Salmonella typhimurium enterotoxin epitopes shared among bacteria

Abstract Selection-induced mutations (SIMS) are mutations that occur as specific and direct responses to environmental challenges, and that occur more often when they are selectively advantageous than when they are selectively neutral. This review includes discussions of how the occurrence of SIMS is measured, acquisitive evolution by SIMS, some of the controversies surrounding SIMS and models that have been advanced to explain the specificity of SIMS, and the requirement of a functional recA gene product for SIMS to occur.     

Lipid imaging with time-of-flight secondary ion mass spectrometry (ToF-SIMS)

Fundamental advances in secondary ion mass spectrometry (SIMS) now allow for the examination and characterization of lipids directly from biological materials. The successful application of SIMS-based imaging in the investigation of lipids directly from tissue and cells are demonstrated. Common complications and technical pitfalls are discussed. In this review, we examine the use of cluster ion sources and cryogenically compatible sample handling for improved ion yields and to expand the application potential of SIMS. Methodological improvements, including pre-treating the sample to improve ion yields and protocol development for 3-dimensional analyses (i.e. molecular depth profiling), are also included in this discussion. New high performance SIMS instruments showcasing the most advanced instrumental developments, including tandem MS capabilities and continuous ion beam compatibility, are described and the future direction for SIMS in lipid imaging is evaluated.     

Biomarkers at the microscopic range: ToF-SIMS molecular imaging of Archaea-derived lipids in a microbial mat

Time‐of‐Flight Secondary Ion Mass Spectrometry (ToF‐SIMS) with a bismuth cluster primary ion source was used for analysing microbial lipid biomarkers in 10‐µm‐thick microscopic cryosections of methanotrophic microbial mats from the Black Sea. Without further sample preparation, archaeal isopranyl glycerol di‐ and tetraether core lipids, together with their intact diglycoside (gentiobiosyl‐) derivatives, were simultaneously identified by exact mass determination. Utilizing the imaging capability of ToF‐SIMS, the spatial distributions of these biomarkers were mapped at a lateral resolution of < 5 µm in 500 × 500 µm² areas on the mat sections. Using cluster projectiles in the burst alignment mode, it was possible to reach a lateral resolution of 1 µm on an area of 233 × 233 µm, thus approaching the typical size of microbial cells. The mappings showed different ‘provenances’ within the sections that are distinguished by individual lipid fingerprints, namely (A) the diethers archaeol and hydroxyarchaeol co‐occurring with glycerol dialkyl glycerol tetraethers (GDGT), (B) hydroxyarchaeol and dihydroxyarchaeol, and (C) GDGT and gentiobiosyl‐GDGT. Because ToF‐SIMS is a virtually nondestructive technique affecting only the outermost layers of the sample surface (typically 10–100 nm), it was possible to further examine the studied areas using conventional microscopy, and associate the individual lipid patterns with specific morphological traits. This showed that provenance (B) was frequently associated with irregular, methane‐derived CaCO₃ crystallites, whereas provenance (C) revealed a population of fluorescent, filamentous microorganisms showing the morphology of known methanotrophic ANME‐1 archaea. The direct coupling of imaging mass spectrometry with microscopic techniques reveals interesting perspectives for the in‐situ study of lipids in geobiology, microbial ecology, and organic geochemistry. After further developing protocols for handling different kinds of environmental samples, ToF‐SIMS could be used as a tool to attack many challenging problems in these fields, such as the attribution of biological source(s) to particular biomarkers in question, or the high‐resolution tracking of biogeochemical processes in modern and ancient natural environments.     

Blood group A-active glycosphingolipids analysis by the combination of TLC-immunostaining assay and TLC/SIMS mass spectrometry

Blood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromatographed by TLC and then blood group A-active glycolipids were detected by TLC-immunostaining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glycolipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glycolipids can be readily analyzed by TLC/SIMS.     

Correlated AFM and NanoSIMS imaging to probe cholesterol-induced changes in phase behavior and non-ideal mixing in ternary lipid membranes

Cholesterol is believed to be an important component in compositionally distinct lipid domains in the cellular plasma membrane, which are referred to as lipid rafts. Insight into how cholesterol influences the interactions that contribute to plasma membrane organization can be acquired from model lipid membranes. Here we characterize the lipid mixing and phase behavior exhibited by (15)N-dilaurolyphosphatidycholine ((15)N-DLPC)/deuterated distearoylphosphatiylcholine (D(70)-DSPC) membranes with various amounts of cholesterol (0, 3, 7, 15 or 19mol%) at room temperature. The microstructures and compositions of individual membrane domains were determined by imaging the same membrane locations with both atomic force microscopy (AFM) and high-resolution secondary ion mass spectrometry (SIMS) performed with a Cameca NanoSIMS 50. As the cholesterol composition increased from 0 to 19mol%, the circular ordered domains became more elongated, and the amount of (15)N-DLPC in the gel-phase domains remained constant at 6-7mol%. Individual and micron-sized clusters of nanoscopic domains enriched in D(70)-DSPC were abundant in the 19mol% cholesterol membrane. AFM imaging showed that these lipid domains had irregular borders, indicating that they were gel-phase domains, and not non-ideally mixed lipid clusters or nanoscopic liquid-ordered domains.     

Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy)

This paper reviews the most recent methodological advances in the field of biological imaging using dynamic secondary ion mass spectrometry (SIMS). After a short reminder of the basic principle of SIMS imaging, the latest high-resolution dynamic SIMS equipment is briefly described. This new ion nanoprobe (CAMECA NanoSIMS 50) has a lateral resolution of less than 50 nm with primary Cs+ ion, the ability to detect simultaneously 5 different ions from the same micro-volume and a very good transmission even at high mass resolution (60% at M/DeltaM=5000). Basic considerations related to sample preparation, mass resolution and primary ion implantation are given. The decisive capability of this new instrument, and more generally of high-resolution dynamic SIMS imaging in biology, are illustrated with the most recent examples of utilization.     

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90-231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90-231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in beta-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of alpha-helical structure. This conformation of PrP with a high content of alpha-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.     

Protein identification with Liquid Chromatography and Matrix Enhanced Secondary Ion Mass Spectrometry (LC-ME-SIMS)

Secondary Ion Mass Spectrometry (SIMS) is a well established method for sensitive surface atomic and molecular analysis. Protein analysis with conventional SIMS has been attempted numerous times; however it delivers exclusively fragment peaks assigned to α-amino acids or immonium ions. In this paper we report experiments where direct sequence information could be measured thanks to a combination of HPLC separation with matrix enhanced SIMS (ME-SIMS) on tryptic digests of intact proteins. We employ peptide mass fingerprinting (PMF) and protein identification through the detection of HPLC-separated digests of Savinase (Sav.) and bovine serum albumin (BSA), followed by MASCOT search. This is the first time that the possibility of full protein identification using LC-ME-SIMS is demonstrated in a classic proteomics workflow and that a 69kDa protein is identified with SIMS. These results demonstrate both the relevance and the potential of LC-ME-SIMS in future high resolution proteomics studies.     

Dietary Modifications of Phospholipid Composition and Biophysical Properties of the Brush Border Membrane Along the Trout Intestine

Brush border membranes (BBM) are isolated from middle and posterior intestine of trout fed either an essential fatty acid-rich diet or a saturated one. The different phospholipid classes are separated, and their fatty acid composition is determined. Fluorescence anisotropy studies are performed using two lipid fluorophores, namely diphenylhexatriene (DPH) and trimethyl-aminodiphenylhexatriene (TMA-DPH). The results indicate that the usual parameters affecting the lipid fluidity such as the phospholipid:protein (PL:PROT), cholesterol:phospholipid (CHOL:PL), and sphingomyelin:phosphatidylcholine (SP:PC) ratios and the unsaturation of the acyl chains are sufficient to explain the fluidity values determined using DPH, but not those obtained with TMA-DPH as a probe. This fluorophore is assessed to be localized only in the external leaflet of the membrane. Hence, it will be affected by the composition of the major phospholipids of this leaflet, sphingomyelin and phosphatidylcholine.     

Insertion kinetics of a denatured alpha helical membrane protein into phospholipid bilayer vesicles

Membrane protein folding has suffered from a lack of detailed kinetic studies, particularly with regard to the insertion of denatured protein into lipid bilayers. We present a detailed in vitro kinetic study of the association of a denatured, transmembrane alpha helical protein with lipid vesicles. The mechanism of folding of Escherichia coli diacylglycerol kinase from a partially denatured state in urea has been investigated. The protein associates with lipid vesicles to give a protein, vesicle complex with an apparent association constant of 2 x 10(6) M(-1) s(-1). This association rate approaches the diffusion limit of the protein, vesicle reaction. The association of the protein with lipid vesicles is followed by a slower process occurring at observed rate of 0.031 s(-1), involving insertion into the bilayer and generation of a functional oligomer of diacylglycerol kinase. Protein aggregation competes with vesicle insertion. The urea-denatured protein monomers begin to aggregate as soon as the urea is diluted. This aggregation is faster than the association of the protein with vesicles so that most protein aggregates before it inserts into a vesicle. Increasing the vesicle concentration favours insertion of protein monomers, but at high vesicle concentrations monomers are primarily in separate vesicles and do not associate to form functional oligomers. Irreversible aggregation limits the yield of functional protein, while the data also suggest that lipid vesicles can reverse another aggregation reaction, leading to the recovery of correctly folded protein.     

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.     

Curvature-based sorting of eight lipid types in asymmetric buckled plasma membrane models

Curvature is a fundamental property of biological membranes and has essential roles in cellular function. Bending of membranes can be induced by their lipid and protein compositions, as well as peripheral proteins, such as those that make up the cytoskeleton. An important aspect of membrane function is the grouping of lipid species into microdomains, or rafts, which serve as platforms for specific biochemical processes. The fluid mosaic model of membranes has evolved to recognize the importance of curvature and leaflet asymmetry, and there are efforts toward evaluating their functional roles. This work investigates the effect of curvature on the sorting of lipids in buckled asymmetric bilayers containing eight lipid types, approximating an average mammalian plasma membrane, through coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field. The simulations reveal that 1) leaflet compositional asymmetry can induce curvature asymmetry, 2) lipids are sorted by curvature to different extents, and 3) curvature-based partitioning trends show moderate to strong correlations with lipid molecular volumes and head to tail bead ratios, respectively. The findings provide unique insights into the role of curvature in membrane organization, and the curvature-based sorting trends should be useful references for later investigations and potentially interpreting the functional roles of specific lipids.     

Detection of fatty acids from intact microorganisms by molecular beam static secondary ion mass spectrometry

We report the use of a surface analysis approach, static secondary ion mass spectrometry (SIMS) equipped with a molecular (ReO(4)(-)) ion primary beam, to analyze the surface of intact microbial cells. SIMS spectra of 28 microorganisms were compared to fatty acid profiles determined by gas chromatographic analysis of transesterfied fatty acids extracted from the same organisms. The results indicate that surface bombardment using the molecular primary beam cleaved the ester linkage characteristic of bacteria at the glycerophosphate backbone of the phospholipid components of the cell membrane. This cleavage enables direct detection of the fatty acid conjugate base of intact microorganisms by static SIMS. The limit of detection for this approach is approximately 10(7) bacterial cells/cm(2). Multivariate statistical methods were applied in a graded approach to the SIMS microbial data. The results showed that the full data set could initially be statistically grouped based upon major differences in biochemical composition of the cell wall. The gram-positive bacteria were further statistically analyzed, followed by final analysis of a specific bacterial genus that was successfully grouped by species. Additionally, the use of SIMS to detect microbes on mineral surfaces is demonstrated by an analysis of Shewanella oneidensis on crushed hematite. The results of this study provide evidence for the potential of static SIMS to rapidly detect bacterial species based on ion fragments originating from cell membrane lipids directly from sample surfaces.     

Analysis of archaeal core ether lipids using Time of Flight–Secondary Ion Mass Spectrometry (ToF-SIMS): Exploring a new prospect for the study of biomarkers in geobiology

The capability of Time of Flight–Secondary Ion Mass Spectrometry (ToF‐SIMS) of analysing molecular archaeal biomarkers in geobiological samples was tested and demonstrated. Using a bismuth cluster primary ion source, isopranyl glycerol di‐ and tetraether core lipids were detected in small amounts of total organic extracts from methanotrophic microbial mats, simultaneously and without further chemical treatment and chromatographic separation. ToF‐SIMS was also employed to track the distribution of fossilized ether lipids in a massive carbonate (aragonite) microbialite that precipitated as a result of the microbial anaerobic oxidation of methane. An unambiguous signal was obtained when analysing a freshly broken rock surface (base of a microdrill core). Though some limitation occurred due to µm‐topographical effects (sample roughness), it was possible to display the abundance of high molecular weight (C₈₆) of tetraethers exposed in particular regions of the rock surface. ‘Molecular mapping’ revealed that a part of these molecules was encased within the rock fabric in a cluster‐like distribution that might trace the arrangement of the calcifying microbial colonies in the once active mat system. The results reveal promising perspectives of ToF‐SIMS for (i) the quasi‐nondestructive analysis of lipids in extremely small geobiological samples at low concentrations; (ii) resolving the spatial distribution of these compounds on a µm²‐ to cm²‐scale; and (iii) the more exact assignment of lipid biomarkers to their biological source.     

Tetrahymena Expresses More than a Hundred Proteins with Lipid‐binding MORN Motifs that can Differ in their Subcellular Localisations

Proteins with membrane occupation and recognition nexus (MORN) motifs are associated with cell fission in apicomplexan parasites, chloroplast division in Arabidopsis and the motility of sperm cells. We found that ciliates are among those that encode the largest variety of MORN proteins. Tetrahymena thermophila expresses 129 MORN protein‐encoding genes, some of which are specifically up‐regulated during conjugation. A lipid‐binding assay underpins the assumption that the predominant function of MORN motifs themselves is to confer the ability of lipid binding. The localisation of four MORN candidate proteins with similar characteristics highlights the functional diversity of this group especially in ciliates.     

High resolution imaging by organic secondary ion mass spectrometry

Secondary-ion mass spectrometry (SIMS) is based on the acceleration of high-energy primary ions onto a target. Secondary electrons, neutrals and ions are emitted from the target, reflecting its chemical composition. This enables simultaneous analysis and localization of target molecules, giving valuable information that is difficult or impossible to obtain with other analytical methods. The secondary ions can be extracted and detected by any type of mass analyzer. SIMS is unique in its ability to detect several target molecules simultaneously in small samples and to image their localization at subcellular resolution. The recent development of bioimaging SIMS opens up new possibilities in biotechnology and biological research with applications in biomedicine and pathology. The current development of this technique has the potential to become as important for biotechnology as the advent of the electron microscope, confocal microscope or in situ hybridization.     

The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition

The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane alpha-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl-l-alpha-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane alpha-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol(-1). The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces.     

Actin-related protein (Arp2) inserts into artificial lipid membranes

Arp2 is localized in the cytoplasm of eukaryotic cells where it controls actin dynamics. Computer analyses have suggested one possible lipid binding site, residues 185-202 of the primary amino acid sequence on Arp2, that could allow for membrane attachment/insertion. We expressed this region as a fusion protein with schistosomal glutathione S-transferase (GST) and investigated the interaction of this fragment with mixtures of dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylglycerol (DOPG) phospholipids in reconstituted lipid bilayers using differential scanning calorimetry (DSC). Calorimetric measurements showed that as the fusion protein increased, the main chain transition enthalpy decreased and the chain-melting temperature shifted, which is indicative of partial protein insertion into the hydrophobic region of the lipid membrane. This was confirmed using the Langmuir Blodgett technique (film balance) on lipid monolayers. The dissociation constant (K(d)) determined by the temperature jump method was approximately 1.1 microM.     

固体支撑的自组装的双层类脂膜

具有通常BLM_s某些相似特性的固体支撑的双层类脂膜(S-BLM)能够通过两步自组装到新劈开的金属丝上面。如:(1)包有聚四氟乙烯的铂丝头部浸在类脂溶液里,用解剖刀把顶部切开;(2)包有类脂溶液的新铂丝末端转移到0.1mol/L KCl溶液里,电测定证实,数分钟后,在金属丝末端自动地形成了稳定的类脂双层。本文报道了这种固体支撑的BLM(S-BLM)在检测Pb²⁺离子中的应用。S-BLM为液晶结构,它可用于基础研究、生物传感器和分子电子器件等技术上的应用。     

The influenza fusion peptide promotes lipid polar head intrusion through hydrogen bonding with phosphates and N‐terminal membrane insertion depth

Influenza infection requires fusion between the virus envelope and a host cell endosomal membrane. The influenza hemagglutinin fusion peptide (FP) is essential to viral membrane fusion. It was recently proposed that FPs would fuse membranes by increasing lipid tail protrusion, a membrane fusion transition state. The details of how FPs induce lipid tail protrusion, however, remain to be elucidated. To decipher the molecular mechanism by which FPs promote lipid tail protrusion, we performed molecular dynamics simulations of the wild‐type (WT) FP, fusogenic mutant F9A, and nonfusogenic mutant W14A in model bilayers. This article presents the peptide–lipid interaction responsible for lipid tail protrusion and a related lipid perturbation, polar head intrusion, where polar heads are sunk under the membrane surface. The backbone amides from the four N‐terminal peptide residues, deeply inserted in the membrane, promoted both perturbations through H bonding with lipid phosphates. Polar head intrusion correlated with peptides N‐terminal insertion depth and activity: the N‐termini of WT and F9A were inserted deeper into the membrane than nonfusogenic W14A. Based on these results, we propose that FP‐induced polar head intrusion would complement lipid tail protrusion in catalyzing membrane fusion by reducing repulsions between juxtaposed membranes headgroups. The presented model provides a framework for further research on membrane fusion and influenza antivirals. Proteins 2014; 82:2118–2127. © 2014 Wiley Periodicals, Inc.