Trichothecene Mycotoxins Inhibit Mitochondrial Translation—Implication for the Mechanism of Toxicity

Fusarium head blight (FHB) reduces crop yield and results in contamination of grains with trichothecene mycotoxins. We previously showed that mitochondria play a critical role in the toxicity of a type B trichothecene. Here, we investigated the direct effects of type A and type B trichothecenes on mitochondrial translation and membrane integrity in Saccharomyces cerevisiae. Sensitivity to trichothecenes increased when functional mitochondria were required for growth, and trichothecenes inhibited mitochondrial translation at concentrations, which did not inhibit total translation. In organello translation in isolated mitochondria was inhibited by type A and B trichothecenes, demonstrating that these toxins have a direct effect on mitochondrial translation. In intact yeast cells trichothecenes showed dose-dependent inhibition of mitochondrial membrane potential and reactive oxygen species, but only at doses higher than those affecting mitochondrial translation. These results demonstrate that inhibition of mitochondrial translation is a primary target of trichothecenes and is not secondary to the disruption of mitochondrial membranes

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

Characterizing the role of mitochondria in the toxicity of trichothecenes produced by Fusarium graminearum

Fusarium graminearum is a toxigenic fungal pathogen infecting economically significant cereal crops. Trichothecenes are a large family of low molecular weight sesquiterpenoid mycotoxins synthesized by F. graminearum and other fungi and are among the most toxic compounds known to man. These mycotoxins and their producers are encountered worldwide in the environment as natural contaminants of cereal grains presenting a high food safety risk for humans and cattle and threaten the global food supply. Trichothecene mycotoxicosis was primarily associated with their inhibitory effects on translation. However, these highly stable toxins also inhibit other cellular processes which contribute to their toxicity. In this work, using yeast as a model organism, a genome wide approach has been applied to obtain a comprehensive understanding of the molecular mechanism of the type A and B trichothecene toxicity. Due to their prevalence and impact, T-2 toxin and diacetoxyscirpenol (DAS) are used as representative type A toxins while trichothecin (Tcin) and deoxynivalenol (DON) are used as representative type B toxins. The yeast knockout collection of nonessential genes was initially used to identify mutant strains that exhibited increased resistance or susceptibility to trichothecenes. This screening led to identification of the role of mitochondria during trichothecene toxicity. The largest group of mutants exhibiting resistance was affected in their mitochondrial functions. Mitochondrial translation was directly inhibited, independent of total translation, and the trichothecene-treated cells exhibited severe fragmentation of mitochondrial membrane. Furthermore, actively respiring cells with functional mitochondria were essential for trichothecene cytotoxicity suggesting a critical role for mitochondria. A large fraction of the highly susceptible strains exhibited very high levels of reactive oxygen species (ROS) upon trichothecene treatment. Antioxidants increased cell survival and reduced mitochondrial membrane damage in trichothecene-treated cells. The direct role for ROS in mediating trichothecene cytotoxicity was confirmed when two novel Arabidopsis nonspecific lipid transfer proteins that mediated resistance to trichothecenes in A. thaliana exhibited antioxidant property and rescued trichothecene-treated yeast cells. Rapamycin-induced mitophagy reduced ROS levels and increased survival in trichothecene-treated cells suggesting mitophagy as a novel prosurvival cellular mechanism during oxidative stress in trichothecene-treated cells.Ph. D.Includes bibliographical referencesby Mohamed Anwar Bin Ume

A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin.

Fusarium head blight (FHB) or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON) accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin), a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1) contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP) genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS) production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione-based antioxidant defense

Overexpression of <i>AtLTP4</i>.<i>4</i> reduces Tcin-induced ROS accumulation in Arabidopsis.

<p>Confocal microscopy analysis of ROS accumulation in wild type <i>Arabidopsis</i> (Col-0) leaves infiltrated with Tcin and the cell-permeable fluorogenic probe 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) after 24 h. (A,B) Mock treatment with buffer, (C,D) treatment with 8 μM Tcin (E,F) treatment with 24 μM Tcin. (G) Intact leaf assay to quantify H₂O₂ levels in transgenic <i>Arabidopsis</i> lines #7 and #16 overexpressing <i>AtLTP4</i>.<i>4</i> using Amplex Red. Treatment with 4 μM Tcin for 24 h induced H₂O₂ accumulation in the vector control (35S:GFP), but not in lines #16 and #7. Error bars indicate S.E where n = 3 independent replicates. One-way ANOVA with post hoc Bonferroni tests, ***P<0.001.</p

IC₅₀ values for trichothecenes in yeast expressing <i>Arabidopsis</i> nsLTPs.

<p>Error indicates S.E where n = 3 independent replicates. Two-sample unpaired t-tests for equal means were performed between the different vectors containing the nsLTPs relative to the vector control for each toxin</p><p>*P<0.05</p><p>***P < 0.001.</p><p>IC₅₀ values for trichothecenes in yeast expressing <i>Arabidopsis</i> nsLTPs.</p

<i>AtLTP4</i>.<i>4</i> overexpression increases glutathione content of <i>Arabidopsis</i> leaves.

<p>(<i>A</i>) Germination of 35S:GFP seedlings using media supplemented with 1 mM cysteine (Cys) or 250 μM glutathione (GSH) and 3 μM Tcin. Comparison of the effect of the addition of cys and GSH with Tcin to the Tcin treatment alone using ANOVA with post hoc Bonferroni tests, ***P<0.001. (<i>B</i>) Germination assay of 35S:GFP seedlings on media supplemented with 250 μM buthionine sulfoximine (BSO) and 2 μM Tcin. Unlike 3 μM Tcin which substantially reduces the germination percentage, 2 μM Tcin did not impact germination percentage. Comparison of the effect of BSO treatment on Tcin treated or untreated samples using ANOVA with post hoc Bonferroni tests, **P < 0.01. (<i>C</i>) GSH and GSSG levels of mature leaves from <i>AtLTP4</i>.<i>4</i> overexpressing line #16 and 35S:GFP control (3 biological replicates each). ANOVA with post hoc Bonferroni tests, *P < 0.05. <i>(D) GSH1</i> expression in lines overexpressing <i>AtLTP4</i>.<i>4</i> relative to 35S:GFP line by qRT-PCR.</p

AtLTP4.4:GFP is localized to the cell wall/apoplast and chloroplasts in <i>Arabidopsis</i> leaves.

<p>(A) Confocal microscopy analysis of line #16 expressing AtLTP4.4:GFP showing expression in the cell wall/apoplast, (B) possibly in the ER, and (C) chloroplasts (D) 35S:GFP (Col-0) control.</p

Characterization of <i>trr1</i>.

<p>(A) Screening of the activation tagged <i>Arabidopsis</i> lines identified a mutant, which was able to form roots/leaves on 4 μM Tcin. (B) The T-DNA tag was inserted into the last exon of the At5G55440 gene in <i>trr1</i>. (C) Expression analysis of the activation tagged AT5G55440 locus and the flanking genes, At5G55420, At5G55430, At5G55450 (<i>AtLTP4</i>.<i>4</i>), At5G55460 (<i>AtLTP4</i>.<i>5</i>) and wild type <i>Arabidopsis</i> Col-0 by qRT-PCR. Expression levels of <i>AtLTP4</i>.<i>4</i> and <i>AtLTP4</i>.<i>5</i> were significantly higher in the activation tagged <i>trr1</i> line compared to the wild type Col-0. (D) Immunoblot analysis of total protein (25 μg) isolated from Col-0 and 3 independent <i>Arabidopsis</i> lines containing <i>AtLTP4</i>.<i>4</i>:<i>GFP</i> or <i>AtLTP4</i>.<i>5</i>:<i>GFP</i>, separated on a 12% SDS polyacrylamide gel and probed with monoclonal anti-His IgG (1:500) (GenScript) followed by Amersham ECL Plex C anti-mouse IgG (1:2500) conjugated with Cy3 and scanned using the Typhoon FLA 9500 (GE Healthcare Life Sciences). The blot was stained with Ponceau S and the ~52 kD band is shown for equal loading. (E) Analysis of <i>AtLTP4</i>.<i>4</i> RNA expression in two independently transformed <i>Arabidopsis</i> lines and in the LTP4.4/LTP4.5 knockout line (SALK_207859c).</p

<i>AtLTP4</i>.<i>4</i> expression attenuates ROS levels and increases glutathione content in yeast.

<p><b>(</b><i>A</i>) Trichothecene treated or untreated wild type yeast (BY4743) cells carrying the empty vector, overexpressing <i>AtLTP4</i>.<i>4</i>, or overexpressing <i>AtLTP4</i>.<i>5</i> were stained for ROS using DCHF and analyzed by flow cytometry. ANOVA with post hoc Bonferroni tests, *P < 0.05, **P < 0.01. (<i>B</i>) GSH and GSSG levels were quantified relative to vector control and normalized to total protein in yeast expressing <i>AtLTP4</i>.<i>4</i> and <i>AtLTP4</i>.<i>5</i> after 3 hour galactose induction. ANOVA with post hoc Bonferroni tests, *P < 0.05, **P < 0.01.</p