Influence of the glycopeptidic moiety of mycobacterial glycopeptidolipids on their lateral organization in phospholipid monolayers.

Glycopeptidolipids (GPLs) from the cell wall of opportunistic pathogenic mycobacteria are potential factors of pathogenicity which can interact with biological membranes. GPL suspensions uncouple oxidative phosphorylation of mitochondria and increase membrane permeability of liposomes. Heavily glycosylated GPLs are less active than lightly glycosylated ones. GPL-phospholipid interactions into preformed mixed films at the air-water interface were investigated in order to understand the permeabilization efficiency differences among GPLs. Polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) was used to determine, in situ, the organization of GPL and of 1,2-di(perdeuteropalmitoyl)phosphatidylcholine (DPPC) molecules in mixed films. Compression isotherms of GPL alone or mixed with DPPC in various proportions showed that the less the GPL was glycosylated the higher its miscibility with DPPC. PMIRRAS studies indicated that low miscibility may result from large self-association of GPL molecules in beta-sheet structures. Low glycosylated GPL molecules increased disorder of DPPC acyl chains. Based on these results, an explanatory model is proposed for membrane permeabilization. Increase of passive permeability may arise from disruption of phospholipid packing induced by GPL molecules. GPL segregation is proposed as the cause of low activity of GPL with high sugar content, by decreasing the number of GPL molecules interacting with phospholipids.

Adjuvant lipopeptide interaction with model membranes.

The cationic lipohexapeptide Pam3Cys-Ser-(Lys)4 is a synthetic model for the triacylated N-terminal part of bacterial lipoproteins, and it is used as an adjuvant and macrophage activator. The amphiphilic lipopeptide was injected below a phosphatidylserine monolayer at the air-water interface. It interacted with the interface, as seen by a decrease in the surface potential (deltaV), and it was inserted in the monolayer, until surface charge neutralization was reached, as seen by the parallel increases of deltaV and of the surface pressure. No insertion occurred above 29 mN/m. The interaction kinetics was sensitive to ionic strength and to the nature of acidic phospholipids and of their acyl chains, but the final equilibrium was independent of these factors. Addition of the lipopeptide to large unilamellar vesicles (LUVs) induced their aggregation, and an exchange of lipids between fluorophor-labelled and non-labelled LUVs. However, no fusion was observed, just as reported for polylysine. The lipopeptide strongly inhibited calcium-induced fusion of PS LUVs, in contrast to the published effect of polylysine. This was probably due to inhibition of calcium fixation on liposomes, since it was observed that the lipopeptide efficiently displaced 45Ca2+ from a PS monolayer. In addition, a phospholipid segregation was observed in SUVs for a few ten micromolar of the lipopeptide.

Mycobacterial glycopeptidolipid interactions with membranes: a monolayer study.

Mycobacterial glycopeptidolipid (GPL) interactions with membranes were analysed with monolayer experiment, using GPLs bearing 3, 1, or 0 carbohydrate residues (GPL3, GPL1, GPL0). Compression isotherms and surface potential determinations suggested that the glycopeptidic moiety of GPL3 permanently dipped in water, while those of GPL1 and GPL0 can lay in the interface. Insertion of GPL molecules into a preformed phospholipid monolayer was observed using GPL3 or GPL1 dispersions, but not from GPL0. It is postulated that the activity of GPL0 is low due to its failure to become inserted into membranes, as is that of GPL3 owing to its insertion only by its acyl chain. GPL1 is likely to disturb membranes by inserting its glycopeptidic moiety into the interface.

Interaction of mycobacterial glycolipids with host cells.

Mycobacteria elaborate a great variety of glycolipids of rather exotic structure. Some of these lipids are abundant cell envelope components and are exposed on the bacterial surface. These comprise the species-specific phenolic glycolipids, glycopeptidolipids, sulfatides, and lipooligosaccharides, and the ubiquitous phosphatidylinositolmannosides. Because pathogenic mycobacterial species are facultative intracellular parasites that infect and reside in host cells, some of them may represent potential virulent factors as they have been shown to inhibit both macrophage antimicrobial activities and lymphoproliferation. These biologic activities may derive, at least in part, from the modulation of the cell functions through the interactions between host membranes and these surface-exposed lipids whose structures are different from those of mammalian cell membrane components. In few cases purified glycolipids have been shown to profoundly affect the physical and functional properties of biologic membranes. Therefore, the enzymes involved in the biosynthesis of the biologically active glycolipids represent potential drug targets. However, definite proofs of their implication in the mycobacterial pathogenicity are lacking. Mutants unable to elaborate defined glycolipids are needed.

Alteration of the lateral organization of the plasma membrane of Chinese hamster ovary cells by synthetic lipopeptide, Pam3Cys-Ser-Lys4.

The cationic lipohexapeptide (S)-[2, 3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)- Lys 4-OH, trihydrochloride (Pam3Cys-Ser-Lys4) is a synthetic analog of the triacylated N-terminal part of bacterial lipoproteins. In this study we addressed the question of whether Pam3Cys-Ser-Lys4 could modify the organization of the plasma membrane of Chinese hamster ovary cells. 1-Acyl-2-[6-(7-nitro-2-1, 3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine (C6-NBD-PC) diffusion was followed by fluorescence recovery after photobleaching experiments carried out on the plasma membrane of Chinese hamster ovary cells. Incubation of cells in the presence of Pam3Cys-Ser-Lys4 induced an increase in the lateral diffusion coefficient and in the immobile fraction of C6-NBD-PC probes. Various control experiments have shown that the increase in the immobile fraction was not due to probe internalization induced by Pam3Cys-Ser-Lys4. Back-exchange experiments showed that a good correlation exists between the fractions of immobilized probes and nonextractable probes in the plasma membrane of Chinese hamster ovary cells. A useful way to analyze the origin of probe immobilization (micrometer-sized domains or aggregated patches of proteins) is to carry out fluorescence recovery after photobleaching experiments at variable observation radii. This type of experiment, carried out on the plasma membrane of Chinese hamster ovary cells incubated with Pam3Cys-Ser-Lys4, confirmed that the lipopeptide induced the aggregation of proteins of Chinese hamster ovary plasma membrane. Lipids which were trapped inside these aggregates were thus prevented from diffusing at long range in the plasma membrane plane and behave as an immobile fraction.

Phagosomal pH determination by dual fluorescence flow cytometry.

Several methods have been developed to measure the pH of phagosomes, using fluorescein derivatives as reporter of pH, and spectrofluorimetry, fluorescence microscopy, or flow cytometry as quantification technique. All have major disadvantages, including either a slow or inaccurate response. In the present study, pH determination was achieved on J774-cell phagosomes containing dual-labeled zymozan particles using dual fluorescence flow cytometry with an argonion laser excitation wavelength at 488 nm. This allowed zymozan-containing macrophages to be distinguished from other cells and their fluorescence to be measured rapidly. The use of a new probe, namely Oregon Green 488 which has a pKa lower than carboxyfluorescein with the same maximum excitation and emission wavelengths, allowed investigation of pH value below 5. The dual labeling with Oregon Green 488 and carboxytetramethylrhodamine as pH-sensitive and pH-insensitive probes, respectively, overcame the absence of an isobestic point in the Oregon Green 488 spectrum. The phagosomal pH was determined using a calibration curve of phagosomal pH established by adding ionophores in phagocyte suspension and measuring the fluorescence intensity ratio (535 nm/585 nm) for different pHs. A phagosomal pH of 4.5 +/- 0.1 can be accurately determined. This method permits pH measurements down to 4, even in the presence of nonengulfed reporter particles.

Regulators of membrane trafficking and Mycobacterium tuberculosis phagosome maturation block.

The biogenesis and maturation of phagosomes is an area of study which has been employing aspects of proteomic analyses and variations on that theme by identifying components on isolated organelles and following their dynamic changes and interactions with the endocytic pathway. In the case of Mycobacterium tuberculosis phagosome, the arrest of its maturation in infected macrophages, referred to in classical texts as the inhibition of phagosome-lysosome fusion, represents a phenomenon that is used to functionally dissect the phagosomal maturation pathway. In this review, we summarize the recent studies on regulators of membrane trafficking and other organelle components in the context of phagosomal biogenesis and mycobacterial phagosome maturation arrest.