Cytotoxicity analysis of three Bacillus thuringiensis Subsp. israelensis d-Endotoxins towards insect and mammalian cells

Three members of the d-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsinactivated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 mg/mL

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Estudo da atividade tóxica para Aedes aegypti das proteínas Cry4Aa e Cry4Ba de Bacillus thuringiensis expressas em baculovírus recombinantes

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2007.Os genes cry4Aa e cry4Ba, pertencentes a diferentes estirpes brasileiras de Bacillus thuringiensis, S1806 e S1989, respectivamente, foram amplificados por PCR e clonados em um vetor de clonagem. A análise do seqüenciamento do gene cry4Aa mostrou alta identidade com outras seqüências do gene já descritas e este foi clonado tanto no vetor de transferência pSynXIVVI+X3, como no pFastBac®1 para expressão e análise da toxicidade da proteína heteróloga a partir dos baculovírus construídos por recombinação homóloga, vSyncry4Aa, e por transposição sítio-específica, vBacCry4Aa. O gene cry4Ba foi introduzido no vetor pSynXIVVI+X3 dando origem, por recombinação homóloga, ao baculovírus recombinante vSyncry4Ba. Os vírus recombinantes, derivados de recombinação homóloga, foram isolados em placas de 96 poços, enquanto o vírus recombiante obtido por transposição foi isolado de colônias de células de Escherichia coli DH10BacTM crescidas em placas de Petri contendo antibióticos, X-Gal e IPTG. Células de inseto BTI-TN5B1-4 foram infectadas com os vírus recombinantes isoladamente e mRNA derivados dos extratos celulares (72 h.p.i.) analisados por RT-PCR, para confirmação da expressão e presença de transcritos específicos para os genes. Extratos de larvas de Spodoptera frugiperda, infectadas com os vírus recombinantes (120 h.p.i.) foram analisados por eletroforese em gel de poliacrilamida (SDS-PAGE), mostrando a presença de bandas de aproximadamente 128 e 130 kDa correspondentes aos tamanhos das proteínas Cry4Aa e Cry4Ba, respectivamente. Possíveis cristais das proteínas recombinantes foram observados por microscopia de luz. Bioensaios com os extratos de insetos infectados mostraram-se tóxicos para larvas de segundo instar de Aedes aegypti. _______________________________________________________________________________ ABSTRACTThe cry4Aa and cry4Ba genes from Brazilian strains of Bacillus thuringiensis, S-1806 and S-1989, respectively, were amplified by PCR, cloned into a plasmid cloning vector and sequenced. Sequence analysis of the cry4Aa gene showed high identity to previous known cry genes and it was cloned into both pSynXIVVI+X3 and pFastBac®1 transfer vectors for expression and toxicity analysis of the heterologous proteins derived from the recombinant baculoviruses constructed by homologous recombination (vSynCry4Aa) and transposition (vBacCry4Aa). The cry4Ba gene was introduced into the transfer vector pSynXIVVI+X3/3 resulting in the construction of the recombinant virus vSynCry4Ba by homologous recombination. The recombinant viruses, derived from homologous recombination, were isolated by serial dilution in 96 well plates while the recombinant virus produced by transposition was isolated from Escherichi coli (DH10BacTM) colonies grown on Petri dishes containing antibiotics, X-Gal and IPTG. Insect cells BTI-TN5B1-4 were separetly infected with the recombinante viruses and mRNA from cell extracts (72 h.p.i.) analysed by RT-PCR, in order to confirm the presence of the genes specific transcripts. Recombinant viruses infected insect extracts (120 h p.i.) were analysed by polyacrylamide gel electrophoresis (SDS-PAGE) showing the presence of polypepitide bands of around 128 and 130 kDa, corresponding, respectively to the sizes of the proteins Cry4Aa and Cry4Ba. Putative crystals from the recombinant proteins were observed by light microscopy. Bioassays with virus-infected insect extracts were shown to be toxic to second instar Aedes aegypti larvae

Avaliação da Toxicidade de proteínas Cry e Cyt de Bacillus thuringiensis subsp. israelensis para diferentes linhagens de células de inseto e de mamífero

Tese (doutorado)—Universidade de Brasília, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2012.Bacillus thuringiensis é uma bactéria Gram-positiva que produz proteínas formadoras de cristal ou δ-endotoxinas que compreendem as toxinas Cry, com atividade inseticida específica, e Cyt, com atividade citolítica inespecífica. Diferentes δ-endotoxinas mostram-se específicas para diferentes insetos-alvos. Esta especificidade deve-se a receptores de membrana distintos nas células do intestino dos insetos e à presença de diferentes proteases próprias dos insetos, necessárias para ativação proteolítica da prótoxina produzida na fase de esporulação do B. thuringiensis. Os genes cry4Aa, cry11A e cyt2Ba foram amplificados por PCR a partir das estirpes de Bacillus thuringiensis subsp. israelensis, S-1989 ou S-1806. Os produtos das PCR foram clonados em um vetor de expressão em B. thuringiensis, pSVP27A. O gene cyt2Ba foi também clonado em vetores de transferência para construção de baculovírus recombinantes para expressão da proteína Cyt2Ba nativa ou fusionada à proteína Poliedrina. Estirpes de B. thuringiensis acristalíferas foram usadas para expressão individual das proteínas. Após purificação e solubilização das proteínas heterólogas, as memsmas foram ativadas com tripsina ou suco gástrico de Spodoptera frugiperda, para a realização de ensaios de atividade em culturas de células de Lepidoptera, Diptera e mamífero. Foram usados, nos ensaios de citotoxicidade, 20 μg/mL de cada toxina Cry individual ou em combinações e 20 μg/mL ou 5 μg/mL da toxina Cyt2Ba. Atividades tóxicas das toxinas heterólogas ativadas por tripsina foram detectadas para todas as linhagens de células de insetos testadas, fato que não ocorreu após ativação das mesmas toxinas com suco gástrico de S. frugiperda. Para células humanas, apenas Cyt2Ba mostrou-se tóxica. Entretanto, a combinação entre Cyt2Ba e as toxinas Cry4Aa e Cry11A apresentou citotóxicidade maior que Cyt2Ba isoladamente, o que pode sugerir que Cyt2Ba funcione como receptor para Cry4Aa e Cry11A em células humanas. _________________________________________________________________________________ ABSTRACTBacillus thuringiensis is a Gram-positive bacterium that produces crystal forming proteins, δ-endotoxins, which consist of Cry toxins harboring specific insecticidal activity, and Cyt toxins that contain non-specific cytolytic activity. Different δ-endotoxins are specific to different target insects. This specificity is due to distinct membrane receptors on the surface of the insect´s midgut cells and to the presence of different host´s proteases, which are necessary to proteolytic activation of the protoxin expressed during B. thuringiensis sporulation phase. The cry4Aa, Cry11A and cyt2Ba genes were amplified from Bacillus thuringiensis subsp. israelensis Brazilian strains, S-1989 or S-1806 by PCR. The PCR products were cloned into the B. thuringiensis expression vector, pSVP27A. The cyt2Ba gene was also cloned into transfer vectors to allow construction of recombinant baculoviruses, in order to express the native Cyt2Ba protein, or Cyt2Ba fusioned to the Polyhedrin protein. Acrystaliferous B. thuringiensis strains were used for the expression of the proteins individually. After purification and solubilization of the heterologous proteins, toxin activation by either trypsin or Spodoptera frugiperda´s gastric juice was carried out to perform citotoxicity assays using Lepidopteran, Dipteran and human cultured cells. Each Cry toxin was used at the concentration of 20 μg/mL, while Cyt2Ba was used at two different concentrations: 20 μg/mL or 5 μg/mL. Cytotoxic activities of the trypsin activated heterologous proteins were detected to all insect cell lines tested, but when the same proteins were digested by S. frugiperda´s gastric juice, no toxic activities were detected. Only Cyt2Ba was shown to be toxic to the human cells tested. Nevertheless, the combination of Cyt2Ba and both Cry4Aa and Cry11A toxins yelded a higher toxicity than that produced by Cyt2Ba isolatedly, which could suggest that Cyt2Ba could be functioning as a receptor for Cry4Aa and Cry11A on the surface of human cells

SDS-PAGE analysis of Cry4Aa and Cry11A proteolytic activation.

<p>The solubilized Cry4Aa and Cry11A protoxins were activated either by standard incubation using trypsin (T) or by using <i>S. frugiperda’</i> gastric juice (GJ). The processed proteins were stained with Coomassie blue. Molecular masses in kDa are shown on the left (Prestained Protein Molecular Weight Marker, Fermentas).</p

Cytotoxicity assays in insect cell cultures and western blot analysis of Cyt2Ba activation.

<p>Panel A: 20 µg/mL of each activated toxin, Cry4Aa and Cry11A, were incubated with different insect cell lines for 30 min. Untreated cells (control) were not incubated with any of the toxins. The percentage of cytotoxicity (cell mortality) was determined by luminometric readings based on luciferase activity. Panel B: Trypsin activation of Cyt2BaHis was proceeded and both protoxin and the activated toxin samples were analyzed by western blot with an anti-His antibody. The arrow shows a band of 27 kDa expected for the non-activated protein. The proteolytic cleavage of Cyt2BaHis resulted in the 6xHis tag loss, making the activated toxin undetectable in western blot analysis using anti-His antibody. Panel C: The activated Cyt2Ba toxin was incubated with insect cells at two different concentrations (5 and 20 µg/mL). Cytotoxicity was determined as for panel A. Panel D: Combinations of Cyt2Ba (5 µg/mL) and the two Cry toxins (20 µg/mL for each toxin) were incubated with insect cell lines. Cytotoxicity percentages were measured according to the same method employed in panel A and C. The toxins concentrations are shown in brackets. One type of dipteran cells (C6/36) and three types of lepidopteran cells (Ld, Sf-21 and Bm) are shown on the cytotoxicity assays graphics.</p

Cytotoxicity of mixtures of Cry and Cyt toxins of <i>Bacillus thuringiensis</i> subsp. <i>israelensis</i> to insect cell lines.

a<p>-mean lethal concentration. Numbers in parentheses are 95% fiducial limits.</p>b<p>-slope.</p>c<p>-chi-square.</p