André Schanck

Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering

We report the engineering of Lactococcus lactis to produce the amino acid l-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH→l-lactate + NAD+) is performed by l-lactate dehydrogenase (l-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (l-AlaDH; pyruvate + NADH + NH4+→l-alanine + NAD+ + H2O). Expression of l-AlaDH in an l-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of l-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.

High-level production of the low-calorie sugar sorbitol by Lactobacillus plantarum through metabolic engineering.

ABSTRACT Sorbitol is a low-calorie sugar alcohol that is largely used as an ingredient in the food industry, based on its sweetness and its high solubility. Here, we investigated the capacity of Lactobacillus plantarum, a lactic acid bacterium found in many fermented food products and in the gastrointestinal tract of mammals, to produce sorbitol from fructose-6-phosphate by reverting the sorbitol catabolic pathway in a mutant strain deficient for both l- and d-lactate dehydrogenase activities. The two sorbitol-6-phosphate dehydrogenase (Stl6PDH) genes (srlD1 and srlD2) identified in the genome sequence were constitutively expressed at a high level in this mutant strain. Both Stl6PDH enzymes were shown to be active, and high specific activity could be detected in the overexpressing strains. Using resting cells under pH control with glucose as a substrate, both Stl6PDHs were capable of rerouting the glycolytic flux from fructose-6-phosphate toward sorbitol production with a remarkably high efficiency (61 to 65% glucose conversion), which is close to the maximal theoretical value of 67%. Mannitol production was also detected, albeit at a lower level than the control strain (9 to 13% glucose conversion), indicating competition for fructose-6-phosphate rerouting by natively expressed mannitol-1-phosphate dehydrogenase. By analogy, low levels of this enzyme were detected in both the wild-type and the lactate dehydrogenase-deficient strain backgrounds. After optimization, 25% of sugar conversion into sorbitol was achieved with cells grown under pH control. The role of intracellular NADH pools in the determination of the maximal sorbitol production is discussed.

Molecular parameters involved in aminoglycoside nephrotoxicity

Aminoglycoside antibiotics are hydrophilic molecules consisting of an animated cyclitol associated with amino sugar. They bind in vivo as well as in vitro to negatively charged membranes. Their use as chemotherapeutic agents is unfortunately accompanied by oto- and nephrotoxic reactions, and the purpose of this review is to examine the role of the molecular interactions between aminoglycosides and membranes in the development of nephrotoxicity. 31P Nuclear magnetic resonance (NMR) and fluorescence depolarization have been used to characterize the effect of aminoglycosides on phosphate heads and fatty acyl chains of phospholipids. 15N NMR has been used to obtain interesting information on regioselective interactions of amino groups of antibiotics with phospholipids. The binding of aminoglycosides with negatively charged membranes is associated with impairment of phospholipid catabolism, change in membrane permeability, and membrane aggregation. Biochemical analysis and 1H NMR spectroscopy have brought information on the molecular mechanism involved in the impairment of phospholipid catabolism. Nephrotoxic aminoglycosides could induce sequestration of phosphatidylinositol and therefore reduce the amount of negative charge available for optimal lysosomal phospholipase activity toward phosphatidylcholine included in liposomes that also contain cholesterol and sphingomyelin. Conformational analysis shows that aminoglycosides, which have a high potency to inhibit lysosomal phospholipase activity, adopt an orientation parallel to the lipid/water interface. This orientation of the aminoglycoside molecule at the interface is also critical to explain the marked increase of membrane permeability induced by less nephrotoxic aminoglycosides such as isepamicin and amikacin. This effect is indeed only observed with aminoglycosides oriented perpendicular to this interface, probably related to the creation of a local condition of disorder. The impairment of phospholipid catabolism, which is considered to be an early and significant step in the development of aminoglycoside toxicity, is therefore not related to the change in membrane permeability. However, the role of this latter phenomenon and of membrane aggregation for aminoglycoside nephrotoxicity could be further investigated.

Piracetam-induced changes to membrane physical properties: A combined approach by 31P nuclear magnetic resonance and conformational analysis

Piracetam, Nootropil (2-oxo-1-pyrrolidine acetamide), is a drug promoting erythrocyte deformability. To establish the mode of action of this compound, we have investigated its influence on the organization of model phospholipid membranes. 31P NMR data show that the drug induces a structural modification in liposomes made of phosphatidylcholine and phosphatidylethanolamine. Our conformational analysis results have allowed the interpretation of the effect of piracetam on these model membranes: the specific interaction between the drug molecules and the phosphate headgroups induces a new organization of the lipids favouring formation of mobile drug-phospholipid complexes that exhibit an isotropic-type signal in the 31P NMR spectra.

Ultrastructural, physico-chemical and conformational study of the interactions of gentamicin and bis(beta-diethylaminoethylether) hexestrol with negatively-charged phospholipid layers

Aminoglycoside antibiotics such as gentamicin, which are fully hydrophilic, and cationic amphiphilic drugs such as bis(beta-diethylaminoethylether)hexestrol (DEH), are both known to inhibit lysosomal phospholipases and induce phospholipidosis. This enzymatic inhibition is probably related to the neutralization of the surface negative charges on which the lysosomal phospholipases A1 and A2 are dependent to express fully their activities (Mingeot-Leclerq et al., Biochem Pharmacol 37: 591-599, 1988). Using negatively charged liposomes, we show by 31P NMR spectroscopy that both gentamicin and DEH cause a significant restriction in the phosphate head mobility and, in sonicated vesicles, the appearance of larger bilayer structures. Both DEH and gentamicin increased the apparent size of sonicated negatively charged liposomes (but not of neutral liposomes) as measured by quasi-elastic light scattering spectroscopy. Examination of replicas from freeze-etched samples, however, revealed that gentamicin caused aggregation of liposomes, whereas DEH induced their fusion and the formation of intramembranous roundly shaped structures. Only DEH caused a significant decrease of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, a fluorescent lipid-soluble probe. In addition, DEH, but not gentamicin, interfered with the bilayer to hexagonal phase transition occurring in dioleoyl- and dielaidoylphosphatidylethanolamine liposomes upon warming, and caused the appearance of an isotropic signal suggestive of the formation of inverted micelles. In computer-aided conformational analysis of the molecules at a simulated air-water interface, gentamicin was shown to display a largely-open crescent shape. When surrounded by phosphatidylinositol molecules, it remained as such at the interface which it locally mis-shaped, establishing close contact with the negatively charged phospho groups. In contrast, DEH could be oriented perpendicularly to the interface, with its two cationic groups associated with the phospho groups, and its phenyl- and diethylethandiyl moieties deeply inserted between and interacting with the aliphatic chains. Thus, although both agents cause lysosomal phospholipases inhibition, the differences in their interactions with negatively-charged bilayers is likely to result in a different organization of the phospholipids accumulated in vivo, which could lead to different toxicities.

Alterations in membrane permeability induced by aminoglycoside antibiotics: studies on liposomes and cultured cells

Aminoglycoside antibiotics bind to negatively-charged membranes in vitro as well as in vivo. We have examined if this binding could be associated with a change in the properties of membrane permeability. We have used a series of aminoglycoside derivatives and two independent test systems, namely (i) the release of calcein and of Mn2+ from phosphatidylinositol-containing large unilamellar vesicles, and (ii) the influx of Ca2+ into cultured macrophages. We found that certain aminoglycosides (e.g., streptomycin, isepamicin) markedly increase the membrane permeability whereas others (e.g., gentamicin) barely or do not influence it. This increase, when it occurs, is slower or less extensive than observed with pore-forming agents (mellitin, nystatin) or a Ca(2+)-ionophore (ionomycin). It is not observed with an agent [bis(beta-diethylaminoethylether)hexestrol] known to cause membrane fusion, and is not associated with any detectable change in membrane fluidity. In computer-aided conformational analysis of mixed monolayers between phosphatidylinositol and the aminoglycosides studied, it was found that those derivatives inducing an increase in membrane permeability in our experiments adopted an orientation rather perpendicular to the interface, whereas those with no or only a moderate effect were placed in a parallel orientation to this interface. The perpendicular orientation might cause a local condition of disorder which could explain the effects observed.

Membrane destabilization induced by beta-amyloid peptide 29-42: importance of the amino-terminus

Increasing evidence implicates interactions between Abeta-peptides and membrane lipids in Alzheimers disease. To gain insight into the potential role of the free amino group of the N-terminus of Abeta29-42 fragment in these processes, we have investigated the ability of Abeta29-42 unprotected and Abeta29-42 N-protected to interact with negatively-charged liposomes and have calculated the interaction with membrane lipids by conformational analysis. Using vesicles mimicking the composition of neuronal membranes, we show that both peptides have a similar capacity to induce membrane fusion and permeabilization. The fusogenic effect is related to the appearance of non-bilayer structures where isotropic motions occur as shown by 31P and 2H NMR studies. The molecular modeling calculations confirm the experimental observations and suggest that lipid destabilization could be due to the ability of both peptides to adopt metastable positions in the presence of lipids. In conclusion, the presence of a free or protected (acetylated) amino group in the N-terminus of Abeta29-42 is therefore probably not crucial for destabilizing properties of the C-terminal fragment of Abeta peptides.

The macrolide antibiotic azithromycin interacts with lipids and affects membrane organization and fluidity: studies on Langmuir-Blodgett monolayers, liposomes and J774 macrophages

The macrolide antibiotic azithromycin was shown to markedly inhibit endocytosis. Here we investigate the interaction of azithromycin with biomembranes and its effects on membrane biophysics in relation to endocytosis. Equilibrium dialysis and 31P NMR revealed that azithromycin binds to lipidic model membranes and decreases the mobility of phospholipid phosphate heads. In contrast, azithromycin had no effect deeper in the bilayer, based on fluorescence polarization of TMA-DPH and DPH, compounds that, respectively, explore the interfacial and hydrophobic domains of bilayers, and it did not induce membrane fusion, a key event of vesicular trafficking. Atomic force microscopy showed that azithromycin perturbed lateral phase separation in Langmuir-Blodgett monolayers, indicating a perturbation of membrane organization in lateral domains. The consequence of azithromycin/phospholipid interaction on membrane endocytosis was next evaluated in J774 macrophages by using three tracers with different insertion preferences inside the biological membranes and intracellular trafficking: C6-NBD-SM, TMA-DPH and N-Rh-PE. Azithromycin differentially altered their insertion into the plasma membrane, slowed down membrane trafficking towards lysosomes, as evaluated by the rate of N-Rh-PE self-quenching relief, but did not affect bulk membrane internalization of C6-NBD-SM and TMA-DPH. Azithromycin also decreased plasma membrane fluidity, as shown by TMA-DPH fluorescence polarization and confocal microscopy after labeling by fluorescent concanavalin A. We conclude that azithromycin directly interacts with phospholipids, modifies biophysical properties of membrane and affects membrane dynamics in living cells. This antibiotic may therefore help to elucidate the physico-chemical properties underlying endocytosis.

Biophysical studies and intracellular destabilization of pH-sensitive liposomes

We examined changes in membrane properties upon acidification of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes and evaluated their potential to deliver entrapped tracers in cultured macrophages. Membrane permeability was determined by the release of entrapped calcein or hydroxypyrene-1,3,6-trisulfonic acid (HPTS)-p-xylene-bis-pyridinium bromide (DPX); membrane fusion, by measuring the change in size of the liposomes and the dequenching of octadecylrhodamine-B fluorescence; and change in lipid organization, by31P nuclear magnetic resonance spectroscopy. Measurement of cell-associated fluorescence and confocal microscopy examination were made on cells incubated with liposomes loaded with HPTS or HPTS-DPX. The biophysical studies showed (i) a lipid reorganization from bilayer to hexagonal phase progressing from pH 8.0 to 5.0, (ii) a membrane permeabilization for pH<6.5, (iii) an increase in the mean diameter of liposomes for pH<6.0, and (iv) a mixing of liposome membranes for pH<5.7. The cellular studies showed (i) an uptake of the liposomes that were brought from pH 7.5–7.0 to 6.5–6.0 and (ii) a release of ∼15% of the endocytosed marker associated with its partial release from the vesicles (diffuse localization). We conclude that the permeabilization and fusion of pH-sensitive liposomes occur as a consequence of a progressive lipid reorganization upon acidification. These changes may develop intracellular after phagocytosis and allow for the release of the liposome content in endosomes associated with a redistribution in the cytosol.

Characterization of the Interactions between Fluoroquinolone Antibiotics and Lipids: a Multitechnique Approach

Probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research and membrane biophysics. Previous studies showed differences in accumulation and intracellular activity between two fluoroquinolones, ciprofloxacin and moxifloxacin, that may actually result from their differential susceptibility to efflux by the ciprofloxacin transporter. In view of the critical role of lipids for the drug cellular uptake and differences observed for the two closely related fluoroquinolones, we investigated the interactions of these two antibiotics with lipids, using an array of complementary techniques. Moxifloxacin induced, to a greater extent than ciprofloxacin, an erosion of the DPPC domains in the DOPC fluid phase (atomic force microscopy) and a shift of the surface pressure-area isotherms of DOPC/DPPC/fluoroquinolone monolayer toward lower area per molecule (Langmuir studies). These effects are related to a lower propensity of moxifloxacin to be released from lipid to aqueous phase (determined by phase transfer studies and conformational analysis) and a marked decrease of all-trans conformation of acyl-lipid chains of DPPC (determined by ATR-FTIR) without increase of lipid disorder and change in the tilt between the normal and the germanium surface (also determined by ATR-FTIR). All together, differences of ciprofloxacin as compared to moxifloxacin in their interactions with lipids could explain differences in their cellular accumulation and susceptibility to efflux transporters.