Molecular-level architecture of Chlamydomonas reinhardtii's glycoprotein-rich cell wall.

Microalgae are a renewable and promising biomass for large-scale biofuel, food and nutrient production. However, their efficient exploitation depends on our knowledge of the cell wall composition and organization as it can limit access to high-value molecules. Here we provide an atomic-level model of the non-crystalline and water-insoluble glycoprotein-rich cell wall of Chlamydomonas reinhardtii. Using in situ solid-state and sensitivity-enhanced nuclear magnetic resonance, we reveal unprecedented details on the protein and carbohydrate composition and their nanoscale heterogeneity, as well as the presence of spatially segregated protein- and glycan-rich regions with different dynamics and hydration levels. We show that mannose-rich lower-molecular-weight proteins likely contribute to the cell wall cohesion by binding to high-molecular weight protein components, and that water provides plasticity to the cell-wall architecture. The structural insight exemplifies strategies used by nature to form cell walls devoid of cellulose or other glycan polymers.

19F solid-state NMR approaches to probe antimicrobial peptide interactions with membranes in whole cells.

To address the global problem of bacterial antibiotic resistance, antimicrobial peptides (AMPs) are considered promising therapeutic candidates due to their broad-spectrum and membrane-lytic activity. As preferential interactions with bacteria are crucial, it is equally important to investigate and understand their impact on eukaryotic cells. In this study, we employed 19F solid-state nuclear magnetic resonance (ssNMR) as a novel approach to examine the interaction of AMPs with whole red blood cells (RBCs). We used RBC ghosts (devoid of hemoglobin) and developed a protocol to label their lipid membranes with palmitic acid (PA) monofluorinated at carbon positions 4, 8, or 14 on the acyl chain, allowing us to probe different locations in model and intact RBC ghost membranes. Our work revealed that changes in the 19F chemical shift anisotropy, monitored through a CF bond order parameter (SCF), can provide insights into lipid bilayer dynamics. This information was also obtained using magic-angle spinning 19F ssNMR spectra with and without 1H decoupling, by studying alterations in the second spectral moment (M2) as well as the 19F isotropic chemical shift, linewidth, T1, and T2 relaxation times. The appearance of an additional isotropic peak with a smaller chemical shift anisotropy, a narrower linewidth, and a shorter T1, induced by the AMP caerin 1.1, supports the presence of high-curvature regions in RBCs indicative of pore formation, analogous to its antimicrobial mechanism. In summary, the straightforward incorporation of monofluorinated FAs and rapid signal acquisition offer promising avenues for the study of whole cells using 19F ssNMR.

Metabolomics and lipidomics reveal the effects of the toxic dinoflagellate Alexandrium catenella on immune cells of the blue mussel, Mytilus edulis.

The increasing occurrence of harmful algal blooms, mostly of the dinoflagellate Alexandrium catenella in Canada, profoundly disrupts mussel aquaculture. These filter-feeding shellfish feed on A. catenella and accumulate paralytic shellfish toxins, such as saxitoxin, in tissues, making them unsafe for human consumption. Algal toxins also have detrimental effects upon several physiological functions in mussels, but particularly on the activity of hemocytes - the mussel immune cells. The objective of this work was to determine the effects of experimental exposure to A. catenella upon hemocyte metabolism and activity in the blue mussel, Mytilus edulis. To do so, mussels were exposed to cultures of the toxic dinoflagellate A. catenella for 120 h. The resulting mussel saxitoxin load had measurable effects upon survival of hemocytes and induced a stress response measured as increased ROS production. The neutral lipid fraction of mussel hemocytes decreased two-fold, suggesting a differential use of lipids. Metabolomic 1H nuclear magnetic resonance (NMR) analysis showed that A. catenella modified the energy metabolism of hemocytes as well as hemocyte osmolyte composition. The modified energy metabolism was reenforced by contrasting plasma metabolomes between control and exposed mussels, suggesting that the blue mussel may reduce feed assimilation when exposed to A. catenella.

Fluorescence Resonance Energy Transfer to Detect Plasma Membrane Perturbations in Giant Plasma Membrane Vesicles.

Disruptions and perturbations of the cellular plasma membrane by peptides have garnered significant interest in the elucidation of biological phenomena. Typically, these complex processes are studied using liposomes as model membranes-either by encapsulating a fluorescent dye or by other spectroscopic approaches, such as nuclear magnetic resonance. Despite incorporating physiologically relevant lipids, no synthetic model truly recapitulates the full complexity and molecular diversity of the plasma membrane. Here, biologically representative membrane models, giant plasma membrane vesicles (GPMVs), are prepared from eukaryotic cells by inducing a budding event with a chemical stressor. The GPMVs are then isolated, and bilayers are labelled with fluorescent lipophilic tracers and incubated in a microplate with a membrane-active peptide. As the membranes become damaged and/or aggregate, the resulting fluorescence resonance energy transfer (FRET) between the two tracers increases and is measured periodically in a microplate. This approach offers a particularly useful way to detect perturbations when the membrane complexity is an important variable to consider. Additionally, it provides a way to kinetically detect damage to the plasma membrane, which can be correlated with the kinetics of peptide self-assembly or structural rearrangements. Key features • Allows testing of various peptide-membrane interaction conditions (peptide:phospholipid ratio, ionic strength, buffer, etc.) at once. • Uses intact plasma membrane vesicles that can be prepared from a variety of cell lines. • Can offer comparable throughput as with traditional synthetic lipid models (e.g., dye-encapsulated liposomes).

Peculiar Phosphonate Modifications of Velvet Worm Slime Revealed by Advanced Nuclear Magnetic Resonance and Mass Spectrometry.

Nature is rich with examples of highly specialized biological materials produced by organisms for functions, including defense, hunting, and protection. Along these lines, velvet worms (Onychophora) expel a protein-based slime used for hunting and defense that upon shearing and dehydration forms fibers as stiff as thermoplastics. These fibers can dissolve back into their precursor proteins in water, after which they can be drawn into new fibers, providing biological inspiration to design recyclable materials. Elevated phosphorus content in velvet worm slime was previously observed and putatively ascribed to protein phosphorylation. Here, we show instead that phosphorus is primarily present as phosphonate moieties in the slime of distantly related velvet worm species. Using high-resolution nuclear magnetic resonance (NMR), natural abundance dynamic nuclear polarization (DNP), and mass spectrometry (MS), we demonstrate that 2-aminoethyl phosphonate (2-AEP) is associated with glycans linked to large slime proteins, while transcriptomic analyses confirm the expression of 2-AEP synthesizing enzymes in slime glands. The evolutionary conservation of this rare protein modification suggests an essential functional role of phosphonates in velvet worm slime and should stimulate further study of the function of this unusual chemical modification in nature.

Development of a novel fluorescence assay for studying lipid bilayer perturbation induced by amyloidogenic peptides using cell plasma membrane vesicles.

Numerous pathophysiological conditions are associated with the misfolding and aggregation of proteins into insoluble amyloid fibrils. The mechanisms by which this process leads to cellular dysfunction remain elusive, though several hypotheses point toward the perturbation of the cell plasma membrane by pre-fibrillar intermediates and/or amyloid growth. However, current models to study membrane perturbations are largely limited to synthetic lipid vesicles and most of experimental approaches cannot be transposed to complex cell-derived plasma membrane systems. Herein, vesicles originating from the plasma membrane of erythrocytes and ß-pancreatic cells were used to study the perturbations induced by an amyloidogenic peptide, the islet amyloid polypeptide (IAPP). These biologically relevant lipid vesicles displayed a characteristic clustering in the presence of the amyloidogenic peptide, which was able to rupture membranes. By exploiting Förster resonance energy transfer (FRET), a rapid, simple, and potentially high-throughput assay to detect membrane perturbations of intact mammalian cell plasma membrane vesicles was implemented. The FRET kinetics of membrane perturbations closely correlated with the kinetics of thioflavin-T fluorescence associated with amyloid formation. This novel kinetics assay expands the toolbox available to study amyloid-associated membrane damage, bridging the gap between synthetic lipid vesicles and living cells.

In situ solid-state NMR study of antimicrobial peptide interactions with erythrocyte membranes.

Antimicrobial peptides are promising therapeutic agents to mitigate the global rise of antibiotic resistance. They generally act by perturbing the bacterial cell membrane and are thus less likely to induce resistance. Because they are membrane-active molecules, it is critical to verify and understand their potential action toward eukaryotic cells to help design effective and safe drugs. In this work, we studied the interaction of two antimicrobial peptides, aurein 1.2 and caerin 1.1, with red blood cell (RBC) membranes using in situ 31P and 2H solid-state NMR (SS-NMR). We established a protocol to integrate up to 25% of deuterated fatty acids in the membranes of ghosts, which are obtained when hemoglobin is removed from RBCs. Fatty acid incorporation and the integrity of the lipid bilayer were confirmed by SS-NMR and fluorescence confocal microscopy. Leakage assays were performed to assess the lytic power of the antimicrobial peptides. The in situ perturbation of the ghost membranes by aurein 1.2 and caerin 1.1 revealed by 31P and 2H SS-NMR is consistent with membrane perturbation through a carpet mechanism for aurein 1.2, whereas caerin 1.1 acts on RBCs via pore formation. These results are compatible with fluorescence microscopy images of the ghosts. The peptides interact with eukaryotic membranes following similar mechanisms that take place in bacteria, highlighting the importance of hydrophobicity when determining such interactions. Our work bridges model membranes and in vitro studies and provides an analytical toolbox to assess drug toxicity toward eukaryotic cells.

Solid-State NMR Investigations of Extracellular Matrixes and Cell Walls of Algae, Bacteria, Fungi, and Plants.

Extracellular matrixes (ECMs), such as the cell walls and biofilms, are important for supporting cell integrity and function and regulating intercellular communication. These biomaterials are also of significant interest to the production of biofuels and the development of antimicrobial treatment. Solid-state nuclear magnetic resonance (ssNMR) and magic-angle spinning-dynamic nuclear polarization (MAS-DNP) are uniquely powerful for understanding the conformational structure, dynamical characteristics, and supramolecular assemblies of carbohydrates and other biomolecules in ECMs. This review highlights the recent high-resolution investigations of intact ECMs and native cells in many organisms spanning across plants, bacteria, fungi, and algae. We spotlight the structural principles identified in ECMs, discuss the current technical limitation and underexplored biochemical topics, and point out the promising opportunities enabled by the recent advances of the rapidly evolving ssNMR technology.

Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications.

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution 31P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase.

Identification and Quantification of Glycans in Whole Cells: Architecture of Microalgal Polysaccharides Described by Solid-State Nuclear Magnetic Resonance.

Microalgae are photosynthetic organisms widely distributed in nature and serve as a sustainable source of bioproducts. Their carbohydrate components are also promising candidates for bioenergy production and bioremediation, but the structural characterization of these heterogeneous polymers in cells remains a formidable problem. Here we present a widely applicable protocol for identifying and quantifying the glycan content using magic-angle-spinning (MAS) solid-state NMR (ssNMR) spectroscopy, with validation from glycosyl linkage and composition analysis deduced from mass-spectrometry (MS). Two-dimensional 13C-13C correlation ssNMR spectra of a uniformly 13C-labeled green microalga Parachlorella beijerinckii reveal that starch is the most abundant polysaccharide in a naturally cellulose-deficient strain, and this polymer adopts a well-organized and highly rigid structure in the cell. Some xyloses are present in both the mobile and rigid domains of the cell wall, with their chemical shifts partially aligned with the flat-ribbon 2-fold xylan identified in plants. Surprisingly, most other carbohydrates are largely mobile, regardless of their distribution in glycolipids or cell walls. These structural insights correlate with the high digestibility of this cellulose-deficient strain, and the in-cell ssNMR methods will facilitate the investigations of other economically important algae species.

Identification of transmissible proteotoxic oligomer-like fibrils that expand conformational diversity of amyloid assemblies.

Protein misfolding and amyloid deposition are associated with numerous diseases. The detailed characterization of the proteospecies mediating cell death remains elusive owing to the (supra)structural polymorphism and transient nature of the assemblies populating the amyloid pathway. Here we describe the identification of toxic amyloid fibrils with oligomer-like characteristics, which were assembled from an islet amyloid polypeptide (IAPP) derivative containing an Asn-to-Gln substitution (N21Q). While N21Q filaments share structural properties with cytocompatible fibrils, including the 4.7 Å inter-strand distance and ß-sheet-rich conformation, they concurrently display characteristics of oligomers, such as low thioflavin-T binding, high surface hydrophobicity and recognition by the A11 antibody, leading to high potency to disrupt membranes and cause cellular dysfunction. The toxic oligomer-like conformation of N21Q fibrils, which is preserved upon elongation, is transmissible to naïve IAPP. These stable fibrils expanding the conformational diversity of amyloid assemblies represent an opportunity to elucidate the structural basis of amyloid disorders.

Interactions between the Cell Membrane Repair Protein S100A10 and Phospholipid Monolayers and Bilayers.

Protein S100A10 participates in different cellular mechanisms and has different functions, especially at the membrane. Among those, it forms a ternary complex with annexin A2 and the C-terminal of AHNAK and then joins the dysferlin membrane repair complex. Together, they act as a platform enabling membrane repair. Both AHNAK and annexin A2 have been shown to have membrane binding properties. However, the membrane binding abilities of S100A10 are not clear. In this paper, we aimed to study the membrane binding of S100A10 in order to better understand its role in the cell membrane repair process. S100A10 was overexpressed by E. coli and purified by affinity chromatography. Using a Langmuir monolayer as a model membrane, the binding parameters and ellipsometric angles of the purified S100A10 were measured using surface tensiometry and ellipsometry, respectively. Phosphorus-31 solid-state nuclear magnetic resonance spectroscopy was also used to study the interaction of S100A10 with lipid bilayers. In the presence of a lipid monolayer, S100A10 preferentially interacts with unsaturated phospholipids. In addition, its behavior in the presence of a bilayer model suggests that S100A10 interacts more with the negatively charged polar head groups than the zwitterionic ones. This work offers new insights on the binding of S100A10 to different phospholipids and advances our understanding of the parameters influencing its membrane behavior.

Investigating the action of the microalgal pigment marennine on Vibrio splendidus by in vivo2H and 31P solid-state NMR.

This work investigates the potential probiotic effect of marennine - a natural pigment produced by the diatom Haslea ostrearia - on Vibrio splendidus. These marine bacteria are often considered a threat for aquaculture; therefore, chemical antibiotics can be required to reduce bacterial outbreaks. In vivo2H solid-state NMR was used to probe the effects of marennine on the bacterial membrane in the exponential and stationary phases. Comparisons were made with polymyxin B (PxB) - an antibiotic used in aquaculture and known to interact with Gram(-) bacteria membranes. We also investigated the effect of marennine using 31P solid-state NMR on model membranes. Our results show that marennine has little effect on phospholipid headgroups dynamics, but reduces the acyl chain fluidity. Our data suggest that the two antimicrobial agents perturb V. splendidus membranes through different mechanisms. While PxB would alter the bacterial outer and inner membranes, marennine would act through a membrane stiffening mechanism, without affecting the bilayer integrity. Our study proposes this microalgal pigment, which is harmless for humans, as a potential treatment against vibriosis.

Investigating Byssogenesis with Proteomic Analysis of Byssus, Foot, and Mantle in Mytilus Mussels by LC-MS/MS.

Mussel byssus represents a fascinating class of biological materials with a unique capacity to adhere onto virtually any solid surface. Proteins expressed in byssus, the byssal-producing organ (foot) as well as mantle tissue from Mytilus edulis or Mytilus californianus are analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The mantle is used as a control tissue to pinpoint unique proteins from the foot samples potentially involved in byssogenesis. This work represents an important step towards identifying biologically important proteins expressed in foot, as well as extending knowledge on the byssus proteome. Considering the minimal proteomics data of the studied species, this data also contributes to a more complete description of M. edulis and M. californianus proteomes.

Extraction Improvement of the Bioactive Blue-Green Pigment "Marennine" from Diatom Haslea ostrearia's Blue Water: A Solid-Phase Method Based on Graphitic Matrices.

The compound "marennine" is a blue-green pigment produced by the benthic microalgae Haslea ostrearia, with pathogenicity reduction activities against some bacteria and promising potential as a natural pigment in seafood industries. After decades of research, the chemical family of this compound still remains unclear, mainly because structural studies were impaired by the presence of co-extracted compounds in marennine isolates. To improve the purity of marennine extract, we developed a novel extraction method using a graphitic stationary phase, which provides various advantages over the previous procedure using tandem ultrafiltration. Our method is faster, more versatile, provides a better crude yield (66%, compared to 57% for ultrafiltration) and is amenable to upscaling with continuous photobioreactor cultivation. Our goal was to take advantage of the modulable surface properties of the graphitic matrix by optimizing its interactions with marennine. As such, the effects of organic modifiers, pH and reducing agents were studied. With this improvement on marennine purification, we achieved altogether the isolation of a fucoidan-related, sulfated polysaccharide from blue water. Characterization of the polysaccharides fraction suggests that roughly half of UV-absorbing compounds could be isolated from the marennine crude extracts. The identification of sulfated polysaccharides could be a major breakthrough for marennine purification, providing targeted isolation techniques. Likewise, the added value of Haslea ostrearia and the role of polysaccharides in previous marennine chemical characterization and bioactivity studies remain to be determined.

Effects of polydopamine-passivation on the optical properties of carbon dots and its potential use in vivo.

Passivation of carbon dots via heteroatom doping has been shown to enhance their optical properties and tune their fluorescence signature. Additionally, the incorporation of polymeric precursors in carbon dot synthesis has gained considerable interest with benefits to biological applications namely bioimaging, drug delivery and sensing, among others. In order to combine the desirable attributes of both, fluorescence enhancement and increased biocompatibility, polymers composed of high aromaticity and nitrogen content can be used as efficient carbon dot passivating agents. Here, the synthesis of fluorescent polymer-passivated carbon dots was developed through a microwave-assisted pyrolysis reaction of galactose, citric acid and polydopamine. Passivation of the dots with polydopamine induces a 90 nm red-shift in the fluorescence maxima from 420 to 510 nm. Moreover, passivation results in excitation-independent fluorescence and a 3.5-fold increase in fluorescence quantum yield, which increases from 1.3 to 4.6%. The application of the carbon dots as imaging probes was investigated in in vitro and in vivo model systems. Cytotoxicity studies in J774 and CHO-K1 cell lines revealed reduced cell toxicity for the polydopamine-passivated carbon dots in comparison to their unpassivated counterpart. In BALB/c mice, biodistribution studies demonstrated that regardless of surface passivation, the dots predominantly remained in the circulatory system 90 minutes post inoculation suggesting their potential use for cardiovascular therapies.

Magnetically-orientable Tween-based model membranes for NMR studies of proteins.

We present a new membrane mimetic system using a membrane softening detergent commonly known as Tween 80 (TW80), to form oriented systems for solid-state NMR applications. TW80 is a fatty acid ester (oleate) of sorbitan polyethoxylate and a mild non-ionic surfactant. Phosphatidylcholine (PC)/TW80 model membrane systems were characterized by solid-state NMR and FTIR spectroscopy. 31P and 2H NMR spectra showed that DMPC (14:0) and DPPC (16:0) self-assemble with TW80 to form oriented structures, and maintain alignment over a wide range of molar ratios and temperatures. The addition of lanthanide ions revealed that the membrane alignment can be flipped from parallel to perpendicular with respect to the magnetic field direction. Using 15N solid-state NMR and a labeled model transmembrane peptide, we showed that TW80-based membranes can be employed to determine the peptide orientation in the magnetic field, which is useful for structural determination. Altogether, our work showed that TW80 could be exploited for direct and efficient membrane protein extraction and to enhance membrane and membrane protein orientation without using a detergent removal step. This approach could be extended to a wide range of membranes including native ones.

AHNAK C-Terminal Peptide Membrane Binding-Interactions between the Residues 5654-5673 of AHNAK and Phospholipid Monolayers and Bilayers.

The dysferlin membrane repair complex contains a small complex, S100A10-annexin A2, which initiates membrane repair by recruiting the protein AHNAK to the membrane, where it interacts via binding sites in the C-terminal region. However, no molecular data are available for the membrane binding of the various proteins involved in this complex. Therefore, the present study investigated the membrane binding of AHNAK to elucidate its role in the cell membrane repair process. A chemically synthesized peptide (pAHNAK), comprising the 20 amino acids in the C-terminal domain of AHNAK, was applied to Langmuir monolayer models, and the binding parameters and insertion angles were measured with surface tensiometry and ellipsometry. The interaction of pAHNAK with lipid bilayers was studied using 31P solid-state nuclear magnetic resonance. pAHNAK preferentially and strongly interacted with phospholipids that comprised negatively charged polar head groups with unsaturated lipids. This finding provides a better understanding of AHNAK membrane behavior and the parameters that influence its function in membrane repair.

Labelling strategy and membrane characterization of marine bacteria Vibrio splendidus by in vivo2H NMR.

Vibrio splendidus is a marine bacterium often considered as a threat in aquaculture hatcheries where it is responsible for mass mortality events, notably of bivalves' larvae. This bacterium is highly adapted to dynamic salty ecosystems where it has become an opportunistic and resistant species. To characterize their membranes as a first and necessary step toward studying bacterial interactions with diverse molecules, we established a labelling protocol for in vivo2H solid-state nuclear magnetic resonance (SS-NMR) analysis of V. splendidus. 2H SS-NMR is a useful tool to study the organization and dynamics of phospholipids at the molecular level, and its application to intact bacteria is further advantageous as it allows probing acyl chains in their natural environment and study membrane interactions. In this study, we showed that V. splendidus can be labelled using deuterated palmitic acid, and demonstrated the importance of surfactant choice in the labelling protocol. Moreover, we assessed the impact of lipid deuteration on the general fitness of the bacteria, as well as the saturated-to-unsaturated fatty acid chains ratio and its impact on the membrane properties. We further characterize the evolution of V. splendidus membrane fluidity during different growth stages and relate it to fatty acid chain composition. Our results show larger membrane fluidity during the stationary growth phase compared to the exponential growth phase under labelling conditions - an information to take into account for future in vivo SS-NMR studies. Our lipid deuteration protocol optimized for V. splendidus is likely applicable other microorganisms for in vivo NMR studies.