Sophie Jan

Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Δ6-desaturase activity

The Δ6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat Δ6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of Δ6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and Δ6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for Δ6-desaturase activity.

Comparative effect of fenofibrate on hepatic desaturases in wild-type and peroxisome proliferator-activated receptor α-deficient mice

In this study is presented the effect of fenofibrate, a prototypical peroxisome proliferator of the fibrate class, on wild-type and peroxisome proliferator-activated receptor α (PPARα)-/-mouse liver FA profile, desaturase mRNA levels, and activities. We established that, following peroxisome proliferator exposure, the hepatic FA profile was greatly modified. These modifications in hepatic FA content required the expression of PPARα, as they are suppressed in transgenic mice deficient in this nuclear receptor. Following peroxisome proliferator exposure, Δ6- and Δ5-desaturase mRNA levels and activities were increased in wild-type but not in PPARα-deficient mouse liver. These results suggest the involvement of PPARα in the control of hepatic Δ6- and Δ5-desaturases in mice. Their roles in minimizing long-chain PUFA depletion in the liver during peroxisome proliferator exposure are discussed.

Isolation and characterization of a psychrotolerant toxin producer, Bacillus weihenstephanensis, in liquid egg products.

A psychrotolerant bacteria of the Bacillus cereus group was found responsible for the spoilage of whole liquid egg products. By sequencing a 16S rRNA region and performing a PCR amplification of specific 16S rRNA and cspA signatures, a Bacillus weihenstephanensis was identified. Characterization of this strain shows its ability to grow in defined medium as well as in whole liquid egg at refrigerated temperatures. The strain isolated possesses genes encoding for hemolysin BL, nonhemolytic enterotoxin, and B. cereus enterotoxins and produces enterotoxins with cytotoxic activity in whole liquid egg, even at refrigerated temperatures. The isolate exhibits a clear ability to stick and form biofilms on stainless steel, the most common material used in egg breaking factories, as well as on model hydrophilic (glass) and hydrophobic (polytetrafluoroethylene) materials. These findings show the necessity to monitor for Bacillus contamination in egg products that are often used in the composition of particularly susceptible finished products such as cream, dessert, dairy, meat, and seafood.

Hen Egg White Lysozyme Permeabilizes Escherichia coli Outer and Inner Membranes

Natural preservatives answer the consumer demand for long shelf life foods, synthetic molecules being perceived as a health risk. Lysozyme is already used because of its muramidase activity against Gram-positive bacteria. It is also described as active against some Gram-negative bacteria; membrane disruption would be involved, but the mechanism remains unknown. In this study, a spectrophotometric method using the mutant Escherichia coli ML-35p has been adapted to investigate membrane disruption by lysozyme for long durations. Lysozyme rapidly increases the permeability of the outer membrane of E. coli due to large size pore formation. A direct delayed activity of lysozyme against the inner membrane is also demonstrated, but without evidence of perforations.

Egg white versus Salmonella Enteritidis! A harsh medium meets a resilient pathogen.

Salmonella enterica serovar Enteritidis is the prevalent egg-product-related food-borne pathogen. The egg-contamination capacity of S. Enteritidis includes its exceptional survival capability within the harsh conditions provided by egg white. Egg white proteins, such as lysozyme and ovotransferrin, are well known to play important roles in defence against bacterial invaders. Indeed, several additional minor proteins and peptides have recently been found to play known or potential roles in protection against bacterial contamination. However, although such antibacterial proteins are well studied, little is known about their efficacy under the environmental conditions prevalent in egg white. Thus, the influence of factors such as temperature, alkalinity, nutrient restriction, viscosity and cooperative interactions on the activities of antibacterial proteins in egg white remains unclear. This review critically assesses the available evidence on the antimicrobial components of egg white. In addition, mechanisms employed by S. Enteritidis to resist egg white exposure are also considered along with various genetic studies that have shed light upon egg white resistance systems. We also consider how multiple, antibacterial proteins operate in association with specific environmental factors within egg white to generate a lethal protective cocktail that preserves sterility.

Native lysozyme and dry-heated lysozyme interactions with membrane lipid monolayers: Lateral reorganization of LPS monolayer, model of the Escherichia coli outer membrane

Lysozyme is mainly described active against Gram-positive bacteria, but is also efficient against some Gram-negative species. Especially, it was recently demonstrated that lysozyme disrupts Escherichia coli membranes. Moreover, dry-heating changes the physicochemical properties of the protein and increases the membrane activity of lysozyme. In order to elucidate the mode of insertion of lysozyme into the bacterial membrane, the interaction between lysozyme and a LPS monolayer mimicking the E. coli outer membrane has been investigated by tensiometry, ellipsometry, Brewster angle microscopy and atomic force microscopy. It was thus established that lysozyme has a high affinity for the LPS monolayer, and is able to insert into the latter as long as polysaccharide moieties are present, causing reorganization of the LPS monolayer. Dry-heating increases the lysozyme affinity for the LPS monolayer and its insertion capacity; the resulting reorganization of the LPS monolayer is different and more drastic than with the native protein.

Dry-heating of lysozyme increases its activity against Escherichia coli membranes.

For food as well as for medical applications, there is a growing interest in novel and natural antimicrobial molecules. Lysozyme is a promising candidate for the development of such molecules. This protein is largely studied and known for its muramidase activity against Gram-positive bacteria, but it also shows antimicrobial activity against Gram-negative bacteria, especially when previously modified. In this study, the activity of dry-heated lysozyme (DH-L) against Escherichia coli has been investigated and compared to that of native lysozyme (N-L). Whereas N-L only delays bacterial growth, DH-L causes an early-stage population decrease. The accompanying membrane permeabilization suggests that DH-L induces either larger pores or more pores in the outer membrane as compared to N-L, as well as more ion channels in the inner membrane. The strong morphological modifications observed by optical microscopy and atomic force microscopy when E. coli cells are treated with DH-L are consistent with the suggested disturbances of membrane integrity. The higher hydrophobicity, surface activity, and positive charge induced by dry-heating could be responsible for the increased activity of DH-L on the E. coli membranes.

Role of Incubation Conditions and Protein Fraction on the Antimicrobial Activity of Egg White against Salmonella Enteritidis and Escherichia coli

The mechanism of egg white antimicrobial activity involves specific molecules and environmental factors. However, it is difficult to compare the data from the literature because of the use of various bacterial strains and incubation conditions. The aim of our study was to determine the effect of temperature, pH, inoculum size, and egg white protein concentration on egg white antimicrobial activity and to investigate the putative interactions among these factors by conducting a complete factorial design analysis. The behavior of Salmonella Enteritidis and Escherichia coli was studied after precultivation in tryptic soy broth and Luria-Bertani broth, respectively, using three different egg white protein concentrations (0, 10, and 100%), five temperatures (37, 40, 42, 45, and 48°C), two pHs (7.8 and 9.3), and six inoculum levels (3 to 8 log CFU/ml). The essential role of temperature was identified. An inverse relationship was observed between bacterial growth and an increase in temperature. The role of egg white proteins was clearly demonstrated. In the absence of egg white proteins, bacterial growth occurred under most incubation conditions, whereas the presence of 10 and 100% protein produced bacteriostatic or bactericidal effects. The interaction between temperature and protein concentration was significant. At the highest tested temperatures, proteins were less involved in the bactericidal effect. Bacterial destruction was higher at pH 9.3 than at pH 7.8. Under our experimental conditions, Salmonella Enteritidis was more resistant to inactivation by egg white than was E. coli.

Ovotransferrin Plays a Major Role in the Strong Bactericidal Effect of Egg White against the Bacillus cereus Group.

Bacillus cereus group bacteria are opportunistically pathogenic spore-forming microorganisms well known in the sector of pasteurized food products because of their involvement in spoilage events. In the sector of egg product processing, these bacteria may lead to important economic losses. It seemed then relevant to study their behavior in egg white, a widely used egg product usually recognized as developing different levels of antimicrobial activities depending on the environmental conditions. A strong bactericidal effect (decrease in the bacterial population of 6.1 ± 0.2 log CFU/ml) was observed for 68 B. cereus group isolates, independently incubated at 30°C in egg white at pH 9.3 (natural egg white pH). To determine which components could explain such a strong bactericidal effect, an experimental strategy was carried out, based on egg white fractionation by ultrafiltration and by anion-exchange liquid chromatography. The role of the protein fraction was thus demonstrated, and subsequent nano-liquid chromatography-tandem mass spectrometry analyses allowed identification of ovotransferrin as a major protein involved. The strong bactericidal effect was confirmed in the presence of commercial ovotransferrin. Such a bactericidal effect (i.e., a decrease in the bacterial population through cell death) had never been described because ovotransferrin is known for its bacteriostatic effect (i.e., inhibition of growth) due to its ability to chelate iron. Surprisingly, the addition of iron did not reverse the bactericidal effect of ovotransferrin under alkaline conditions (pH 9.3), whereas it completely reversed this effect at pH 7.3. Ovotransferrin was shown to provoke a perturbation of the electrochemical potential of the cytoplasmic membrane. A membrane disturbance mechanism could, hence, be involved, leading to the lysis of B. cereus group bacteria incubated in egg white.

Biochemical and micrographic evidence of Escherichia coli membrane damage during incubation in egg white under bactericidal conditions.

Bacterial membranes are often thought to be the main targets of the antimicrobial activity of egg white. In order to test this hypothesis, the state of the membranes of Escherichia coli K-12 cells during either bactericidal (45°C) or bacteriostatic (30°C) incubation in egg white at natural alkaline pH was studied by biochemical methods. Namely, the permeability of the outer membrane was evaluated through its ability to incorporate a hydrophobic fluorescent probe (1-N-phenylnaphthylamine), and the permeability of the cytoplasmic membrane was evaluated through the release of a specific intracellular enzyme (β-galactosidase). The bacteria were observed by atomic force microscopy in order to support the biochemical results. At 45°C, the outer membrane of E. coli K-12 incorporated the hydrophobic probe, suggesting that it was disrupted. In addition, the cytoplasmic β-galactosidase was released at this temperature. The atomic force microscopy analysis revealed the formation of spheroplasts, which provided further evidence of the cell wall disruption and a progressive release of cellular contents. At 30°C, biochemical and micrographic experiments confirmed that membrane integrity was preserved. These techniques provide a useful approach for studying the mechanisms of bacterial cell death in egg white.