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The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier to protect against toxic compounds. By nature, the OM is asymmetric with the highly packed lipopolysaccharide (LPS) at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla system, in which is responsible for the retrograde transport of glycerophospholipids from the OM to the inner membrane. This system is comprised of six Mla proteins, including MlaA, an OM lipoprotein involved in the removal of glycerophospholipids that are mis-localized at the outer leaflet of the OM. Interestingly, MlaA was initially identified - and called VacJ - based on its role in the intracellular spreading of Shigella flexneri.Many open questions remain with respect to the Mla system and the mechanism involved in the translocation of mislocated glycerophospholipids at the outer leaflet of the OM, by MlaA. After summarizing the current knowledge on MlaA, we focus on the impact of mlaA deletion on OM lipid composition and biophysical properties of the OM. How changes in OM lipid composition and biophysical properties can impact the generation of membrane vesicles and membrane permeability is discussed. Finally, we explore whether and how MlaA might be a candidate for improving the activity of antibiotics and as a vaccine candidate.Efforts dedicated to understanding the relationship between the OM lipid composition and the mechanical strength of the bacterial envelope and, in turn, how such properties act against external stress, are needed for the design of new targets or drugs for Gram-negative infections., (© 2023. The Author(s).)

Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital acquired infections poses threat by its ability for adaptation to various growth modes and environmental conditions and by its intrinsic resistance to antibiotics. The latter is mainly due to the outer membrane (OM) asymmetry which is maintained by the Mla pathway resulting in the retrograde transport of glycerophospholipids from the OM to the inner membrane. It comprises six Mla proteins, including MlaA, an OM lipoprotein involved in the removal of glycerophospholipids mislocalized at the outer leaflet of OM. To investigate the role of P. aeruginosa OM asymmetry especially MlaA, this study investigated the effect of mlaA deletion on (i) the susceptibility to antibiotics, (ii) the secretion of virulence factors, the motility, biofilm formation, and (iii) the inflammatory response. mlaA deletion in P. aeruginosa ATCC27853 results in phenotypic changes including, an increase in fluoroquinolones susceptibility and in PQS (Pseudomonas Quinolone Signal) and TNF-α release and a decrease in rhamnolipids secretion, motility and biofilm formation. Investigating how the mlaA knockout impacts on antibiotic susceptibility, bacterial virulence and innate immune response will help to elucidate the biological significance of the Mla system and contribute to the understanding of MlaA in P. aeruginosa OM asymmetry., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Pseudomonas aeruginosa is a versatile pathogen that resists environmental stress, such as suboptimal pH. As a result of exposure to environmental stress, P. aeruginosa shows an altered virulence-related phenotype. This study investigated the modifications that P. aeruginosa undertakes at a mildly low pH (pH 5.0) compared with the bacteria grown in a neutral medium (pH 7.2). Results indicated that in a mildly acidic environment, expression of two-component system genes ( phoP / phoQ and pmrA / pmrB ), lipid A remodeling genes such as arnT and pagP and virulence genes, i.e., pqsE and rhlA , were induced. Moreover, lipid A of the bacteria grown at a mildly low pH is modified by adding 4-amino-arabinose (l-Ara4N). Additionally, the production of virulence factors such as rhamnolipid, alginate, and membrane vesicles is significantly higher in a mildly low-pH environment than in a neutral medium. Interestingly, at a mildly low pH, P. aeruginosa produces a thicker biofilm with higher biofilm biomass. Furthermore, studies on inner membrane viscosity and permeability showed that a mildly low pH causes a decrease in the inner membrane permeability and increases its viscosity. Besides, despite the importance of PhoP, PhoQ, PmrA, and PmrB in Gram-negative bacteria for responding to low pH stress, we observed that the absence of each of these two-component systems does not meaningfully impact the remodeling of the P. aeruginosa envelope. Given that P. aeruginosa is likely to encounter mildly acidic environments during infection in its host, the alterations that the bacterium undertakes under such conditions must be considered in designing antibacterial strategies against P. aeruginosa. IMPORTANCE P. aeruginosa encounters environments with acidic pH when establishing infections in hosts. The bacterium develops an altered phenotype to tolerate a moderate decrease in the environmental pH. At the level of the bacterial envelope, modified lipid A composition and a reduction of the bacterial inner membrane permeability and fluidity are among the changes P. aeruginosa undergoes at a mildly low pH. Also, the bacterium is more likely to form biofilm in a mildly acidic environment. Overall, these alterations in the P. aeruginosa phenotype put obstacles in the way of antibacterial activities. Thus, considering physiological changes in the bacterium at low pH helps design and implement antimicrobial approaches against this hostile microorganism., Competing Interests: The authors declare no conflict of interest.

Despite growing attention, antibiotics (such as streptomycin, oxytetracycline or kasugamycin) are still used worldwide for the control of major bacterial plant diseases. This raises concerns on their potential, yet unknown impact on antibiotic and multidrug resistances and the spread of their genetic determinants among bacterial pathogens. Antibiotic resistance genes (ARGs) have been identified in plant pathogenic bacteria (PPB), with streptomycin resistance genes being the most commonly reported. Therefore, the contribution of mobile genetic elements (MGEs) to their spread among PPB, as well as their ability to transfer to other bacteria, need to be further explored. The only well-documented example of ARGs vector in PPB, Tn 5393 and its highly similar variants (carrying streptomycin resistance genes), is concerning because of its presence outside PPB, in Salmonella enterica and Klebsiella pneumoniae , two major human pathogens. Although its structure among PPB is still relatively simple, in human- and animal-associated bacteria, Tn 5393 has evolved into complex associations with other MGEs and ARGs. This review sheds light on ARGs and MGEs associated with PPB, but also investigates the potential role of antibiotic use in resistance selection in plant-associated bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Verhaegen, Bergot, Liebana, Stancanelli, Streissl, Mingeot-Leclercq, Mahillon and Bragard.)

Leishmaniasis is a neglected tropical disease that still infects thousands of people per year throughout the world. The occurrence of resistance against major treatments for this disease causes a healthcare burden in low-income countries. Eugenol is a phenylpropanoid that has shown in vitro antileishmanial activity against Leishmania mexicana mexicana ( Lmm ) promastigotes with an IC 50 of 2.72 µg/mL and a high selectivity index. Its specific mechanism of action has yet to be studied. We prepared large unilamellar vesicles (LUVs), mimicking Lmm membranes, and observed that eugenol induced an increase in membrane permeability and a decrease in membrane fluidity at concentrations much higher than IC 50 . The effect of eugenol was similar to the current therapeutic antibiotic, amphotericin B, although the latter was effective at lower concentrations than eugenol. However, unlike amphotericin B, eugenol also affected the permeability of LUVs without sterol. Its effect on the membrane fluidity of Lmm showed that at high concentrations (≥22.5× IC 50 ), eugenol increased membrane fluidity by 20-30%, while no effect was observed at lower concentrations. Furthermore, at concentrations below 10× IC 50 , a decrease in metabolic activity associated with the maintenance of membrane integrity revealed a leishmaniostatic effect after 24 h of incubation with Lmm promastigotes. While acidocalcisomes distribution and abundance revealed by Trypanosoma brucei vacuolar H + pyrophosphatase (TbVP1) immunolabeling was not modified by eugenol, a dose-dependent decrease of lipid droplets assessed by the Nile Red assay was observed. We hereby demonstrate that the antileishmanial activity of eugenol might not directly involve plasma membrane sterols such as ergosterol, but rather target the lipid storage of Lmm .

Tumor cells exhibit altered cholesterol content. However, cholesterol structural subcellular distribution and implication in cancer cell invasion are poorly understood mainly due to difficulties to investigate cholesterol both quantitatively and qualitatively and to compare isogenic cell models. Here, using the MCF10A cell line series (non-tumorigenic MCF10A, pre-malignant MCF10AT and malignant MCF10CAIa cells) as a model of breast cancer progression and the highly invasive MDA-MB-231 cell line which exhibits the common TP53 mutation, we investigated if cholesterol contributes to cancer cell invasion, whether the effects are specific to cancer cells and the underlying mechanism. We found that partial membrane cholesterol depletion specifically and reversibly decreased invasion of the malignant cell lines. Those cells exhibited dorsal surface cholesterol-enriched submicrometric domains and narrow ER-plasma membrane and ER-intracellular organelles contact sites. Dorsal cholesterol-enriched domains can be endocytosed and reach the cell ventral face where they were involved in invadopodia formation and extracellular matrix degradation. In contrast, non-malignant cells showed low cell invasion, low surface cholesterol exposure and cholesterol-dependent focal adhesions. The differential cholesterol distribution and role in breast cancer cell invasion provide new clues for the understanding of the molecular events underlying cellular mechanisms in breast cancer., (© 2022. The Author(s).)

Pseudomonas aeruginosa is an opportunistic pathogen capable of resisting environmental insults by applying various strategies, including regulating membrane fluidity and producing membrane vesicles (MVs). This study examined the difference in membrane fluidity between planktonic and biofilm modes of growth in P. aeruginosa and whether the ability to alter membrane rigidity in P. aeruginosa could be transferred via MVs. To this end, planktonic and biofilm P. aeruginosa were compared with respect to the lipid composition of their membranes and their MVs and the expression of genes contributing to alteration of membrane fluidity. Additionally, viscosity maps of the bacterial membrane in planktonic and biofilm lifestyles and under the effect of incubation with bacterial MVs were obtained. Further, the growth rate and biofilm formation capability of P. aeruginosa in the presence of MVs were compared. Results showed that the membrane of the biofilm bacteria is significantly less fluid than the membrane of the planktonic bacteria and is enriched with saturated fatty acids. Moreover, the enzymes involved in altering the structure of existing lipids and favoring membrane rigidification are overexpressed in the biofilm bacteria. MVs of biofilm P. aeruginosa elicit membrane rigidification and delay the bacterial growth in the planktonic lifestyle; conversely, they enhance biofilm development in P. aeruginosa. Overall, the study describes the interplay between the planktonic and biofilm bacteria by shedding light on the role of MVs in altering membrane fluidity. IMPORTANCE Membrane rigidification is a survival strategy in Pseudomonas aeruginosa exposed to stress. Despite various studies dedicated to the mechanism behind this phenomenon, not much attention has been paid to the contribution of the bacterial membrane vesicles (MVs) in this regard. This study revealed that P. aeruginosa rigidifies its membrane in the biofilm mode of growth. Additionally, the capability of decreasing membrane fluidity is transferable to the bacterial population via the bacterial MVs, resulting in reprogramming of bacterial membrane fluidity. Given the importance of membrane rigidification for decreasing the pathogen's susceptibility to antimicrobials, elucidation of the conditions leading to such biophysicochemical modulation of the P. aeruginosa membrane should be considered for the purpose of developing therapeutic approaches against this resistant pathogen.

Staphylococcus aureus is an opportunistic pathogen and the major causative agent of life-threatening hospital- and community-acquired infections. A combination of antibiotics could be an opportunity to address the widespread emergence of antibiotic-resistant strains, including Methicillin-Resistant S. aureus (MRSA). We here investigated the potential synergy between ampicillin and plant-derived antibiotics (pentacyclic triterpenes, ursolic acid (UA) and oleanolic acid (OA)) towards MRSA (ATCC33591 and COL) and the mechanisms involved. We calculated the Fractional Inhibitory Concentration Index (FICI) and demonstrated synergy. We monitored fluorescence of Bodipy-TR-Cadaverin, propidium iodide and membrane potential-sensitive probe for determining the ability of UA and OA to bind to lipoteichoic acids (LTA), and to induce membrane permeabilization and depolarization, respectively. Both pentacyclic triterpenes were able to bind to LTA and to induce membrane permeabilization and depolarization in a dose-dependent fashion. These effects were not accompanied by significant changes in cellular concentration of pentacyclic triterpenes and/or ampicillin, suggesting an effect mediated through lipid membranes. We therefore focused on membranous effects induced by UA and OA, and we investigated on models of membranes, the role of specific lipids including phosphatidylglycerol and cardiolipin. The effect induced on membrane fluidity, permeability and ability to fuse were studied by determining changes in fluorescence anisotropy of DPH/generalized polarization of Laurdan, calcein release from liposomes, fluorescence dequenching of octadecyl-rhodamine B and liposome-size, respectively. Both UA and OA showed a dose-dependent effect with membrane rigidification, increase of membrane permeabilization and fusion. Except for the effect on membrane fluidity, the effect of UA was consistently higher compared with that obtained with OA, suggesting the role of methyl group position. All together the data demonstrated the potential role of compounds acting on lipid membranes for enhancing the activity of other antibiotics, like ampicillin and inducing synergy. Such combinations offer an opportunity to explore a larger antibiotic chemical space.

Development of novel therapeutics to treat antibiotic-resistant infections, especially those caused by ESKAPE pathogens, is urgent. One of the most critical pathogens is P. aeruginosa , which is able to develop a large number of factors associated with antibiotic resistance, including high level of impermeability. Gram-negative bacteria are protected from the environment by an asymmetric Outer Membrane primarily composed of lipopolysaccharides (LPS) at the outer leaflet and phospholipids in the inner leaflet. Based on a large hemi-synthesis program focusing on amphiphilic aminoglycoside derivatives, we extend the antimicrobial activity of 3',6-dinonyl neamine and its branched isomer, 3',6-di(dimethyloctyl) neamine on clinical P. aeruginosa , ESBL, and carbapenemase strains. We also investigated the capacity of 3',6-homodialkyl neamine derivatives carrying different alkyl chains (C7-C11) to interact with LPS and alter membrane permeability. 3',6-Dinonyl neamine and its branched isomer, 3',6-di(dimethyloctyl) neamine showed low MICs on clinical P. aeruginosa , ESBL, and carbapenemase strains with no MIC increase for long-duration incubation. In contrast from what was observed for membrane permeability, length of alkyl chains was critical for the capacity of 3',6-homodialkyl neamine derivatives to bind to LPS. We demonstrated the high antibacterial potential of the amphiphilic neamine derivatives in the fight against ESKAPE pathogens and pointed out some particular characteristics making the 3',6-dinonyl- and 3',6-di(dimethyloctyl)-neamine derivatives the best candidates for further development.

RNA therapeutics are poised to revolutionize medicine. To unlock the full potential of RNA drugs, safe and efficient (nano)formulations to deliver them inside target cells are required. Endosomal sequestration of nanocarriers represents a major bottleneck in nucleic acid delivery. Gaining more detailed information on the intracellular behavior of RNA nanocarriers is crucial to rationally develop delivery systems with improved therapeutic efficiency. Surfactant protein B (SP-B) is a key component of pulmonary surfactant (PS), essential for mammalian breathing. In contrast to the general belief that PS should be regarded as a barrier for inhaled nanomedicines, we recently discovered the ability of SP-B to promote gene silencing by siRNA-loaded and lipid-coated nanogels. However, the mechanisms governing this process are poorly understood. The major objective of this work was to obtain mechanistic insights into the SP-B-mediated cellular delivery of siRNA. To this end, we combined siRNA knockdown experiments, confocal microscopy, and focused ion beam scanning electron microscopy imaging in an in vitro non-small-cell lung carcinoma model with lipid mixing assays on vesicles that mimic the composition of (intra)cellular membranes. Our work highlights a strong correlation between SP-B-mediated fusion with anionic endosomal membranes and cytosolic siRNA delivery, a mode of action resembling that of certain viruses and virus-derived cell-penetrating peptides. Building on these gained insights, we optimized the SP-B proteolipid composition, which dramatically improved delivery efficiency. Altogether, our work provides a mechanistic understanding of SP-B-induced perturbation of intracellular membranes, offering opportunities to fuel the rational design of SP-B-inspired RNA nanoformulations for inhalation therapy.

Membrane vesicles are the nano-sized vesicles originating from membranes. The production of membrane vesicles is a common feature among bacteria. Depending on the bacterial growth phase and environmental conditions, membrane vesicles show diverse characteristics. Various physiological and ecological roles have been attributed to membrane vesicles under both homeostatic and stressful conditions. Pathogens encounter several stressors during colonization in the hostile environment of host tissues. Nutrient deficiency, the presence of antibiotics as well as elements of the host's immune system are examples of stressors threatening pathogens inside their host. To combat stressors and survive, pathogens have established various defensive mechanisms, one of them is production of membrane vesicles. Pathogens produce membrane vesicles to alleviate the destructive effects of antibiotics or other types of antibacterial treatments. Additionally, membrane vesicles can also provide benefits for the wider bacterial community during infections, through the transfer of resistance or virulence factors. Hence, given that membrane vesicle production may affect the activities of antibacterial agents, their production should be considered when administering antibacterial treatments. Besides, regarding that membrane vesicles play vital roles in bacteria, disrupting their production may suggest an alternative strategy for battling against pathogens. Here, we aim to review the stressors encountered by pathogens and shed light on the roles of membrane vesicles in increasing pathogen adaptabilities in the presence of stress-inducing factors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Mozaheb and Mingeot-Leclercq.)

Synthetic glucocorticoids such as budesonide (BUD) are potent anti-inflammatory drugs commonly used to treat patients suffering from chronic inflammatory diseases. A previous animal study reported a higher anti-inflammatory activity with a 2-hydroxypropyl-β-cyclodextrin (HPβCD)-based formulation of BUD (BUD:HPβCD). This study investigated, on cellular models (A549 and A-THP-1), the effect of BUD:HPβD in comparison with BUD and HPβCD on the effects induced by oxidative and inflammatory stress as well as the role of cholesterol. We demonstrated the protective effect afforded by BUD:HPβCD against cytotoxicity and ROS generation induced by oxidative and inflammatory stress. The effect observed for BUD:HPβCD was comparable to that observed with HPβCD with no major effect of cholesterol content. We also demonstrated (i) the involvement of the canonical molecular pathway including ROS generation, a decrease in PI3K/Akt activation, and decrease in phosphorylated/unphosphorylated HDAC2 in the effect induced by BUD:HPβCD, (ii) the maintenance of IL-8 decrease with BUD:HPβCD, and (iii) the absence of improvement in glucocorticoid insensitivity with BUD:HPβCD in comparison with BUD, in conditions where HDAC2 was inhibited. Resulting from HPβCD antioxidant and anticytotoxic potential and protective capacity against ROS-induced PI3K/Akt signaling and HDAC2 inhibition, BUD:HPβCD might be more beneficial than BUD alone in a context of concomitant oxidative and inflammatory stress.

The conjugation of hydrophobic group(s) to the polycationic hydrophilic core of the antibiotic drugs aminoglycosides (AGs), targeting ribosomal RNA, has led to the development of amphiphilic aminoglycosides (AAGs). These drugs exhibit numerous biological effects, including good antibacterial effects against susceptible and multidrug-resistant bacteria due to the targeting of bacterial membranes. In the first part of this review, we summarize our work in identifying and developing broad-spectrum antibacterial AAGs that constitute a new class of antibiotic agents acting on bacterial membranes. The target-shift strongly improves antibiotic activity against bacterial strains that are resistant to the parent AG drugs and to antibiotic drugs of other classes, and renders the emergence of resistant Pseudomonas aeruginosa strains highly difficult. Structure-activity and structure-eukaryotic cytotoxicity relationships, specificity and barriers that need to be crossed in their development as antibacterial agents are delineated, with a focus on their targets in membranes, lipopolysaccharides (LPS) and cardiolipin (CL), and the corresponding mode of action against Gram-negative bacteria. At the end of the first part, we summarize the other recent advances in the field of antibacterial AAGs, mainly published since 2016, with an emphasis on the emerging AAGs which are made of an AG core conjugated to an adjuvant or an antibiotic drug of another class (antibiotic hybrids). In the second part, we briefly illustrate other biological and biochemical effects of AAGs, i.e., their antifungal activity, their use as delivery vehicles of nucleic acids, of short peptide (polyamide) nucleic acids (PNAs) and of drugs, as well as their ability to cleave DNA at abasic sites and to inhibit the functioning of connexin hemichannels. Finally, we discuss some aspects of structure-activity relationships in order to explain and improve the target selectivity of AAGs.

Tumor cells present profound alterations in their composition, structural organization, and functional properties. A landmark of cancer cells is an overall altered mechanical phenotype, which so far are linked to changes in their cytoskeletal regulation and organization. Evidence exists that the plasma membrane (PM) of cancer cells also shows drastic changes in its composition and organization. However, biomechanical characterization of PM remains limited mainly due to the difficulties encountered to investigate it in a quantitative and label-free manner. Here, the biomechanical properties of PM of a series of MCF10 cell lines, used as a model of breast cancer progression, are investigated. Notably, a strong correlation between the cell PM elasticity and oncogenesis is observed. The altered membrane composition under cancer progression, as emphasized by the PM-associated cholesterol levels, leads to a stiffening of the PM that is uncoupled from the elastic cytoskeletal properties. Conversely, cholesterol depletion of metastatic cells leads to a softening of their PM, restoring biomechanical properties similar to benign cells. As novel therapies based on targeting membrane lipids in cancer cells represent a promising approach in the field of anticancer drug development, this method contributes to deciphering the functional link between PM lipid content and disease., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Published by Wiley‐VCH GmbH.)

In this review, we will focus on the activity of ginsenosides on membranes and their related effects, from physicochemical, biophysical, and pharmacological viewpoints. Ginsenosides are a class of saponins with a large structural diversity and a wide range of pharmacological effects. These effects can at least partly be related to their activity on membranes which results from their amphiphilic character. Some ginsenosides are able to interact with membrane lipids and associate into nanostructures, making them possible adjuvants for vaccines. They are able to modulate membrane biophysical properties such as membrane fluidity, permeability or the formation of lateral domains with some degree of specificity towards certain cell types such as bacteria, fungi, or cancer cells. In addition, they have shown antioxidant properties which protect membranes from lipid oxidation. They further displayed some activity on membrane proteins either through direct or indirect interaction. We investigate the structure activity relationship of ginsenosides on membranes and discuss the implications and potential use as anticancer, antibacterial, and antifungal agents., (Copyright © 2020 Verstraeten, Lorent and Mingeot-Leclercq.)

2-aminothiophene derivatives (2AT) in which the thiophene ring is fused with a cycloalkyl or a N-acylated piperidine ring by positions 5 and 6 and carrying a 3-carbethoxy group were synthesized and their bacterial growth and enzyme inhibitory effects against efflux proteins of Staphylococcus aureus leading to resistance to fluoroquinolones and erythromycin (ERY) were investigated. Compounds that most effectively decreases the minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) were assayed for their dose and time effects on the accumulation and efflux of ethidium bromide (EtBr) in the SA-1 strain. None of the compounds displayed antibacterial activity however, three derivatives carrying 2-amino, 2-aminoacetyl and 2-aminotrifluoroacetyl group enhanced the activity of CIP and ERY by 8- and 16-fold, respectively, and were able to restore the sensitivity of resistant strains, acting as typical efflux pump inhibitors (EPIs). The 2-aminoacetyl and 2-aminotrifluoroacetyl derivatives and two other piperidinyl 2-aminotrifluoroacetyl derivatives increased EtBr accumulation in a dose- and time-dependent manner, and one of them was also able to inhibit the EtBr efflux. Taken together, these results represent an important advance in the development of new EPIs, and demonstrate that 2AT represent a good scaffold for developing new antibiotic adjuvants., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Amphiphilic aminoglycoside derivatives are potential new antimicrobial agents mostly developed to fight resistant bacteria. The mechanism of action of the 3',6-dinonyl neamine, one of the most promising derivative, has been investigated on Gram-negative bacteria, including P. aeruginosa. In this study, we have assessed its mechanism of action against Gram-positive bacteria, S. aureus and B. subtilis. By conducting time killing experiments, we assessed the bactericidal effect induced by 3',6-dinonyl neamine on S. aureus MSSA and MRSA. By measuring the displacement of BODIPY™-TR cadaverine bound to lipoteichoic acids (LTA), we showed that 3',6-dinonyl neamine interacts with these bacterial surface components. We also highlighted the ability of 3',6-dinonyl neamine to enhance membrane depolarization and induce membrane permeability, by using fluorescent probes, DiSC 3 C(5) and propidium iodide, respectively. These effects are observed for both MSSA and MRSA S. aureus as well as for B. subtilis. By electronic microscopy, we imaged the disruption of membrane integrity of the bacterial cell wall and by fluorescence microscopy, we demonstrated changes in the localization of lipids from the enriched-septum region and the impairment of the formation of septum. At a glance, we demonstrated that 3',6-dinonyl neamine interferes with multiple targets suggesting a low ability of bacteria to acquire resistance to this agent. In turn, the amphiphilic neamine derivatives are promising candidates for development as novel multitarget therapeutic antibiotics., (Copyright © 2019. Published by Elsevier B.V.)

The membrane activity of some saponins, such as digitonin or alpha-hederin, is usually attributed to their interaction with membrane cholesterol (Chol). This contrasts with our recent publication showing that Chol, contrary to sphingomyelin (SM), can delay the cytotoxicity of the saponin ginsenoside Rh2, challenging the usual view that most saponins mediate their membrane effects through interaction with Chol. The aim of the present study was to elucidate the respective importance of Chol and SM as compared to phosphatidylcholine (PC) species in the membrane-related effects of Rh2. On simple lipid monolayers, Rh2 interacted more favorably with eggSM and DOPC than with Chol and eggPC. Using Large Unilamellar Vesicles (LUVs) of binary or ternary lipid compositions, we showed that Rh2 increased vesicle size, decreased membrane fluidity and induced membrane permeability with the following preference: eggSM:eggPC > eggSM:eggPC:Chol > eggPC:Chol. On Giant Unilamellar Vesicles (GUVs), we evidenced that Rh2 generated positive curvatures in eggSM-containing GUVs and small buds followed by intra-luminal vesicles in eggSM-free GUVs. Altogether, our data indicate that eggSM promotes and accelerates membrane-related effects induced by Rh2 whereas Chol slows down and depresses these effects. This study reconsiders the theory that Chol is the only responsible for the activity of saponins.

Human African trypanosomiasis (HAT), known as sleeping sickness and caused by Trypanosoma brucei , is threatening low-income populations in sub-Saharan African countries with 61 million people at risk of infection. In order to discover new natural products against HAT, thirty-seven Vietnamese essential oils (EOs) were screened for their activity in vitro on Trypanosoma brucei brucei ( Tbb ) and cytotoxicity on mammalian cells (WI38, J774). Based on the selectivity indices (SIs), the more active and selective EOs were analyzed by gas chromatography. The anti-trypanosomal activity and cytotoxicity of some major compounds (isolated or commercial) were also determined. Our results showed for the first time the selective anti-trypanosomal effect of four EOs, extracted from three Zingiberaceae species ( Curcuma longa , Curcuma zedoaria , and Zingiber officinale ) and one Lauraceae species ( Litsea cubeba ) with IC 50 values of 3.17 ± 0.72, 2.51 ± 1.08, 3.10 ± 0.08, and 2.67 ± 1.12 nL/mL respectively and SI > 10. Identified compounds accounted for more than 85% for each of them. Among the five major components of Curcuma longa EO, curlone is the most promising anti-trypanosomal candidate with an IC 50 of 1.38 ± 0.45 µg/mL and SIs of 31.7 and 18.2 compared to WI38 and J774 respectively.

The adsorption of a peptide (NFL-TBS.40-63 peptide (NFL)) known to induce neural stem cells (NSC) differentiation in vitro, at the surface of lipid nanocapsules (LNC) provides a targeting drug delivery system (NFL-LNC) that penetrates subventricular zone-neural stem cells (SVZ-NSC) but not central canal-NSC (CC-NSC). We hypothesized preferential interactions could explaine, at least partially, the different properties of SVZ- and CC-NSC plasma membranes. The objective of this work was to compare SVZ- and CC-NSC plasma membrane lipid composition, fluidity and permeability. Plasma membranes of SVZ- and CC-NSC were isolated and analyzed by LC-MS for their lipid content. Membrane fluidity was evaluated by measuring the generalized polarization (GP) of Laurdan and membrane permeability by fluorescent dextran penetration. Liposomes with different lipid compositions and steady state fluidities were prepared. ΔGP was measured after incubation with NFL-LNC. A significantly higher proportion of cholesterol, ceramides, sphingomyelins, phosphatidylethanolamines and a lower proportion of phosphatidylcholines and sulfatides were observed in SVZ- compared to CC-NSC. Fluidity, probably more than lipid composition, drove NFL-LNC and NSC interactions, and SVZ-NSC were more sensitive to NFL permeabilization than CC-NSC. We demonstrated that NSC membrane lipid composition and fluidity depended of NSC origin and that these features could play a role in the specific interactions with NFL-LNC., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Amphiphilic aminoglycosides (AAGs) constitute a new class of antibacterial compounds targeting the bacterial membranes. We have identified the 3',6-dinonyl neamine 9 as a broad spectrum antibacterial AAG. Here, we report on the synthesis, antibacterial activity and eukaryotic cytotoxicity of new 3',6-dialkyl neamines designed in order to finely delineate the structure-activity relationships relating their activity to a lipophilicity window. New broad-spectrum antibacterial derivatives were obtained carrying two identical linear or branched alkyl groups or two different linear alkyl groups. Two fluorescent antibacterial 3',6-heterodialkyl neamines carrying a pyrenylbutyl fluorophore were also identified as potential tools for mechanistic study. Homodialkyl and heterodialkyl neamines appeared to be more active on Gram-negative bacteria than dinaphthylalkyl neamines. However, branched dialkyl neamines or heterodialkyl derivatives were found to be more cytotoxic on mammalian cells than 9. The exposure of P. aeruginosa over one month to half-MIC of one of the most active derivatives 9 demonstrated the high difficulty of resistance emergence to AAGs., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Amphiphilic aminoglycoside derivatives are promising new antibacterials active against Gram-negative bacteria such as Pseudomonas aeruginosa, including colistin resistant strains. In this study, we demonstrated that addition of cardiolipin to the culture medium delayed growth of P. aeruginosa, favored asymmetrical growth and enhanced the efficiency of a new amphiphilic aminoglycoside derivative, the 3',6-dinonylneamine. By using membrane models mimicking P. aeruginosa plasma membrane composition (POPE:POPG:CL), we demonstrated the ability of 3'6-dinonylneamine to induce changes in the biophysical properties of membrane model lipid systems in a cardiolipin dependent manner. These changes include an increased membrane permeability associated with a reduced hydration and a decreased ability of membrane to mix and fuse as shown by monitoring calcein release, Generalized Polarization of Laurdan and fluorescence dequenching of octadecyl rhodamine B, respectively. Altogether, results shed light on how cardiolipin may be critical for improving antibacterial action of new amphiphilic aminoglycoside derivatives., Competing Interests: The authors have declared that no competing interests exist.

Saponins exhibit several biological and pharmacological activities, such as antibacterial, anti-inflammatory and anticancer effects. Many studies attribute their activities to their interactions with cholesterol. In this study, we focus on the steroid saponin ginsenoside Rh2, one of the active principles of Panax ginseng root. Some evidence suggests that lipid rafts, defined as nanodomains enriched in cholesterol and sphingolipids, could be involved in the Rh2-induced apoptosis. However, the role of membrane lipids, especially cholesterol, in this process is still poorly understood. Here, we demonstrate that (i) A549, THP-1 and U937 cells are all susceptible to the Rh2-induced apoptosis but to a differential extent and (ii) the cytotoxic effect inversely correlates with the cell membrane cholesterol content. Upon cholesterol depletion via methyl-β-cyclodextrin, those three cells lines become more sensitive to Rh2-induced apoptosis. Then, focusing on the cholesterol-auxotroph U937 cell line, we showed that Rh2 alters plasma membrane fluidity by compacting the hydrophobic core of lipid bilayer (DPH anisotropy) and relaxing the interfacial packaging of the polar head of phospholipids (TMA-DPH anisotropy). The treatment with Rh2 conducts to the dephosphorylation of Akt and the activation of the intrinsic pathway of apoptosis (loss of mitochondrial membrane potential, caspase-9 and -3 activation). All these features are induced faster in cholesterol-depleted cells, which could be explained by faster cell accumulation of Rh2 in these conditions. This work is the first reporting that membrane cholesterol could delay the activity of ginsenoside Rh2, renewing the idea that saponin cytotoxicity is ascribed to an interaction with membrane cholesterol., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background/aims: Transient nanometric cholesterol- and sphingolipid-enriched domains, called rafts, are characterized by higher lipid order as compared to surrounding lipids. Here, we asked whether the seminal concept of highly ordered rafts could be refined with the presence of lipid domains exhibiting different enrichment in cholesterol and sphingomyelin and association with erythrocyte curvature areas. We also investigated how differences in lipid order between domains and surrounding membrane (bulk) are regulated and whether changes in order differences could participate to erythrocyte deformation and vesiculation., Methods: We used the fluorescent hydration- and membrane packing-sensitive probe Laurdan to determine by imaging mode the Generalized Polarization (GP) values of lipid domains vs the surrounding membrane., Results: Laurdan revealed the majority of sphingomyelin-enriched domains associated to low erythrocyte curvature areas and part of the cholesterol-enriched domains associated with high curvature. Both lipid domains were less ordered than the surrounding lipids in erythrocytes at resting state. Upon erythrocyte deformation (elliptocytes and stimulation of calcium exchanges) or membrane vesiculation (storage at 4°C), lipid domains became more ordered than the bulk. Upon aging and in membrane fragility diseases (spherocytosis), an increase in the difference of lipid order between domains and the surrounding lipids contributed to the initiation of domain vesiculation., Conclusion: The critical role of domain-bulk differential lipid order modulation for erythrocyte reshaping is discussed in relation with the pressure exerted by the cytoskeleton on the membrane., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Nystatin (Nys) is a pore forming broad-spectrum and efficient antifungal drug with significant toxicity in mammalian organisms. In order to develop a non-toxic and more effective Nys formulation, its molecular mechanism of action at the cell membrane needs to be better understood. It is widely accepted that Nys activity and toxicity depend on the presence and type of membrane sterols. Taking advantage of multiple biophysical methodologies, we now show that the formation and stabilization of Nys aqueous pores, which are associated with Nys cytotoxicity, occur in the absence of membrane sterols. Our results suggest that the Nys mechanism of action is driven by the presence of highly ordered membrane domains capable of stabilizing the Nys oligomers. Moreover, Nys pore formation is accompanied by strong Nys-induced membrane reorganization that depends on membrane lipid composition and seems to underlie the Nys cytotoxic effect. Accordingly, in membranes enriched in a gel-phase forming phospholipid, Nys incorporates within the phospholipid-enriched gel domains, where it forms pores able to expand the gel domains. In contrast, in membranes enriched in gel domain forming sphingolipids, Nys-induced pore formation occurs through the destabilization of the gel phase. These results show that the Nys mechanism of action is complex and not only dependent on membrane sterols, and provide further insight into the molecular details governing Nys activity and toxicity.

Budesonide (BUD), a poorly soluble anti-inflammatory drug, is used to treat patients suffering from asthma and COPD (Chronic Obstructive Pulmonary Disease). Hydroxypropyl-β-cyclodextrin (HPβCD), a biocompatible cyclodextrin known to interact with cholesterol, is used as a drug-solubilizing agent in pharmaceutical formulations. Budesonide administered as an inclusion complex within HPβCD (BUD:HPβCD) required a quarter of the nominal dose of the suspension formulation and significantly reduced neutrophil-induced inflammation in a COPD mouse model exceeding the effect of each molecule administered individually. This suggests the role of lipid domains enriched in cholesterol for inflammatory signaling activation. In this context, we investigated the effect of BUD:HPβCD on the biophysical properties of membrane lipids. On cellular models (A549, lung epithelial cells), BUD:HPβCD extracted cholesterol similarly to HPβCD. On large unilamellar vesicles (LUVs), by using the fluorescent probes diphenylhexatriene (DPH) and calcein, we demonstrated an increase in membrane fluidity and permeability induced by BUD:HPβCD in vesicles containing cholesterol. On giant unilamellar vesicles (GUVs) and lipid monolayers, BUD:HPβCD induced the disruption of cholesterol-enriched raft-like liquid ordered domains as well as changes in lipid packing and lipid desorption from the cholesterol monolayers, respectively. Except for membrane fluidity, all these effects were enhanced when HPβCD was complexed with budesonide as compared with HPβCD. Since cholesterol-enriched domains have been linked to membrane signaling including pathways involved in inflammation processes, we hypothesized the effects of BUD:HPβCD could be partly mediated by changes in the biophysical properties of cholesterol-enriched domains., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Some bacterial proteins involved in cell division and oxidative phosphorylation are tightly bound to cardiolipin. Cardiolipin is a non-bilayer anionic phospholipid found in bacterial inner membrane. It forms lipid microdomains located at the cell poles and division plane. Mechanisms by which microdomains are affected by membrane-acting antibiotics and the impact of these alterations on membrane properties and protein functions remain unclear. In this study, we demonstrated cardiolipin relocation and clustering as a result of exposure to a cardiolipin-acting amphiphilic aminoglycoside antibiotic, the 3',6-dinonyl neamine. Changes in the biophysical properties of the bacterial membrane of P. aeruginosa, including decreased fluidity and increased permeability, were observed. Cardiolipin-interacting proteins and functions regulated by cardiolipin were impacted by the amphiphilic aminoglycoside as we demonstrated an inhibition of respiratory chain and changes in bacterial shape. The latter effect was characterized by the loss of bacterial rod shape through a decrease in length and increase in curvature. It resulted from the effect on MreB, a cardiolipin dependent cytoskeleton protein as well as a direct effect of 3',6-dinonyl neamine on cardiolipin. These results shed light on how targeting cardiolipin microdomains may be of great interest for developing new antibacterial therapies.

Although lipid domains have been evidenced in several living cell plasma membranes, their roles remain largely unclear. We here investigated whether they could contribute to function-associated cell (re)shaping. To address this question, we used erythrocytes as cellular model since they (i) exhibit a specific biconcave shape, allowing for reversible deformation in blood circulation, which is lost by membrane vesiculation upon aging; and (ii) display at their outer plasma membrane leaflet two types of submicrometric domains differently enriched in cholesterol and sphingomyelin. We here reveal the specific association of cholesterol- and sphingomyelin-enriched domains with distinct curvature areas of the erythrocyte biconcave membrane. Upon erythrocyte deformation, cholesterol-enriched domains gathered in high curvature areas. In contrast, sphingomyelin-enriched domains increased in abundance upon calcium efflux during shape restoration. Upon erythrocyte storage at 4 °C (to mimick aging), lipid domains appeared as specific vesiculation sites. Altogether, our data indicate that lipid domains could contribute to erythrocyte function-associated (re)shaping.

Leishmania mexicana is one of the pathogens causing cutaneous leishmaniasis which is associated with patient morbidity. In our researches for new safe and effective treatments, thirty-seven essential oils (EOs) extracted from Vietnamese plants were screened in vitro for the first time on Leishmania mexicana mexicana (Lmm) promastigotes at the maximum concentration of 50 nL/mL. Active EOs were also analyzed for cytotoxicity on mammalian cell lines (WI38, J774) and their selectivity indices (SI) were calculated. Their composition was determined by GC-MS and GC-FID. Our results indicated that EOs extracted from Cinnamomum cassia , Zingiber zerumbet , Elsholtzia ciliata and Amomum aromaticum , possessed a moderate anti-leishmanial activity, with IC 50 values of 2.92 ± 0.08, 3.34 ± 0.34, 8.49 ± 0.32 and 9.25 ± 0.64 nL/mL respectively. However, they also showed cytotoxicity with SI < 10. The most promising EO was extracted from Ocimum gratissimum , displaying an IC 50 of 4.85 ± 1.65 nL/mL and SI > 10. It contained 86.5% eugenol, which was demonstrated to be effective on Lmm with IC 50 of 2.57 ± 0.57 nL/mL and not toxic on mammalian cells, explaining the observed activity., Competing Interests: The authors declare no conflict of interest.

Type 3 secretion systems (T3SSs) are major virulence factors in Gram-negative bacteria. Pseudomonas aeruginosa expresses two T3SSs, namely, an injectisome (iT3SS) translocating effector proteins in the host cell cytosol and a flagellum (fT3SS) ensuring bacterial motility. Inhibiting these systems is an appealing therapeutic strategy for acute infections. This study examines the protective effects of the salicylidene acylhydrazide INP0341 and of the hydroxyquinoline INP1750 (previously described as T3SS inhibitors in other species) toward cytotoxic effects of P. aeruginosa in vitro Both compounds reduced cell necrosis and inflammasome activation induced by reference strains or clinical isolates expressing T3SS toxins or only the translocation apparatus. INP0341 inhibited iT3SS transcriptional activation, including in strains with constitutive iT3SS expression, and reduced the total expression of toxins, suggesting it targets iT3SS gene transcription. INP1750 inhibited toxin secretion and flagellar motility and impaired the activity of the YscN ATPase from Yersinia pseudotuberculosis (homologous to the ATPase present in the basal body of P. aeruginosa iT3SS and fT3SS), suggesting that it rather targets a T3SS core constituent with high homology among iT3SS and fT3SS. This mode of action is similar to that previously described for INP1855, another hydroxyquinoline, against P. aeruginosa Thus, although acting by different mechanisms, INP0341 and INP1750 appear as useful inhibitors of the virulence of P. aeruginosa Hydroxyquinolines may have a broader spectrum of activity by the fact they act upon two virulence factors (iT3SS and fT3SS)., (Copyright © 2017 American Society for Microbiology.)

In perspective of reducing the mortality of cancer, there is a high interest in compounds which act on multiple cellular targets and therefore prevent the appearance of cancer resistances. Saponins and α -hederin, an oleanane-type saponin, induce cancer cell death through different pathways, including apoptosis and membrane permeabilization. Unfortunately, the mechanism by which cell death is induced is unknown. We hypothesized that the activity of α -hederin mainly depends on its interaction with membrane cholesterol and therefore investigated the cholesterol and saponin-structure dependency of apoptosis and membrane permeabilization in two malignant monocytic cell lines. Apoptotic cell death and membrane permeabilization were significantly reduced in cholesterol-depleted cells. Permeabilization further depended upon the osidic side chain of α -hederin and led to extracellular calcium influx and nuclear fragmentation, with only the latter being susceptible to caspase inhibitors. Membrane order, measured by laurdan generalized polarization imaging, was neither reduced by α -hederin nor its aglycone hederagenin suggesting that their activity was not related to membrane cholesterol extraction. However, a radical change in morphology, including the disappearance of pseudopodes was observed upon incubation with α -hederin. Our results suggest that the different activities of α -hederin mainly depend on its interaction with membrane cholesterol and consequent pore formation., (Georg Thieme Verlag KG Stuttgart · New York.)

Biofilms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against S. aureus biofilms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with β-1-3-glucan synthase (caspofungin's pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biofilms, and increases fluoroquinolone penetration inside biofilms. Our study identifies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biofilm-related infections., Competing Interests: F.V.B. and P.M.T. have received research grants from Melinta Therapeutics for projects unrelated to the present work. The remaining authors declare no competing financial interests.

Aminoglycosides (AGs) constitute a major family of potent and broad-spectrum antibiotics disturbing protein synthesis through binding to the A site of 16S rRNA. Decades of widespread clinical use of AGs strongly reduced their clinical efficacy through the selection of resistant bacteria. Recently, conjugation of lipophilic groups to AGs generated a novel class of potent antibacterial amphiphilic aminoglycosides (AAGs) with significant improved activities against various sensitive and resistant bacterial strains. We have identified amphiphilic 3',6-dialkyl derivatives of the small aminoglycoside neamine as broad spectrum antibacterial agents targeting bacterial membranes. Here, we report on the synthesis and the activity against sensitive and resistant Gram-negative and/or Gram-positive bacteria of new amphiphilic 3',4'-dialkyl neamine derivatives and of their smaller analogues in the 6-aminoglucosamine (neosamine) series prepared from N-acetylglucosamine.

Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines., (Copyright © 2016 Elsevier Inc. All rights reserved.)

With the rise of multidrug resistance, Pseudomonas aeruginosa infections require alternative therapeutics. The injectisome (iT3SS) and flagellar (fT3SS) type III secretion systems are 2 virulence factors associated with poor clinical outcomes. iT3SS translocates toxins, rod, needle, or regulator proteins, and flagellin into the host cell cytoplasm and causes cytotoxicity and NLRC4-dependent inflammasome activation, which induces interleukin 1β (IL-1β) release and reduces interleukin 17 (IL-17) production and bacterial clearance. fT3SS ensures bacterial motility, attachment to the host cells, and triggers inflammation. INP1855 is an iT3SS inhibitor identified by in vitro screening, using Yersinia pseudotuberculosis Using a mouse model of P. aeruginosa pulmonary infection, we show that INP1855 improves survival after infection with an iT3SS-positive strain, reduces bacterial pathogenicity and dissemination and IL-1β secretion, and increases IL-17 secretion. INP1855 also modified the cytokine balance in mice infected with an iT3SS-negative, fT3SS-positive strain. In vitro, INP1855 impaired iT3SS and fT3SS functionality, as evidenced by a reduction in secretory activity and flagellar motility and an increase in adenosine triphosphate levels. As a result, INP1855 decreased cytotoxicity mediated by toxins and by inflammasome activation induced by both laboratory strains and clinical isolates. We conclude that INP1855 acts by dual inhibition of iT3SS and fT3SS and represents a promising therapeutic approach., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

The injectisome type three secretion system (T3SS) is a major virulence factor in Pseudomonas aeruginosa. This bacterium is responsible for severe infections in immunosuppressed or cystic fibrosis patients and has become resistant to many antibiotics. Inhibitors of T3SS may therefore constitute an innovative therapeutic target. After a brief description of the T3SS and its regulation, this review presents strategies to inhibit T3SS-mediated toxicity and describes the main families of existing inhibitors. Over the past few years, 12 classes of small-molecule inhibitors and two types of antibody have been discovered and evaluated in vitro for their capacity to inhibit T3SS expression or function, and to protect host cells from T3SS-mediated cytotoxicity. While only one small molecule has been tested in vivo, a bifunctional antibody targeting both the translocation apparatus of the T3SS and a surface polysaccharide is currently in Phase II clinical trials., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Bacterial membranes are highly organized, containing specific microdomains that facilitate distinct protein and lipid assemblies. Evidence suggests that cardiolipin molecules segregate into such microdomains, probably conferring a negative curvature to the inner plasma membrane during membrane fission upon cell division. 3',6-Dinonyl neamine is an amphiphilic aminoglycoside derivative active against Pseudomonas aeruginosa, including strains resistant to colistin. The mechanisms involved at the molecular level were identified using lipid models (large unilamellar vesicles, giant unilamelllar vesicles, and lipid monolayers) that mimic the inner membrane of P. aeruginosa The study demonstrated the interaction of 3',6-dinonyl neamine with cardiolipin and phosphatidylglycerol, two negatively charged lipids from inner bacterial membranes. This interaction induced membrane permeabilization and depolarization. Lateral segregation of cardiolipin and membrane hemifusion would be critical for explaining the effects induced on lipid membranes by amphiphilic aminoglycoside antibiotics. The findings contribute to an improved understanding of how amphiphilic aminoglycoside antibiotics that bind to negatively charged lipids like cardiolipin could be promising antibacterial compounds., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Type III secretion system (T3SS) in Pseudomonas aeruginosa is associated with poor clinical outcome in acute infections. T3SS allows for injection of bacterial exotoxins (e.g. ExoU or ExoS) into the host cell, causing cytotoxicity. It also activates the cytosolic NLRC4 inflammasome, activating caspase-1, inducing cytotoxicity and release of mature IL-1β, which impairs bacterial clearance. In addition, flagellum-mediated motility has been suggested to also modulate inflammasome response and IL-1β release. Yet the capacity of clinical isolates to induce IL-1β release and its relation with cytotoxicity have never been investigated. Using 20 clinical isolates from acute infections with variable T3SS expression levels and human monocytes, our aim was to correlate IL-1β release with toxin expression, flagellar motility and cytotoxicity. ExoU-producing isolates caused massive cell death but minimal release of IL-1β, while those expressing T3SS but not ExoU (i.e. expressing ExoS or no toxins) induced caspase-1 activation and IL-1β release, the level of which was correlated with cytotoxicity. Both effects were prevented by a specific caspase-1 inhibitor. Flagellar motility was not correlated with cytotoxicity or IL-1β release. No apoptosis was detected. Thus, T3SS cytotoxicity is accompanied by a modification in cytokine balance for P. aeruginosa clinical isolates that do not express ExoU., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Saponins, amphiphiles of natural origin with numerous biological activities, are widely used in the cosmetic and pharmaceutical industry. Some saponins exhibit relatively selective cytotoxic effects on cancer cells but the tendency of saponins to induce hemolysis limits their anticancer potential. This review focused on the effects of saponin activity on membranes and consequent implications for red blood and cancer cells. This activity seems to be strongly related to the amphiphilic character of saponins that gives them the ability to self-aggregate and interact with membrane components such as cholesterol and phospholipids. Membrane interactions of saponins with artificial membrane models, red blood and cancer cells are reviewed with respect to their molecular structures. The review considered the mechanisms of these membrane interactions and their consequences including the modulation of membrane dynamics, interaction with membrane rafts, and membrane lysis. We summarized current knowledge concerning the mechanisms involved in the interactions of saponins with membrane lipids and examined the structure activity relationship of saponins regarding hemolysis and cancer cell death. A critical analysis of these findings speculates on their potential to further develop new anticancer compounds.

Biofilm-related infections remain a scourge. In an in vitro model of biofilms using Staphylococcus aureus reference strains, delafloxacin and daptomycin were found to be the most active among the antibiotics from 8 different pharmacological classes (J. Bauer, W. Siala, P. M. Tulkens, and F. Van Bambeke, Antimicrob. Agents Chemother. 57:2726-2737, 2013, doi:10.1128/AAC.00181-13). In this study, we compared delafloxacin to daptomycin and vancomycin using biofilms produced by 7 clinical strains (S. aureus epidemic clones CC5 and CC8) in order to rationalize the differences observed between the antibiotics and strains. The effects of the antibiotics on bacterial viability (resazurin reduction assay) and biomass (crystal violet staining) were measured and correlated with the proportion of polysaccharides in the matrix, the local microenvironmental pH (micro-pH), and the antibiotic penetration in the biofilm. At clinically meaningful concentrations, delafloxacin, daptomycin, and vancomycin caused a ≥25% reduction in viability against the biofilms formed by 5, 4, and 3 strains, respectively. The antibiotic penetration within the biofilms ranged from 0.6 to 52% for delafloxacin, 0.2 to 10% for daptomycin, and 0.2 to 1% for vancomycin; for delafloxacin, this was inversely related to the polysaccharide proportion in the matrix. Six biofilms were acidic, explaining the high potency of delafloxacin (lower MICs at acidic pH). Norspermidine and norspermine (disassembling the biofilm matrix) drastically increased delafloxacin potency and efficacy (50% reduction in viability for 6 biofilms at clinically meaningful concentrations) in direct correlation with its increased penetration within the biofilm, while they only modestly improved daptomycin efficacy (50% reduction in viability for 2 biofilms) and penetration, and they showed marginal effects with vancomycin. Delafloxacin potency and efficacy against biofilms are benefited by its penetration into the matrix and the local acidic micro-pH., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The development of novel antimicrobial agents is urgently required to curb the widespread emergence of multidrug-resistant bacteria like colistin-resistant Pseudomonas aeruginosa. We previously synthesized a series of amphiphilic neamine derivatives active against bacterial membranes, among which 3',6-di-O-[(2"-naphthyl)propyl]neamine (3',6-di2NP), 3',6-di-O-[(2"-naphthyl)butyl]neamine (3',6-di2NB), and 3',6-di-O-nonylneamine (3',6-diNn) showed high levels of activity and low levels of cytotoxicity (L. Zimmermann et al., J. Med. Chem. 56:7691-7705, 2013). We have now further characterized the activity of these derivatives against colistin-resistant P. aeruginosa and studied their mode of action; specifically, we characterized their ability to interact with lipopolysaccharide (LPS) and to alter the bacterial outer membrane (OM). The three amphiphilic neamine derivatives were active against clinical colistin-resistant strains (MICs, about 2 to 8 μg/ml), The most active one (3',6-diNn) was bactericidal at its MIC and inhibited biofilm formation at 2-fold its MIC. They cooperatively bound to LPSs, increasing the outer membrane permeability. Grafting long and linear alkyl chains (nonyl) optimized binding to LPS and outer membrane permeabilization. The effects of amphiphilic neamine derivatives on LPS micelles suggest changes in the cross-bridging of lipopolysaccharides and disordering in the hydrophobic core of the micelles. The molecular shape of the 3',6-dialkyl neamine derivatives induced by the nature of the grafted hydrophobic moieties (naphthylalkyl instead of alkyl) and the flexibility of the hydrophobic moiety are critical for their fluidifying effect and their ability to displace cations bridging LPS. Results from this work could be exploited for the development of new amphiphilic neamine derivatives active against colistin-resistant P. aeruginosa., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Purpose: To evaluate pharmacological properties (antibacterial activity; accumulation in phagocytic cells; activity against intracellular bacteria; susceptibility to fluoroquinolone efflux transporters) of ciprofloxacin derivatives modified at C-7 of the piperazine ring., Methods: N-acetyl- (1), N-benzoyl- (2), N-ethyl- (3), and N-benzyl- (4) ciprofloxacin were synthesized. MICs against Escherichia coli and Staphylococcus aureus were determined following CLSI guidelines. Cellular accumulation, subcellular distribution, and intracellular activity (towards S. aureus and Listeria monocytogenes) were determined in J774 mouse macrophages. Efflux in bacteria (NorA [S. aureus], Lde [L. monocytogenes]) and in macrophages (Mrp4) was assessed using the corresponding inhibitors reserpine and gemfibrozil, respectively., Results: All derivatives were active, though less than ciprofloxacin. 2 and 3 accumulated 2-3 fold more than ciprofloxacin in mouse macrophages but remained substrates for efflux by Mrp4. 4 was insensitive to NorA and Lde, accumulated approx 50-fold more than ciprofloxacin in macrophages, was barely affected by Mrp4, localized in the soluble fraction of cells, and was equipotent to ciprofloxacin against intracellular bacteria., Conclusions: Benzyl substitution at C7 markedly affects the pharmacological profile of ciprofloxacin with respect to recognition by efflux transporters and cellular accumulation. N-benzyl-ciprofloxacin may serve as basis for designing molecules with higher intrinsic activity while remaining poorly susceptible to efflux.

Saponins and triterpenic acids have been shown to be able to interact with lipid membranes and domains enriched with cholesterol (rafts). How saponins are able to modulate lipid phase separation in membranes and the role of the sugar chains for this activity is unknown. We demonstrate in a binary membrane model composed of DMPC/Chol (3:1 mol/mol) that the saponin α-hederin and its aglycone presenting no sugar chain, the triterpenic acid hederagenin, are able to induce the formation of lipid domains. We show on multilamellar vesicles (MLV), giant unilamellar vesicles (GUV), and supported planar bilayers (SPB) that the presence of sugar units on the sapogenin accelerates domain formation and increases the proportion of sterols within these domains. The domain shape is also influenced by the presence of sugars because α-hederin and hederagenin induce the formation of tubular and spherical domains, respectively. These highly curved structures should result from the induction of membrane curvature by both compounds. In addition to the formation of domains, α-hederin and hederagenin permeabilize GUV. The formation of membrane holes by α-hederin comes along with the accumulation of lipids into nonbilayer structures in SPB. This process might be responsible for the permeabilizing activity of both compounds. In LUV, permeabilization by α-hederin was sterol-dependent. The biological implications of our results and the mechanisms involved are discussed in relation to the activity of saponins and triterpenic acids on membrane rafts, cancer cells, and hemolysis.

Oritavancin, a lipoglycopeptide antibiotic in development, accumulates to high levels in the lysosomes of eukaryotic cells. We examined specific functions of macrophages (phagocytic capacity, lysosomal integrity, metabolic activity, and production of reactive oxygen species [ROS]) in correlation with the cellular accumulation of the drug, using J774 mouse macrophages and THP-1 human monocytes differentiated into macrophages using phorbol 12-myristate 13-acetate. Oritavancin did not affect Pseudomonas aeruginosa phagocytosis, lysosomal integrity, or metabolic activity in cells incubated for 3 h with extracellular concentrations ranging from 5 to 50 μg/ml. At extracellular concentrations of ≥25 μg/ml, oritavancin reduced latex bead phagocytosis by approximately 50% and doubled ROS production in J774 macrophages only. This may result from the fact that the cellular accumulation of oritavancin was 15 times higher in J774 cells than in activated THP-1 cells at 3 h. Human pharmacokinetic studies estimate that the concentration of oritavancin in alveolar macrophages could reach approximately 560 μg/ml after administration of a cumulative dose of 4 g, which is below the cellular concentration needed in the present study to impair latex bead phagocytosis (1,180 μg/ml) or to stimulate ROS production (15,000 μg/ml) by J774 cells. The data, therefore, suggest that, in spite of its substantial cellular accumulation, oritavancin is unlikely to markedly affect macrophage functions under the conditions of use investigated in current phase III trials (a single dose of 1,200 mg).

Aminoglycosides are antibiotic drugs that act through binding to rRNA. In the search for antimicrobial amphiphilic aminoglycosides targeting bacterial membranes, we report here on the discovery of three dialkyl derivatives of the small aminoglycoside neamine active against susceptible and resistant Gram-positive and Gram-negative bacteria. One of these derivatives (R = 2-naphthylpropyl), which has good activity against MRSA and VRSA, showed a low toxicity in eukaryotic cells at 10 μM. The synthesis of amphiphilic paromamine and neamine homologous derivatives pointed out the role of the 6'-amine function of the neamine core in the antibacterial effects. The optimal number of lipophilic substituents to be attached to the neamine core and the corresponding required lipophilicity determined here should permit a more selective targeting of bacterial membranes relative to eukaryotic membranes. This work revealed the existence of windows of lipophilicity necessary for obtaining strong antibacterial effects that should be of interest in the field of antibacterial amphiphilic aminoglycosides.

The lipopeptide surfactin exhibits promising antimicrobial activities which are hampered by haemolytic toxicity. Rational design of new surfactin molecules, based on a better understanding of membrane:surfactin interaction, is thus crucial. We here performed bioimaging of lateral membrane lipid heterogeneity in adherent living human red blood cells (RBCs), as a new relevant bioassay, and explored its potential to better understand membrane:surfactin interactions. RBCs show (sub)micrometric membrane domains upon insertion of BODIPY analogs of glucosylceramide (GlcCer), sphingomyelin (SM) and phosphatidylcholine (PC). These domains exhibit increasing sensitivity to cholesterol depletion by methyl-β-cyclodextrin. At concentrations well below critical micellar concentration, natural cyclic surfactin increased the formation of PC and SM, but not GlcCer, domains, suggesting preferential interaction with lipid assemblies with the highest vulnerability to methyl-β-cyclodextrin. Surfactin not only reversed disappearance of SM domains upon cholesterol depletion but further increased PC domain abundance over control RBCs, indicating that surfactin can substitute cholesterol to promote micrometric domains. Surfactin sensitized excimer formation from PC and SM domains, suggesting increased lipid recruitment and/or diffusion within domains. Comparison of surfactin congeners differing by geometry, charge and acyl chain length indicated a strong dependence on acyl chain length. Thus, bioimaging of micrometric lipid domains is a visual powerful tool, revealing that intrinsic lipid domain organization, cholesterol abundance and drug acyl chain length are key parameters for membrane:surfactin interaction. Implications for surfactin preferential location in domains or at their boundaries are discussed and may be useful for rational design of better surfactin molecules., (Copyright © 2013 Elsevier B.V. All rights reserved.)