a likely effector of the SGK and Akt pathways. Mass spectrometry identified phosphorylated peptides in relevant proteins. GC-SGK1 potently regulates CFTR in the intestine and is implicated in diarrheal disease.NEW & NOTEWORTHY This is the first study to examine the mechanisms of glucocorticoid, serum- and glucocorticoid-inducible kinase 1, and nongenomic kinase signaling of CFTR in the native intestine. We identified unique and druggable intestine-specific factors of the pathway that are targets for treating stress-induced diarrhea.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dexametasona/toxicidade , Diarreia/etiologia , Dimetil Sulfóxido/toxicidade , Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animais , Toxinas Bacterianas/toxicidade , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Diarreia/induzido quimicamente , Enterotoxinas/toxicidade , Proteínas de Escherichia coli/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces/genética , Masculino , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinase Piruvato Desidrogenase (Transferência de Acetil)/genética , Quinase Piruvato Desidrogenase (Transferência de Acetil)/metabolismo , Ratos , Ratos Sprague-Dawley , Trocador 3 de Sódio-Hidrogênio/genética , Trocador 3 de Sódio-Hidrogênio/metabolismo
3.
Nat Commun ; 11(1): 2694, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483155

RESUMO

Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.


Assuntos
Toxinas Bacterianas/toxicidade , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Polissacarídeos/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacocinética , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Heparina/química , Heparina/metabolismo , Humanos , Insetos/microbiologia , Antígenos CD15/química , Antígenos CD15/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Morganella morganii/patogenicidade , Photorhabdus/patogenicidade , Polissacarídeos/química , Xenorhabdus/patogenicidade
4.
Medicine (Baltimore) ; 99(20): e19617, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32443285

RESUMO

To describe the epidemiological, clinical, laboratory, and radiological features and the management of adult patients who experienced a relapse between 2003 and 2015 of an acute hematogenous osteomyelitis acquired in childhood.A retrospective multicentric cohort study was conducted in 5 centers in France.Thirty-seven patients were included. The median age was 40 years (28-56), and 26 (70%) were male. The first site of infection was the distal femur (n = 23, 62%). The median time between the osteomyelitis in childhood and the relapse in adulthood was 26 years (13-45). Thirty-four (92%) patients reported inflammatory local clinical manifestations, 17 (46%) draining fistula, 10 (27%) fever. Most patients had intramedullary gadolinium deposition (with or without abscess) on magnetic resonance imaging. Most relapses were monomicrobial infections (82%). Staphylococcus aureus was the most commonly found microorganism (82%), expressing a small colony variant phenotype in 3 cases. Most patients (97%) had a surgical treatment, and the median duration of antibiotics for the relapse was 12 weeks. All patients had a favorable outcome, no patient died and no further relapse occurred. We count 2 femoral fractures on osteotomy site.Osteomyelitis in childhood can relapse later in adulthood, especially in patients with lack of care during the initial episode. Osteotomy and prolonged antimicrobial therapy are required for clinical remission.


Assuntos
Osteomielite/epidemiologia , Adulto , Idoso , Toxinas Bacterianas/toxicidade , Exotoxinas/toxicidade , Feminino , França/epidemiologia , Humanos , Leucocidinas/toxicidade , Masculino , Pessoa de Meia-Idade , Osteomielite/diagnóstico por imagem , Osteomielite/microbiologia , Osteomielite/terapia , Recidiva , Estudos Retrospectivos , Infecções Estafilocócicas/complicações , Adulto Jovem
5.
Molecules ; 25(10)2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32455942

RESUMO

Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2') cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species and the level of pathogenicity. Recent comparisons of the genomes of the highly pathogenic SARS-CoV2 and MERS-CoV, with less pathogenic strains (e.g., Bat-RaTG13, the bat homologue of SARS-CoV2) identified possible mutations in the receptor binding domain and in the S1 and S2' cleavage sites of their spike glycoprotein. However, there remains some confusion on the relative roles of the possible serine proteases involved for priming. Using anthrax toxin as a model system, we show that in vivo inhibition of priming by pan-active serine protease inhibitors can be effective at suppressing toxicity. Hence, our studies should encourage further efforts in developing either pan-serine protease inhibitors or inhibitor cocktails to target SARS-CoV2 and potentially ward off future pandemics that could develop because of additional mutations in the S-protein priming sequence in coronaviruses.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/farmacologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Antígenos de Bactérias/toxicidade , Antivirais/uso terapêutico , Toxinas Bacterianas/toxicidade , Betacoronavirus/patogenicidade , Sítios de Ligação , Sistemas de Liberação de Medicamentos , Feminino , Furina/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Pandemias , Células RAW 264.7 , Inibidores de Serino Proteinase/uso terapêutico , Glicoproteína da Espícula de Coronavírus/química
6.
PLoS One ; 15(5): e0233854, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470006

RESUMO

Alpha-toxin (Hla) is a major virulence factor of Staphylococcus aureus (S. aureus) and plays an important role in S. aureus-induced pneumonia. It binds as a monomer to the cell surface of eukaryotic host cells and forms heptameric transmembrane pores. Sensitivities toward the toxin of various types of potential host cells have been shown to vary substantially, and the reasons for these differences are unclear. We used three human model airway epithelial cell lines (16HBE14o-, S9, A549) to correlate cell sensitivity (measured as rate of paracellular gap formation in the cell layers) with Hla monomer binding, presence of the potential Hla receptors ADAM10 or α5ß1 integrin, presence of the toxin-stabilizing factor caveolin-1 as well as plasma membrane lipid composition (phosphatidylserine/choline, sphingomyelin). The abundance of ADAM10 correlated best with gap formation or cell sensitivities, respectively, when the three cell types were compared. Caveolin-1 or α5ß1 integrin did not correlate with toxin sensitivity. The relative abundance of sphingomyelin in plasma membranes may also be used as a proxi for cellular sensitivity against alpha-toxin as sphingomyelin abundances correlated well with the intensities of alpha-toxin mediated gap formation in the cell layers.


Assuntos
Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidade , Interações Hospedeiro-Patógeno , Sistema Respiratório/patologia , Células A549 , Caveolina 1/metabolismo , Membrana Celular/efeitos dos fármacos , Tamanho Celular , Células Epiteliais/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Modelos Biológicos , Fosfolipídeos/metabolismo , Ligação Proteica , Receptores de Superfície Celular/metabolismo
7.
Mol Phylogenet Evol ; 148: 106824, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32294544

RESUMO

Raphidiopsis (Cylindrospermopsis) raciborskii, a globally distributed bloom-forming cyanobacterium, produces either the cytotoxin cylindrospermopsin (CYL) in Oceania, Asia and Europe or the neurotoxin saxitoxin (STX) and analogues (paralytic shellfish poison, PSP) in South America (encoded by sxt genetic cluster) and none of them in Africa. Nevertheless, this particular geographic pattern is usually overlooked in current hypotheses about the species dispersal routes. Here, we combined genomics, phylogenetic analyses, toxicity data and a literature survey to unveil the evolutionary history and spread of the species. Phylogenies based on 354 orthologous genes from all the available genomes and ribosomal ITS sequences of the taxon showed two well-defined clades: the American, having the PSP producers; and the Oceania/Europe/Asia, including the CYL producers. We propose central Africa as the original dispersion center (non-toxic populations), reaching North Africa and North America (in former Laurasia continent). The ability to produce CYL probably took place in populations that advanced to sub-Saharan Africa and then to Oceania and South America. According to the genomic context of the sxt cluster found in PSP-producer strains, this trait was acquired once by horizontal transfer in South America, where the ability to produce CYL was lost.


Assuntos
Toxinas Bacterianas/toxicidade , Cylindrospermopsis/classificação , Cylindrospermopsis/genética , Genômica , Filogenia , Filogeografia , Saxitoxina/toxicidade , Uracila/análogos & derivados , Sequência Conservada/genética , Funções Verossimilhança , Família Multigênica , Sintenia/genética , Uracila/toxicidade
8.
PLoS Negl Trop Dis ; 14(3): e0008060, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163415

RESUMO

The northeast (NE) region of Brazil commonly goes through drought periods, which favor cyanobacterial blooms, capable of producing neurotoxins with implications for human and animal health. The most severe dry spell in the history of Brazil occurred between 2012 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus (ZIKV) outbreak took place in the NE region of Brazil during the same years. In this work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance showed higher cyanobacteria amounts and STX occurrence in human drinking water supplies of NE compared to other regions of Brazil. Experimentally, we described that STX doubled the quantity of ZIKV-induced neural cell death in progenitor areas of human brain organoids, while the chronic ingestion of water contaminated with STX before and during gestation caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a public health concern regarding the consequences of arbovirus outbreaks happening in areas with droughts and/or frequent freshwater cyanobacterial blooms.


Assuntos
Morte Celular/efeitos dos fármacos , Microcefalia/patologia , Envenenamento/complicações , Envenenamento/patologia , Saxitoxina/toxicidade , Infecção por Zika virus/complicações , Infecção por Zika virus/patologia , Animais , Toxinas Bacterianas/análise , Toxinas Bacterianas/toxicidade , Encéfalo/patologia , Brasil/epidemiologia , Células Cultivadas , Modelos Animais de Doenças , Surtos de Doenças , Feminino , Humanos , Incidência , Toxinas Marinhas/análise , Toxinas Marinhas/toxicidade , Camundongos Endogâmicos C57BL , Microcistinas/análise , Microcistinas/toxicidade , Modelos Teóricos , Neurotoxinas/análise , Neurotoxinas/toxicidade , Saxitoxina/análise , Água/química
9.
PLoS One ; 15(3): e0219275, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163417

RESUMO

Pathogenic bacteria often damage tissues by secreting toxins that form pores in cell membranes, and the most common pore-forming toxins are cholesterol-dependent cytolysins. During bacterial infections, glutamine becomes a conditionally essential amino acid, and glutamine is an important nutrient for immune cells. However, the role of glutamine in protecting tissue cells against pore-forming toxins is unclear. Here we tested the hypothesis that glutamine supports the protection of tissue cells against the damage caused by cholesterol-dependent cytolysins. Stromal and epithelial cells were sensitive to damage by the cholesterol-dependent cytolysins, pyolysin and streptolysin O, as determined by leakage of potassium and lactate dehydrogenase from cells, and reduced cell viability. However, glutamine deprivation increased the leakage of lactate dehydrogenase and reduced the viability of cells challenged with cholesterol-dependent cytolysins. Without glutamine, stromal cells challenged with pyolysin leaked lactate dehydrogenase (control vs. pyolysin, 2.6 ± 0.6 vs. 34.4 ± 4.5 AU, n = 12), which was more than three-fold the leakage from cells supplied with 2 mM glutamine (control vs. pyolysin, 2.2 ± 0.3 vs. 9.4 ± 1.0 AU). Glutamine cytoprotection did not depend on glutaminolysis, replenishing the Krebs cycle via succinate, changes in cellular cholesterol, or regulators of cell metabolism (AMPK and mTOR). In conclusion, although the mechanism remains elusive, we found that glutamine supports the protection of tissue cells against the damage caused by cholesterol-dependent cytolysins from pathogenic bacteria.


Assuntos
Colesterol/metabolismo , Citoproteção/efeitos dos fármacos , Citotoxinas/toxicidade , Glutamina/farmacologia , Animais , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Bovinos , Células HeLa , Proteínas Hemolisinas/toxicidade , Humanos , L-Lactato Desidrogenase/metabolismo , Estreptolisinas/toxicidade , Células Estromais/efeitos dos fármacos
10.
Ecotoxicol Environ Saf ; 194: 110444, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32169726

RESUMO

Nodularin (NOD) is a kind of cyanobacterial toxins. It is of concern due to elicit severe genotoxicity in humans and animals. The comprehensive evaluation of NOD-induced adverse effects in living organisms is urgently needed. This study is aimed to report the developmental toxicity and molecular mechanism using zebrafish embryos exposed to NOD. The embryonic toxicity induced by NOD is demonstrated by inhibition of embryo hatching, increase in mortality rate, abnormal heart rate, embryonic malformation as well as defects in angiogenesis and common cardinal vein remodeling. NOD triggered a decreased rate of angiogenesis through inhibiting endothelial cells migration. NOD induced embryonic cell apoptosis and DNA damage, which can be alleviated by antioxidant N-acetyl-L-cysteine. NOD significantly caused oxidative damage as indicated by changes in reactive oxygen species, superoxide dismutase, catalase, glutathione and malondialdehyde. NOD also altered the expression of vascular development-genes (DLL4, CDH5, VEGFA, VEGFC) and apoptosis-related genes (BAX, BCL-2, P53, CASPASE 3). Taken together, NOD induced adverse effect on zebrafish embryos development, which may be associated with oxidative stress and apoptosis through the activation of P53-BAX/BCL-2-CASPASE 3-mediated pathway.


Assuntos
Toxinas Bacterianas/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Microcistinas/toxicidade , Peptídeos Cíclicos/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/embriologia , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Dano ao DNA , Células Endoteliais/metabolismo , Humanos , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra/metabolismo
11.
Arch Insect Biochem Physiol ; 104(2): e21673, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32212396

RESUMO

Three-domain Cry toxins from the bacterium Bacillus thuringiensis (Bt) are increasingly used in agriculture to replace chemical insecticides in pest control. Most chemical insecticides kill pest insects swiftly, but are also toxic to beneficial insects and other species in the agroecosystem. Cry toxins enjoy the advantages of high selectivity and the possibility of the application by sprays or transgenic plants. However, these benefits are offset by the limited host range and the evolution of resistance to Bt toxins by insect pests. Understanding how Bt toxins kill insects will help to understand the nature of both problems. The recent realization that ABC transporters play a central role in the killing mechanism will play an important role in devising solutions.


Assuntos
Bacillus thuringiensis/química , Toxinas Bacterianas/farmacologia , Agentes de Controle Biológico/farmacologia , Evolução Biológica , Insetos/efeitos dos fármacos , Controle Biológico de Vetores , Animais , Toxinas Bacterianas/toxicidade , Agentes de Controle Biológico/toxicidade , Insetos/microbiologia , Plantas Geneticamente Modificadas/microbiologia
12.
Proc Natl Acad Sci U S A ; 117(14): 8064-8073, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198200

RESUMO

Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, PLoS One 8, e73204 (2013); S. Kozar et al., Cell Stem Cell 13, 626-633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen Clostridioides difficile, we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Infecções por Clostridium/patologia , Clostridium difficile/patogenicidade , Colo/patologia , Mucosa Intestinal/patologia , Células-Tronco/patologia , Animais , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Células Cultivadas , Infecções por Clostridium/microbiologia , Clostridium difficile/metabolismo , Colo/citologia , Colo/microbiologia , Modelos Animais de Doenças , Feminino , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Camundongos , Organoides , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células-Tronco/microbiologia
13.
PLoS One ; 15(2): e0228959, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084148

RESUMO

Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77, O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Toxinas Bacterianas/toxicidade , Células CACO-2 , Citotoxinas/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/toxicidade , Humanos , Serina Endopeptidases/metabolismo , Sistemas de Secreção Tipo V/metabolismo , Fatores de Virulência/metabolismo
14.
Environ Res ; 183: 109236, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32062183

RESUMO

Cylindrospermopsin (CYN) is a widely distributed cyanobacterial toxin in water bodies and is considered to pose growing threats to human and environmental health. Although its potential toxicity has been reported, its effects on the vascular system are poorly understood. In this study, we examined the toxic effects of CYN on vascular development and the possible mechanism of vascular toxicity induced by CYN using zebrafish embryos and human umbilical vein endothelial cells (HUVECs). CYN exposure induced abnormal vascular development and led to an increase in the growth of common cardinal vein (CCV), in which CCV remodeling was delayed as reflected by the larger CCV area and wider ventral diameter. CYN decreased HUVECs viability, inhibited HUVECs migration, promoted HUVECs apoptosis, destroyed cytoskeleton, and increased intracellular ROS levels. Additionally, CYN could promote the expression of Bax, Bcl-2, and MLC-1 and inhibit the expression of ITGB1, Rho, ROCK, and VIM-1. Taken together, CYN may induce cytoskeleton damage and promote vascular endothelial cell apoptosis by the Rho/ROCK signaling pathway, leading to abnormal vascular development. The current results provide potential insight into the mechanism of CYN toxicity in angiocardiopathy and are beneficial for understanding the environmental risks of CYN for aquatic organisms and human health.


Assuntos
Apoptose , Toxinas Bacterianas , Uracila/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Citoesqueleto/efeitos dos fármacos , Humanos , Transdução de Sinais , Cordão Umbilical/citologia , Uracila/toxicidade
15.
Vet Res ; 51(1): 27, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093740

RESUMO

The pore-forming protein epsilon toxin (Etx) from Clostridium perfringens produces acute perivascular edema affecting several organs, especially the brain and lungs. Despite the toxin evident effect on microvasculature and endothelial cells, the underlying molecular and cellular mechanisms remain obscure. Moreover, no Etx-sensitive endothelial cell model has been identified to date. Here, we characterize the mouse lung endothelial cell line 1G11 as an Etx-sensitive cell line and compare it with the well-characterized Etx-sensitive Madin-Darby canine kidney epithelial cell line. Several experimental approaches, including morphological and cytotoxic assays, clearly demonstrate that the 1G11 cell line is highly sensitive to Etx and show the specific binding, oligomerization, and pore-forming activity of the toxin in these cells. Recently, the myelin and lymphocyte (MAL) protein has been postulated as a putative receptor for Etx. Here, we show the presence of Mal mRNA in the 1G11 cell line and the presence of the MAL protein in the endothelium of some mouse lung vessels, supporting the hypothesis that this protein is a key element in the Etx intoxication pathway. The existence of an Etx-sensitive cell line of endothelial origin would help shed light on the cellular and molecular mechanisms underlying Etx-induced edema and its consequences.


Assuntos
Toxinas Bacterianas/toxicidade , Células Endoteliais/efeitos dos fármacos , Animais , Linhagem Celular , Infecções por Clostridium/metabolismo , Clostridium perfringens/fisiologia , Pulmão/efeitos dos fármacos , Camundongos
16.
Int J Med Sci ; 17(2): 145-152, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32038097

RESUMO

The azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model is commonly used to study colitis-associated cancer. The human commensal bacterium, enterotoxigenic Bacteroides fragilis (ETBF) secretes the Bacteroides fragilis toxin (BFT) which is necessary and sufficient to cause colitis. We report that BALB/c mice infected with WT-ETBF and administered three cycles of AOM/DSS developed numerous, large-sized polyps predominantly in the colorectal region. In addition, AOM/DSS-treated BALB/c mice orally inoculated with wild-type nontoxigenic Bacteroides fragilis (WT-NTBF) overexpressing bft (rETBF) developed numerous polyps whereas mice infected with WT-NTBF overexpressing a biologically inactive bft (rNTBF) did not promote polyp formation. Unexpectedly, the combination of AOM+ETBF did not induce polyp formation whereas ETBF+DSS did induce polyp development in a subset of BALB/c mice. In conclusion, WT-ETBF promoted polyp development in AOM/DSS murine model with increased colitis in BALB/c mice. The model described herein provides an experimental platform for understanding ETBF-induced colonic tumorigenesis and studying colorectal cancer in wild-type mice.


Assuntos
Infecções por Bacteroides/patologia , Carcinogênese/genética , Colite/patologia , Neoplasias Colorretais/patologia , Animais , Azoximetano/toxicidade , Toxinas Bacterianas/toxicidade , Infecções por Bacteroides/induzido quimicamente , Infecções por Bacteroides/complicações , Infecções por Bacteroides/microbiologia , Bacteroides fragilis/patogenicidade , Carcinogênese/induzido quimicamente , Colite/induzido quimicamente , Colite/complicações , Colite/microbiologia , Colo/efeitos dos fármacos , Colo/patologia , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/complicações , Neoplasias Colorretais/microbiologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Humanos , Metaloendopeptidases/toxicidade , Camundongos , Pólipos/induzido quimicamente
17.
Aquat Toxicol ; 220: 105399, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31896464

RESUMO

There is little information in scientific literature as to how conditions created by a microcystin (MC) producing cyanobacterial bloom affect the oxidant/antioxidant, biotransformation and neurotoxicity parameters in adult frogs in situ. We investigated biochemical parameters in the skin and muscle of Pelophylax kl. esculentus from Lake Ludas (Serbia) by comparing frogs that live on the northern bloom side (BS) of the lake with those that inhabit the southern no-bloom side (NBS). A higher protein carbonylation level and lower antioxidant defense system capability in the skin of frogs living in conditions of the cyanobacterial bloom were observed. Inhibition of glutathione-dependent machinery was the major mechanism responsible for the induction of cyanobacterial bloom-mediated oxidative stress in frog skin. On the other hand, the detected higher ability of muscle to overcome bloom prooxidant toxicity was linked to a higher efficiency of the biotransformation system through glutathione-S-transferase activity and/or was the consequence of indirect exposure of the tissue to the bloom. Our results have also revealed that the cyanobacterial bloom conditions induced the cholinergic neurotransmitter system in both tissues. This study provides a better understanding of the ecotoxicological impact of the MC producing cyanobacterial bloom on frogs in situ. However, further investigations of the complex mechanism involved in cyanobacterial bloom toxicity in real environmental conditions are required.


Assuntos
Toxinas Bacterianas/toxicidade , Cianobactérias/metabolismo , Eutrofização , Toxinas Marinhas/toxicidade , Microcistinas/toxicidade , Músculos/efeitos dos fármacos , Rana esculenta/metabolismo , Pele/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Antioxidantes/metabolismo , Toxinas Bacterianas/metabolismo , Biotransformação , Cianobactérias/crescimento & desenvolvimento , Monitoramento Ambiental , Lagos/química , Toxinas Marinhas/metabolismo , Microcistinas/metabolismo , Músculos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Rana esculenta/crescimento & desenvolvimento , Sérvia , Pele/metabolismo , Poluentes Químicos da Água/metabolismo
18.
Environ Pollut ; 259: 113890, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31918145

RESUMO

Eutrophication and warming lead to frequent occurrence of cyanobacterial blooms, which significantly impact on zooplankton. Freshwater zooplankton Daphnia adopts two distinct ways of reproduction: asexual (parthenogenetic) reproduction for rapidly reproducing many offspring in favorable environment and sexual reproduction for producing resting eggs as seed bank to survive in harsh environments. Daphnia pulex has worse performance in growth and reproduction under the exposure to toxic cyanobacteria Microcystis aeruginosa and tends to allocate less energy to reproduction in the case of insufficient food. However, the relative reproductive allocation strategy (energy allocation) of D. pulex individuals exposed to toxic M. aeruginosa is still unclear. Here we tested the relative reproductive performance of D. pulex fed on solely Chlorella pyrenoidosa (high quality food) or Chlorella mixed with toxic M. aeruginosa (low quality food), based on the parthenogenetic reproduction (life-history experiments) and sexual reproduction (population experiments). The results showed that under low quality food conditions, D. pulex reproduced fewer offspring which were also smaller and thus led to a reduced absolute output in parthenogenetic reproduction, but produced ephippia in the same size and quantity compared to those cultured under high quality food conditions. However, as the body size of maternal D. pulex cultured under low quality food conditions decreased, the relative reproductive allocation significantly increased in both parthenogenetic and sexual reproduction, compared to those cultured under high quality food conditions. In conclusion, D. pulex tend to allocate relatively more energy to reproduction under Microcystis conditions, which is a reasonable strategy for it to decentralize the risks from low-quality food.


Assuntos
Toxinas Bacterianas , Chlorella , Daphnia , Toxinas Marinhas , Microcistinas , Microcystis , Animais , Toxinas Bacterianas/toxicidade , Daphnia/efeitos dos fármacos , Daphnia/metabolismo , Toxinas Marinhas/toxicidade , Microcistinas/toxicidade , Microcystis/química , Reprodução/efeitos dos fármacos
19.
Ecotoxicol Environ Saf ; 191: 110222, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31982683

RESUMO

Cylindrospermopsin (CYN) is a cyanotoxicant which occurrence is increasing due to climate change. Cylindrospermopsin is able to exert damage in the organism at several levels, among them, in the nervous system. Moreover, it is important to take into account that it is not usually present isolated in nature, but in combination with some other pollutants, being the case of the pesticide chlorpyrifos (CPF). Thus, the aim of the present work was to assess the effects of the interaction of CYN in combination with CPF in the human neuroblastoma cell line SH-SY5Y by evaluating cytotoxicity and mechanistic endpoints. The mixtures 0.25 + 21, 0.5 + 42, 1 + 84 µg/mL of CYN + CPF based on cytotoxicity results, were evaluated, and the isobologram method detected an antagonistic effect after 24 and 48 h of exposure. Moreover, although no alterations of reactive oxygen species were detected, a significant decrease of glutathione levels was observed after exposure to both, CPF alone and the combination, at all the concentrations and times of exposure assayed. In addition, CYN + CPF caused a marked decrease in the acetylcholinesterase activity, providing similar values to CPF alone. However, these effects were less severe than expected. All these findings, together with the morphological study results, point out that it is important to take into account the interaction of CYN with other pollutants. Further research is required to contribute to the risk assessment of CYN and other contaminants considering more realistic exposure scenarios.


Assuntos
Toxinas Bacterianas/toxicidade , Clorpirifos/toxicidade , Inibidores da Colinesterase/toxicidade , Poluentes Ambientais/toxicidade , Inseticidas/toxicidade , Uracila/análogos & derivados , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Uracila/toxicidade
20.
Artif Cells Nanomed Biotechnol ; 48(1): 452-462, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31918570

RESUMO

Clostridium difficile (C. difficile) infection results in toxin-induced epithelial injury and marked colonic inflammation. Mitogen-activated protein kinase (MAPK) and NF-κB which regulated by MAP kinase phosphatase (MKP, also known as dual specificity phosphatases, DUSP) are fundamental signalling pathways that mediate multiple cellular processes. However, the regulation of DUSP/MAPKs and NF-κB pathway in C. difficile-induced colonic inflammation remains unclear. Here, we report that TcdB significantly inhibits cell viability and induces production of IL-1ß and TNF-α and activation of MAPKs and NF-κB. An E3-ubiquitin ligase, TRIM46, ubiquitinates DUSP1, and its knockdown significantly inhibit TcdB-induced activation of MAPKs and NF-κB and production of IL-1ß and TNF-α. Moreover, TRIM46 overexpression induced production of IL-1ß and TNF-α also reversed by DUSP1 overexpression. We further found that promoter of TRIM46 also demonstrated binding to NF-κBp65, leading to regulate TRIM46 expression. In addition, the increased colonic inflammation induced by C. difficile administration was inhibited by TRIM46 knockdown in vivo. Taken together, the present study shows that TRIM46, as a new regulator of DUSP1/MAPKs and NF-κB signalling pathway, plays an important role in TcdB-induced colonic inflammation.


Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Clostridium difficile/metabolismo , Colo/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Fator de Transcrição RelA/metabolismo , Linhagem Celular , Colo/patologia , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia
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