Membrane Peptides Research Articles

Membrane interactions of the anuran antimicrobial peptide HSP1-NH2: Different aspects of the association to anionic and zwitterionic biomimetic systems

Studies have suggested that antimicrobial peptides act by different mechanisms, such as micellisation, self-assembly of nanostructures and pore formation on the membrane surface. This work presents an extensive investigation of the membrane interactions of the 14 amino-acid antimicrobial peptide hylaseptin P1-NH2 (HSP1-NH2), derived from the tree-frog Hyla punctata, which has stronger antifungal than antibacterial potential. Biophysical and structural analyses were performed and the correlated results were used to describe in detail the interactions of HSP1-NH2 with zwitterionic and anionic detergent micelles and phospholipid vesicles. HSP1-NH2 presents similar well-defined helical conformations in both zwitterionic and anionic micelles, although NMR spectroscopy revealed important structural differences in the peptide N-terminus. 2H exchange experiments of HSP1-NH2 indicated the insertion of the most N-terminal residues (1–3) in the DPC-d38 micelles. A higher enthalpic contribution was verified for the interaction of the peptide with anionic vesicles in comparison with zwitterionic vesicles. The pore formation ability of HSP1-NH2 (examined by dye release assays) and its effect on the size and surface charge as well as on the lipid acyl chain ordering (evaluated by Fourier-transform infrared spectroscopy) of anionic phospholipid vesicles showed membrane disruption even at low peptide-to-phospholipid ratios, and the effect increases proportionately to the peptide concentration. On the other hand, these biophysical investigations showed that a critical peptide-to-phospholipid ratio around 0.6 is essential for promoting disruption of zwitterionic membranes. In conclusion, this study demonstrates that the binding process of the antimicrobial HSP1-NH2 peptide depends on the membrane composition and peptide concentration.

Antimicrobial peptides in human synovial membrane as (low-grade) periprosthetic joint infection biomarkers

BackgroundSafe diagnosis of periprosthetic joint infection (PJI) is of utmost importance for successful exchange arthroplasty. However, current diagnostic tools show insufficient accuracy in the clinically common and challenging chronic low-grade infections. To close this diagnostic gap, reliable (bio)markers display the most promising candidates. Antimicrobial peptides (AMPs) are part of the innate immune response towards microbial growth. Recently we could show significant intraarticular levels of human cathelicidin LL-37 and β-defensin-3 (HBD-3) with high diagnostic accuracy in PJI synovial fluid. Consequently, these promising biomarkers were evaluated in PJI synovial membrane and synoviocytes, which may significantly facilitate histological diagnosis of PJI to improve outcome of septic joint replacement.MethodsIn this prospective single-center controlled clinical study (diagnostic level II), consecutive patients with total hip (THR) and knee (TKR) replacements were included undergoing primary arthroplasty (n = 8), surgical revision due to aseptic loosening (n = 9) and septic arthroplasty with coagulase-negative staphylococci (n = 8) according to the criteria of the Musculoskeletal Infection Society (MSIS). Semiquantitative immunohistochemical (IHC) analysis of LL-37, HBD-3 and HBD-2 in synovial membrane and isolated synoviocytes based on Total Allred Score (TS) and Immunoreactive Remmele and Stegner score (IRS) was performed. For statistical analysis, SPSS 26.0/R3.6.3 (p < 0.05) was used.ResultsThe AMPs LL-37 and HBD-3 were significantly elevated (up to 20×) in synovial membranes from PJI compared to aseptic loosening or primary arthroplasty. The area under the curve (AUC) in a receiver operating characteristic curve analysis was equal to 1.0 for both scores revealing excellent diagnostic accuracy. Isolated synoviocytes as cellular AMP source showed comparable results with a significant LL-37/HBD-3-increase up to 3 × in PJI. In contrast, local HBD-2 levels were negligible (p > 0.23) upon PJI with a lower diagnostic accuracy (AUC = 0.65) in analogy to our previous findings with synovial fluid.ConclusionsOur results implicate AMPs as promising and specific biomarkers for the histological diagnosis of PJI.

Chlorotoxin fusion protein regulates miR-374a and TNFAIP8 expression and inhibits glioma cell proliferation and promotes apoptosis.

Glioblastoma multiforme is the most common primary central nervous system malignancy, accounting for half of all intracranial primary tumors. In this study we constructed a multifunctional chlorotoxin fusion protein E-CHP that combines enhanced green fluorescent protein (E), glioma-targeting peptide chlorotoxin (C), destabilizing lipid membrane peptide riHA2 (H), and C-terminal and mouse double minute domains of p53 (P). E-CHP was expressed in Escherichia coli and purified by His affinity chromatography. Fluorescence microscopy observation showed that E-CHP could effectively target glioma cells; real-time quantitative PCR revealed that E-CHP increased miR-374a expression; and the dual luciferase reporter assay showed that tumor necrosis factor alpha-induced protein (TNFAIP)8 is a direct target of miR-374a. E-CHP and miR-374a inhibited the proliferation and migration of glioma cells, and Western blot analysis indicated that they suppressed TNFAIP8 expression in glioma cells and promoted the expression of caspase-3 and -8. Finally, E-CHP and miR-374a stimulated the apoptosis of glioma cells, as determined by flow cytometry analysis. These results suggest that miR-374a is a new candidate target for glioma therapy, whereas E-CHP fusion protein has the potential to be developed as a multifunctional carrier for targeted drug delivery and therapy.

Unsaturated Lipid Accelerates Formation of Oligomeric β-Sheet Structure of GP41 Fusion Peptide in Model Cell Membrane.

Membrane fusion of the viral and host cell membranes is the initial step of virus infection and is catalyzed by fusion peptides. Although the β-sheet structure of fusion peptides has been proposed to be the most important fusion-active conformation, it is still very challenging to experimentally identify different types of β-sheet structures at the cell membrane surface in situ and in real time. In this work, we demonstrate that the interface-sensitive amide II spectral signals of protein backbones, generated by the sum frequency generation vibrational spectroscopy, provide a sensitive probe for directly capturing the formation of oligomeric β-sheet structure of fusion peptides. Using human immunodeficiency virus (HIV) glycoprotein GP41 fusing peptide (FP23) as the model, we find that formation speed of oligomeric β-sheet structure depends on lipid unsaturation. The unsaturated lipid such as POPG can accelerate formation of oligomeric β-sheet structure of FP23. The β-sheet structure is more deeply inserted into the hydrophobic region of the POPG bilayer than the α-helical segment. This work will pave the way for future researches on capturing intermediate structures during membrane fusion processes and revealing the fusion mechanism.

Ultrafast Spectroscopy of Lipid-Water Interfaces: Transmembrane Crowding Drives H-Bond Dynamics.

Biology takes place in crowded, heterogeneous environments, and it is therefore essential to account for crowding effects in our understanding of biophysical processes at the molecular level. Comparable to the cytosol, proteins occupy approximately 30% of the plasma membrane surface; thus, crowding should have an effect on the local structure and dynamics at the lipid-water interface. Using a combination of ultrafast two-dimensional infrared spectroscopy and molecular dynamics simulations, we quantify the effects of membrane peptide concentration on the picosecond interfacial H-bond dynamics. The measurements reveal a nonmonotonic dependence of water orientation and dynamics as a function of transmembrane peptide:lipid ratio. We observe three dynamical regimes: a "pure lipid-like" regime at low peptide concentrations, a bulk-like region at intermediate peptide concentrations where dynamics are faster by ∼20% compared to those of the pure lipid bilayer, and a crowded regime where high peptide concentrations slow dynamics by ∼50%.

Role of lipopolysaccharides and lipoteichoic acids on C-Chrysophsin-1 interactions with model Gram-positive and Gram-negative bacterial membranes.

Antimicrobial peptides (AMPs) are attractive as biomaterial coatings because they have broad spectrum activity against different microbes, with a low likelihood of incurring antimicrobial resistance. Direct action against the bacterial membrane is the most common mechanism of action (MOA) of AMPs, with specific MOAs dependent on membrane composition, peptide concentration, and environmental factors that include temperature. Chrysophsin-1 (CHY1) is a broad spectrum salt-tolerant AMP that is derived from a marine fish. A cysteine modification was made to the peptide to facilitate attachment to a surface, such as a biomedical device. The authors used quartz crystal microbalance with dissipation monitoring to study how temperature (23 and 37 °C) and lipid composition influence the MOA of cysteine-modified peptide (C-CHY1) with model membranes comprised of supported lipid bilayers (SLBs). These two temperatures were used so that the authors could better understand the differences in behavior between typical lab temperatures and physiologic conditions. The authors created model membranes that mimicked properties of Gram-negative and Gram-positive bacteria in order to understand how the mechanisms might differ for different types of bacterial systems. SLB models of Gram-positive bacterial membranes were formed using combinations of phosphatidylcholine, phosphatidylglycerol (PG), and S. aureus-derived lipoteichoic acid (LTA). SLB models of Gram-negative bacterial membranes were formed using combinations of phosphatidylethanolamine (PE), PG, and E. coli-derived lipopolysaccharides (LPS). The molecules that distinguish Gram-positive and Gram-negative membranes (LTA and LPS) have the potential to alter the MOA of C-CHY1 with the SLBs. The authors' results showed that the MOA for the Gram-positive SLBs was not sensitive to temperature, but the LTA addition did have an effect. Specifically, similar trends in frequency and dissipation changes across all overtones were observed, and the same mechanistic trends were observed in the polar plots at 23 and 37 °C. However, when LTA was added, polar plots showed an association between C-CHY1 and LTA, leading to SLB saturation. This was demonstrated by significant changes in dissipation, while the frequency (mass) was not increasing after the saturation point. For the Gram-negative SLBs, the composition did not have a significant effect on MOA, but the authors saw more differences between the two temperatures studied. The authors believe this is due to the fact that the gel-liquid crystal transition temperature of PE is 25 °C, which means that the bilayer is more rigid at 23 °C, compared to temperatures above the transition point. At 23 °C, a significant energetic shift would be required to allow for additional AMP insertion. This could be seen in the polar plots, where there was a steep slope but there was very little mass addition. At 37 °C, the membrane is more fluid and there is less of an energetic requirement for insertion. Therefore, the authors observed greater mass addition and fewer changes in dissipation. A better understanding of C-CHY1 MOA using different SLB models will allow for the more rational design of future therapeutic solutions that make use of antimicrobial peptides, including those involving biomaterial coatings.

Synergistic Biophysical Techniques Reveal Structural Mechanisms of Engineered Cationic Antimicrobial Peptides in Lipid Model Membranes.

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d-enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.

Structure of a Parkinson's Disease-Involved α-Synuclein Peptide Is Modulated by Membrane Composition and Physical State.

α-Synuclein (AS), the protein responsible for Parkinson's disease, contains a 12-residue-long sequence, AS71-82, that is thought to play a crucial role in the α-synuclein aggregation process. Neuronal membranes are direct interacting partners of α-synuclein and play a role in fibrillogenesis by providing a charged catalytic surface, notably from anionic phospholipids. However, details are lacking regarding the impact of membrane composition and the driving forces leading to membrane anchorage and peptide structure conversion. To decipher the interplay of α-synuclein with neuronal membranes, the structure of AS71-82 was investigated in the presence of anionic model membranes. Infrared (IR) spectroscopy and solid-state nuclear magnetic resonance data show that AS71-82 adopts a perfectly in-register parallel β-sheet structure with fibrillar morphology upon interactions with anionic model membranes. IR thermotropism experiments conducted with several membrane compositions revealed that the phospholipids' phase transition induces a rearrangement of the AS71-82 β-sheet structure. In contrast, membranes are not significantly affected by the presence of AS71-82, which advocates for the amyloid fibrils to lie loosely on the membrane surface. The results bring new arguments for the lipid-sensing capabilities of AS71-82 and revealed its protofibrillar structure. The striking similarities between AS71-82 and α-synuclein make it a potential good aggregation inhibitor upon chemical modifications.

Chiral supramolecular architecture of stable transmembrane pores formed by an \u03b1-helical antibiotic peptide in the presence of lyso-lipids

The amphipathic α-helical antimicrobial peptide MSI-103 (aka KIA21) can form stable transmembrane pores when the bilayer takes on a positive spontaneous curvature, e.g. by the addition of lyso-lipids. Solid-state 31P- and 15N-NMR demonstrated an enrichment of lyso-lipids in these toroidal wormholes. Anionic lyso-lipids provided additional stabilization by electrostatic interactions with the cationic peptides. The remaining lipid matrix did not affect the nature of the pore, as peptides maintained the same orientation independent of lipid charge, and a change in membrane thickness did not considerably affect their tilt angle. Under optimized conditions (i.e. in the presence of lyso-lipids and appropriate bilayer thickness), stable and well-aligned pores could be obtained for solid-state 2H-NMR analysis. These data revealed for the first time the complete 3D alignment of this representative amphiphilic peptide in fluid membranes, which is compatible with either monomeric helices as constituents, or left-handed supercoiled dimers as building blocks from which the overall toroidal wormhole is assembled.

PH-responsive high stability polymeric nanoparticles for targeted delivery of anticancer therapeutics

The practical application of nanoparticles (NPs) as chemotherapeutic drug delivery systems is often hampered by issues such as poor circulation stability and targeting inefficiency. Here, we have utilized a simple approach to prepare biocompatible and biodegradable pH-responsive hybrid NPs that overcome these issues. The NPs consist of a drug-loaded polylactic-co-glycolic acid (PLGA) core covalently ‘wrapped’ with a crosslinked bovine serum albumin (BSA) shell designed to minimize interactions with serum proteins and macrophages that inhibit target recognition. The shell is functionalized with the acidity-triggered rational membrane (ATRAM) peptide to facilitate internalization specifically into cancer cells within the acidic tumor microenvironment. Following uptake, the unique intracellular conditions of cancer cells degrade the NPs, thereby releasing the chemotherapeutic cargo. The drug-loaded NPs showed potent anticancer activity in vitro and in vivo while exhibiting no toxicity to healthy tissue. Our results demonstrate that the ATRAM-BSA-PLGA NPs are a promising targeted cancer drug delivery platform.

Attempted “Rescue” of Glutamic Acid by Arginine in a Transmembrane Helix

Membrane proteins are vital for biological function and are complex to study, as they must be investigated in lipid membranes. While studies of small proteins or model peptides in lipid membranes have begun to reveal some of the important lipid interactions of single residues or individual functional groups, the combined effects or interactions of even pairs of protein or peptide side chains still are not well understood. The goal of this research is to understand the combined effects of a glutamic acid and an arginine spaced two residues apart - giving 200° radial separation - within a hydrophobic transmembrane alpha-helix. To this end, we incorporated E16 and R14 the transmembrane helix of acetyl-GGALW5(LA)6LW19LAGA-amide (GWALP23) and labeled selected core alanines with deuterium for detection using 2H NMR spectroscopy. The singly substituted R14-GWALP23 shows remarkably narrow 2H NMR resonances (J Am Chem Soc 132, 5803), whereas the singly substituted E16-GWALP23 shows very broad spectra (BBA 1859, 484). Initial results for the doubly substituted R14,E16-GWALP23 show multiple populations of states for the helix in DOPC bilayers, which is very different from the effects of either residue alone. There is furthermore early indication that a major preferred helix orientation emerges when the pH is lowered from 6 to 3 in DOPC. Ongoing experiments are addressing the multi-state behavior and the pH dependence of the properties of the R14,E16-GWALP23 helix in DOPC membranes. Additional experiments will address the behavior of this helix in DLPC, DMPC and DEiPC bilayer membranes.

The N-Terminal Region of a Ph-Responsive Peptide Controls Its Interaction with Phosphatidylserine-Containing Bilayers

Local acidosis is common in solid tumors, and provides a promising target for peptide-based anticancer treatments. To exploit this characteristic, the acidity-triggered rational membrane (ATRAM) peptide was designed to insert selectively into lipid bilayers when the surrounding media is acidic. Insertion of ATRAM into the bilayer as a transmembrane α-helix involves translocation of the C-terminus across the membrane, such that the N-terminus is free to interact with lipids on the outer leaflet. Since many cancers overexpose on the outer membrane leaflet the negatively charged lipid phosphatidylserine (PS), we propose that mutations at the non-inserting N-terminus would modify ATRAM's interactions with model cancer cell membranes. Here, we used two ATRAM variants, termed K2-ATRAM (L2K/L5K) and Y-ATRAM (L2Y), to explore the effects of positively charged or mildly hydrophobic amino acids near the N-terminus. The presence of PS in the membrane enhanced membrane partitioning for K2-ATRAM but decreased it for Y-ATRAM. However, screening with high-salt conditions showed that the effects of PS on the pK of insertion are multifactorial and cannot explained by electrostatics alone. These results suggest that studies using only neutral bilayers may fail to indicate the true interactions of anticancer peptides with tumor cells, which contain negatively charged phospholipids. Additionally, the different behaviors of the ATRAM variants demonstrate how the non-inserting N-terminal region can be tailored to adjust the specificity of ATRAM for target membranes. This information will aid the design of new ATRAMs and other peptides that are tailored to the pH and lipid compositions of a variety of diseases.

A Trifunctional Linker for Palmitoylation and Peptide and Protein Localization in Biological Membranes.

Attachment of lipophilic groups is an important post‐translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids, or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine‐based trifunctional linker. The linker allows the facile incorporation of two different functionalities at a cysteine residue in a single step. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α‐helical model peptide WALP with or without here and in all cases below; palmitoyl groups in giant unilamellar vesicles that contain a liquid‐ordered (Lo) and liquid‐disordered (Ld) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the Ld phase. These data demonstrate that the Lo domain in model membranes is highly unfavorable for a single membrane‐spanning peptide.

Structural changes introduced by cholesterol and melatonin to the model membranes mimicking preclinical conformational diseases.

The structure and dynamics of membranes depend on many external and internal factors that in turn determine their biological functions. One of the widely accepted and studied characteristics of biomembranes is their fluidity. We research a simple system with variable fluidity tweakable via its composition. The addition of cholesterol is employed to increase the order of lipid chains, thus decreasing the membrane fluidity, while melatonin is shown to elevate the chain disorder, thus also the membrane fluidity. We utilize the densitometric measurements to show a shift of studied systems closer or further from the gel-to-fluid phase transition. The structural changes represented by changes to membrane thickness are evaluated from small angle neutron scattering. Finally, we look at the ability of the two additives to control the interactions between membrane and amyloid-beta peptides. Our results suggest that fluidizing effect of melatonin can promote an insertion of peptide within the membrane interior. Intriguingly, the latter structure relates possibly to an Alzheimer's disease preventing mechanism postulated in the case of melatonin.

Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells.

Studies focused on development of an attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis (Ptb) in cattle and other species, revealed that deletion of relA, a global gene regulator, abrogates the ability of Map to establish a persistent infection. In the absence of relA, cattle develop CD8 cytotoxic T cells (CTL) with the ability to kill intracellular bacteria. Analysis of the recall response to a relA mutant, Map/ΔrelA, with cells from a vaccinated steer demonstrated that a 35-kDa membrane peptide (MMP) is one of the targets of the response. This observation suggested that it might be possible to develop a peptide-based vaccine. As reported here, the gene encoding the hypothetical MMP ORF, MAP2121c, was modified for expression in mammalian cells as a first step in developing an expression cassette for incorporation into a mammalian expression vector. The modified sequence of MMP, tPA-MMP, was mutated to generate two additional sequences for the study, one with substitutions to replace five potential residues that could be glycosylated, tPA-MMP-5mut, and one with substitutions to replace the first two potential residues that could be glycosylated, tPA-MMP-2mut. The sequences were placed in an expression cassette to produce peptides for analysis. An ex vivo platform was used with flow cytometry and a bacterium viability assay to determine if modifications in the gene encoding MMP for expression in mammalian cells altered its capacity to elicit development of CD8 CTL, essential for its use in a peptide-based vaccine. Monocyte-depleted PBMC (mdPBMC) were stimulated with antigen-presenting cells (APC) pulsed with different MMP constructs. CD4 and CD8 T cells proliferated in response to stimulation with MMP (control) expressed in Escherichia coli (eMMP), tPA-MMP, and tPA-MMP-2mut. CD8 T cells retained the capacity to kill intracellular bacteria. The tPA-MMP-5mut failed to elicit a proliferative response and was not included in further studies. The data show that the expression cassettes containing MMP and MMP-2mut can be used to screen and select a mammalian expression vector for the development of an efficacious peptide-based vaccine against Ptb.

Membrane interactions of antimicrobial peptide-loaded microgels

In the present study, lipid membrane interactions of anionic poly(ethyl acrylate-co-methacrylic acid) (MAA) microgels as carriers for the cationic antimicrobial peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) were investigated. In doing so, neutron reflectometry (NR), Fourier-transform infrared spectroscopy with attenuated total reflection (FTIR-ATR), zeta potential, ellipsometry, and circular dichroism spectroscopy (CD) experiments were employed to investigate the relative importance of membrane interactions of peptide-loaded microgel particles and of released peptide. For the free peptide, NR results showed membrane binding occurring preferentially in the tail region in a concentration-dependent manner. At low peptide concentration (0.3 μM) only peptide insertion in the outer leaflet was seen, however, pronounced membrane defects and peptide present in both leaflets was observed at higher peptide concentration (5.0 μM). LL-37 loaded into MAA microgels qualitatively mirrored these effects regarding both peptide localization within the membrane and concentration-dependent defect formation. In addition, very limited membrane binding of microgel particles was observed, in agreement with FTIR-ATR and liposome leakage results. FTIR-ATR showed LL-37 to undergo α-helix formation on membrane insertion, also supported by CD results, the kinetics of which was substantially reduced for microgel-loaded LL-37 due to sustained peptide release. Together, these findings demonstrate that membrane interactions for microgel-loaded LL-37 are dominated by released peptide, but also that slow release of microgel-loaded LL-37 translates into kinetic effects on peptide-membrane interactions, relating to both peptide localization within the bilayer, and to bilayer structure.

Serotyping of Toxoplasma gondii Infection Using Peptide Membrane Arrays

The intracellular parasite Toxoplasma gondii can cause chronic infections in most warm-blooded animals, including humans. In the USA, strains belonging to four different Toxoplasma clonal lineages (types 1, 2, 3, and 12) are commonly isolated, whereas strains not belonging to these lineages are predominant in other continents such as South America. Strain type plays a pivotal role in determining the severity of Toxoplasma infection. Therefore, it is epidemiologically relevant to develop a non-invasive and inexpensive method for determining the strain type in Toxoplasma infections and to correlate the genotype with disease outcome. Serological typing is based on the fact that many host antibodies are raised against immunodominant parasite proteins that are highly polymorphic between strains. However, current serological assays can only reliably distinguish type 2 from non-type 2 infections. To improve these assays, mouse, rabbit, and human infection serum were reacted against 950 peptides from 62 different polymorphic Toxoplasma proteins by using cellulose membrane peptide arrays. This allowed us to identify the most antigenic peptides and to pinpoint the most relevant polymorphisms that determine strain specificity. Our results confirm the utility of previously described peptides and identify novel peptides that improve and increase the specificity of the assay. In addition, a large number of novel proteins showed potential to be used for Toxoplasma diagnosis. Among these, peptides derived from several rhoptry, dense granule, and surface proteins represented promising candidates that may be used in future experiments to improve Toxoplasma serotyping. Moreover, a redesigned version of the published GRA7 typing peptide performed better and specifically distinguished type 3 from non-type 3 infections in sera from mice, rabbits, and humans.

Energy-Representation Theory of Solutions: Its Formulation and Application to Soft, Molecular Aggregates

Abstract The energy-representation theory of solutions is developed to address the dissolution of a molecule in homogeneous fluid as well as the partitioning into such nanoscale structures as micelle and lipid membrane and the physisorption onto gas-liquid and solid-liquid interfaces in a unified manner as solvation in an extended sense. The present review describes the formulation of the solution theory with illustrative applications to the peptide configuration in lipid membrane, the water dissolution into polymer, and the physisorption on urea crystal in contact with liquid water. The solution theory in the energy representation is a density-functional scheme formulated by adopting the solute-solvent pair interaction energy as a one-dimensional coordinate for distribution functions and provides an approximate functional for the solvation free energy in terms of energy distribution functions in the reference-solvent and solution systems of interest. Each of the solute and solvent molecules is treated as a single unit as a whole, and due to this feature, a species with intramolecular flexibility and a solvent system with nano-scale inhomogeneity or interface can be analyzed in a common framework. The role of water is pointed out in determining the configuration of a peptide in lipid membrane, and the dissolution of water into polymer medium is described at chemical accuracy. Some directions of future developments are also discussed.

Identification of twelve Aeromonas spp. species with monoclonal antibody-based immunoassay in water and fish samples

Aeromonas spp. are the causative agents of motile Aeromonas septicemia (MAS) in fish, as well as gastroenteritis and infections in humans. The antigen diversity between Aeromonas spp. and serotypes greatly challenges the identification of this zoonotic pathogen in environmental, food, and clinical samples. In the present study, six conserved peptides in the outer membrane of Aeromonas were selected and used as immunogens after conjugation to produce monoclonal antibodies (mAbs). Peptides 1 and 4 with the amino sequences of “VYDKDGTTFD” and “GGFKGKLSYQTND”, effectively elicited cross-reactive antibodies against Aeromonas in mice. Based on the mAbs 1B1 and 14H5, the developed sandwich immunoassay detected twelve Aeromonas spp. species, including A. hydrophila (8/8), A. veronii (7/7), A. caviae, A. sobria, A. dhakensis, A. media, A. popoffii, A. punctata subsp. caviae, A. bivalvium, A. lacus, A. jandaei, A. bestiarum and A. salmonicida (1/2). The detection limit of these strains primarily ranged from 1.69 × 104 to 4.57 × 105 CFU mL−1. No cross-reactivity was observed with the other tested strains. The sensitivity and specificity of this method was 96% (26/27) and 100% (15/15), respectively. Furthermore, real sample analyses detected river water samples containing 105 CFU mL−1 of A. hydrophila or A. caviae, and detected Carassius auratus samples showing hemorrhagic septicemia after enrichment in buffered peptone water. The established immunoassay may be promising as an effective screening method for monitoring Aeromonas spp. in environmental and food samples.

Membrane-mediated fibrillation and toxicity of the tau hexapeptide PHF6

The aggregation of the tau protein into neurofibrillary tangles is believed to correlate with cognitive decline in several neurodegenerative disorders, including Alzheimer's disease. Recent studies suggest that tau's interactions with the cell membrane could serve as a toxicity pathway and also enhance fibrillation into paired helical filaments (PHFs). Conformational changes associated with tau-membrane interactions are poorly understood, and their characterization could improve our understanding of tau pathogenicity. In this study, we investigated the molecular level structural changes associated with the interaction of the tau hexapeptide PHF6 with model lipid membranes and characterized the effects of these interactions on membrane stability and peptide fibrillation. We used two PHF6 forms, the aggregation-prone PHF6 with N-terminal acetylation (Ac-PHF6) and the non-aggregation prone PHF6 with a standard N terminus (NH3+-PHF6). We found that both PHF6 peptides are neurotoxic and exhibit similar membrane-mediated changes, consisting of: 1) favorable interactions with anionic membranes, 2) membrane destabilization through lipid extraction, and 3) membrane-mediated fibrillation. The rate at which these changes occurred was the main difference between the two peptides. NH3+-PHF6 displayed slow membrane-mediated fibrillation after 6 days of incubation, whereas Ac-PHF6 adopted a β-sheet conformation at the surface of the membrane within hours. Ac-PHF6 interactions with the membrane were also accompanied by membrane invagination and rapid membrane destabilization. Overall, our results reveal that membrane interactions could play a critical role in tau toxicity and fibrillation, and highlight that unraveling these interactions is important for significantly advancing the development of therapeutic strategies to manage tau-associated neurodegenerative diseases.