β-barrel membrane proteins perform important functions in the outer membranes (OMs) of Gram-negative bacteria and of the mitochondria and chloroplasts of eukaryotes. The protein complexes that ...assemble these proteins in their respective membranes have been identified and shown to contain a component that has been conserved from bacteria to humans. β-barrel proteins are handled differently from α-helical membrane proteins in the cell in order to efficiently transport them to their final locations in unfolded but folding-competent states. The mechanism by which the assembly complex then binds, folds, and inserts β-barrels into the membrane is not well understood, but recent structural, biochemical, and genetic studies have begun to elucidate elements of how the complex provides a facilitated pathway for β-barrel assembly. Ultimately, studies of the mechanism of β-barrel assembly and comparison to the better-understood process of α-helical membrane protein assembly will reveal whether there are general principles that guide the folding and insertion of all membrane proteins.
The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor compound, has not yet been fully elucidated. Here, we show that human cancer cells have markedly lower levels of ...sphingomyelin (SM) than nontumor (MRC-5) cells. In this context, 2OHOA treatment strongly augments SM mass (4.6-fold), restoring the levels found in MRC-5 cells, while a loss of phosphatidylethanolamine and phosphatidylcholine is observed (57 and 30%, respectively). The increased SM mass was due to a rapid and highly specific activation of SM synthases (SMS). This effect appeared to be specific against cancer cells as it did not affect nontumor MRC-5 cells. Therefore, low SM levels are associated with the tumorigenic transformation that produces cancer cells. SM accumulation occurred at the plasma membrane and caused an increase in membrane global order and lipid raft packing in model membranes. These modifications would account for the observed alteration by 2OHOA in the localization of proteins involved in cell apoptosis (Fas receptor) or differentiation (Ras). Importantly, SMS inhibition by D609 diminished 2OHOA effect on cell cycle. Therefore, we propose that the regulation of SMS activity in tumor cells is a critical upstream event in 2OHOA antitumor mechanism, which also explains its specificity for cancer cells, its potency, and the lack of undesired side effects. Finally, the specific activation of SMS explains the ability of this compound to trigger cell cycle arrest, cell differentiation, and autophagy or apoptosis in cancer cells.
In recent years, hopanoids, a group of pentacyclic compounds found in bacterial membranes, are in the spotlight since it was proposed that they induce order in lipid membranes in a similar way ...cholesterol do in eukaryotes, despite their structural differences. We studied here whether diplopterol (an abundant hopanoid) promoted similar effects on model membranes as sterols do. We analyzed the compaction, dynamics, phase segregation, permeability and compressibility of model membranes containing diplopterol, and compared with those containing sterols from animals, plants and fungi. We also tested the effect that the incubation with diplopterol had on hopanoid-lacking bacteria. Our results show that diplopterol induces phase segregation, increases lipid compaction, and decreases permeability on phospholipid membranes, while retaining membrane fluidity and compressibility. Furthermore, the exposition to this hopanoid decreases the permeability of the opportunistic pathogen Pseudomonas aeruginosa and increases the resistance to antibiotics. All effects promoted by diplopterol were similar to those generated by the sterols. Our observations add information on the functional significance of hopanoids as molecules that play an important role in membrane organization and dynamics in model membranes and in a bacterial system.
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•The hopanoid diplopterol reduces membrane permeability.•Diplopterol condense monolayers without affecting fluidity and compressibility.•Diplopterol increases resistance to antibiotics of P. aeruginosa.•This hopanoid induces liquid-liquid phase segregation.
Treatment of Staphylococcus aureus infections is complicated by the development of antibiotic tolerance, a consequence of the ability of S. aureus to enter into a nongrowing, dormant state in which ...the organisms are referred to as persisters. We report that the clinically approved anthelmintic agent bithionol kills methicillin-resistant S. aureus (MRSA) persister cells, which correlates with its ability to disrupt the integrity of Gram-positive bacterial membranes. Critically, bithionol exhibits significant selectivity for bacterial compared with mammalian cell membranes. All-atom molecular dynamics (MD) simulations demonstrate that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterol-rich mammalian-mimic lipid bilayers. In addition to causing rapid membrane permeabilization, the insertion of bithionol increases membrane fluidity. By using bithionol and nTZDpa (another membrane-active antimicrobial agent), as well as analogs of these compounds, we show that the activity of membrane-active compounds against MRSA persisters positively correlates with their ability to increase membrane fluidity, thereby establishing an accurate biophysical indicator for estimating antipersister potency. Finally, we demonstrate that, in combination with gentamicin, bithionol effectively reduces bacterial burdens in a mouse model of chronic deep-seated MRSA infection. This work highlights the potential repurposing of bithionol as an antipersister therapeutic agent.
Summary
Outer membrane vesicles (OMVs) are asymmetrical single bilayer membranous nanostructures produced by Gram‐negative bacteria important for bacterial interaction with the environment. ...Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces OMVs that act as a virulence factor secretion system contributing to its pathogenicity. Despite their biological importance, the mechanisms of OMV biogenesis have not been fully elucidated. The ~14 times more curvature of the OMV membrane than cell outer membrane (OM) indicates that OMV biogenesis requires energy expenditure for significant curvature of the OMV membrane. In P. gingivalis, we propose that this may be achieved by upregulating the production of certain inner or outer leaflet lipids, which causes localized outward curvature of the OM. This results in selection of anionic lipopolysaccharide (A‐LPS) and associated C‐terminal domain (CTD) ‐family proteins on the outer surface due to their ability to accommodate the curvature. Deacylation of A‐LPS may further enable increased curvature leading to OMV formation. Porphyromonas gingivalis OMVs that are selectively enriched in CTD‐family proteins, largely the gingipains, can support bacterial coaggregation, promote biofilm development and act as an intercessor for the transport of non‐motile bacteria by motile bacteria. The P. gingivalis OMVs are also believed to contribute to host interaction and colonization, evasion of immune defense mechanisms, and destruction of periodontal tissues. They may be crucial for both micro‐ and macronutrient capture, especially heme and probably other assimilable compounds for its own benefit and that of the wider biofilm community.
Due to recent technical developments in microscopy, huge advances have been made in our understanding of the architecture of the cell membrane. It is now well appreciated that nanoscale clustering is ...a common feature of membrane proteins. Many of these clusters have been implicated in signal initiation and integration platforms. However, the mechanisms that mediate the dynamic nanoscale arrangement of membrane proteins are not fully understood and could involve lipid domains, electrostatic interactions between proteins and lipid, protein scaffolding as well as purely mechanical processes. In this review we summarise these mechanisms giving rise to dynamic nanoscale protein reorganisation in the plasma membrane with reference to recent examples of immune receptor clustering to illustrate general principles.
Plant flavonoids are not only known as powerful antioxidants, but also as cell metabolism regulators. It has been postulated that they are able to control cell signal pathways by targeting receptors ...on the cell surface or by intercalating the lipid bilayer of membranes. Some flavonoids can increase lipid viscosity and decrease the cooperativity of hydrocarbon chain melting, while others can considerably decrease the lipid melting temperature, thus providing additional freedom for lipid diffusion. Here we discuss the ability of flavonoids to influence phase transition and lateral segregation of lipids, responsible for the formation of membrane compartments known as lipid rafts. The thermodynamic parameters of the bilayer determined by lipid packing characteristics and by lateral segregation of the bilayer are expected to depend on the location of flavonoid molecules in the bilayer. Flavonoid molecules preferably located in the hydrophobic region of the bilayer can initiate formation of raft-like domains (raft-making effect), while the molecules located in the polar interface region of the bilayer can fluidize membranes (raft-breaking effect), or initiate formation of interdigitated or micellar structures. Accordingly, we expect that in cellular membranes flavonoids can influence the appearance and development of rafts or raft-like membrane domains and thus influence the lateral diffusion of lipid molecules. Because rafts participate in cellular signal transduction, endocytosis and transmembrane translocation of different compounds, flavonoids may control cell metabolism by modulating the bilayer state.
In Gram-negative bacteria, a synergistic relationship between slow passive uptake of antibiotics across the outer membrane and active efflux transporters creates a permeability barrier, which ...efficiently reduces the effective concentrations of antibiotics in cells and, hence, their activities. To analyze the relative contributions of active efflux and the passive barrier to the activities of antibiotics, we constructed Escherichia coli strains with controllable permeability of the outer membrane. The strains expressed a large pore that does not discriminate between compounds on the basis of their hydrophilicity and sensitizes cells to a variety of antibacterial agents. We found that the efficacies of antibiotics in these strains were specifically affected by either active efflux or slow uptake, or both, and reflect differences in the properties of the outer membrane barrier, the repertoire of efflux pumps, and the inhibitory activities of antibiotics. Our results identify antibiotics which are the best candidates for the potentiation of activities through efflux inhibition and permeabilization of the outer membrane.
An enigmatic step in de novo formation of the autophagosome membrane compartment is the expansion of the precursor membrane phagophore, which requires the acquisition of lipids to serve as building ...blocks. Autophagy-related 2 (ATG2), the rod-shaped protein that tethers phosphatidylinositol 3-phosphate (PI3P)-enriched phagophores to the endoplasmic reticulum (ER), is suggested to be essential for phagophore expansion, but the underlying mechanism remains unclear. Here, we demonstrate that human ATG2A is a lipid transfer protein. ATG2A can extract lipids from membrane vesicles and unload them to other vesicles. Lipid transfer by ATG2A is more efficient between tethered vesicles than between untethered vesicles. The PI3P effectors WIPI4 and WIPI1 associate ATG2A stably to PI3P-containing vesicles, thereby facilitating ATG2A-mediated tethering and lipid transfer between PI3P-containing vesicles and PI3P-free vesicles. Based on these results, we propose that ATG2-mediated transfer of lipids from the ER to the phagophore enables phagophore expansion.
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