Protein-Protein Interaction: Bacterial Two-Hybrid

International audienceThe bacterial two-hybrid (BACTH, for "Bacterial Adenylate Cyclase-Based Two-Hybrid") system is a simple and fast genetic approach to detecting and characterizing protein-protein interactions in vivo. This system is based on the interaction-mediated reconstitution of a cyclic adenosine monophosphate (cAMP) signaling cascade in Escherichia coli. As BACTH uses a diffusible cAMP messenger molecule, the physical association between the two interacting chimeric proteins can be spatially separated from the transcription activation readout, and therefore it is possible to analyze protein-protein interactions that occur either in the cytosol or at the inner membrane level as well as those that involve DNA-binding proteins. Moreover, proteins of bacterial origin can be studied in an environment similar (or identical) to their native one. The BACTH system may thus permit a simultaneous functional analysis of proteins of interest-provided the hybrid proteins retain their activity and their association state. This chapter describes the principle of the BACTH genetic system and the general procedures to study protein-protein interactions in vivo in E. coli

Hyperthermophilic archaea produce membrane vesicles that can transfer DNA.

International audienceThermococcales are hyperthermophilic archaea found in deep-sea hydrothermal vents. They have been recently reported to produce membrane vesicles (MVs) into their culture medium. Here, we have characterized the mode of production and determined the biochemical composition of MVs from two species of Thermococcales, Thermococcus gammatolerans and Thermococcus kodakaraensis. We observed that MVs are produced by a budding process from the cell membrane reminiscent of ectosome (microparticle) formation in eukaryotes. MVs and cell membranes from the same species have a similar protein and lipid composition, confirming that MVs are produced from cell membranes. The major protein present in cell membranes and MVs of both species is the oligopeptide binding protein OppA. This protein is also abundant in MVs from cells grown in minimal medium, suggesting that OppA could be involved in processes other than peptides scavenging. We have previously shown that MVs from Thermococcales harbour DNA and protect DNA against thermodegradation. Here, we show that T. kodakaraensis cells transformed with the shuttle plasmid pLC70 release MVs harbouring this plasmid. Notably, these MVs can be used to transfer pLC70 into plasmid-free cells, suggesting that MVs could be involved in DNA transfer between cells at high temperature

Membrane vesicles, nanopods and/or nanotubes produced by hyperthermophilic archaea of the genus Thermococcus.

International audienceThermococcus species produce MVs (membrane vesicles) into their culture medium. These MVs are formed by a budding process from the cell envelope, similar to ectosome formation in eukaryotic cells. The major protein present in MVs of Thermococci is a peptide-binding receptor of the OppA (oligopeptide-binding protein A) family. In addition, some of them contain a homologue of stomatin, a universal membrane protein involved in vesiculation. MVs produced by Thermococcus species can recruit endogenous or exogenous plasmids and plasmid transfer through MVs has been demonstrated in Thermococcus kodakaraensis. MVs are frequently secreted in clusters surrounded by S-layer, producing either big protuberances (nanosphere) or tubular structures (nanotubes). Thermococcus gammatolerans and T. kodakaraensis produce nanotubes containing strings of MVs, resembling the recently described nanopods in bacteria, whereas Thermococcus sp. 5-4 produces filaments whose internal membrane is continuous. These nanotubes can bridge neighbouring cells, forming cellular networks somehow resembling nanotubes recently observed in Firmicutes. As suggested for bacteria, archaeal nanopods and/or nanotubes could be used to expand the metabolic sphere around cells and/or to promote intercellular communication

Membrane vesicles, nanopods and/or nanotubes produced by hyperthermophilic archaea of the genus Thermococcus. Biochem Soc Trans 41:436–442

Abstract Thermococcus species produce MVs (membrane vesicles) into their culture medium. These MVs are formed by a budding process from the cell envelope, similar to ectosome formation in eukaryotic cells. The major protein present in MVs of Thermococci is a peptide-binding receptor of the OppA (oligopeptide-binding protein A) family. In addition, some of them contain a homologue of stomatin, a universal membrane protein involved in vesiculation. MVs produced by Thermococcus species can recruit endogenous or exogenous plasmids and plasmid transfer through MVs has been demonstrated in Thermococcus kodakaraensis. MVs are frequently secreted in clusters surrounded by S-layer, producing either big protuberances (nanosphere) or tubular structures (nanotubes). Thermococcus gammatolerans and T. kodakaraensis produce nanotubes containing strings of MVs, resembling the recently described nanopods in bacteria, whereas Thermococcus sp. 5-4 produces filaments whose internal membrane is continuous. These nanotubes can bridge neighbouring cells, forming cellular networks somehow resembling nanotubes recently observed in Firmicutes. As suggested for bacteria, archaeal nanopods and/or nanotubes could be used to expand the metabolic sphere around cells and/or to promote intercellular communication

Extracellular membrane vesicles harbouring viral genomes

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Impact of milk polar lipid supplementation on postprandial bile acid composition

International audienceBile acids (BA) are the end products of cholesterol catabolism and may act as signalling molecules and metabolic regulators of energy homeostasis. Disorders in BA metabolism can lead to liver and cardiovascular diseases. In a 4-week double-blind RCT, we demonstrated that the daily consumption of a cream cheese enriched with 3 or 5g of milk polar lipids (PL) improved lipid metabolism by reducing hypercholesterolemia in overweight postmenopausal women. Postprandial (pp) metabolic explorations (0-480 min) were performed before and after the intervention, including a standardized high fat-high sucrose breakfast at fasting and a standardized lunch containing the test cream cheese at 240min. We aimed to determine the effect of milk PL on circulating BA in the fasting and pp state. Before intervention, serum concentration of total fasting BA in the 3 groups was within physiological concentrations (<8µM). The milk PL intervention slightly increased total fasting BA concentrations (After-Before) (PPL=0.03), with no significant effect on BA species profile (% of total BA). Total BA concentration was not impacted during the pp period, but the primary/secondary BA ratio was significantly decreased in both milk PL groups versus control (Pgroup<0.001, PPL<0.001). Milk PL decreased the relative abundance of primary BA (PPL=0.02), increased Tauro-conjugated BA (Pgroup=0.02) and highly decreased Glyco-conjugated BA (Pgroup<0.001). Proportions of GCA (glycocholic acid), GCDCA (glycochenodeoxycholic acid) and GLCA (glycolithocholic acid) were also decreased during the pp period. The latter is derived from the LCA (litocholic acid) whose accumulation is toxic. In conclusion, the 4-week milk PL supplementation did not alter the overall circulating levels of BA in neither the fasting nor the pp state, but impacted the BA profile by decreasing some of deleterious species. Such results provide new insights in the knowledge of BA metabolism, and a potential link with the cholesterol-lowering effects of milk PL deserves to be investigated

Impact of milk polar lipid supplementation on postprandial bile acid composition

International audienceBile acids (BA) are the end products of cholesterol catabolism and may act as signalling molecules and metabolic regulators of energy homeostasis. Disorders in BA metabolism can lead to liver and cardiovascular diseases. In a 4-week double-blind RCT, we demonstrated that the daily consumption of a cream cheese enriched with 3 or 5g of milk polar lipids (PL) improved lipid metabolism by reducing hypercholesterolemia in overweight postmenopausal women. Postprandial (pp) metabolic explorations (0-480 min) were performed before and after the intervention, including a standardized high fat-high sucrose breakfast at fasting and a standardized lunch containing the test cream cheese at 240min. We aimed to determine the effect of milk PL on circulating BA in the fasting and pp state. Before intervention, serum concentration of total fasting BA in the 3 groups was within physiological concentrations (<8µM). The milk PL intervention slightly increased total fasting BA concentrations (After-Before) (PPL=0.03), with no significant effect on BA species profile (% of total BA). Total BA concentration was not impacted during the pp period, but the primary/secondary BA ratio was significantly decreased in both milk PL groups versus control (Pgroup<0.001, PPL<0.001). Milk PL decreased the relative abundance of primary BA (PPL=0.02), increased Tauro-conjugated BA (Pgroup=0.02) and highly decreased Glyco-conjugated BA (Pgroup<0.001). Proportions of GCA (glycocholic acid), GCDCA (glycochenodeoxycholic acid) and GLCA (glycolithocholic acid) were also decreased during the pp period. The latter is derived from the LCA (litocholic acid) whose accumulation is toxic. In conclusion, the 4-week milk PL supplementation did not alter the overall circulating levels of BA in neither the fasting nor the pp state, but impacted the BA profile by decreasing some of deleterious species. Such results provide new insights in the knowledge of BA metabolism, and a potential link with the cholesterol-lowering effects of milk PL deserves to be investigated

Impact of milk polar lipid supplementation on postprandial bile acid composition

International audienceBile acids (BA) are the end products of cholesterol catabolism and may act as signalling molecules and metabolic regulators of energy homeostasis. Disorders in BA metabolism can lead to liver and cardiovascular diseases. In a 4-week double-blind RCT, we demonstrated that the daily consumption of a cream cheese enriched with 3 or 5g of milk polar lipids (PL) improved lipid metabolism by reducing hypercholesterolemia in overweight postmenopausal women. Postprandial (pp) metabolic explorations (0-480 min) were performed before and after the intervention, including a standardized high fat-high sucrose breakfast at fasting and a standardized lunch containing the test cream cheese at 240min. We aimed to determine the effect of milk PL on circulating BA in the fasting and pp state. Before intervention, serum concentration of total fasting BA in the 3 groups was within physiological concentrations (<8µM). The milk PL intervention slightly increased total fasting BA concentrations (After-Before) (PPL=0.03), with no significant effect on BA species profile (% of total BA). Total BA concentration was not impacted during the pp period, but the primary/secondary BA ratio was significantly decreased in both milk PL groups versus control (Pgroup<0.001, PPL<0.001). Milk PL decreased the relative abundance of primary BA (PPL=0.02), increased Tauro-conjugated BA (Pgroup=0.02) and highly decreased Glyco-conjugated BA (Pgroup<0.001). Proportions of GCA (glycocholic acid), GCDCA (glycochenodeoxycholic acid) and GLCA (glycolithocholic acid) were also decreased during the pp period. The latter is derived from the LCA (litocholic acid) whose accumulation is toxic. In conclusion, the 4-week milk PL supplementation did not alter the overall circulating levels of BA in neither the fasting nor the pp state, but impacted the BA profile by decreasing some of deleterious species. Such results provide new insights in the knowledge of BA metabolism, and a potential link with the cholesterol-lowering effects of milk PL deserves to be investigated