Impact of pH on the Stability and the Cross-Reactivity of Ochratoxin A and Citrinin

Mycotoxins are secondary metabolites produced by several fungi contaminating crops. In several countries, the maximum permitted levels of mycotoxins are found in foodstuffs and feedstuffs. The common strategy of mycotoxin analysis involves extraction, clean-up and quantification by chromatography. In this paper, we analyzed the reasons of underestimation of ochratoxin A (OTA) content in wine, and overestimation of OTA in wheat, depending on the pH of the clean-up step and the simultaneous presence of citrinin (CIT). We demonstrated that the increase of pH by adding polyethylene glycol (PEG) to wine led to an underestimation of OTA by conversion of OTA into open ring ochratoxin A OP-OA. In comparing three methods of extraction and clean-up for the determination of OTA and CIT in wheat—(i) an inter-laboratory validated method for OTA in cereals using immunoaffinity column clean-up (IAC) and extraction by acetonitrile/water; (ii) a validated method using IAC and extraction with 1% bicarbonate Na; and (iii) an in-house validated method based on acid liquid/liquid extraction—we observed an overestimation of OTA after immunoaffinity clean-up when CIT is also present in the sample, whereas an underestimation was observed when OTA was alone. Under neutral and alkaline conditions, CIT was partially recognized by OTA antibodie

Molecular analysis of cry1A genes of a Bacillus thuringiensis strain and study of the interaction of the corresponding toxins with a biomimetic membrane system

Bacillus thuringiensis (Bt) est une bactérie produisant des inclusions protéiques cristallines à pouvoir insecticide et elle est largement exploitée à l'échelle industrielle. Dans cette étude, des souches de Bt ont été isolées du sol libanais. Nous avons étudié en premier la présence des principaux gènes cry1A codant pour des δ-endotoxines actives sur les lépidoptères. Les souches possédant ces gènes ont été testées pour leur toxicité sur des larves d'Ephestia kuehniella (E. kuehniella). Une souche nommée Lip, étant quatre fois plus toxique sur ces larves que la référence mondiale Bt subsp. kurstaki HD1, fut sélectionnée pour une étude plus approfondie. Après clonage et séquençage, nous avons identifié une nouvelle toxine de type Cry1Aa : Cry1Aa22 et une nouvelle variante de la toxine Cry1Ac. Ces dernières se sont montrées plus toxiques sur des larves d'E. kuehniella, et plus stables en présence des protéases intestinales de ces larves que Cry1Aa et Cry1Ac de HD1 permettant d'expliquer la toxicité élevée de la souche sauvage. D'autre part, nous avons optimisé la construction d'un modèle de membrane biomimétique incluant la membrane de la bordure en brosse intestinale (BBM) des larves d'E. kuehniella. Ces membranes nous ont servi à l'étude de l'interaction des toxines Cry1Aa et Cry1Ac de Lip et celles de HD1. Les toxines de Lip ont interagit différemment et avec une plus grande affinité avec ces modèles que celles de HD1.Tous ces résultats montrent que Lip est une souche intéressante pour une exploitation industrielle et que le modèle de membrane biomimétique est une alternative permettant la prédiction de l'affinité des toxines Cry.Bacillus thuringiensis (Bt) is a bacterium that synthesizes insecticidal proteic crystallin inclusions and is widely used at an industrial scale. In this study, Bt strains were isolated from Lebanese soil. We studied the presence of the main cry1A genes encoding for δ-endotoxins active on Lepidoptera. Strains harboring these genes were tested for their toxicity against Ephestia kuehniella (E. kuehniella) larvae. The strain named Lip, being four folds more toxic to the larvae than the reference strain Bt subsp. kurstaki HD1, was selected for further study. We identified a novel Cry1Aa toxin, Cry1Aa22, and a variety of the Cry1Ac toxin after cloning and sequencing of the corresponding genes. These toxins were more toxic to E. kuehniella larvae and more stable in the presence of these larvae's intestinal midgut juice than Cry1Aa and Cry1Ac of HD1. Moreover, we optimized the construction of a biomimetic membrane model based on the intestinal brush border membrane (BBM) of E. kuehniella larvae. These models were used to study the interaction of Cry1Aa and Cry1Ac of Lip and HD1. Toxins of Lip interacted differently and with a greater affinity with these model membranes than toxins of HD1.These results show that Lip is an interesting Bt strain that could be exploited at an industrial scale. On another hand, the biomimetic membrane constructed in this study could be an alternative allowing the prediction of the Cry toxin's affinity

Analyse moléculaire des gènes cry1A d'une souche de Bacillus thuringiensis et étude de l'interaction des toxines correspondantes dans une modèle de membrane biomimétique

Bacillus thuringiensis (Bt) est une bactérie produisant des inclusions protéiques cristallines à pouvoir insecticide et elle est largement exploitée à l'échelle industrielle. Dans cette étude, des souches de Bt ont été isolées du sol libanais. Nous avons étudié en premier la présence des principaux gènes cry1A codant pour des -endotoxines actives sur les lépidoptères. Les souches possédant ces gènes ont été testées pour leur toxicité sur des larves d'Ephestia kuehniella (E. kuehniella). Une souche nommée Lip, étant quatre fois plus toxique sur ces larves que la référence mondiale Bt subsp. kurstaki HD1, fut sélectionnée pour une étude plus approfondie. Après clonage et séquençage, nous avons identifié une nouvelle toxine de type Cry1Aa : Cry1Aa22 et une nouvelle variante de la toxine Cry1Ac. Ces dernières se sont montrées plus toxiques sur des larves d'E. kuehniella, et plus stables en présence des protéases intestinales de ces larves que Cry1Aa et Cry1Ac de HD1 permettant d'expliquer la toxicité élevée de la souche sauvage. D'autre part, nous avons optimisé la construction d'un modèle de membrane biomimétique incluant la membrane de la bordure en brosse intestinale (BBM) des larves d'E. kuehniella. Ces membranes nous ont servi à l'étude de l'interaction des toxines Cry1Aa et Cry1Ac de Lip et celles de HD1. Les toxines de Lip ont interagit différemment et avec une plus grande affinité avec ces modèles que celles de HD1.Tous ces résultats montrent que Lip est une souche intéressante pour une exploitation industrielle et que le modèle de membrane biomimétique est une alternative permettant la prédiction de l'affinité des toxines Cry.Bacillus thuringiensis (Bt) is a bacterium that synthesizes insecticidal proteic crystallin inclusions and is widely used at an industrial scale. In this study, Bt strains were isolated from Lebanese soil. We studied the presence of the main cry1A genes encoding for -endotoxins active on Lepidoptera. Strains harboring these genes were tested for their toxicity against Ephestia kuehniella (E. kuehniella) larvae. The strain named Lip, being four folds more toxic to the larvae than the reference strain Bt subsp. kurstaki HD1, was selected for further study. We identified a novel Cry1Aa toxin, Cry1Aa22, and a variety of the Cry1Ac toxin after cloning and sequencing of the corresponding genes. These toxins were more toxic to E. kuehniella larvae and more stable in the presence of these larvae's intestinal midgut juice than Cry1Aa and Cry1Ac of HD1. Moreover, we optimized the construction of a biomimetic membrane model based on the intestinal brush border membrane (BBM) of E. kuehniella larvae. These models were used to study the interaction of Cry1Aa and Cry1Ac of Lip and HD1. Toxins of Lip interacted differently and with a greater affinity with these model membranes than toxins of HD1.These results show that Lip is an interesting Bt strain that could be exploited at an industrial scale. On another hand, the biomimetic membrane constructed in this study could be an alternative allowing the prediction of the Cry toxin's affinity.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Measurements and Simulations of Surfactant's Impact on the Conductivity and the <formula formulatype="inline"><tex Notation="TeX">1/f\hbox{1}/f</tex></formula> Noise in Percolation Carbon Nanotube Networks

International audienc

Effect of cardiolipin on the antimicrobial activity of a new amphiphilic aminoglycoside derivative on Pseudomonas aeruginosa.

Amphiphilic aminoglycoside derivatives are promising new antibacterials active against Gram-negative bacteria such as Pseudomonas aeruginosa, including colistin resistant strains. In this study, we demonstrated that addition of cardiolipin to the culture medium delayed growth of P. aeruginosa, favored asymmetrical growth and enhanced the efficiency of a new amphiphilic aminoglycoside derivative, the 3',6-dinonylneamine. By using membrane models mimicking P. aeruginosa plasma membrane composition (POPE:POPG:CL), we demonstrated the ability of 3'6-dinonylneamine to induce changes in the biophysical properties of membrane model lipid systems in a cardiolipin dependent manner. These changes include an increased membrane permeability associated with a reduced hydration and a decreased ability of membrane to mix and fuse as shown by monitoring calcein release, Generalized Polarization of Laurdan and fluorescence dequenching of octadecyl rhodamine B, respectively. Altogether, results shed light on how cardiolipin may be critical for improving antibacterial action of new amphiphilic aminoglycoside derivatives

Targeting Bacterial Cardiolipin Enriched Microdomains: An Antimicrobial Strategy Used by Amphiphilic Aminoglycoside Antibiotics.

Some bacterial proteins involved in cell division and oxidative phosphorylation are tightly bound to cardiolipin. Cardiolipin is a non-bilayer anionic phospholipid found in bacterial inner membrane. It forms lipid microdomains located at the cell poles and division plane. Mechanisms by which microdomains are affected by membrane-acting antibiotics and the impact of these alterations on membrane properties and protein functions remain unclear. In this study, we demonstrated cardiolipin relocation and clustering as a result of exposure to a cardiolipin-acting amphiphilic aminoglycoside antibiotic, the 3',6-dinonyl neamine. Changes in the biophysical properties of the bacterial membrane of P. aeruginosa, including decreased fluidity and increased permeability, were observed. Cardiolipin-interacting proteins and functions regulated by cardiolipin were impacted by the amphiphilic aminoglycoside as we demonstrated an inhibition of respiratory chain and changes in bacterial shape. The latter effect was characterized by the loss of bacterial rod shape through a decrease in length and increase in curvature. It resulted from the effect on MreB, a cardiolipin dependent cytoskeleton protein as well as a direct effect of 3',6-dinonyl neamine on cardiolipin. These results shed light on how targeting cardiolipin microdomains may be of great interest for developing new antibacterial therapies

Negatively Charged Lipids as a Potential Target for New Amphiphilic Aminoglycoside Antibiotics: A BIOPHYSICAL STUDY*

Bacterial membranes are highly organized, containing specific microdomains that facilitate distinct protein and lipid assemblies. Evidence suggests that cardiolipin molecules segregate into such microdomains, probably conferring a negative curvature to the inner plasma membrane during membrane fission upon cell division. 3',6-Dinonyl neamine is an amphiphilic aminoglycoside derivative active against Pseudomonas aeruginosa, including strains resistant to colistin. The mechanisms involved at the molecular level were identified using lipid models (large unilamellar vesicles, giant unilamelllar vesicles, and lipid monolayers) that mimic the inner membrane of P. aeruginosa The study demonstrated the interaction of 3',6-dinonyl neamine with cardiolipin and phosphatidylglycerol, two negatively charged lipids from inner bacterial membranes. This interaction induced membrane permeabilization and depolarization. Lateral segregation of cardiolipin and membrane hemifusion would be critical for explaining the effects induced on lipid membranes by amphiphilic aminoglycoside antibiotics. The findings contribute to an improved understanding of how amphiphilic aminoglycoside antibiotics that bind to negatively charged lipids like cardiolipin could be promising antibacterial compounds

Antimicrobial activity of amphiphilic neamine derivatives: Understanding the mechanism of action on Gram-positive bacteria.

Amphiphilic aminoglycoside derivatives are potential new antimicrobial agents mostly developed to fight resistant bacteria. The mechanism of action of the 3',6-dinonyl neamine, one of the most promising derivative, has been investigated on Gram-negative bacteria, including P. aeruginosa. In this study, we have assessed its mechanism of action against Gram-positive bacteria, S. aureus and B. subtilis. By conducting time killing experiments, we assessed the bactericidal effect induced by 3',6-dinonyl neamine on S. aureus MSSA and MRSA. By measuring the displacement of BODIPY™-TR cadaverine bound to lipoteichoic acids (LTA), we showed that 3',6-dinonyl neamine interacts with these bacterial surface components. We also highlighted the ability of 3',6-dinonyl neamine to enhance membrane depolarization and induce membrane permeability, by using fluorescent probes, DiSCC(5) and propidium iodide, respectively. These effects are observed for both MSSA and MRSA S. aureus as well as for B. subtilis. By electronic microscopy, we imaged the disruption of membrane integrity of the bacterial cell wall and by fluorescence microscopy, we demonstrated changes in the localization of lipids from the enriched-septum region and the impairment of the formation of septum. At a glance, we demonstrated that 3',6-dinonyl neamine interferes with multiple targets suggesting a low ability of bacteria to acquire resistance to this agent. In turn, the amphiphilic neamine derivatives are promising candidates for development as novel multitarget therapeutic antibiotics

New Broad-spectrum Antibacterial Amphiphilic Aminoglycosides Active Against Resistant Bacteria: From Neamine Derivatives to Smaller Neosamine Analogues

Aminoglycosides (AGs) constitute a major family of potent and broad-spectrum antibiotics disturbing protein synthesis through binding to the A site of 16S rRNA. Decades of widespread clinical use of AGs strongly reduced their clinical efficacy through the selection of resistant bacteria. Recently, conjugation of lipophilic groups to AGs generated a novel class of potent antibacterial amphiphilic aminoglycosides (AAGs) with significant improved activities against various sensitive and resistant bacterial strains. We have identified amphiphilic 3’,6-dialkyl derivatives of the small aminoglycoside neamine as broad spectrum antibacterial agents targeting bacterial membranes. Here, we report on the synthesis and the activity against sensitive and resistant Gram-negative and/or Gram-positive bacteria of new amphiphilic 3’,4’-dialkyl neamine derivatives and of their smaller analogues in the 6- aminoglucosamine (neosamine) series prepared from N-acetylglucosamine

Involvement of the Prion Protein in the Protection of the Human Bronchial Epithelial Barrier Against Oxidative Stress

International audienceAIM: Bronchial epithelium acts as a defensive barrier against inhaled pollutants and microorganisms. This barrier is often compromised in inflammatory airway diseases that are characterized by excessive oxidative stress responses, leading to bronchial epithelial shedding, barrier failure, and increased bronchial epithelium permeability. Among proteins expressed in the junctional barrier and participating to the regulation of the response to oxidative and to environmental stresses is the cellular prion protein (PrPC). However, the role of PrPC is still unknown in the bronchial epithelium. Herein, we investigated the cellular mechanisms by which PrPC protein participates into the junctional complexes formation, regulation, and oxidative protection in human bronchial epithelium.RESULTS: Both PrPC messenger RNA and mature protein were expressed in human epithelial bronchial cells. PrPC was localized in the apical domain and became lateral, at high degree of cell polarization, where it colocalized and interacted with adherens (E-cadherin/γ-catenin) and desmosomal (desmoglein/desmoplakin) junctional proteins. No interaction was detected with tight junction proteins. Disruption of such interactions induced the loss of the epithelial barrier. Moreover, we demonstrated that PrPC protection against copper-associated oxidative stress was involved in multiple processes, including the stability of adherens and desmosomal junctional proteins.INNOVATION: PrPC is a pivotal protein in the protection against oxidative stress that is associated with the degradation of adherens and desmosomal junctional proteins.CONCLUSION: Altogether, these results demonstrate that the loss of the integrity of the epithelial barrier by oxidative stress is attenuated by the activation of PrPC expression, where deregulation might be associated with respiratory diseases