Jordi H. Borrell

Molecular study of quinolone resistance mechanisms and clonal relationship of Salmonella enterica clinical isolates

In the last few years, the number of Salmonella enterica strains resistant to nalidixic acid has steadily increased. In a previous study, the quinolone susceptibility phenotype and genotype of 38 S. enterica clinical isolates (19 S. enterica serovar Typhimurium and 19 S. enterica serovar Enteritidis) were determined. Forty-two percent of the isolates showed nalidixic acid resistance associated with a mutation in gyrA together with putative overexpression of efflux pump(s). In this study, mutations in the quinolone resistance-determining region (QRDR) of parE and the regulators of AcrAB (acrR, marRAB, soxRS and ramR) were analysed. Intracellular accumulation of ciprofloxacin and nalidixic acid was determined. Gene expression of the efflux pump components acrB, tolC, acrF and emrB was also assessed. In addition, an epidemiological study of the isolates by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) was performed. No mutations were detected in parE, whereas two amino acid substitutions were found in two susceptible strains in MarR (I84L) and AcrR (N214T) in one strain each, although both were suggested to be polymorphisms. No changes in the gene expression of acrB, tolC, acrF and emrB were detected between nalidixic-acid-resistant and -susceptible strains. Intracellular accumulation was not useful to reveal differences. Epidemiological analysis showed an important clonal relatedness among the S. Enteritidis isolates, whereas major divergence was seen for S. Typhimurium. Altogether, these results suggest the presence of previously undiscovered drug efflux pump(s) and confirm the high clonality of S. Enteritidis and the genetic divergence of S. Typhimurium.

Enhanced topical delivery of hyaluronic acid encapsulated in liposomes: A surface-dependent phenomenon

In the present study, we investigated the release and permeation of hyaluronic acid (HA) encapsulated in liposomes when deposited onto two surfaces: cellulose, a model widely used for investigating transport of drugs; and human skin, a natural biointerface used for transdermal drug delivery. We prepared and characterised liposomes loaded with HA and liposomes incorporating two penetration enhancers (PEs): the non-ionic surfactant Tween 80, and Transcutol P, a solubilising agent able to mix with polar and non-polar solvents. In vitro and ex vivo permeation assays showed that PEs indeed enhance HA-release from liposomes. Since one of the possible mechanisms postulated for the action of liposomes on skin is related to its adsorption onto the stratum corneum (SC), we used atomic force microscopy (AFM) topography and force volume (FV) analysis to investigate the structures formed after deposition of liposome formulations onto the investigated surfaces. We explored the possible relationship between the formation of planar lipid structures on the surfaces and the permeation of HA.

Unspecific membrane protein–lipid recognition: combination of AFM imaging, force spectroscopy, DSC and FRET measurements†

In this work, we will describe in quantitative terms the unspecific recognition between lactose permease (LacY) of Escherichia coli, a polytopic model membrane protein, and one of the main components of the inner membrane of this bacterium. Supported lipid bilayers of 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE) and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoglycerol (POPG) (3:1, mol/mol) in the presence of Ca2+ display lateral phase segregation that can be distinguished by atomic force microscopy (AFM) as well as force spectroscopy. LacY shows preference for fluid (Lα) phases when it is reconstituted in POPE : POPG (3:1, mol/mol) proteoliposomes at a lipid‐to‐protein ratio of 40. When the lipid‐to‐protein ratio is decreased down to 0.5, two domains can be distinguished by AFM. While the upper domain is formed by self‐segregated units of LacY, the lower domain is constituted only by phospholipids in gel (Lβ) phase. On the one hand, classical differential scanning calorimetry (DSC) measurements evidenced the segregation of a population of phospholipids and point to the existence of a boundary region at the lipid–protein interface. On the other hand, Förster Resonance Energy Transfer (FRET) measurements in solution evidenced that POPE is selectively recognized by LacY. A binary pseudophase diagram of POPE : POPG built from AFM observations enables to calculate the composition of the fluid phase where LacY is inserted. These results are consistent with a model where POPE constitutes the main component of the lipid–LacY interface segregated from the fluid bulk phase where POPG predominates. Copyright

Membrane Protein Lipid Interactions: Physics and Chemistry in the Bilayer

This book has been conceives as a brief introduction to biomembranes physical chemistry for undergraduate students of sciences, and it is particularly dedicated to the lipid-protein membrane interactions. A general introduction is presented in Chapters 1 and 2. The following Chapters, 3 and 4, describe the most accepted theories on lipid-membrane protein interactions as well as the new experimental approaches, in particular, these arose from nano sciences as atomic for microscopy and single molecule force spectroscopy. The book emphasizes the relevance of physical parameters as the lateral surface pressure and the lipid curvature as actors for understanding the physicochemical properties of the biomembranes

Lateral Distribution of Membrane Components and Transient Lipid-Protein Structures

The individual physicochemical properties of membrane phospholipids, acyl chain composition and headgroup charge, are major determinants of lipid phase separation into domains. In this chapter we will review the general procedure to reconstitute integral membrane proteins (IMPs) into supported lipid bilayers (SLBs). Second, a brief introduction to lipid phase diagrams is presented. Then we will discuss the wide variety of lipid-protein structures reported in the literature. Protein affinity for lipids is discussed on the basis of temporal and spatial residence of lipids at the lipid-protein boundary, leading to definitions of boundary, associated non-boundary-lipid, and the remainder of lipids that are distributed in the bulk of the bilayer. We present some examples to illustrate the hydrophobic matching between lipids and IMPs, and based on physical properties of the lipid, stretching and bending, we introduce the surface flexible model (SFM) of the membrane which is consistent with the continuum theory of matter applied to membranes. This model accounts for the experimentally observed physicochemical behaviours to interpret the insertion of IMPs into specific lipid domains of the membrane.

Mapping phase diagrams of supported lipid bilayers by atomic force microscopy.

In this work, we present the method followed to construct a pseudophase diagram of two phospholipids: 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol). Two different techniques, DSC and AFM, have been used based in the determination of the onset (Tonset) and completion (Toffset) temperatures of the gel‐to‐liquid crystalline phases (Lβ→Lα), the first from the endotherms from liposomes and the second from the topographic images of supported lipid bilayers. The features of both phase diagrams are discussed emphasizing the influence of Ca2+ presence and the substrate (mica) on the transition undergone by the phospholipid mixture. Microsc. Res. Tech. 80:4–10, 2017.

Critical Temperature of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine Monolayers and Its Possible Biological Relevance

Because transmembrane proteins (TMPs) can be obtained with sufficient purity for X-ray diffraction studies more frequently than decades ago, their mechanisms of action may now be elucidated. One of the pending issues is the actual interplay between transmembrane proteins and membrane lipids. There is strong evidence of the involvement of specific lipids with some membrane proteins, such as the potassium crystallographically sited activation channel (KcsA) of Streptomyces lividans and the secondary transporter of lactose LacY of Escherichia coli, the activities of which are associated with the presence of anionic phospholipids such as the phosphatidylglycerol (PG) and phosphatidyethanolamine (PE), respectively. Other proteins such as the large conductance mechanosensitive channel (MscL) of E. coli seem to depend on the adaptation of specific phospholipids to the irregular surface of the integral membrane protein. In this work we investigated the lateral compressibility of two homoacid phosphatidylethanolamines (one with both acyl chains unsaturated (DOPE), the other with the acyl chains saturated (DPPE)) and the heteroacid phosphatidyletanolamine (POPE) and their mixtures with POPG. The liquid expanded (LE) to liquid condensed (LC) transition was observed in POPE at a temperature below its critical temperature (Tc = 36 °C). Because Tc lies below the physiological temperature, the occurrence of this phase transition may have something to do with the functioning of LacY. This magnitude is discussed within the context of the experiments carried out at temperatures below the Tc of POPE at which the activity of Lac Y and other TMPs are frequently studied.

Physicochemical Properties of Lipids and Macromolecules in Higher Level Organization

This chapter relates in a very concise way, how the physicochemical properties of membrane lipids determine the formation of self-segregated structures. The most common model methods used to understand the influence of lipid organization in membranes, lipid monolayers, liposomes and supported lipid bilayers, are reviewed as well for their suitability in the investigation of lipid-membrane protein interactions.

Molecular Membrane Biochemistry

In this chapter, a description of the present view of the structure of the cell membrane is presented. This includes a basic introduction to the chemistry and physics of lipids and proteins with special attention to properties that are relevant to understanding lipid-protein interactions within the membrane. It covers protein folding in membranes by describing the interaction forces involved in the process and by focusing on known cases where lipids are involved. By revisiting the fluid mosaic model for membranes the chapter finishes with a visual description of membrane structure at the nanoscale level.

Dependence of Protein Membrane Mechanisms on Specific Physicochemical Lipid Properties

In Chap. 3 we have shown some examples of how lipid-protein interactions lead to laterally segregated structures in membranes, and how the activity of proteins is related to physical properties of the phospholipids. In this chapter we will first discuss the relationship between membrane structure and bioenergetics, emphasizing that lipids may be part of the machinery involved in proton transport between protein components of the respiratory chain. Second we will present selected examples that relate membrane protein activity with specific phospholipids and we will discuss how this can be rationalized theoretically by introducing the concept of a lateral pressure profile of the membrane. Since the magnitude of lateral pressure within the membrane cannot be experimentally measured, we will show how using atomic force microscopy in force mode and single-molecule force spectroscopy, we can extract nanomechanical properties of the membranes related to protein packing. These properties, in particular the unfolding force or the force required to extract a membrane protein from a bilayer, are related to both the lateral pressure of pure lipid monolayers and the intrinsic surface curvature of monolayers. Finally, we will discuss the application of FRET to identify the phospholipid species present at the lipid-protein interface.