Bis-Indole Alkaloids Isolated from the Sponge Spongosorites calcicola Disrupt Cell Membranes of MRSA

Antimicrobial resistance (AMR) is a global health challenge with methicillin resistant Staphylococcus aureus (MRSA), a leading cause of nosocomial infection. In the search for novel antibiotics, marine sponges have become model organisms as they produce diverse bioactive compounds. We investigated and compared the antibacterial potential of 3 bis-indole alkaloids—bromodeoxytopsentin, bromotopsentin and spongotine A—isolated from the Northeastern Atlantic sponge Spongosorites calcicola. Antimicrobial activity was determined by MIC and time-kill assays. The mechanism of action of bis-indoles was assessed using bacterial cytological profiling via fluorescence microscopy. Finally, we investigated the ability of bis-indole alkaloids to decrease the cytotoxicity of pathogens upon co-incubation with HeLa cells through the measurement of mammalian cell lysis. The bis-indoles were bactericidal to clinically relevant Gram-positive pathogens including MRSA and to the Gram-negative gastroenteric pathogen Vibrio parahaemolyticus. Furthermore, the alkaloids were synergistic in combination with conventional antibiotics. Antimicrobial activity of the bis-indole alkaloids was due to rapid disruption and permeabilization of the bacterial cell membrane. Significantly, the bis-indoles reduced pathogen cytotoxicity toward mammalian cells, indicating their ability to prevent bacterial virulence. In conclusion, sponge bis-indole alkaloids are membrane-permeabilizing agents that represent good antibiotic candidates because of their potency against Gram-positive and Gram-negative bacterial pathogens.     

The Topology,in Model Membranes,of the Core Peptide Derived from the T‐Cell Receptor Transmembrane Domain

The T‐cell receptor–CD3 complex (TCR–CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of α‐ and β‐chains, which are responsible for antigen recognition. Within the transmembrane domain of the α‐subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T‐cell‐mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phosphatidylcholine/cholesterol (9:1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15‐residue segments of TCRα, containing the CP, were synthesized and spin‐labeled at different locations with a nitroxide derivative. Electron spin‐echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron–electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane.     

Understanding the Biophysical Interaction of LTX-315 with Tumoral Model Membranes

Host defense peptides are found primarily as natural antimicrobial agents among all lifeforms. These peptides and their synthetic derivatives have been extensively studied for their potential use as therapeutic agents. The most accepted mechanism of action of these peptides is related to a nonspecific mechanism associated with their interaction with the negatively charged groups present in membranes, inducing bilayer destabilization and cell death through several routes. Among the most recently reported peptides, LTX-315 has emerged as an important oncolytic peptide that is currently in several clinical trials against different cancer types. However, there is a lack of biophysical studies regarding LTX-315 and its interaction with membranes. This research focuses primarily on the understanding of the molecular bases of LTX-315′s interaction with eukaryotic lipids, based on two artificial systems representative of non-tumoral and tumoral membranes. Additionally, the interaction with individual lipids was studied by differential scanning calorimetry and Fourier-transformed infrared spectroscopy. The results showed a strong interaction of LTX-315 with the negatively charged phosphatidylserine. The results are important for understanding and facilitating the design and development of improved peptides with anticancer activity.     

Probing the Role of Chirality in Phospholipid Membranes

Nucleotides, amino acids, sugars, and lipids are almost ubiquitously homochiral within individual cells on Earth. While oligonucleotides and proteins exist as one natural chirality throughout the tree of life, two stereoisomers of phospholipids have separately emerged in archaea and bacteria, an evolutionary divergence known as “the lipid divide”. Within this review, we focus on the emergence of phospholipid homochirality and compare the stability of synthetic homochiral and heterochiral membranes in vitro. We discuss chemical probes designed to study the stereospecific interactions of lipid membranes in vitro. Overall, we aim to highlight studies that help elucidate the determinants of stereospecific interactions between lipids, peptides, and small molecule ligands. Continued work in understanding the drivers of favorable interactions between chiral molecules and biological membranes will lead to the design of increasingly selective chemical tools for bioorthogonal labeling of lipid membranes and safer membrane-associating pharmaceuticals.     

The Antimicrobial Peptide 1018-K6 Interacts Distinctly with Eukaryotic and Bacterial Membranes,the Basis of Its Specificity and Bactericidal Activity

Since penicillin was discovered, antibiotics have been critical in the fight against infections. However, antibiotic misuse has led to drug resistance, which now constitutes a serious health problem. In this context, antimicrobial peptides (AMPs) constitute a natural group of short proteins, varying in structure and length, that act against certain types of bacterial pathogens. The antimicrobial peptide 1018-K6 (VRLIVKVRIWRR- NH2) has significant bactericidal and antibiofilm activity against Listeria monocytogenes isolates, and against different strains and serotypes of Salmonella. Here, the mechanism of action of 1018-K6 was explored further to understand the peptide–membrane interactions relevant to its activity, and to define their determinants. We combined studies with model synthetic membranes (liposomes) and model biological membranes, assessing the absorption maximum and the quenching of 1018-K6 fluorescence in aqueous and lipid environments, the self-quenching of carboxyfluorescein, as well as performing lipid sedimentation assays. The data obtained reflect the differential interactions of the 1018-K6 peptide with eukaryotic and prokaryotic membranes, and the specific interactions and mechanisms of action in the three prokaryotic species studied: Salmonella Typhimurium^2GN, Escherichia coli^3GN, and Staphylococcus aureus^3GP. The AMP 1018-K6 is a candidate to prevent (food preservation) or treat (antibiotic use) infections caused by certain pathogenic bacteria, especially some that are resistant to current antibiotics.     

Inward Translocation of the Phospholipid Analogue Miltefosine across Caco-2 Cell Membranes Exhibits Characteristics of a Carrier-mediated Process

Miltefosine (hexadecylphosphocholine, HePC) is the first effective oral agent for the treatment of visceral leishmaniasis. The characteristics of HePC incorporation into the human intestinal epithelial cell line Caco-2 were investigated in order to understand its oral absorption mechanism. The results provide evidence for the involvement of a carrier-mediated mechanism, since the association of HePC at the apical pole of Caco-2 cells was (1) saturable as a function of time with a rapid initial incorporation over 5 min followed by a more gradual increase; (2) saturable as a function of concentration over the range studied (2–200 μM) with a saturable component which followed Michaelis–Menten kinetics (apparent K _m 15.7 μmol/L, V _max 39.2 nmol/mg protein/h) and a nonspecific diffusion component; (3) partially inhibited by low temperature and ATP depletion, indicating the temperature and energy-dependence of the uptake process. Moreover, we demonstrated, by an albumin back-extraction method, that HePC is internalized via translocation from the outer to the inner leaflet of the plasma membrane and that HePC may preferentially diffuse through intact raft microdomains. In conclusion, our results suggest that incorporation of HePC at the apical membrane of Caco-2 cells may occur through a passive diffusion followed by a translocation in the inner membrane leaflet through an active carrier-mediated mechanism.     

The Molecular Bases of the Interaction between a Saponin from the Roots of Gypsophila paniculata L. and Model Lipid Membranes

In view of the possible medical applications of saponins, the molecular structure of a GOTCAB saponin from the roots of Gypsophila paniculata L. was determined by NMR. The biological activity of saponins may depend on the interaction with cell membranes. To obtain more insight in the mechanism of membrane-related saponin function, an experimental and theoretical study was conducted. Ternary lipid systems composed of sphingomyelin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and cholesterol were used as models of mammalian cell membranes. The membrane–saponin interaction was studied experimentally by monitoring surface pressure in the monomolecular films formed at the air–aqueous subphase interface. The behavior of GOTCAB saponin in a water box and model monolayer systems was characterized by molecular dynamics simulations. The results obtained showed that, in the systems used, cholesterol had a decisive effect on the interaction between GOTCAB and phosphocholine or sphingomyelin as well as on its location within the lipid film.     

The Small Heat Shock Protein,HSPB1, Interacts with and Modulates the Physical Structure of Membranes

Small heat shock proteins (sHSPs) have been demonstrated to interact with lipids and modulate the physical state of membranes across species. Through these interactions, sHSPs contribute to the maintenance of membrane integrity. HSPB1 is a major sHSP in mammals, but its lipid interaction profile has so far been unexplored. In this study, we characterized the interaction between HSPB1 and phospholipids. HSPB1 not only associated with membranes via membrane-forming lipids, but also showed a strong affinity towards highly fluid membranes. It participated in the modulation of the physical properties of the interacting membranes by altering rotational and lateral lipid mobility. In addition, the in vivo expression of HSPB1 greatly affected the phase behavior of the plasma membrane under membrane fluidizing stress conditions. In light of our current findings, we propose a new function for HSPB1 as a membrane chaperone.     

Effects of Lipidation on a Proline-Rich Antibacterial Peptide

The emergence of multidrug-resistant bacteria is a worldwide health problem. Antimicrobial peptides have been recognized as potential alternatives to conventional antibiotics, but still require optimization. The proline-rich antimicrobial peptide Bac7(1-16) is active against only a limited number of Gram-negative bacteria. It kills bacteria by inhibiting protein synthesis after its internalization, which is mainly supported by the bacterial transporter SbmA. In this study, we tested two different lipidated forms of Bac7(1-16) with the aim of extending its activity against those bacterial species that lack SbmA. We linked a C12-alkyl chain or an ultrashort cationic lipopeptide Lp-I to the C-terminus of Bac7(1-16). Both the lipidated Bac-C12 and Bac-Lp-I forms acquired activity at low micromolar MIC values against several Gram-positive and Gram-negative bacteria. Moreover, unlike Bac7(1-16), Bac-C12, and Bac-Lp-I did not select resistant mutants in E. coli after 14 times of exposure to sub-MIC concentrations of the respective peptide. We demonstrated that the extended spectrum of activity and absence of de novo resistance are likely related to the acquired capability of the peptides to permeabilize cell membranes. These results indicate that C-terminal lipidation of a short proline-rich peptide profoundly alters its function and mode of action and provides useful insights into the design of novel broad-spectrum antibacterial agents.     

Marine Antimicrobial Peptides: Nature Provides Templates for the Design of Novel Compounds against Pathogenic Bacteria

The discovery of antibiotics for the treatment of bacterial infections brought the idea that bacteria would no longer endanger human health. However, bacterial diseases still represent a worldwide treat. The ability of microorganisms to develop resistance, together with the indiscriminate use of antibiotics, is mainly responsible for this situation; thus, resistance has compelled the scientific community to search for novel therapeutics. In this scenario, antimicrobial peptides (AMPs) provide a promising strategy against a wide array of pathogenic microorganisms, being able to act directly as antimicrobial agents but also being important regulators of the innate immune system. This review is an attempt to explore marine AMPs as a rich source of molecules with antimicrobial activity. In fact, the sea is poorly explored in terms of AMPs, but it represents a resource with plentiful antibacterial agents performing their role in a harsh environment. For the application of AMPs in the medical field limitations correlated to their peptide nature, their inactivation by environmental pH, presence of salts, proteases, or other components have to be solved. Thus, these peptides may act as templates for the design of more potent and less toxic compounds.     

界面聚合制备复合膜过程的数学模型

基于高分子物理化学、质量传递和相分离成膜理论,研究在复合膜制备过程中采用界面聚合反应成膜的机理,建立了非稳态条件下反应．扩散联合控制的数学模型;通过有针对性地简化,该模型可适用于反应控制和扩散控制。模型中无量纲参数有明确的物理意义,较好地反映了界面聚合反应成膜过程的机理。无量纲化处理使模型解析解形式更为简单、实用,模型与实验数据吻合良好,且优于现有模型。通过模拟计算,可得出单体(A组分)浓度、膜的厚度、膜厚增长率随时间的变化关系,并可考察聚合反应速率常数、单体(A组分)在复合层中的扩散系数、单体初始浓度等参数对成膜过程的影响．理论结果可用于指导界面聚合反应成膜实践。     

Improving the Therapeutic Index of Smp24, a Venom-Derived Antimicrobial Peptide: Increased Activity against Gram-Negative Bacteria

Antimicrobial peptides (AMPs) are naturally occurring compounds which possess a rapid killing mechanism and low resistance potential. Consequently, they are being viewed as potential alternatives to traditional antibiotics. One of the major factors limiting further development of AMPs is off-target toxicity. Enhancements to antimicrobial peptides which can maximise antimicrobial activity whilst reducing mammalian cytotoxicity would make these peptides more attractive as future pharmaceuticals. We have previously characterised Smp24, an AMP derived from the venom of the scorpion Scorpio maurus palmatus. This study sought to better understand the relationship between the structure, function and bacterial selectivity of this peptide by performing single amino acid substitutions. The antimicrobial, haemolytic and cytotoxic activity of modified Smp24 peptides was determined. The results of these investigations were compared with the activity of native Smp24 to determine which modifications produced enhanced therapeutic indices. The structure–function relationship of Smp24 was investigated by performing N-terminal, mid-chain and C-terminal amino acid substitutions and determining the effect that they had on the antimicrobial and cytotoxic activity of the peptide. Increased charge at the N-, mid- and C-termini of the peptide resulted in increased antimicrobial activity. Increased hydrophobicity at the N-terminus resulted in reduced haemolysis and cytotoxicity. Reduced antimicrobial, haemolytic and cytotoxic activity was observed by increased hydrophobicity at the mid-chain. Functional improvements have been made to modified peptides when compared with native Smp24, which has produced peptides with enhanced therapeutic indices.     

Piloting the Membranolytic Activities of Peptides with a Self‐organizing Map

Antimicrobial peptides (AMPs) show remarkable selectivity toward lipid membranes and possess promising antibiotic potential. Their modes of action are diverse and not fully understood, and innovative peptide design strategies are needed to generate AMPs with improved properties. We present a de novo peptide design approach that resulted in new AMPs possessing low‐nanomolar membranolytic activities. Thermal analysis revealed an entropy‐driven mechanism of action. The study demonstrates sustained potential of advanced computational methods for designing peptides with the desired activity.     

Identification of Bacterial Membrane Selectivity of Romo1-Derived Antimicrobial Peptide AMPR-22 via Molecular Dynamics

The abuse or misuse of antibiotics has caused the emergence of extensively drug-resistant (XDR) bacteria, rendering most antibiotics ineffective and increasing the mortality rate of patients with bacteremia or sepsis. Antimicrobial peptides (AMPs) are proposed to overcome this problem; however, many AMPs have attenuated antimicrobial activities with hemolytic toxicity in blood. Recently, AMPR-11 and its optimized derivative, AMPR-22, were reported to be potential candidates for the treatment of sepsis with a broad spectrum of antimicrobial activity and low hemolytic toxicity. Here, we performed molecular dynamics (MD) simulations to clarify the mechanism of lower hemolytic toxicity and higher efficacy of AMPR-22 at an atomic level. We found four polar residues in AMPR-11 bound to a model mimicking the bacterial inner/outer membranes preferentially over eukaryotic plasma membrane. AMPR-22 whose polar residues were replaced by lysine showed a 2-fold enhanced binding affinity to the bacterial membrane by interacting with bacterial specific lipids (lipid A or cardiolipin) via hydrogen bonds. The MD simulations were confirmed experimentally in models that partially mimic bacteremia conditions in vitro and ex vivo. The present study demonstrates why AMPR-22 showed low hemolytic toxicity and this approach using an MD simulation would be helpful in the development of AMPs.     

Conformational Changes of Anoplin,W-MreB1–9, and (KFF)3K Peptides near the Membranes

Many peptides interact with biological membranes, but elucidating these interactions is challenging because cellular membranes are complex and peptides are structurally flexible. To contribute to understanding how the membrane-active peptides behave near the membranes, we investigated peptide structural changes in different lipid surroundings. We focused on two antimicrobial peptides, anoplin and W-MreB_1–9, and one cell-penetrating peptide, (KFF)₃K. Firstly, by using circular dichroism spectroscopy, we determined the secondary structures of these peptides when interacting with micelles, liposomes, E. coli lipopolysaccharides, and live E. coli bacteria. The peptides were disordered in the buffer, but anoplin and W-MreB_1–9 displayed lipid-induced helicity. Yet, structural changes of the peptide depended on the composition and concentration of the membranes. Secondly, we quantified the destructive activity of peptides against liposomes by monitoring the release of a fluorescent dye (calcein) from the liposomes treated with peptides. We observed that only for anoplin and W-MreB_1–9 calcein leakage from liposomes depended on the peptide concentration. Thirdly, bacterial growth inhibition assays showed that peptide conformational changes, evoked by the lipid environments, do not directly correlate with the antimicrobial activity of the peptides. However, understanding the relation between peptide structural properties, mechanisms of membrane disruption, and their biological activities can guide the design of membrane-active peptides.     

Expanding the Landscape of Amino Acid-Rich Antimicrobial Peptides: Definition,Deployment in Nature,Implications for Peptide Design and Therapeutic Potential

Unlike the α-helical and β-sheet antimicrobial peptides (AMPs), our knowledge on amino acid-rich AMPs is limited. This article conducts a systematic study of rich AMPs (>25%) from different life kingdoms based on the Antimicrobial Peptide Database (APD) using the program R. Of 3425 peptides, 724 rich AMPs were identified. Rich AMPs are more common in animals and bacteria than in plants. In different animal classes, a unique set of rich AMPs is deployed. While histidine, proline, and arginine-rich AMPs are abundant in mammals, alanine, glycine, and leucine-rich AMPs are common in amphibians. Ten amino acids (Ala, Cys, Gly, His, Ile, Lys, Leu, Pro, Arg, and Val) are frequently observed in rich AMPs, seven (Asp, Glu, Phe, Ser, Thr, Trp, and Tyr) are occasionally observed, and three (Met, Asn, and Gln) were not yet found. Leucine is much more frequent in forming rich AMPs than either valine or isoleucine. To date, no natural AMPs are simultaneously rich in leucine and lysine, while proline, tryptophan, and cysteine-rich peptides can simultaneously be rich in arginine. These findings can be utilized to guide peptide design. Since multiple candidates are potent against antibiotic-resistant bacteria, rich AMPs stand out as promising future antibiotics.     

Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro.     

Contribution of Invariant Natural Killer T Cells to the Clearance of Pseudomonas aeruginosa from Skin Wounds

Chronic infections are considered one of the most severe problems in skin wounds, and bacteria are present in over 90% of chronic wounds. Pseudomonas aeruginosa is frequently isolated from chronic wounds and is thought to be a cause of delayed wound healing. Invariant natural killer T (iNKT) cells, unique lymphocytes with a potent regulatory ability in various inflammatory responses, accelerate the wound healing process. In the present study, we investigated the contribution of iNKT cells in the host defense against P. aeruginosa inoculation at the wound sites. We analyzed the re-epithelialization, bacterial load, accumulation of leukocytes, and production of cytokines and antimicrobial peptides. In iNKT cell–deficient (Jα18KO) mice, re-epithelialization was significantly decreased, and the number of live colonies was significantly increased, when compared with those in wild-type (WT) mice on day 7. IL-17A, and IL-22 production was significantly lower in Jα18KO mice than in WT mice on day 5. Furthermore, the administration of α-galactosylceramide (α-GalCer), a specific activator of iNKT cells, led to enhanced host protection, as shown by reduced bacterial load, and to increased production of IL-22, IL-23, and S100A9 compared that of with WT mice. These results suggest that iNKT cells promote P. aeruginosa clearance during skin wound healing.     

相关向量机方法预测药膜的释药规律

鉴于药膜释药过程的高度非线性,采用相关向量机方法为药膜的释药过程建模,得到药膜累积释放率与时间的定量关系。通过优化建模参数,所建相关向量机模型具有较高的拟合能力,且预测误差小,稳健性好。研究结果表明,相关向量机建立的模型可对释放进行有效预测。