Cécile Mangavel

Structure and Function of a Mitochondrial Late Embryogenesis Abundant Protein Are Revealed by Desiccation

Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into α-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes.

Exploring the interactions of gliadins with model membranes: Effect of confined geometry and interfaces

Mechanisms leading to the assembly of wheat storage proteins into proteins bodies within the endoplasmic reticulum (ER) of endosperm cells are unresolved today. In this work, physical chemistry parameters which could be involved in these processes were explored. To model the confined environment of proteins within the ER, the dynamic behavior of γ‐gliadins inserted inside lyotropic lamellar phases was studied using FRAP experiments. The evolution of the diffusion coefficient as a function of the lamellar periodicity enabled to propose the hypothesis of an interaction between γ‐gliadins and membranes. This interaction was further studied with the help of phospholipid Langmuir monolayers. γ‐ and ω‐gliadins were injected under DMPC and DMPG monolayers and the two‐dimensional (2D) systems were studied by Brewster angle microscopy (BAM), polarization modulation infrared reflection‐absorption spectroscopy (PM‐IRRAS), and surface tension measurements. Results showed that both gliadins adsorbed under phospholipid monolayers, considered as biological membrane models, and formed micrometer‐sized domains at equilibrium. However, their thicknesses, probed by reflectance measurements, were different: ω‐gliadins aggregates displayed a constant thickness, consistent with a monolayer, while the thickness of γ‐gliadins aggregates increased with the quantity of protein injected. These different behaviors could find some explanations in the difference of aminoacid sequence distribution: an alternate repeated ‐ unrepeated domain within γ‐gliadin sequence, while one unique repeated domain was present within ω‐gliadin sequence. All these findings enabled to propose a model of gliadins self‐assembly via a membrane interface and to highlight the predominant role of wheat prolamin repeated domain in the membrane interaction. In the biological context, these results would mean that the repeated domain could be considered as an anchor for the interaction with the ER membrane and a nucleus point for the formation and growth of protein bodies within endosperm cells.

Infrared Spectroscopy as Applied to Glycinin Film and Gel Formation Kinetics

Secondary structural changes of glycinin during film and gel formation were studied by infrared spectroscopy. Two complementary data treatments—i.e., principal component analysis and two-dimensional correlation spectroscopy—were applied to the infrared spectra in order to determine the time evolution of conformational changes. Kinetic curves, assessed by taking scores of the first principal components into account, revealed more pronounced changes in the case of film formation. The development of aggregates in film and gel, through intermolecular β-sheets, was identified by an increase of the absorbance band at around 1620 cm−1. Two-dimensional infrared correlation spectroscopy made it possible to observe a decrease in both α-helices and disordered structures prior to the appearance of aggregates. Changes in intramolecular β-sheets took place in a second step. The more drastic changes observed in the case of film formation were believed to result from dehydration and aggregation coupled phenomena.

Oxidative stability of DHA phenolic ester

Docosahexaenoic acid vanillyl ester (DHA-VE) was synthesized from docosahexaenoic acid ethyl ester (DHA-EE) and vanillyl alcohol by a solvent-free alcoholysis process catalysed by Candida antarctica lipase B. Oxidative stability of pure DHA-VE and the crude reaction medium consisting of 45% DHA-VE and 55% DHA-EE were compared with that of DHA-EE under various storage conditions. Oxidation progress was followed by determination of conjugated dienes and FTIR measurements. Analyses showed that DHA-EE was rapidly oxidised under all storage conditions in comparison with DHA-VE and crude reaction medium, whatever the temperature and the storage time. The grafting of vanillyl alcohol appeared as a powerful way to stabilize DHA against oxidation. Thanks to their stability, both DHA-VE and the crude reaction medium, allowing the production of the ester, offer huge potential as functional ingredients.

Recombinant pediocin in Lactococcus lactis : increased production by propeptide fusion and improved potency by co-production with PedC

We describe the impact of two propeptides and PedC on the production yield and the potency of recombinant pediocins produced in Lactococcus lactis. On the one hand, the sequences encoding the propeptides SD or LEISSTCDA were inserted between the sequence encoding the signal peptide of Usp45 and the structural gene of the mature pediocin PA‐1. On the other hand, the putative thiol‐disulfide oxidoreductase PedC was coexpressed with pediocin. The concentration of recombinant pediocins produced in supernatants was determined by enzyme‐linked immunosorbent assay. The potency of recombinant pediocins was investigated by measuring the minimal inhibitory concentration by agar well diffusion assay. The results show that propeptides SD or LEISSTCDA lead to an improved secretion of recombinant pediocins with apparently no effect on the antibacterial potency and that PedC increases the potency of recombinant pediocin. To our knowledge, this study reveals for the first time that pediocin tolerates fusions at the N‐terminal end. Furthermore, it reveals that only expressing the pediocin structural gene in a heterologous host is not sufficient to get an optimal potency and requires the accessory protein PedC. In addition, it can be speculated that PedC catalyses the correct formation of disulfide bonds in pediocin.

Enzymatic Crosslinking Enhance Film Properties of Deamidated Gluten

Covalent bonds were introduced in deamidated gluten films by transglutaminase. The type of cross-links was modulated by adding diamines in the film-forming solution. Biochemical and mechanical properties of the resulting films were investigated. Film properties of deamidated gluten were compared to those of native gluten. Plasticizer content modified considerably the film properties and a ratio of 0.35 was chosen for the following experiments. At this ratio, the addition of increasing diamine concentration in the film solution was shown to have a slight positive plasticizer effect. As shown by SDS PAGE of the film proteins, transglutaminase was efficient in catalysing the formation of polymers in the film solution. The loss of solubility was related to the formation of high molecular weight polymers in the film. In all cases, the action of transglutaminase induced simultaneously an increase in strain and stress of the films. The addition of diamines in the film solution affected more the strain than the stress properties. These diamines, able to react at their two extremities, probably acted as spacers between gluten proteins. The IR study didn’t show any modification of the secondary structure of the proteins in the film. Transglutaminase was efficient in introducing new cross-links in gluten films. This modification enhanced their mechanical properties and modified their solubility properties.

MultiLocus Sequence Typing of Carnobacterium maltaromaticum strains associated with fish disease and dairy products

Carnobacterium maltaromaticum is a lactic acid bacterium of technological interest in the field of dairy ripening and food bioprotection and is generally recognized as safe in the United States. As it is associated with fish infections, the European Food Safety Agency did not include this species in the qualified presumption safety list of micro‐organisms. This implies that the risk assessment for the species has to be performed at the strain level.

High-Throughput Identification of Candidate Strains for Biopreservation by Using Bioluminescent Listeria monocytogenes

This article describes a method for high-throughput competition assays using a bioluminescent strain of L. monocytogenes. This method is based on the use of the luminescent indicator strain L. monocytogenes EGDelux. The luminescence of this strain is correlated to growth, which make it suitable to monitor the growth of L. monocytogenes in mixed cultures. To this aim, luminescence kinetics were converted into a single numerical value, called the Luminescence Disturbance Indicator (LDI), which takes into account growth inhibition phenomena resulting in latency increase, decrease in the luminescence rate, or reduction of the maximum luminescence. The LDI allows to automatically and simultaneously handle multiple competition assays which are required for high-throughput screening (HTS) approaches. The method was applied to screen a collection of 1810 strains isolated from raw cow’s milk in order to identify non-acidifying strains with anti-L. monocytogenes bioprotection properties. This method was also successfully used to identify anti-L. monocytogenes candidates within a collection of Lactococcus piscium, a species where antagonism was previously described as non-diffusible and requiring cell-to-cell contact. In conclusion, bioluminescent L. monocytogenes can be used in HTS to identify strains with anti-L. monocytogenes bioprotection properties, irrespectively of the inhibition mechanism.

Repeat-based Sequence Typing of Carnobacterium maltaromaticum

Carnobacterium maltaromaticum is a Lactic Acid Bacterium (LAB) of technological interest for the food industry, especially the dairy as bioprotection and ripening flora. The industrial use of this LAB requires accurate and resolutive typing tools. A new typing method for C. maltaromaticum inspired from MLVA analysis and called Repeat-based Sequence Typing (RST) is described. Rather than electrophoresis analysis, our RST method is based on sequence analysis of multiple loci containing Variable-Number Tandem-Repeats (VNTRs). The method described here for C. maltaromaticum relies on the analysis of three VNTR loci, and was applied to a collection of 24 strains. For each strain, a PCR product corresponding to the amplification of each VNTR loci was sequenced. Sequence analysis allowed delineating 11, 11, and 12 alleles for loci VNTR-A, VNTR-B, and VNTR-C, respectively. Considering the allele combination exhibited by each strain allowed defining 15 genotypes, ending in a discriminatory index of 0.94. Comparison with MLST revealed that both methods were complementary for strain typing in C. maltaromaticum.

Film formation from wheat gluten proteins by FTIR spectroscopy

The potential of wheat gluten proteins in the preparation of protein-based films in alkaline conditions has already been demonstrated [1], [2]. However, little information is available concerning protein network formation and polypeptide interactions in the film. The aim of this study was then to obtain basic informations about the modification of protein structures induced by film-forming processes.