Nathalie Eynard

Electropermeabilization of cell membranes

A position dependent modulation of the membrane potential difference is induced when an electric field is applied to a cell. When cells are submitted to short lived electric field pulses with an overcritical intensity, a local membrane alteration is induced, which may reseal. Its molecular definition remains unknown. A free exchange of hydrophilic molecules takes place across the membrane. A leakage of cytosolic metabolites is present. However, a loading of polar drugs into the cytoplasm is obtained. A short description of the processes affecting the cell membrane organization is given. Lipids appear as the primary target of the field effect as in the case of liposomes. Nevertheless membrane proteins appear to be affected by a direct or by a back effect. The permeabilized state is long lived. The cell metabolism plays indeed a critical role in the recovery. The cell viability can be nevertheless preserved.

Recent biotechnological developments of electropulsation. A prospective review

During the last 25 years, basic research has improved our knowledge on the molecular mechanisms triggered at the membrane level by electric pulses. Applied aspects may now be used under safe conditions. Electropulsation is known as a very efficient tool for obtaining gene transfer in many species to produce genetically modified organisms (GMO). This is routinely used for industrial purposes to transfer exogenous activities in bacteria, yeasts and plants. The method is simple and of a low cost. But electropulsation is not limited to this application for biotechnological purposes. It is known that the field-associated membrane alterations can be irreversible. The pulsed species cannot recover after the treatment. Their viability is strongly affected. This appears as a very promising technology for the eradication of pathogenic microorganisms. Recent developments are proposed for sterilization purposes. New flow technologies of field generation allow the treatment of large volumes of solution. When high flow rates are used, microorganisms are submitted both to a hydromechanical and to an electrical stress. The synergy of the two effects may be present when suitable pulsing conditions are chosen. Several examples for the treatment of domestic water and in the food industry are described. Walled microorganisms are affected not only at the membrane level. We observed that alterations are present on the cell wall. A very promising technology is the associated controlled leakage of the cytoplasmic soluble proteins. Large dimeric proteins such as beta-galactosidases can be extracted at a high yield. High volumes can be treated by using a flow process. Extraction of proteins is obtained with many systems including mammalian cells.

ELECTROTRANSFORMATION PATHWAYS OF PROCARYOTIC AND EUCARYOTIC CELLS : RECENT DEVELOPMENTS

Abstract This report describes synthetic conclusions obtained from studies of electrotransformation made on 4 cell types: mammalian cells (CHO), bacteria (E. coli), yeast (S. Cerevisiae) and intact plant cells (maize). A similar multistep pathway is always observed in spite of the significant differences between cell types. Electrotransfer of DNA through the cell membrane and envelope is controlled by the amount of plasmids which is bound before the pulse. Electrotransformation seemed to be controlled by the electric parameters in a similar way for all species, even walled cells. Results on intact plant cells showed that the wall may prevent the transfer. Nevertheless, maize cells were electrotransformed with a simple pretreatment. DNA electrotransfer was obtained after a prepulse plasmolysis, which induced a contact between plasmid and membrane before the pulse. No transformation was detected when plasmid was added after the pulse. Electrooptics experiments on bacteria showed that a plasmid dependent orientation took place during the pulse along permeabilization. A key second step in gene transfer appeared to be a strong anchoring of DNA to the permeabilised membrane or envelope during the pulse. This is clearly associated to a direct field effect. Different electrophoretic effects may be involved. In the third step of electrotransformation, the plasmid slowly crossed the cell envelope by a cell dependent, but electrophoresis independent, process. For all species we studied, a delay was always observed between the pulse and the time of total protection of the plasmid against DNAse action. The fourth and final step of electrotransformation was dependent on plasmid functionality in the cell. Replication and gene expression should occur to detect the transfered activity.

Optimized conditions for electrotransformation of bacteria are related to the extent of electropermeabilization

Electropulsation is a simple and efficient way to introduce cloned genes into a variety of cell types, even with walled species. In the case of bacteria, we observed no direct correlation between survival rate and transformation yield. In the present work, we show that the yield of transformation is directly related to the level of the electric-field induced level of cell permeabilization. From experiments on Escherichia coli, it was confirmed that the extent of associated ATP leakage was a reliable assay. This approach was extended to other strains, such as Salmonella typhimurium, which to date had not been electrotransformed by plasmids.

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of Tm (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.

The changes in mycolic acid structures caused by hadC mutation have a dramatic effect on the virulence of Mycobacterium tuberculosis.

Understanding the molecular strategies used by Mycobacterium tuberculosis to invade and persist within the host is of paramount importance to tackle the tuberculosis pandemic. Comparative genomic surveys have revealed that hadC, encoding a subunit of the HadBC dehydratase, is mutated in the avirulent M. tuberculosis H37Ra strain. We show here that mutation or deletion of hadC affects the biosynthesis of oxygenated mycolic acids, substantially reducing their production level. Additionally, it causes the loss of atypical extra‐long mycolic acids, demonstrating the involvement of HadBC in the late elongation steps of mycolic acid biosynthesis. These events have an impact on the morphotype, cording capacity and biofilm growth of the bacilli as well as on their sensitivity to agents such as rifampicin. Furthermore, deletion of hadC leads to a dramatic loss of virulence: an almost 4‐log drop of the bacterial load in the lungs and spleens of infected immunodeficient mice. Both its unique function and importance for M. tuberculosis virulence make HadBC an attractive therapeutic target for tuberculosis drug development.

The Non-Essential Mycolic Acid Biosynthesis Genes hadA and hadC Contribute to the Physiology and Fitness of Mycobacterium smegmatis

Gram positive mycobacteria with a high GC content, such as the etiological agent of tuberculosis Mycobacterium tuberculosis, possess an outer membrane mainly composed of mycolic acids (MAs), the so-called mycomembrane, which is essential for the cell. About thirty genes are involved in the biosynthesis of MAs, which include the hadA, hadB and hadC genes that encode the dehydratases Fatty Acid Synthase type II (FAS-II) known to function as the heterodimers HadA-HadB and HadB-HadC. The present study shows that M. smegmatis cells remain viable in the absence of either HadA and HadC or both. Inactivation of HadC has a dramatic effect on the physiology and fitness of the mutant strains whereas that of HadA exacerbates the phenotype of a hadC deletion. The hadC mutants exhibit a novel MA profile, display a distinct colony morphology, are less aggregated, are impaired for sliding motility and biofilm development and are more resistant to detergent. Conversely, the hadC mutants are significantly more susceptible to low- and high-temperature and to selective toxic compounds, including several current anti-tubercular drugs.

A novel mycolic acid species defines two novel genera of the Actinobacteria, Hoyosella and Amycolicicoccus.

Corynebacterineae are characterized by the presence of long-chain lipids, notably mycolic acids (α-alkyl, β-hydroxy fatty acids), the structures of which are genus-specific. Mycolic acids from two environmental strains, Amycolicicoccus subflavus and Hoyosella altamirensis, were isolated and their structures were established using a combination of mass spectrometry analysis, (1)H-NMR spectroscopy and chemical degradations. The C(2)-C(3) cleavage of these C(30)-C(36) acids led to the formation of two fragments: saturated C(9)-C(11) acids, and saturated and unsaturated C(20)-C(25) aldehydes. Surprisingly, the fatty acids at the origin of the two fragments making up these mycolic acids were present in only minute amounts in the fatty acid pool. Moreover, the double bond in the main C(24) aldehyde fragment was located at position ω16, whereas that found in the ethylenic fatty acids of the bacteria was at ω9. These data question the biosynthesis of these new mycolic acids in terms of the nature of the precursors, chain elongation and desaturation. Nevertheless, they are consistent with the occurrence of the key genes of mycolic acid biosynthesis, including those encoding proteins of the fatty acid synthase II system, identified in the genome of A. subflavus. Altogether, while the presence of mycolic acids and analysis of their 16S rDNA sequences would suggest that these strains belong to the Mycobacteriaceae family, the originality of their structures reinforces the recent description of the novel genera Amycolicicoccus and Hoyosella.

Direct monitoring of the field strength during electropulsation

Abstract The field strength which is applied on the sample during electropulsation can be affected by electrochemical process at the level of the electrodes. A previous study [U. Pliquett, E.A. Gift, J.C. Weaver, Determination of the electric field and anomalous heating caused by exponential pulses with aluminium electrodes in electropulsation experiments, Bioelectrochemistry and Bioenergetics Vol. 39 (1996) pp. 39–53] showed that post pulse effects were present. The present work investigates the magnitude of the field by following at a submillisecond time resolution its associated sample Joule heating. When using stainless steel electrodes, the field present on the sample is what is set on the pulse generator. Problems are present with aluminium cuvettes.

Definition of a duration threshold in the first step for energy transduction by Escherichia coli by use of a microsecond electric field pulse

Abstract The total ATP content increased in Escherichia coli when the bacteria were submitted to square wave electric pulses with field intensities of about 1 kV/cm and duration larger than 20 μs. Nothing was observed when duration was shorter. The transduction of energy from its electrical form (membrane potential) to another, stored in the membrane which was converted later in the terminal P bond of ATP, required a key-step which had to last for more than this threshold of 20 μs. Square wave electric-field pulses appeared as a new non-invasive approach for the determination of the kinetics of fast energy-transduction events.