Interactions of Brominated Flame Retardants with Membrane Models of Dehalogenating Bacteria: Langmuir Monolayer and Grazing Incidence X-ray Diffraction Studies.

Brominated flame retardants (BFRs) are small organic molecules containing several bromine substituents added to plastics to limit their flammability. BFRs can constitute up to 30% of the weight of some plastics, which is why they are produced in large quantities. Along with plastic waste and microplastic particles, BFRs end up in the soil and can easily leach causing contamination. As polyhalogenated molecules, multiple BFRs were classified as persistent organic pollutants (POPs), meaning that their biodegradation in the soils is especially challenging. However, some anaerobic bacteria as Dehaloccocoides can dehalogenate BFRs, which is important in the bioremediation of contaminated soils. BFRs are hydrophobic, can accumulate in plasma membranes, and disturb their function. On the other hand, limited membrane accumulation is necessary for BFR dehalogenation. To study the BFR-membrane interaction, we created membrane models of soil dehalogenating bacteria and tested their interactions with seven legacy and novel BFRs most common in soils. Phospholipid Langmuir monolayers with appropriate composition were used as membrane models. These membranes were doped in the selected BFRs, and the incorporation of BFR molecules into the phospholipid matrix and also the effects of BFR presence on membrane physical properties and morphology were studied. It turned out that the seven BFRs differed significantly in their membrane affinity. For some, the incorporation was very limited, and others incorporated effectively and could affect membrane properties, while one of the tested molecules induced the formation of bilayer domains in the membranes. Thus, Langmuir monolayers can be effectively used for pretesting BFR membrane activity.

Statin Action Targets Lipid Rafts of Cell Membranes: GIXD/PM-IRRAS Investigation of Langmuir Monolayers.

Lipid rafts are condensed regions of cell membranes rich in cholesterol and sphingomyelin, which constitute the target for anticholesterolemic drugs - statins. In this work, we use for the first time a combined grazing-incidence X-ray diffraction (GIXD)/polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS)/Brewster angle microscopy (BAM) approach to show the statin effect on model lipid rafts and its components assembled in Langmuir monolayers at the air-water interface. Two representatives of these drugs, fluvastatin (FLU) and cerivastatin (CER), of different hydrophobicity were chosen, while cholesterol (Chol) and sphingomyelin (SM), and their 1:1 mixture were selected to form condensed monolayers of lipid rafts. The effect of statins on the single components of lipid rafts indicated that both the hydrophobicity of the drugs and the organization of the layer determined the drug-lipid interaction. For cholesterol monolayers, only the most hydrophobic CER was effectively changing the film structure, while for the less organized sphingomyelin, the biggest effect was observed for FLU. This drug affected both the polar headgroup region as shown by PM-IRRAS results and the 2D crystalline structure of the SM monolayer as evidenced by GIXD. Measurements performed for Chol/SM 1:1 models proved also that the statin effect depends on the presence of Chol-SM complexes. In this case, the less hydrophobic FLU was not able to penetrate the binary layer at all, while exposure to the hydrophobic CER resulted in the phase separation and formation of ordered assemblies. The changes in the membrane properties were visualized by BAM images and GIXD patterns and confirmed by thermodynamic parameters of hysteresis in the Langmuir monolayer compression-decompression experiments.

Persistent organic pollutants in model fungal membranes. Effects on the activity of phospholipases.

Soils are the final sink for multiple organic pollutants emitted to the environment. Some of these chemicals which are toxic, recalcitrant and can bioaccumulate in living organism and biomagnify in trophic chains are classified persistent organic pollutants (POP). Vast areas of arable land have been polluted by POPs and the only economically possible means of decontamination is bioremediation, that is the utilization of POP-degrading microbes. Especially useful can be non-ligninolytic fungi, as their fast-growing mycelia can reach POP molecules strongly bond to soil minerals or humus fraction inaccessible to bacteria. The mobilized POP molecules are incorporated into the fungal plasma membrane where their degradation begins. The presence of POP molecules in the membranes can change their physical properties and trigger toxic effects to the cell. To avoid these phenomena fungi can quickly remodel the phospholipid composition of their membrane with employing different phospholipases and acyltransferases. However, if the presence of POP downregulates the phospholipases, toxic effects and the final death of microbial cells are highly probable. In our studies we applied multicomponent Langmuir monolayers with their composition mimicking fungal plasma membranes and studied their interactions with two different microbial phospholipases: phospholipase C (α-toxin) and phospholipase A1 (Lecitase ultra). The model membranes were doped with selected POPs that are frequently found in contaminated soils. It turned out that most of the employed POPs do not downregulate considerably the activity of phospholipases, which is a good prognostics for the application of non-ligninolytic fungi in bioremediation.

Membrane composition and successful bioaugmentation. Studies of the interactions of model thylakoid and plasma cyanobacterial and bacterial membranes with fungal membrane-lytic enzyme Lecitase ultra.

Cyanobacterial/bacterial consortia are frequently inoculated to soils to increase the soil fertility and to accelerate the biodegradation of organic pollutants. Moreover, such consortia can also be successfully applied in landfills especially for the biodegradation of plastic wastes. However, the bioaugmentation techniques turn out frequently inefficient due to the competition of the indigenous microorganisms attacking directly these inoculated or secreting to their surroundings cell wall and membrane-lytic enzymes. It can be hypothesized that the resistance of the microbial membrane to the enzymatic degradation is correlated with its lipid composition. To verify this hypothesis glycolipid and phospholipid Langmuir monolayers were applied as models of thylakoid and plasma cyanobacterial and bacterial membranes. Hybrid fungal enzyme Lecitase ultra joining the activity of lipase and phospholipase A1 was applied as the model of fungal membrane-lytic enzyme. It turned out that anionic thylakoid lipids sulfoquinovosyldiacylglycerol and phosphatidylglycerols were the main targets of Lecitase ultra in the model multicomponent thylakoid membranes. The resistance of the model plasma bacterial membranes to enzymatic degradation depended significantly to their composition. The resistance increased generally when the unsaturated lipids were exchanged to their saturated counterparts. However, most resistant turned out the membranes composed of unsaturated phosphatidylamine and saturated anionic phospholipids.

Interactions of fungal phospholipase Lecitase ultra with phospholipid Langmuir monolayers - Search for substrate specificity and structural factors affecting the activity of the enzyme.

Inoculation of selected microbial species into the soils is one of the most effective means of bioremediation of soils polluted by persistent organic pollutants as well as of biocontrol of plant pests. However, this procedure turns out frequently to be ineffective due to the membrane-destructive enzymes secreted to the soil by the autochthonous microorganisms. Especial role play here phospholipases and among them phospholipase A1 (PLA1), Therefore, to explain the interactions of microbial membranes and PLA1 at molecular level and to find the correlation between the composition of the membrane and its resistance to PLA1 action we applied phospholipid Langmuir monolayers as model microbial membranes. As a representative soil extracellular PLA1 we applied Lecitase ultra which is a commercially available hybrid enzyme of PLA1 activity. With the application of specific sn1-ether-sn2-ester phospholipids we proved that Lecitase ultra has solely PLA1 activity; thus, can be applied as an effective model of soil PLA1s. Our studies proved that this enzyme has vast substrate specificity and can hydrolyze structural phospholipids regardless the structure of their polar headgroup. It turned out that the hydrolysis rate was controlled by the condensation of the model membranes. These built of the phospholipids with long saturated fatty acid chains were especially resistant to the action of this enzyme, whereas these formed by the 1-saturated-2-unsaturated-sn-glycero-3-phospholipids were readily degraded. Regarding the polar headgroup we proposed the following row of substrate preference of Lecitase ultra: phosphatidylglycerols > phosphatidylcholines > phosphatidylethanolamines > cardiolipins.

Incorporation of simvastatin into lipid membranes: Why deliver a statin in form of inclusion complex with hydrophilic cyclodextrin.

In this work, the effects of simvastatin (SIM), (2-hydroxypropyl)-ß-cyclodextrin (HPßCD) and their complex (SIM:HPßCD) on the structure and properties of lipid membranes were investigated for the first time by Langmuir technique combined with PM-IRRAS spectroscopy. An improved understanding of the differences of the interactions between free SIM, and SIM in the form of an inclusion complex with HPßCD with the lipid membrane will improve the development of preparation methods for in vivo applications. Monolayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol (Chol) and their mixture DMPC:Chol (7:3) served as simple models of one leaflet of the cell membrane. The penetration of well-organized lipid layers by simvastatin lead to their fluidization but the extent of this unwanted effect was smaller when the drug was delivered in the form of the SIM:HPßCD complex. Surface pressure vs. time dependencies showed that the drug encapsulated with cyclodextrin dissociated from the complex upon contact with the lipid layer and the weak interactions between the exterior polar part of the HPßCD and the polar headgroups of the lipid layer facilitated smooth incorporation of the released lipophilic drug into the membrane. At a longer time-scale, the HPßCD ligand released from the complex removed some cholesterol, but not DMPC, from the lipid layer, hence, similarly to the enzyme inhibiting action of statins - it lead to the decrease of the amount of cholesterol in the membrane. Delivery of simvastatin in the form of an inclusion complex with HPßCD is proposed as an approach improving its bioavailability in the cholesterol-lowering therapies.

Simultaneous action of microbial phospholipase C and lipase on model bacterial membranes - Modeling the processes crucial for bioaugmentation.

Bioaugmentation is a promising method of the remediation of soils polluted by persistent organic pollutants (POP). Unfortunately, it happens frequently that the microorganisms inoculated into the soil die out due to the presence of enzymes secreted by autochthonous microorganisms. Especially destructive are here phospholipases C (PLC) and lipases which destruct the microorganism's cellular membrane. The composition of bacterial membranes differs between species, so it is highly possible that depending on the membrane constitution some bacteria are more resistant to PLCs and lipases than other. To shed light on these problems we applied phospholipid Langmuir monolayers as model microbial membranes and studied their interactions with α-toxin (model bacterial PLC) and the lipase isolated from soil fungus Candida rugosa. Membrane phospholipids differing in their headgroup (phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols and cardiolipins) and in their tail structure were applied. The monolayers were characterized by the Langmuir technique, visualized by Brewster angle microscopy, and the packing mode of the phospholipid molecules was verified by the application of the diffraction of synchrotron radiation. We also studied the mutual miscibility of diacylglycerols and the native phospholipids as their interaction is crucial for the understanding of the PLC and lipase activity. It turned out that all the investigated phospholipid classes can be hydrolyzed by PLC; however, they differ profoundly in the hydrolysis degree. Depending on the effects of the initial PLC action and the mutual organization of the diacylglycerol and phospholipid molecules the lipase can ruin the model membranes or can be completely neutral to them.

The composition of phospholipid model bacterial membranes determines their endurance to secretory phospholipase A2 attack - The role of cardiolipin.

Soil bacteria are decomposer organisms crucial for the biodegradation of organic pollutants, mineralization of dead organic matter and the turnover of biogenic elements. In their environment they are constantly exposed to membrane-lytic enzymes emitted to the soil by other microorganisms competing for the same niche. Therefore, the composition and structure of their membranes is of utmost importance for survival in the harsh environment. Although soil bacteria species can be Gram-negative or Gram-positive and their membranes differ significantly, they are formed by phospholipids belonging mainly to three classes: phosphatidylethanolamines (PE), phosphatidylglycerols (PG) and cardiolipins (CL). The correlation of the membrane phospholipid composition and its susceptibility to secretory membrane-lytic enzymes is widely unknown; thus, to shed light on these phenomena we applied the Langmuir monolayer technique to construct models of soil bacteria membranes differing in the mutual proportion of the main phospholipids. To characterize the systems we studied their elasticity, mesoscopic texture, 2D crystalline structure and discussed the thermodynamics of the interactions between their components. The model membranes were exposed to secretory phospholipase A2. It turned out that in spite of the structural similarities the model membranes differed significantly in their susceptibility to s-PLA2 attack. The membranes devoid of cardiolipin were completely degraded, whereas, these containing cardiolipin were much more resistant to the enzymatic hydrolysis. It also turned out that the sole presence of cardiolipin in the model membrane did not guarantee the membrane durability and that the interplay between cardiolipin and the zwitterionic phosphatidylethanolamine was here of crucial importance.

The role of phospholipid composition and ergosterol presence in the adaptation of fungal membranes to harsh environmental conditions-membrane modeling study.

Soil fungi play an important role in the environment decomposing dead organic matter and degrading persistent organic pollutants (POP). The presence of hydrophobic POP in the soil and membrane-lytic substances excreted by competing microorganism to the soil solution is the constant threat to these organisms. To survive in the harsh environment and counteract these hazards the fungal cells have to strictly control the composition of the lipids in their cellular membranes. However, in the case of fungal membranes the correlation between their composition and physical properties is not fully understood. In our studies we applied Langmuir monolayers formed by phospholipids typical to fungal membranes and ergosterol as versatile model membranes. These membranes were characterized by the Langmuir technique, Brewster Angle Microscopy and Grazing Incidence X-ray Diffraction, as well as were exposed to the action of phospholipase A2 treated as a model membrane-lytic protein. We started our studies from the equimolar mixture of phosphatidylethanolamine with phosphatidylcholine and doped this matrix with phosphatidylserine (PS) or phosphatidylinositol (PI). It turned out that the membranes with PS were much more condensed at the mesoscale and periodically organized at the molecular level. Starting from these models we derived two families of model fungal membranes adding to these phospholipid matrices ergosterol. It turned out that the level of ergosterol content is of crucial importance for the model membrane structure and its durability. Changing the ergosterol mole ratio from 0 to 0.5 we defined and described in detail four different 2D crystalline phases.

The effect of chlorination degree and substitution pattern on the interactions of polychlorinated biphenyls with model bacterial membranes.

Polychlorinated biphenyls (PCB) are persistent organic pollutants that due to their chemical resistivity and inflammability found multiple applications. In spite of the global ban for PCB production, due to their long half-lives periods, PCB accumulate in the soils, so effective bioremediation of the polluted lands is of crucial importance. Some of the 209 PCB congeners exhibit increased toxicity to soil bacteria and their presence impoverish the soil decomposer community and slows down the degradation of environmental pollutants in the soils. The exact mechanism of PCB antimicrobial activity is unknown, but it is strictly related with the membrane activity of PCB. Therefore, to shed light on these interactions we applied Langmuir monolayers formed by selected phospholipids as model bacterial membranes. In our studies we tested 5 PCB congeners differing in the degree of chlorination and the distribution of the chlorine substituents around the biphenyl frame. Special attention was paid to tetra-substituted PCB because of their increased presence in the environment and disubstituted PCB being their degradation products. To characterize the model membranes as Langmuir monolayers, we used surface pressure measurements, Brewster angle microscopy and Grazing Incidence X-ray Diffraction. It turned out that among the tetra-substituted PCB the ortho-substituted non-dioxin like compound was much more membrane destructive than the flat dioxin-like congener. On the contrary, among the di-substituted PCB the flat para-substituted 2,2'-dichlorobiphenyl turned out to exhibit high membrane activity.

The influence of terpinen-4-ol and eucalyptol - The essential oil components - on fungi and plant sterol monolayers.

Antifungal and herbicidal activity of terpenes, being the components of the essential oils, is directly related to the incorporation of these compounds into cellular membranes. Thus, the differences in the lipid composition of various pathogenic membranes may be the factor determining the activity of these molecules. One of the class of lipids, which form the membrane environment are sterols. The aim of this work was to compare the effect of two terpenes: terpinen-4-ol and eucalyptol on the monolayers formed by ergosterol and ß - sitosterol, which are the components of fungi and plant membranes, respectively. The modifications in the sterol monolayer properties were investigated in the surface pressure-area measurements and penetration studies as well as in a micrometer scale (Brewster angle microscopy experiments) and in nanoscale (GIXD technique). It was evidenced that although at higher surface pressure the terpene molecules are in part removed from the interface, they are able to substantially modify the condensation, morphology and molecular organization of the sterol film. It was also found that the incorporation of terpenes into sterol films is comparable for both sterols, however, ß - sitosterol monolayers properties are affected more strongly than ergosterol films. Finally, the analysis of the results of the studies performed on model membrane systems and the results of antimicrobial studies reported in literature, enabled us to suggest that the activity of terpenes depends on the membrane composition and that the sterol concentration may be important from the point of view of antifungal effect of terpinen-4-ol and eucalyptol.

Effects of Polychlorinated Pesticides and Their Metabolites on Phospholipid Organization in Model Microbial Membranes.

Polychlorinated pesticides (PPs) were classified as persistent organic pollutants because of their toxicity, limited degradability in the environment, bioaugmentation, and accumulation in animal tissues. PPs accumulate in the environment mainly in the soils and water sediments where they are toxic to the decomposer organisms including soil bacteria and fungi. Therefore, there is an urgent need to search for the microorganisms capable of PP biodegradation which could be applied for soil bioremediation. The exact mechanism of PP microbial toxicity is unknown; however, there is evidence that it can be membrane related. To shed light on the interactions of PPs with microbial membranes, we applied Langmuir monolayers formed by phospholipids as model biomembranes. The model membranes were formed by phospholipids typical to microbial membranes: cardiolipins and phosphatidylglycerols the main components of Gram positive bacteria membranes, phosphatidylcholine typical to fungal membranes, as well as phosphatidylethanolamine found in the inner membranes of Gram negative bacteria. For the studies, the most ecotoxic PPs and their water-soluble metabolites were chosen. The monolayers were studied with the application of mutually complementary techniques: Langmuir technique, grazing incidence X-ray diffraction, and PM-IRRAS spectroscopy. It turned out that the cyclodiene PPs are more membrane active than monocyclic PPs and that the possibility of their incorporation is strictly related to the phospholipid structure. The membranes prepared with cardiolipin turned out to be especially resistant to PP incorporation. Regarding the metabolites, pentachlorophenol turned out to be especially structure breaking, affecting the molecular organization of all of the investigated phospholipids.

Effects of water soluble perfluorinated pollutants on phospholipids in model soil decomposer membranes.

Water soluble perfluorinated compounds (PFCs) as perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA) and their shorter chain homologues are persistent organic pollutants widely distributed in the environment. PFCs accumulate in soils and sediments and because of their toxicity endanger the decomposer organisms. PFCs are toxic to a wide spectrum of soil bacteria and their biocide activity was related with their membrane activity; however, the exact mechanism of PFCs - bacterial membrane interactions is unknown. Therefore, to shed light on these questions we applied phospholipid Langmuir monolayers as simplified models of bacterial membranes and studied their interactions with selected environmentally relevant PFCs. The mechanical properties of the monolayers were characterized by surface pressure-mean molecular area isotherms and the analysis of compression modulus. The effects of PFC on the texture of the model membranes were studied with Brewster angle microscopy, whereas their influence on molecular packing in the 2D crystal lattice was searched by the Grazing Incidence X-ray diffraction technique. The effects of PFCs on the phospholipid polar heargroup conformation were studied by PM-IRRAS spectroscopy, whereas the effectivenes of the incorporation of PFCs into the model membrane was monitored in penetration tests. It turned out that the membranes rich in phosphatidylethanolamine typical to Gram negative bacteria are much PFCs susceptible than the cardiolipin rich membranes imitating Gram positive species. Moreover, the studies indicated that the switch from eight­carbon atom perfluorinated chains to shorter chain homologues is not necessarily environmentally benign as perfluorobutane sulfonate caused also significant structural changes in the model membranes.

Interactions of Long-Chain Perfluorotelomer Alcohol and Perfluorinated Hydrocarbons with Model Decomposer Membranes.

Perfluorinated hydrocarbons and their polar derivatives are produced annually in high quantities and find multiple industrial and technological applications due to their chemical and physical durability, significant hydro- and lipophobicity and excellent surface activity. Unfortunately, multiple perfluorinated compounds are recognized as persistent organic pollutants as they are completely nonbiodegradable and accumulate in soils and sediments. In our studies, we applied Langmuir monolayers formed by different structural phospholipids as models of soil bacteria and fungi membranes and investigated the effects exerted by long-chain perfluorinated pollutants, perfluorotelomer alcohol and two structurally different perfluorinated hydrocarbons, on the artificial membranes. Various mutually complemental methods such as surface pressure-mean molecular area isotherm registration, Brewster angle microscopy (BAM), and grazing incidence X-ray diffraction (GIXD) were applied. It turned out that the presence of the perfluorinated chemicals profoundly affected the phospholipid monolayers. The miscibility of the phospholipid with the perfluorotelomer alcohol depended strongly on the size and charge of the polar headgroup. Additionally, it was observed by BAM that the presence of the perfluorinated molecules significantly changed the texture of all the investigated phospholipid monolayers. On the basis of the BAM and GIXD results and other studies described in the scientific literature, we postulated that the perfluorinated hydrocarbons form an additional monolayer anchored on top of the phospholipid film. Our studies prove that both polar and nonpolar perfluorinated pollutants can be toxic to decomposer organisms and that their toxicity is strictly correlated with the phospholipid composition of the cellular membrane.

Influence of Parabens on Bacteria and Fungi Cellular Membranes: Studies in Model Two-Dimensional Lipid Systems.

Langmuir monolayers were used to study the influence of four commercially applied parabens on multicomponent systems composed of lipid species characteristic of the cellular membrane of microorganisms found in carbohydrates and proteins reaching products, including food and cosmetics. The aim of the undertaken studies was to shed new light on the problem of parabens' interactions with membrane lipids and their affinity for monolayers differing with regard to the composition, mutual lipid ratios, and physicochemical properties. The discussion is based on the π-A isotherm characteristics, surface morphology observation performed with BAM, and analysis of the diffraction data collected for the periodically ordered lipid domains present in the investigated multicomponent films. Our studies revealed that the selected parabens are capable of surface film modification and that the magnitude of this effect increases with the number of methylene groups in the ester part of paraben molecules. We found that the strongest destructive effect was observed for model 1 (Staphylococcus aureus), a lower effect was observed for model 2 (Pseudomonas aeruginosa), and the lowest effect was observed for model 3 (Candida albicans). It was inferred that such a trend appears due to the composition of the artificial membranes, i.e., above all, in the presence or lack of sterol molecules and the content of negatively charged lipids.

Polycyclic aromatic hydrocarbons in model bacterial membranes - Langmuir monolayer studies.

High molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) are persistent organic pollutants which due to their limited biodegradability accumulate in soils where their increased presence can lead to the impoverishment of the decomposer organisms. As very hydrophobic PAHs easily penetrate cellular membranes of soil bacteria and can be incorporated therein, changing the membrane fluidity and other functions which in consequence can lead to the death of the organism. The structure and size of PAH molecule can be crucial for its membrane activity; however the correlation between PAH structure and its interaction with phospholipids have not been investigated so far. In our studies we applied phospholipid Langmuir monolayers as model bacterial membranes and investigated how the incorporation of six structurally different PAH molecules change the membrane texture and physical properties. In our studies we registered surface pressure and surface potential isotherms upon the monolayer compression, visualized the monolayer texture with the application of Brewster angle microscopy and searched the ordering of the film-forming molecules with molecular resolution with the application of grazing incidence X-ray diffraction (GIXD) method. It turned out that the phospholipid-PAH interactions are strictly structure dependent. Four and five-ring PAHs of the angular or cluster geometry can be incorporated into the model membranes changing profoundly their textures and fluidity; whereas linear or large cluster PAHs cannot be incorporated and separate from the lipid matrix. The observed phenomena were explained based on structural similarities of the applied PAHs with membrane steroids and hopanoids.

Studies on the Behavior of Eucalyptol and Terpinen-4-ol-Natural Food Additives and Ecological Pesticides-in Model Lipid Membranes.

Effective application of the essential oils requires detailed exploration of their mechanism of action and the origin of diverse activity of their components. In this work, the influence of eucalyptol and terpinen-4-ol on artificial membranes was studied to verify whether the differences in the activity of these compounds are related to their effect on membranes. The properties of monolayers formed from structurally different lipids in the presence of terpenes were examined based on the results of the surface pressure-area measurements, penetration studies, and Brewster angle microscopy experiments. Both compounds were able to incorporate into the membrane and alter lipid/lipid interactions, making the monolayer less stable and more fluid. These effects were determined by monolayer composition (but not by its condensation per se) and the resulting rheological properties and were stronger in the presence of terpinen-4-ol. These findings confirm the hypothesis that differences in the antimicrobial potency of these terpenes are membrane-related, and membrane composition may determine their selectivity.

Interactions of two structurally related anionic phospholipids cardiolipin and phosphatidylglycerol with phospholipase A2. Langmuir monolayer studies.

Anionic phospholipids cardiolipins (CL) and phosphatidylglycerols (PG) dominate in the biomembranes of the majority of soil bacteria. CL to PG ratio differs between the species and is also dependent on the external conditions. CL/PG ratio is different in polluted than in unspoiled soils and it was hypothesized that it is connected with the activity of the membranelytic enzymes from the phospholipase A2 class (PLA2) as it was proved that persistent soil pollutants can activate PLA2. In our studies we applied the Langmuir monolayer technique and Brewster angle microscopy to elucidate the mechanism of the interactions of PLA2 with the model membranes formed by anionic phospholipids. It turned out that there are significant differences between CL and PG. The monolayer of PG is hydrolyzed readily and entirely, whereas for CL approximately 30% of the phospholipid molecules are hydrolyzed after which the enzyme is inhibited. The observed differences between PG and CL are strictly connected with the hydrophobicity of the generated lysolipids: lyso-PG and lyso-CL. Lyso-PG is water soluble and leaves the interface whereas lyso-CL is water-insoluble remains at the interface and modifies the monolayer properties. The second hydrolysis product - myristic acid (MA) forms crystallites of calcium myristate when generated from PG, whereas when generated from CL it is shielded by the lysolipid and does not interact with calcium. Therefore, on the basis of our study it can be concluded that the increase in CL content protects the soil bacteria from PLA2 activity and from the loss of calcium homeostasis.

Sterol-Phospholipid Hybrids at the Air/Water Interface: Studies on Properties and Interactions with Parent Lipid Molecules.

The two synthetic sterol-phospholipid hybrids DCholPC and PCholPC were investigated in monolayers at the air/water interface. Study was based on π-A isotherm analysis complemented with application of grazing incidence X-ray diffraction. It was found that both compounds are capable of forming stable, highly condensed monolayers of a surface characteristics typical for sterols. GIXD studies show that the crystallographic area for DCholPC monolayer is very similar to that found for cholesterol (37.1 vs 38.0 Å(2)), while for PCholPC (28.8 Å(2)) it is significantly smaller as compared to area for the mixed Chol/DPPC 2/1 monolayer (33.4 Å(2)). In our study the problem of interactions between investigated sterol-phospholipid hybrids and natural membrane lipid components was for the first time analyzed in planar lipid systems. Studies on mixed monolayers showed that both hybrids, similarly to cholesterol, reveal a condensing effect toward DPPC acyl chains; however, DCholPC having two steroid moieties in the molecule was found to be more efficient. On the other hand, the sterol moiety and the hydrocarbon chain of PCholPC molecule are packed in the 2D crystalline phase extremely tight. Our studies showed that the investigated compounds can be applied as biocompatible components of stable liposomes.

Studies on the interactions between parabens and lipid membrane components in monolayers at the air/aqueous solution interface.

The interactions between parabens (PBs) and lipid components of mammalian and bacterial cell membranes were investigated in model systems of Langmuir monolayers. Me-, Et-, Pr- and Bu-paraben studied in this paper are frequently applied as cosmetics and food preservatives, since they possess broad antimicrobial activity. The mode of PB action is connected with their incorporation into the membrane of bacterial organisms, however; it is not known what is the role of the respective lipid species in this mechanism. This problem is crucial to understand the differences in paraben activity toward individual microorganisms and to shed the light onto the problem of PB cytotoxicity reported in studies on mammalian cells. In this paper, the mentioned aspects were investigated with application of the Langmuir monolayer technique complemented with BAM and GIXD. Our experiments revealed that the influence of PBs depends on their chemical structure, solution concentration and on the class of lipid. The strongest modification of the monolayer characteristics, leading to its collapse at low surface pressure, occurred in the presence of BuPB, having the largest chain. PBs interact preferentially with the monolayers possessing low degree of condensation, whereas for LC state, the effect was weaker and observed only as modification of the 2D unit cells. In the model systems, PBs interact with phospholipids characteristic for mammalian membranes (phosphatidylcholine) stronger than with bacterial (phosphatidylglycerol and cardiolipin). This strong influence of parabens on the model systems composed of animal lipids may explain cytotoxic activity of these preservatives.