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1.
BMC Infect Dis ; 19(1): 940, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31699037

RESUMO

BACKGROUND: Bacillus anthracis causes a highly lethal infectious disease primarily due to toxin-mediated injury. Antibiotics are no longer effective to treat the accumulation of anthrax toxin, thereby new strategies of antibody treatment are essential. Two anti- anthrax protective antigen (PA) antibodies, hmPA6 and PA21, have been reported by our lab previously. METHODS: The mechanisms of the two antibodies were elucidated by Electrophoresis, Competitive Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test, and in vitro, in vivo (F344 rats) treatment test. The epitopes of the two antibodies were proved by Western blot and Enzyme-linked immune sorbent assay with different domains of PA. RESULTS: In this study, we compared affinity and neutralization of these two antibodies. PA21 was better in protecting cells and rats, whereas hmPA6 had higher affinity. Furthermore, the neutralization mechanisms of the two antibodies and their recognition domains of PA were studied. The results showed that hmPA6 recognized domain IV, thus PA could not bind to cell receptors. Conversely, PA21 recognized domain II, thereby limiting heptamer oligomerization of PA63 in cells. CONCLUSIONS: Our studies elucidated the mechanisms and epitopes of hmPA6 and PA21. The present investigation can advance future use of the two antibodies in anthrax treatment or prophylaxis, and potentially as a combination treatment as the antibodies target different epitopes.


Assuntos
Anticorpos Antibacterianos/metabolismo , Anticorpos Neutralizantes/metabolismo , Bacillus anthracis/imunologia , Animais , Antraz/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eletroforese , Epitopos/análise , Epitopos/imunologia , Imunoensaio , Ratos , Ratos Endogâmicos F344
2.
PLoS Pathog ; 15(9): e1007921, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31568537

RESUMO

Humans are frequently exposed to bacterial genotoxins involved in digestive cancers, colibactin and Cytolethal Distending Toxin (CDT), the latter being secreted by many pathogenic bacteria. Our aim was to evaluate the effects induced by these genotoxins on nuclear remodeling in the context of cell survival. Helicobacter infected mice, coculture experiments with CDT- and colibactin-secreting bacteria and hepatic, intestinal and gastric cells, and xenograft mouse-derived models were used to assess the nuclear remodeling in vitro and in vivo. Our results showed that CDT and colibactin induced-nuclear remodeling can be associated with the formation of deep cytoplasmic invaginations in the nucleus of giant cells. These structures, observed both in vivo and in vitro, correspond to nucleoplasmic reticulum (NR). The core of the NR was found to concentrate ribosomes, proteins involved in mRNA translation, polyadenylated RNA and the main components of the complex mCRD involved in mRNA turnover. These structures are active sites of mRNA translation, correlated with a high degree of ploidy, and involve MAPK and calcium signaling. Additional data showed that insulation and concentration of these adaptive ribonucleoprotein particles within the nucleus are dynamic, transient and protect the cell until the genotoxic stress is relieved. Bacterial genotoxins-induced NR would be a privileged gateway for selected mRNA to be preferably transported therein for local translation. These findings offer new insights into the context of NR formation, a common feature of many cancers, which not only appears in response to therapies-induced DNA damage but also earlier in response to genotoxic bacteria.


Assuntos
Toxinas Bacterianas/toxicidade , Helicobacter/patogenicidade , Ribonucleoproteínas/metabolismo , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Sobrevivência Celular , Dano ao DNA , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mutagênicos/toxicidade , Peptídeos/toxicidade , Policetídeos/toxicidade , RNA Mensageiro/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-31522662

RESUMO

A cluster of gastrointestinal illness was detected following receipt of a complaint of becoming ill after a multi-course dinner at a restaurant in Canberra, Australian Capital Territory (ACT), Australia. The complaint led to an investigation by ACT Health. Food samples retained by the restaurant for microbiological analysis returned an unsatisfactory level of Bacillus cereus in beef (19,000 colony forming units/gram [cfu/g]) and a satisfactory level in arancini (50 cfu/g). These positive samples underwent whole genome sequencing and genes encoding diarrhoeal toxins were detected with no laboratory evidence of the emetic toxin. No stool specimens were collected. A cohort study was undertaken and 80% (33/41) of patrons took part in a structured interview. There was no significant difference in age or sex between those ill and not ill. Due to universal exposure most foods were unable to be statistically analysed and no significant results were found from the food history. The ill cohort diverged into two distinct groups based on incubation period and symptoms suggesting this outbreak involved B. cereus intoxication with both diarrhoeal and potentially emetic toxins. Some hygiene practices during food preparation were noted to be inadequate and heating and cooling procedures were unverified when questioned. A combination of the incubation periods and symptom profile, food laboratory evidence, and genomic sequencing of the B. cereus diarrhoeal gene suggest a probable aetiology of B. cereus intoxication. Public health action included the restaurant rectifying hygiene practices and documenting heating/cooling procedures.


Assuntos
Bacillus cereus/isolamento & purificação , Toxinas Bacterianas/toxicidade , Surtos de Doenças , Doenças Transmitidas por Alimentos/epidemiologia , Gastroenterite/epidemiologia , Carne Vermelha/microbiologia , Animais , Território da Capital Australiana/epidemiologia , Bacillus cereus/genética , Bovinos , Estudos de Coortes , Diarreia/epidemiologia , Diarreia/microbiologia , Diarreia/mortalidade , Eméticos , Feminino , Contaminação de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/mortalidade , Gastroenterite/microbiologia , Gastroenterite/mortalidade , Humanos , Masculino , Restaurantes , Estudos Retrospectivos , Inquéritos e Questionários
4.
Insect Biochem Mol Biol ; 112: 103209, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31422154

RESUMO

Insecticidal proteins from Bacillus thuringiensis (Bt) are the primary recombinant proteins expressed in transgenic crops (Bt-crops) to confer insect resistance. Development of resistance to Bt toxins in insect populations threatens the sustainable application of Bt-crops in agriculture. The Bt toxin Cry2Ab is a major insecticidal protein used in current Bt-crops, and resistance to Cry2Ab has been selected in several insects, including the cabbage looper, Trichoplusia ni. In this study, the Cry2Ab resistance gene in T. ni was mapped to Chromosome 17 by genetic linkage analyses using a whole genome resequencing approach, and was then finely mapped using RNA-seq-based bulked segregant analysis (BSA) and amplicon sequencing (AmpSeq)-based fine linkage mapping to a locus containing two genes, ABCA1 and ABCA2. Mutations in ABCA1 and ABCA2 in Cry2Ab resistant T. ni were identified by both genomic DNA and cDNA sequencing. Analysis of the expression of ABCA1 and ABCA2 in T. ni larvae indicated that ABCA2 is abundantly expressed in the larval midgut, but ABCA1 is not a midgut-expressed gene. The mutation in ABCA2 in Cry2Ab resistant T. ni was identified to be an insertion of a transposon Tntransib in ABCA2. For confirmation of ABCA2 as the Cry2Ab-resistance gene, T. ni mutants with frameshift mutations in ABCA1 and ABCA2 were generated by CRISPR/Cas9 mutagenesis. Bioassays of the T. ni mutants with Cry2Ab verified that the mutations of ABCA1 did not change larval susceptibility to Cry2Ab, but the ABCA2 mutants were highly resistant to Cry2Ab. Genetic complementation test of the ABCA2 allele in Cry2Ab resistant T. ni with an ABCA2 mutant generated by CRISPR/Cas9 confirmed that the ABCA2 mutation in the Cry2Ab resistant strain confers the resistance. The results from this study confirmed that ABCA2 is essential for the toxicity of Cry2Ab in T. ni and mutation of ABCA2 confers the resistance to Cry2Ab in the resistant T. ni strain derived from a Bt resistant greenhouse population.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Resistência a Inseticidas/genética , Mariposas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bacillus thuringiensis , Toxinas Bacterianas/toxicidade , Endotoxinas/toxicidade , Expressão Gênica , Ligação Genética , Proteínas Hemolisinas/toxicidade , Inseticidas , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Mariposas/efeitos dos fármacos , Mariposas/metabolismo , Mutação
5.
Aquat Toxicol ; 215: 105269, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31408752

RESUMO

Blooms of the dominant cyanobacterium Aphanizomenon flosaquae are frequently encountered in natural waters, and their secretion of neurotoxic paralytic shellfish toxins called aphantoxins threatens environmental safety and human health worldwide. The liver is the primary detoxification organ in animals, and its pro- and anti-inflammatory responses are important functions in the detoxification of toxins. Therefore, we investigated the response of these inflammatory factors to aphantoxins in the liver of zebrafish (Danio rerio). A. flosaquae DC-1 was sampled during blooms in Dianchi Lake, China and cultured, and the toxin was extracted and analyzed using high performance liquid chromatography. The primary constituents were gonyautoxins 1 (34.04%) and 5 (21.28%) and neosaxitoxin (12.77%). Zebrafish were injected intraperitoneally with 5.3 µg (low dose) or 7.61 µg (high dose) of saxitoxin equivalents [equivalents (eq.)]/kg body weight of A. flosaquae DC-1 aphantoxins. Hyperemia, the hepatosomatic index (HSI), and physiological and molecular responses of pro- and anti-inflammatory cytokines in the zebrafish liver were investigated at different time points 1-24 h post-exposure. Aphantoxins significantly enhanced hepatic hyperemia and altered the HSI 3-24 h post-exposure, suggesting that inflammation caused morphological changes. Subsequent investigations using the enzyme-linked immunosorbent assay showed that the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1ß (IL-1ß), IL-6, and IL-8 and anti-inflammatory cytokines IL-10 and transforming growth factor ß were higher in the liver of zebrafish exposed to aphantoxins, which indicated physiological inflammatory responses. Further analysis by real-time fluorescence quantitative polymerase chain reaction demonstrated upregulated mRNA expression of these cytokines, suggesting molecular inflammatory responses in the zebrafish liver. These changes showed dose- and time-dependent patterns. These results indicated that aphantoxins induced hyperemia and altered the HSI, and subsequently increased the levels of proinflammatory cytokines TNF-α, IL-1ß, IL-6 and IL-8 to induce physiological inflammatory responses. These changes activated the anti-inflammatory cytokines IL-10 and TGF-ß to suppress inflammatory damage. The induced changes were the result of upregulated mRNA expression of these inflammatory cytokines caused by aphantoxins. Aphantoxins resulted in hepatic immunotoxicity and response by inducing pro-inflammatory cytokines. Zebrafish liver in turn suppressed the inflammatory damage by upregulating the activities of anti-inflammatory cytokines. In the future, these pro- and anti-inflammatory cytokines in the zebrafish liver may be prove to be useful biomarkers of aphantoxins and blooms in nature.


Assuntos
Anti-Inflamatórios/metabolismo , Aphanizomenon/química , Toxinas Bacterianas/toxicidade , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Fígado/metabolismo , Toxinas Marinhas/toxicidade , Peixe-Zebra/metabolismo , Animais , Citocinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperemia/genética , Hiperemia/patologia , Fígado/efeitos dos fármacos , Masculino , Poluentes Químicos da Água/toxicidade
6.
Int J Mol Sci ; 20(14)2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315248

RESUMO

Host defense against infection is based on two crucial mechanisms: the inflammatory response and the activation of coagulation. Platelets are involved in both hemostasis and immune response. These mechanisms work together in a complex and synchronous manner making the contribution of platelets of major importance in sepsis. This is a summary of the pathophysiology of sepsis-induced thrombocytopenia, microvascular consequences, platelet-endothelial cells and platelet-pathogens interactions. The critical role of platelets during sepsis and the therapeutic implications are also reviewed.


Assuntos
Plaquetas/imunologia , Sepse/sangue , Trombocitopenia/sangue , Animais , Toxinas Bacterianas/toxicidade , Plaquetas/patologia , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Humanos , Sepse/complicações , Trombocitopenia/etiologia
7.
Food Chem Toxicol ; 132: 110664, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31279043

RESUMO

Cylindrospermopsin (CYN) is a potent cyanotoxin recognized as an emerging human threat due to its cytotoxicity and potential carcinogenicity. Although the genotoxicity of CYN has been extensively studied in vitro, limited data are available on its in vivo genotoxicity. The aim of this study was to evaluate the in vivo genotoxicity of pure CYN (7.5-75 µg/kg body weight) after oral exposure of rats through a combined assay of the micronucleus test (MN) in bone marrow, and the standard and modified comet assay in stomach, liver and blood. Also, histopathological changes in stomach and liver were evaluated. Positive results in the MN test were observed in bone marrow in the exposed rats at all the tested concentrations. However, the comet assay revealed that CYN did not induce DNA strand breaks nor oxidative DNA damage in any of the tissues investigated. Finally, histopathological changes were observed in stomach and liver (7.5-75 µg/kg) in intoxicated rats. These results could indicate that CYN is able to induce irritation in stomach before its biotransformation in rats orally exposed, and genotoxicity in bone marrow.


Assuntos
Toxinas Bacterianas/toxicidade , Ensaio Cometa , Testes para Micronúcleos , Mutagênicos/toxicidade , Uracila/análogos & derivados , Animais , Medula Óssea/efeitos dos fármacos , Dano ao DNA , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Estômago/efeitos dos fármacos , Uracila/toxicidade
8.
Nat Med ; 25(7): 1082-1088, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270506

RESUMO

Salmonella Typhi is a human host-restricted pathogen that is responsible for typhoid fever in approximately 10.9 million people annually1. The typhoid toxin is postulated to have a central role in disease pathogenesis, the establishment of chronic infection and human host restriction2-6. However, its precise role in typhoid disease in humans is not fully defined. We studied the role of typhoid toxin in acute infection using a randomized, double-blind S. Typhi human challenge model7. Forty healthy volunteers were randomized (1:1) to oral challenge with 104 colony-forming units of wild-type or an isogenic typhoid toxin deletion mutant (TN) of S. Typhi. We observed no significant difference in the rate of typhoid infection (fever ≥38 °C for ≥12 h and/or S. Typhi bacteremia) between participants challenged with wild-type or TN S. Typhi (15 out of 21 (71%) versus 15 out of 19 (79%); P = 0.58). The duration of bacteremia was significantly longer in participants challenged with the TN strain compared with wild-type (47.6 hours (28.9-97.0) versus 30.3(3.6-49.4); P ≤ 0.001). The clinical syndrome was otherwise indistinguishable between wild-type and TN groups. These data suggest that the typhoid toxin is not required for infection and the development of early typhoid fever symptoms within the context of a human challenge model. Further clinical data are required to assess the role of typhoid toxin in severe disease or the establishment of bacterial carriage.


Assuntos
Toxinas Bacterianas/toxicidade , Salmonella typhi/patogenicidade , Febre Tifoide/etiologia , Doença Aguda , Adolescente , Adulto , Animais , Método Duplo-Cego , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Febre Tifoide/imunologia , Febre Tifoide/patologia , Adulto Jovem
9.
PLoS Negl Trop Dis ; 13(7): e0007590, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31306427

RESUMO

BACKGROUND: The entomopathogenic fungus Beauveria bassiana has been widely used to kill mosquito larvae and adults in the laboratory and field. However, its slow action of killing has hampered its widespread application. In our study, the B. bassiana fungus was genetically modified to express the Bacillus thuringiensis (Bt) toxin Cyt2Ba to improve its efficacy in killing mosquitoes. METHODOLOGY/PRINCIPAL FINDINGS: The efficacy of the wild type (WT) of B. bassiana and a transgenic strain expressing Cyt2Ba toxin (Bb-Cyt2Ba) was evaluated against larval and adult Aedes mosquitoes (Aedes aegypti and Aedes albopictus) using insect bioassays. The Bb-Cyt2Ba displayed increased virulence against larval and adult Aedes mosquitoes compared with the WT: for Ae. aegypti adults, the median lethal time (LT50) was decreased by 33% at the concentration of 1× 108 conidia/ml, 19% at 1× 107 conidia/ml and 47% at 1× 106 conidia/ml. The LT50 for Ae. albopictus adults was reduced by 20%, 23% and 29% at the same concentrations, respectively. The LT50 for Ae. aegypti larvae was decreased by 42% at 1× 107 conidia/ml and 25% at 1× 106 conidia/ml, and that for Ae. albopictus larvae was reduced by 33% and 31% at the same concentrations, respectively. In addition, infection with Bb-Cyt2Ba resulted in a dramatic reduction in the fecundity of Aedes mosquitoes. CONCLUSIONS/SIGNIFICANCE: In conclusion, our study demonstrated that the virulence of B. bassiana against mosquitoes can be significantly improved by introducing the Bt toxin gene Cyt2Ba into the genome to express the exogenous toxin in the fungus. The transgenic strain Bb-Cyt2Ba significantly reduced the survival and fecundity of Ae. aegypti and Ae. albopictus compared with the WT strain, which suggested that this recombinant B. bassiana has great potential for use in mosquito control.


Assuntos
Aedes/microbiologia , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Beauveria/genética , Endotoxinas/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Controle Biológico de Vetores/métodos , Animais , Feminino , Fertilidade , Regulação Fúngica da Expressão Gênica , Instabilidade Genômica , Larva/microbiologia , Controle de Mosquitos/métodos , Proteínas Recombinantes , Esporos Fúngicos , Virulência/genética
10.
Nat Microbiol ; 4(10): 1760-1769, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31160825

RESUMO

Clostridium difficile toxin A (TcdA) is a major exotoxin contributing to disruption of the colonic epithelium during C. difficile infection. TcdA contains a carbohydrate-binding combined repetitive oligopeptides (CROPs) domain that mediates its attachment to cell surfaces, but recent data suggest the existence of CROPs-independent receptors. Here, we carried out genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated screens using a truncated TcdA lacking the CROPs, and identified sulfated glycosaminoglycans (sGAGs) and low-density lipoprotein receptor (LDLR) as host factors contributing to binding and entry of TcdA. TcdA recognizes the sulfation group in sGAGs. Blocking sulfation and glycosaminoglycan synthesis reduces TcdA binding and entry into cells. Binding of TcdA to the colonic epithelium can be reduced by surfen, a small molecule that masks sGAGs, by GM-1111, a sulfated heparan sulfate analogue, and by sulfated cyclodextrin, a sulfated small molecule. Cells lacking LDLR also show reduced sensitivity to TcdA, although binding between LDLR and TcdA are not detected, suggesting that LDLR may facilitate endocytosis of TcdA. Finally, GM-1111 reduces TcdA-induced fluid accumulation and tissue damage in the colon in a mouse model in which TcdA is injected into the caecum. These data demonstrate in vivo and pathological relevance of TcdA-sGAGs interactions, and reveal a potential therapeutic approach of protecting colonic tissues by blocking these interactions.


Assuntos
Toxinas Bacterianas/metabolismo , Clostridium difficile/química , Enterotoxinas/metabolismo , Glicosaminoglicanos/metabolismo , Receptores de LDL/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Membrana Celular/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Endocitose , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/toxicidade , Glicosaminoglicanos/deficiência , Células HeLa , Heparitina Sulfato/análogos & derivados , Heparitina Sulfato/farmacologia , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Mutação , Oligopeptídeos/genética , Ligação Proteica , Receptores de LDL/deficiência
11.
Emerg Microbes Infect ; 8(1): 934-945, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31237474

RESUMO

Cytoskeletal rearrangement and acute cytotoxicity occur in Vibrio vulnificus-infected host cells. RtxA1 toxin, a multifunctional autoprocessing repeats-in-toxin (MARTX), is essential for the pathogenesis of V. vulnificus and the programmed necrotic cell death. In this study, HeLa cells expressing RtxA1 amino acids 1491-1971 fused to GFP were observed to be rounded. Through yeast two-hybrid screening and subsequent immunoprecipitation validation assays, we confirmed the specific binding of a RtxA11491-1971 fragment with host-cell filamin A, an actin cross-linking scaffold protein. Downregulation of filamin A expression decreased the cytotoxicity of RtxA1 toward host cells. Furthermore, the phosphorylation of JNK and p38 MAPKs was induced by the RtxA1-filamin A interaction during the toxin-mediated cell death. However, the phosphorylation of these MAPKs was not observed during the RtxA1 intoxication of filamin A-deficient M2 cells. In addition, the depletion of pak1, which appeared to be activated by the RtxA1-filamin A interaction, inhibited RtxA1-induced phosphorylation of JNK and p38, and the cells treated with a pak1 inhibitor exhibited decreased RtxA1-mediated cytoskeletal rearrangement and cytotoxicity. Thus, the binding of filamin A by the RtxA11491-1971 domain appears to be a requisite to pak1-mediated MAPK activation, which contributes to the cytoskeletal reorganization and host cell death.


Assuntos
Toxinas Bacterianas/metabolismo , Citoesqueleto/metabolismo , Filaminas/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Vibrioses/metabolismo , Vibrio vulnificus/metabolismo , Quinases Ativadas por p21/metabolismo , Motivos de Aminoácidos , Toxinas Bacterianas/toxicidade , Morte Celular , Citoesqueleto/genética , Filaminas/genética , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Ligação Proteica , Vibrioses/genética , Vibrioses/microbiologia , Vibrioses/fisiopatologia , Vibrio vulnificus/química , Vibrio vulnificus/genética , Quinases Ativadas por p21/genética
12.
Int J Toxicol ; 38(3): 163-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31179828

RESUMO

A recombinant protective antigen (rPA) anthrax vaccine candidate (rPA7909) was developed as a next-generation vaccine indicated for postexposure prophylaxis of disease resulting from suspected or confirmed Bacillus anthracis exposure. The lyophilized form of rPA7909-vaccinated candidate contains 75 µg purified rPA, 750 µg aluminum (as Alhydrogel adjuvant), and 250 µg of an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide CpG 7909 in a 0.5 mL phosphate-buffered suspension. General toxicity and local reactogenicity were evaluated in Sprague Dawley rats vaccinated with the full human dose of rPA7909 by intramuscular injection. Animals were immunized on study days 1, 15, and 29. Control groups were administered diluent only or adjuvant control (excipients, CpG 7909, and Alhydrogel adjuvant in diluent) intramuscularly at the same dose volume and according to the same schedule used for rPA7909. Toxicity was assessed based on the results of clinical observations, physical examinations, body weights, injection site reactogenicity, ophthalmology, clinical pathology (hematology, coagulation, and serum chemistry), organ weights, and macroscopic and microscopic pathology evaluation. The immune response to rPA7909 vaccination was confirmed by measuring serum anti-PA immunoglobulin G levels. The rPA7909 vaccine produced no apparent systemic toxicity and only transient reactogenicity at the injection site. The injection site reaction from animals receiving the adjuvant control was very similar to those receiving rPA7909 with respect to the inflammation. The inflammatory response observed in the injection site and the draining lymph nodes was consistent with expected immune stimulation. The overall results indicated a favorable safety profile for rPA7909.


Assuntos
Adjuvantes Imunológicos/toxicidade , Vacinas contra Antraz/toxicidade , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Oligodesoxirribonucleotídeos/toxicidade , Animais , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Feminino , Liofilização , Imunoglobulina G/sangue , Masculino , Ratos Sprague-Dawley , Proteínas Recombinantes/toxicidade
13.
Chemosphere ; 234: 139-147, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31207420

RESUMO

Cylindrospermopsin (CYN) is an alkaloid biosynthesized by selected cyanobacteria, the cyto- and genotoxic properties of which have been studied extensively by in vitro and in vivo experimental models. Various studies have separately established the role of uracil, guanidine and hydroxyl groups in CYN-induced toxicity. In the present study, we have prepared five synthetic analogues that all possess a uracil group but had variations in the other functionality found in CYN. We compared the in vitro toxicity of these analogues in common carp hepatocytes by assessing oxidative stress markers, DNA fragmentation and apoptosis. All the analogues tested induced generation of reactive oxygen species, lipid peroxidation (LPO) and DNA fragmentation. However, the greatest increase in LPO and increase in caspase-3 activity, an apoptosis marker, was demonstrated by an analogue containing guanidine, hydroxyl and uracil functionalities similar to those found in CYN but lacking the complex tricyclic structure of CYN. We also report a crystal structure of an analogue lacking the hydroxyl group found in CYN which does not show intramolecular H-bonding interactions between the guanidine and the uracil functionalities. The observations made in this work supports the hypothesis that CYN toxicity is a result of an interplay between both of the uracil, hydroxyl and guanidine functional groups.


Assuntos
Toxinas Bacterianas/toxicidade , Uracila/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Carpas , Cianobactérias , Dano ao DNA/efeitos dos fármacos , Guanidina/química , Hepatócitos/metabolismo , Radical Hidroxila/química , Peroxidação de Lipídeos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio , Uracila/toxicidade
14.
Emerg Microbes Infect ; 8(1): 707-716, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119985

RESUMO

Staphylococcus aureus (S. aureus) is one of the most serious human pathogens. α-Hemolysin (Hla) secreted by S. aureus is a key toxin for various infections. We herein report that Honokiol, a natural plant polyphenol, inhibits the secretion and hemolytic activity of staphylococcal Hla with concomitant growth inhibition of S. aureus and protection of S. aureus-mediated cell injury within subinhibitory concentrations. In parallel, Honokiol attenuates the staphylococcal Hla-induced inflammatory response by inhibiting NLRP3 inflammasome activation in vitro and in vivo. Consequently, the biologically active forms of the inflammatory cytokines IL-1ß and IL-18 are reduced significantly in response to Honokiol in mice infected with S. aureus. Experimentally, we confirm that Honokiol binds to monomeric Hla with a modest affinity without impairing its oligomerization. Based on molecular docking analyses in silico, we make a theoretical discovery that Honokiol is located outside of the triangular region of monomeric Hla. The binding model restricts the function of the residues related to membrane channel formation, which leads to the functional disruption of the assembled membrane channel. This research creates a new paradigm for developing therapeutic agents against staphylococcal Hla-mediated infections.


Assuntos
Toxinas Bacterianas/metabolismo , Compostos de Bifenilo/administração & dosagem , Proteínas Hemolisinas/metabolismo , Inflamassomos/antagonistas & inibidores , Lignanas/administração & dosagem , Receptores de Superfície Celular/antagonistas & inibidores , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Células A549 , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Toxinas Bacterianas/toxicidade , Compostos de Bifenilo/metabolismo , Compostos de Bifenilo/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Proteínas Hemolisinas/toxicidade , Histocitoquímica , Humanos , Lignanas/metabolismo , Lignanas/farmacologia , Fígado/patologia , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Ligação Proteica , Infecções Estafilocócicas/patologia , Staphylococcus aureus/metabolismo , Resultado do Tratamento
15.
Anaerobe ; 59: 61-67, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31125604

RESUMO

Around the world, Clostridium perfringens type A is known to be a common foodborne pathogen. Therefore, the control and treatment of food poisoning caused by this pathogen are important. This study investigated, in vitro, the effects of Bacillus coagulans and its culture extracts on alpha toxin gene expression, growth inhibition, cytotoxicity, and apoptosis induced by C. perfringens spore, germinated spore and its enterotoxin. Flow cytometry was used to evaluate the apoptosis rate, and MTT test was used to evaluate cytotoxicity. Minimum inhibitory concentration was also used to measure the percentage of inhibition in the broth medium. Finally, RT-qPCR was used to evaluate alpha toxin gene expression. The results showed that the B. coagulans culture extract was able to inhibit the growth of the germinated spore of C. perfringens. Moreover, treating the extract with pepsin can reduce growth in the broth medium. MTT and flow cytometry showed that both B. coagulans and its extract can significantly reduce the cytotoxicity and apoptosis rate induced by C. perfringens type A. In addition, it was shown that the co-culture of B. coagulans and C. perfringens decreases alpha toxin gene expression. The findings of this study indicate that B. coagulans, with growth inhibition and reduced expression of alpha toxin in C. perfringens, can reduce the cytotoxicity and apoptosis rate induced on HT-29 cells.


Assuntos
Antibiose , Bacillus coagulans/crescimento & desenvolvimento , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/toxicidade , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/toxicidade , Clostridium perfringens/crescimento & desenvolvimento , Clostridium perfringens/metabolismo , Probióticos , Fosfolipases Tipo C/biossíntese , Fosfolipases Tipo C/toxicidade , Apoptose , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase em Tempo Real
16.
Anaerobe ; 59: 76-91, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31145997

RESUMO

Epsilon toxin (ETX) is the major virulence determinant of C. perfringens type B or type D strains, causing diseases in animals, besides being a listed biological and toxin warfare (BTW) agent. Keeping in mind the high lethality and the rapid onset of clinical manifestations, early diagnosis of epsilon toxin exposure is of paramount importance for implementation of appropriate medical countermeasures. Using a 2DE-MS approach, the present study is the first comprehensive proteomic elucidation of ETX-induced protein markers in the mouse model, providing putative targets for early diagnosis of ETX exposure. A total of 52 unique proteins showing ETX-induced modulations were identified in plasma and urine samples. Fibrinogen, apolipoprotein, serum amyloid protein, plasminogen, serum albumin, glutathione peroxidase, transferrin, major urinary protein 2, haptoglobin, transthyretin, and vitamin D-binding protein were among the proteins observed in more than one dataset with altered abundance after the ETX-intoxication. The predicted localization, function, and interaction of the ETX-modulated proteins in the plasma and urine indicated involvement of multiple pathways; extracellular proteins, followed by macromolecular complexes associated with blood coagulation and plasminogen activating cascade, being the most prominent among others. The putative markers elucidated here warrants further validation and can be of immense value for the early diagnosis of ETX exposure.


Assuntos
Toxinas Bacterianas/toxicidade , Biomarcadores/sangue , Biomarcadores/urina , Envenenamento/patologia , Proteínas/análise , Animais , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Feminino , Espectrometria de Massas , Camundongos Endogâmicos BALB C , Plasma/química , Urina/química
17.
J Fish Dis ; 42(8): 1125-1132, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31115066

RESUMO

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.


Assuntos
Imunização Passiva , Imunoglobulinas/imunologia , Penaeidae/imunologia , Vibrio parahaemolyticus/efeitos dos fármacos , Ração Animal/análise , Animais , Toxinas Bacterianas/toxicidade , Galinhas , Dieta , Suplementos Nutricionais/análise , Gema de Ovo/química , Imunoglobulinas/administração & dosagem , Penaeidae/efeitos dos fármacos , Penaeidae/microbiologia , Vacinação , Vibrio parahaemolyticus/fisiologia
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.
Toxins (Basel) ; 11(4)2019 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013617

RESUMO

Clostridium perfringens strains B and C cause fatal intestinal diseases in animals. The secreted pore-forming toxin delta-toxin is one of the virulence factors of the strains, but the mechanism of intestinal pathogenesis is unclear. Here, we investigated the effects of delta-toxin on the mouse ileal loop. Delta-toxin caused fluid accumulation and intestinal permeability to fluorescein isothiocyanate (FITC)-dextran in the mouse ileal loop in a dose- and time-dependent manner. Treatment with delta-toxin induced significant histological damage and shortening of villi. Delta-toxin activates a disintegrin and metalloprotease (ADAM) 10, leading to the cleavage of E-cadherin, the epithelial adherens junction protein, in human intestinal epithelial Caco-2 cells. In this study, E-cadherin immunostaining in mouse intestinal epithelial cells was almost undetectable 1 h after toxin treatment. ADAM10 inhibitor (GI254023X) blocked the toxin-induced fluid accumulation and E-cadherin loss in the mouse ileal loop. Delta-toxin stimulated the shedding of intestinal epithelial cells. The shedding cells showed the accumulation of E-cadherin in intracellular vesicles and the increased expression of active caspase-3. Our findings demonstrate that delta-toxin causes intestinal epithelial cell damage through the loss of E-cadherin cleaved by ADAM10.


Assuntos
Toxinas Bacterianas/toxicidade , Intestino Delgado/efeitos dos fármacos , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Caderinas/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos ICR
20.
Toxins (Basel) ; 11(4)2019 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-31013880

RESUMO

Absorption and accumulation of bioavailable cyanobacterial metabolites (including cyanotoxins) are likely in fish after senescence and the rupturing of cells during bloom episodes. We determined the toxicity of cyanopeptides identified from two strains of Microcystis (M. panniformis MIRS-04 and M. aeruginosa NPDC-01) in a freshwater tropical fish, Astyanax altiparanae (yellowtail tetra, lambari). Aqueous extracts of both Microcystis strains were prepared in order to simulate realistic fish exposure to these substances in a freshwater environment. Both strains were selected because previous assays evidenced the presence of microcystins (MCs) in MIRS-04 and lack of cyanotoxins in NPDC-01. Identification of cyanobacterial secondary metabolites was performed by LC-HR-QTOF-MS and quantification of the MC-LR was carried out by LC-QqQ-MS/MS. MIRS-04 produces the MCs MC-LR, MC-LY and MC-HilR as well as micropeptins B, 973, 959 and k139. NPCD-01 biosynthetizes microginins FR1, FR2/FR4 and SD-755, but does not produce MCs. Larval fish survival and changes in morphology were assessed for 96 h exposure to aqueous extracts of both strains at environmentally relevant concentrations from 0.1 to 0.5 mg (dry weight)/mL, corresponding to 0.15 to 0.74 µg/mL of MC-LR (considering dried amounts of MIRS-04 for comparison). Fish mortality increased with concentration and time of exposure for both strains of Microcystis. The frequencies of morphological abnormalities increased with concentration in both strains, and included abdominal and pericardial oedema, and spinal curvature. Results demonstrate that toxicity was not solely caused by MCs, other classes of cyanobacterial secondary metabolites contributed to the observed toxicity.


Assuntos
Toxinas Bacterianas/toxicidade , Characidae/anormalidades , Larva/efeitos dos fármacos , Microcystis , Peptídeos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Larva/crescimento & desenvolvimento
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