Nascent teichoic acids insertion into the cell wall directs the localization and activity of the major pneumococcal autolysin LytA.

The bacterial cell wall is in part composed of the peptidoglycan (PG) layer that maintains the cell shape and sustains the basic cellular processes of growth and division. The cell wall of Gram-positive bacteria also carries teichoic acids (TAs). In this work, we investigated how TAs contribute to the structuration of the PG network through the modulation of PG hydrolytic enzymes in the context of the Gram-positive Streptococcus pneumoniae bacterium. Pneumococcal TAs are decorated by phosphorylcholine residues which serve as anchors for the Choline-Binding Proteins, some of them acting as PG hydrolases, like the major autolysin LytA. Their binding is non covalent and reversible, a property that allows easy manipulation of the system. In this work, we show that the release of LytA occurs independently from its amidase activity. Furthermore, LytA fused to GFP was expressed in pneumococcal cells and showed different localization patterns according to the growth phase. Importantly, we demonstrate that TAs modulate the enzymatic activity of LytA since a low level of TAs present at the cell surface triggers LytA sensitivity in growing pneumococcal cells. We previously developed a method to label nascent TAs in live cells revealing that the insertion of TAs into the cell wall occurs at the mid-cell. In conclusion, we demonstrate that nascent TAs inserted in the cell wall at the division site are the specific receptors of LytA, tuning in this way the positioning of LytA at the appropriate place at the cell surface.

Specific and spatial labeling of choline-containing teichoic acids in Streptococcus pneumoniae by click chemistry.

Propargyl-choline was efficiently incorporated into teichoic acid (TA) polymers on the surface of Streptococcus pneumoniae. The introduction of a fluorophore by click chemistry enabled sufficient labeling of the pneumococcus, as well as its specific detection when mixed with other bacterial species. The labeling is localized at the septal site, suggesting a similar location of the TA and peptidoglycan (PG) synthetic machineries. This method is a unique opportunity to improve our understanding of the spatial location of pneumococcal TA biosynthesis.

Structure of the fiber head of Ad3, a non-CAR-binding serotype of adenovirus.

Adenoviruses of serotype Ad3 (subgenus B) use a still-unknown host cell receptor for viral attachment, whereas viruses from all other known subgenera use the coxsackie and adenovirus receptor (CAR). The receptor binding domain (head) of the Ad3 fiber protein has been expressed in Escherichia coli inclusion bodies. After denaturation and renaturation using a rapid dilution method, crystals of trimeric head were obtained. The 1.6 A resolution X-ray structure shows a strict conservation of the beta-sheet scaffold of the protein very similar to the head structures of the CAR-binding serotypes Ad2, Ad5, and Ad12. The conformation of the loops is different, with the exception of the AB loop, which forms the center of the interface in the Ad12-CAR complex structure. The structure explains why a mutation in Ad5 of one residue in the AB loop to glutamic acid, as in Ad3, abrogates binding to CAR. It is possible that the Ad3 receptor binding site is nevertheless situated similar to the CAR binding site, although it cannot be excluded that other regions of the relatively hydrophobic head surface may be used.

Differential involvement of peroxisome-proliferator-activated receptors alpha and delta in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine.

Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (alpha, gamma and delta) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARalpha-null mice, we have found that PPARalpha in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARdelta/alpha agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARalpha-null mice. These findings demonstrate that PPARdelta can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARdelta target gene in the small intestine. We propose that PPARdelta contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.

Formation of glycine receptor clusters and their accumulation at synapses.

The glycine receptor is highly enriched in microdomains of the postsynaptic neuronal surface apposed to glycinergic afferent endings. There is substantial evidence suggesting that the selective clustering of glycine receptor at these sites is mediated by the cytoplasmic protein gephyrin. To investigate the formation of postsynaptic glycine receptor domains, we have examined the surface insertion of epitope-tagged receptor alpha subunits in cultured spinal cord neurons after gene transfer by polyethylenimine-adenofection. Expression studies were also carried out using the non-neuronal cell line COS-7. Immunofluorescence microscopy was performed using wild-type isoforms and an alpha mutant subunit bearing the gephyrin-binding motif of the beta subunit. In COS-7 cells, transfected glycine receptor alpha subunits had a diffuse surface distribution. Following cotransfection with gephyrin, only the mutant subunit formed cell surface clusters. In contrast, in neurons all subunits were able to form cell surface clusters after transfection. These clusters were not colocalized with detectable endogenous gephyrin, and the GlyR beta subunit could not be detected in transfected cells. Therefore, exogenous receptors were not assembled as heteromeric complexes. A quantitative analysis demonstrated that newly synthesized glycine receptor progressively populated endogenous gephyrin clusters, since association of both proteins increased as a function of time after the onset of receptor synthesis. This phenomenon was accelerated when glycine receptor contained the gephyrin-binding domain. Together with previous results, these data support a two-step model for glycinergic synaptogenesis whereby the gephyrin-independent formation of cell surface clusters precedes the gephyrin-mediated postsynaptic accumulation of clusters.

Mechanism of adenovirus improvement of cationic liposome-mediated gene transfer.

Substantial effort has been focused on the development of highly efficient gene transfer strategies. Although viral and non-viral methods have been elaborated, mechanisms of gene delivery are still poorly understood. We exploited our recent observation that replication-deficient type 5 adenovirus dramatically enhances lipofectAMINE-mediated gene transfer (lipoadenofection) in differentiated cells to elucidate the mechanism of adenovirus action in this process. Heat-induced denaturation of viral capsid abolishes adenovirus action whereas inactivation of viral genome by short treatment with UV has no effect. Electron microscopic observations reveal the formation of a complex containing adenovirus and lipofectAMINE which probably carries DNA into cells via endocytosis. Anti-adenovirus antiserum or monoclonal anti-alpha(v)beta3 integrin antibody inhibits lipoadenofection, at least partially. Neutralization of endosomal compartments with chloroquine, ammonium chloride or monensin does not prevent adenovirus improvement of gene transfer. Hence, adenovirus-lipofectAMINE-DNA complexes in which viral particles are each encompassed by three lipid layers, penetrate cells via an endocytic pathway involving probably the adenovirus receptor and alpha(v)beta3 integrin. The resulting efficient transfer and expression of plasmid DNA proceeds from a mechanism in which adenoviral endosomolytic activity appears to be required while viral genome is not essential.

Adenovirus enhancement of polyethylenimine-mediated transfer of regulated genes in differentiated cells.

Efficient gene transfer is a prerequisite for analysing regulation of transfected promoters. We combined the DNA binding property of the cationic polymer polyethylenimine (PEI) and the potent endocytic activity of adenovirus in a PEI-DNA-adenovirus complex which provided efficient plasmid delivery in differentiated cultured cells. We transfected 3T3-F442A adipocytes, C2.7 myocytes and FAO hepatoma cells with a construct containing the simian virus 40 promoter fused to the chloramphenicol acetyltransferase (CAT) gene, using a combination of PEI and 200 p.f.u. per cell of replication-deficient type 5 adenovirus. Resulting CAT activities varied according to the cell type reaching about 0.6, 8 and 38 units/mg protein for respectively 3T3-F442A, FAO and C2.7 cells. Increases in transfection efficiencies were 140- to 300-fold when compared with those obtained with PEI alone. Then we tested physiologically regulated promoters: the phosphoenolpyruvate carboxykinase gene promoter in 3T3-F442A or FAO cells and the hexokinase II gene promoter in C2.7 myocytes. Gene expression was appropriately increased by clofibrate, dexamethasone and insulin for 3T3-F442A, FAO and C2.7 cells, respectively. Thus, the combination of PEI and adenovirus is a simple, efficient, inexpensive and versatile method of gene transfer which is applicable to several differentiated cells and provides a physiologically coherent transgene regulation. We name this method PEI-adenofection.

Up-regulation of the expression of the gene for liver fatty acid-binding protein by long-chain fatty acids.

The role of fatty acids in the expression of the gene for liver fatty acid-binding protein (L-FABP) was investigated in the well-differentiated FAO rat hepatoma cell line. Cells were maintained in serum-free medium containing 40 microM BSA/320 microM oleate. Western blot analysis showed that oleate triggered an approx. 4-fold increase in the cytosolic L-FABP level in 16 h. Oleate specifically stimulated L-FABP mRNA in time-dependent and dose-dependent manners with a maximum 7-fold increase at 16 h in FAO cells. Preincubation of FAO cells with cycloheximide prevented the oleate-mediated induction of L-FABP mRNA, showing that protein synthesis was required for the action of fatty acids. Run-on transcription assays demonstrated that the control of L-FABP gene expression by oleate was, at least in part, transcriptional. Palmitic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid were similarly potent whereas octanoic acid was inefficient. This regulation was also found in normal hepatocytes. Therefore long-chain fatty acids are strong inducers of L-FABP gene expression. FAO cells constitute a useful tool for studying the underlying mechanism of fatty acid action.

Efficient transfer of regulated genes in adipocytes and hepatoma cells by the combination of liposomes and replication-deficient adenovirus.

Efficient transfer of genes maintaining a correct hormonal control in transfected cells is the prerequisite for gene regulation studies and for gene therapy. Differentiated cells, like adipocytes or hepatocytes, are difficult to transfect. In an attempt to improve gene transfer, we first transiently transfected cultured 3T3-F442A adipocytes with a construct containing the simian virus 40 (SV40) promoter fused to the chloramphenicol acetyltransferase (CAT) gene (pSV2-CAT), using various cationic liposomes. Among these, only lipofectAMINE was five times more efficient than the standard calcium phosphate procedure. To further augment efficiency, we transfected 3T3-F442A adipocytes and FAO hepatoma cells with the lipofectAMINE/pSV2-CAT complex in the presence of replication-deficient recombinant type-5 adenovirus at 200 pfu/cell. CAT activity of transiently transfected cells was increased about 50-fold when compared to the calcium phosphate procedure. To determine whether this methodology would be useful for obtaining stable transfectants and would not interfere with correct gene regulation, we used a construct containing -2100 to +69 bp of the phosphoenolpyruvate carboxykinase gene fused to the CAT gene (pPL1-CAT). This construct was shown previously to be cAMP-responsive after calcium-phosphate-mediated transfection of adipocytes and hepatoma cells. 3T3-F442A or FAO cells in which pPL1-CAT was either transiently or stably transferred by lipofectAMINE and adenovirus responded to isoproterenol or cAMP, respectively, with a 2-3-fold increase in CAT activity. Therefore the association of liposomes and adenovirus is an efficient method for transient or stable transfer of regulated genes in adipocytes and hepatoma cells.