Martine Rouvet

Cell surface-associated elongation factor Tu mediates the attachment of Lactobacillus johnsonii NCC533 (La1) to human intestinal cells and mucins

ABSTRACT The aim of this work was to identify Lactobacillus johnsonii NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins. Incubation of Caco-2 intestinal epithelial cells with an L. johnsonii La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor. The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria. Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule. Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent. Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity. In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14. Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.

Incorporation of Caseinoglycomacropeptide and Caseinophosphopeptide into the Salivary Pellicle Inhibits Adherence of Mutans Streptococci

The protective effects of milk and milk products against dental caries have been demonstrated in many animal studies. We have shown that this effect was mediated by micellar casein or caseinopeptide derivatives. A reduction in the Streptococcus sobrinus population in the oral microbiota of animals fed diets supplemented with these milk components was consistently observed. A possible explanation for these findings is that milk components are incorporated into the salivary pellicle, thereby reducing the adherence of S. sobrinus. This hypothesis was tested in vitro by the incubation of bovine enamel discs with unstimulated saliva. The resulting pellicle was washed and incubated with caseinoglycomacropeptide (CGMP) and/or caseinophosphopeptide (CPP) labeled with 17- and 12-nm gold particles. All samples were prepared for electron microscopy by high-pressure freezing followed by freeze-substitution. It was demonstrated by high-resolution scanning electron microscopy with back-scattered electron imaging, as well as by transmission electron microscopy, that both peptides were incorporated into the pellicle in exchange for albumin, confirming previous findings. This protein was identified with a mouse anti-human serum albumin followed by goat anti-mouse IgG labeled with 25-nm gold particles. Incorporation of CGMP and/or CPP into sali\ arv pellicles reduced the adherence of both S. sobrinus and S. mutans significantly. It is suggested that the calcium-and phosphate-rich micellar casein or caseinopeptides are incorporated into the pellicle. The resulting ecological shifts, together with the increased remineralization potential of this biofilm, may explain its modified cariogenic potential.

Internal structure and colloidal behaviour of covalent whey protein microgels obtained by heat treatment

Covalently cross-linked whey protein microgels (WPM) were produced without the use of a chemical cross-linking agent. The hierarchical structure of WPM is formed by a complex interplay of heat denaturation, aggregation, electrostatic repulsion, and formation of disulfide bonds. Therefore, well-defined spherical particles with a diameter of several hundreds of nanometers and with relatively low polydispersity are formed in a narrow pH regime (5.8–6.2) only. WPM production was carried out on large scale by heating a protein solution in a plate-plate heat exchanger. Thereafter, the microgels were concentrated by microfiltration and spray dried into a powder. The spherical structure of the WPM was conserved in the powder. After re-dispersion, the microgel dispersions fully recovered their initial structure and size distribution. Due to the formation of disulfide bonds the particles were internally covalently cross-linked and were remarkably stable in a large pH range. Because of the pH dependent charge of the constituents the particles underwent significant size changes upon shifting the pH. Small angle X-ray scattering experiments were used to reveal their internal structure, and we report on the pH-induced structural changes occurring on different length scale. Our experiments showed that close analogies could be drawn to internally cross-linked and pH-responsive microgels based on weak polyelectrolytes. WPM also exhibited a pronounced swelling at pH values below the isoelectric point (IEP), and a collapse at the IEP. However, in contrast to classical microgels, WPM are not build up by simple polymer chains but possess a complex hierarchical structure consisting of strands formed by clusters of aggregated denatured proteins that act as primary building blocks. They were flexible enough to respond to changes of the environment, and were stable enough to tolerate pH values where the proteins were highly charged and the strands were stretched.

Multiscale Characterization of Individualized β-Lactoglobulin Microgels Formed upon Heat Treatment under Narrow pH Range Conditions

Aqueous dispersions of demineralized beta-lactoglobulin (beta-lg) were held at 85 degrees C for 15 min at a constant protein concentration of 1 wt % in the pH range of 3.0-7.0. This led to denatured protein content ranging from 20% (pH 3.0) to 90% (pH 5.0). The protein aggregates formed were characterized as to their stability to sedimentation (turbidity), morphology, size, surface charge, ANS surface hydrophobicity, and content in accessible thiol groups. Additionally, the changes in secondary structures of the protein upon heating were followed by Fourier transform infrared spectroscopy (FTIR). Stable dispersions (no sedimentation for 10 min) of individualized beta-lg microgels were obtained at specific pH 4.6 and 5.8, corresponding to an aggregation yield of about 80%. The width of the pH region leading to these microgels was 0.3 pH unit below or above the two specific pH values. Microgels were characterized by a spherical shape and remarkably low polydispersity in size (<0.2). Their z-average hydrodynamic diameter determined by dynamic light scattering (DLS) was between 160 and 220 nm, and their zeta-potential was +30 or -40 mV, depending on the initial pH before heating. Microgels obtained at pH 4.6 displayed a lower binding capacity for ANS and a lower content of accessible thiol groups as compared to those obtained at pH 5.8. Both types of microgels might therefore differ in their internal and interfacial structures. Between pH 4.6 and 5.8, large sedimenting protein particulates were obtained, whereas soluble aggregates were formed at pH <4.6 or >5.8. Interestingly, DLS experiments showed that before heating, beta-lg was mainly present in an oligomeric state at pH 4.6 and 5.8. This result was confirmed by FTIR measurements indicating the stronger contribution of the 1616-1624 cm(-1) spectral band corresponding to intermolecular beta-sheets in the pH range of 4.0-6.0. Upon heating, FTIR spectroscopy revealed that individualized microgels were obtained under pH conditions where a balance between attractive forces arising from protein unfolding leading to the formation of intermolecular beta-sheets (1616-1624 cm(-1 )band) and the repulsive electrostatic forces due to the initial protein net charge was achieved.

Inhibition of Giardia intestinalis by Extracellular Factors from Lactobacilli: an In Vitro Study

ABSTRACT The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent.Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G1 phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation ofGiardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.

Contribution of Aggregation-Promoting Factor to Maintenance of Cell Shape in Lactobacillus gasseri 4B2

Aggregation-promoting factor (APF) was originally described as a protein involved in the conjugation and autoaggregation of Lactobacillus gasseri 4B2, whose corresponding apf gene was cloned and sequenced. In this report, we identified and sequenced an additional apf gene located in the region upstream of the previously published one. Inactivation of both apf genes was unsuccessful, indicating that APF function may be essential for the cell. Overproduction of APF proteins caused drastic alteration in the cell shape of this strain. These cells were irregular, twisted, enlarged, and tightly bound in unbreakable clumps of chains. Down-regulation of APF synthesis was achieved by cloning of the apf2 promoter region on a high-copy-number plasmid, which recruited a putative apf activator. As a consequence, the shape of the corresponding recombinant cells was elongated (filamentous) and cell division sites were no longer visible. None of the induced changes in APF production levels was clearly correlated with modifications of the aggregation phenotype. This report shows, for the first time, that APF proteins are mainly critical for L. gasseri 4B2 cell shape maintenance.

Monocomponent hexa- and dodecaethylene glycol succinyl-tocopherol esters: Self-assembly structures, cellular uptake and sensitivity to enzyme hydrolysis

We have chemically synthesized two water-soluble forms of tocopherol succinate linked via an ester bond to hexaethylene glycol and dodecaethylene glycol. The self-assembly structure of the former in water is vesicular, whereas the latter forms elongated micelles. We treated Caco-2 cells with these compounds in these physical forms, in addition to a mixed micelle form. The intact compounds were taken up into the cells, influenced by both the chain length and the physical structure. In addition, the tocopherol derivatives were also metabolized into tocopherol succinate and tocopherol inside the cell. The total hydrolysis and uptake into the cells was two-fold higher from tocopherol hexaethylene glycol succinate in the form of mixed micelles than in vesicular form as assessed by analyzing intracellular tocopherol and tocopherol succinate. The longer polyethylene glycol chain gave a higher intracellular tocopherol succinate/tocopherol ratio. The major intracellular esterase in Caco-2 cells is carboxyl esterase 1 (EC 3.1.1.1), and in silico modelling studies show that the position of docking and hence the site of hydrolysis is influenced by the chain length. The in silico prediction is consistent with the in vitro data, since a longer chain length is predicted to favour hydrolysis of the ester bond between the succinate and polyethylene glycol moieties.

Tuneable thickness barriers for composite o/w and w/o capsules, films, and their decoration with particles

Oil-in-water and water-in-oil capsules, and flat membranes of tuneable thickness and composition were prepared in one single facile step, based on the interfacial complexation between chitosan and anionic phosphatidic fatty acids. The phosphatidic acid molecules were introduced via the oil phase. The thickness of the capsule shell or the membrane grows by a diffusion-controlled mechanism, hence can be tuned using e.g. concentration and formation time parameters. A mechanism is proposed to explain the observed behavior. The capsule size is set by the emulsification conditions applied. Microfluidic methods proved useful for the generation of hollow capsules of uniform size and thickness in one step. The capsules and membranes display remarkable integrity over several years in a pH window 2–14. The thickness can easily reach several micrometres within an hour for the wet capsule shell or membrane, which explains the high interfacial rheological properties measured. Hence various processes can be envisaged after their formation. The simple preparation opens the way to tailored, environment-responsive composite systems for fabricating biopolymer-based materials for various applications. The capsules could be washed from the surrounding continuous phase and placed into one of arbitrary choice. Furthermore, the surface of the w/o capsules and of the membranes could be decorated by particles that attach to the water/oil interface with a high energy. The choice of the particle functionality is left open.