mastoparan-X Research Articles

Oral Administration of the Antimicrobial Peptide Mastoparan X Alleviates Enterohemorrhagic Escherichia coli-Induced Intestinal Inflammation and Regulates the Gut Microbiota.

The gut microbiota plays an important role in intestinal immune system development and in driving inflammation. Antibiotic administration for therapeutic purposes causes an imbalance in the gut microbiota. Antimicrobial peptides can regulate the gut microbiota and maintain intestinal homeostasis. The aim of this study was to investigate the anti-inflammatory effects and regulation of the gut microbiota by the orally administered antimicrobial peptide mastoparan X (MPX). In this study, Escherichia coli was used to induce intestinal inflammation, and the results showed that MPX+ E. coli alleviated weight loss and intestinal pathological changes in necropsy specimens of E. coli-infected mice. MPX+ E. coli reduced the serum levels of the inflammation-related proteins interleukin-2, interleukin-6, tumour necrosis factor-α, myeloperoxidase, and lactate dehydrogenase on days 7 and 28. Furthermore, MPX+ E. coli increased the length of villi and reduced the infiltration of inflammatory cells into the jejunum and colon post infection. Scanning electron microscopy and transmission electron microscopy results showed that MPX could improve the morphology of jejunum villi and microvilli and increase tight junction protein levels. 16S rRNA sequencing analysis of caecal content samples showed that the species diversity and richness were lower in the E. coli-infected group. At the genus level, MPX+ E. coli significantly reduced the abundance of Bacteroidales and Alistipes and enhanced the relative abundance of Muribaculaceae. Alpha-diversity analyses (Shannon index) showed that MPX significantly increased the microbial diversity of mice. Overall, this study is the first to investigate the effects of oral administration of MPX on intestinal inflammation and the gut microbiota, providing a new perspective regarding the prevention of enteritis and maintenance of intestinal homeostasis.

The Antimicrobial Peptide Mastoparan X Protects Against Enterohemorrhagic Escherichia coli O157:H7 Infection, Inhibits Inflammation, and Enhances the Intestinal Epithelial Barrier.

Escherichia coli can cause intestinal diseases in humans and livestock, destroy the intestinal barrier, exacerbate systemic inflammation, and seriously threaten human health and animal husbandry development. The aim of this study was to investigate whether the antimicrobial peptide mastoparan X (MPX) was effective against E. coli infection. BALB/c mice infected with E. coli by intraperitoneal injection, which represents a sepsis model. In this study, MPX exhibited no toxicity in IPEC-J2 cells and notably suppressed the levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), myeloperoxidase (MPO), and lactate dehydrogenase (LDH) released by E. coli. In addition, MPX improved the expression of ZO-1, occludin, and claudin and enhanced the wound healing of IPEC-J2 cells. The therapeutic effect of MPX was evaluated in a murine model, revealing that it protected mice from lethal E. coli infection. Furthermore, MPX increased the length of villi and reduced the infiltration of inflammatory cells into the jejunum. SEM and TEM analyses showed that MPX effectively ameliorated the jejunum damage caused by E. coli and increased the number and length of microvilli. In addition, MPX decreased the expression of IL-2, IL-6, TNF-α, p-p38, and p-p65 in the jejunum and colon. Moreover, MPX increased the expression of ZO-1, occludin, and MUC2 in the jejunum and colon, improved the function of the intestinal barrier, and promoted the absorption of nutrients. This study suggests that MPX is an effective therapeutic agent for E. coli infection and other intestinal diseases, laying the foundation for the development of new drugs for bacterial infections.

Side Chain Hydrophobicity Modulates Therapeutic Activity and Membrane Selectivity of Antimicrobial Peptide Mastoparan-X

The discovery of new anti-infective compounds is stagnating and multi-resistant bacteria continue to emerge, threatening to end the “antibiotic era”. Antimicrobial peptides (AMPs) and lipo-peptides such as daptomycin offer themselves as a new potential class of antibiotics; however, further optimization is needed if AMPs are to find broad use as antibiotics. In the present work, eight analogues of mastoparan-X (MPX) were investigated, having side chain modifications in position 1, 8 and 14 to modulate peptide hydrophobicity. The self-association properties of the peptides were characterized, and the peptide-membrane interactions in model membranes were compared with the bactericidal and haemolytic properties. Alanine substitution at position 1 and 14 resulted in higher target selectivity (red blood cells versus bacteria), but also decreased bactericidal potency. For these analogues, the gain in target selectivity correlated to biophysical parameters showing an increased effective charge and reduction in the partitioning coefficient for membrane insertion. Introduction of an unnatural amino acid, with an octyl side chain by amino acid substitution, at positions 1, 8 and 14 resulted in increased bactericidal potency at the expense of radically reduced membrane target selectivity. Overall, optimized membrane selectivity or bactericidal potency was achieved by changes in side chain hydrophobicity of MPX. However, enhanced potency was achieved at the expense of selectivity and vice versa in all cases.

Effect of the aspartic acid D2 on the affinity of Polybia-MP1 to anionic lipid vesicles

Polybia-MP1 (IDWKKLLDAAKQIL-NH2), a helical peptide extracted from the venom of a Brazilian wasp, has broad-spectrum antimicrobial activities without being hemolytic or cytotoxic. This peptide has also displayed anticancer activity against cancer cell cultures. Despite its high selectivity, MP1 has an unusual low net charge (Q = +2). The aspartic residue (D2) in the N-terminal region plays an important role in its affinity and selectivity; its substitution by asparagine (D2N mutant) led to a less selective peptide. Aiming to explore the importance of this residue for the peptides' affinity, we compared the zwitterionic and anionic vesicle adsorption activity of Polybia-MP1 versus its D2N mutant and also mastoparan X (MPX). The adsorption, electrostatic, and conformational free energies were assessed by circular dichroism (CD) and fluorescence titrations using large unilamellar vesicles (LUVs) at the same conditions in association with measurement of the zeta potential of LUVs in the presence of the peptides. The adsorption free energies of the peptides, determined from the partition coefficients, indicated higher affinity of MP1 to anionic vesicles compared with the D2N mutant and MPX. The electrostatic and conformational free energies of MP1 in anionic vesicles are less favorable than those found for the D2N mutant and MPX. Therefore, the highest affinity of MP1 to anionic vesicles is likely due to other energetic contributions. The presence of D2 in MP1 makes these energetic components 1.2 and 1.5 kcal/mol more favorable compared with the D2N mutant and MPX, respectively.

Optimizing Antimicrobial Peptide Activity Through Balancing of Charge and Hydrophobicity

Antimicrobial peptides (AMPs) offer themselves as potential drugs for treating bacterial, fungal, and viral infections and thus as a new class of antibiotics. However, AMPs have not yet been able to fulfill early hopes for substituting classical antibiotics in the treatment of infections by multi-resistant bacteria, since greater bacterial membrane selectivity and in vivo stability have to be achieved. Several strategies for optimization of AMPs’ therapeutic potential have been pursued. Previously, we discovered that improved selectivity toward negatively charged liposomes could be achieved by short chain acylation of mastoparan-X (MPX), whereas acylation by a longer chain impaired the selectivity. In the present work, this hypothesis was further explored through an extended library of novel MPX analogues having C1, C4 or C8 carbon chain substitution at three individual positions along the MPX backbone. The peptides were synthesized exchanging selected hydrophobic residues with Ala, Leu or 2-amino-decanoic acid at position 1, 8 and 14 of the native MPX sequence. Isothermal titration calorimetry was employed for gauging the partitioning into liposomes and the pore formation properties of the MPX analogues. Steady-state fluorescence was utilized for addressing the aggregation state of the peptides in aqueous solution. Bacteriocidal and hemolytic properties of the peptides were measured (on E.coli, L.lactis and human red blood cells) and correlated to the biophysical parameters. The results show that both the position of acylation on the peptide backbone and length of the acyl-chain affect the therapeutic window of the peptide analogue. Two MPX analogues with reduced hemolytic activity and retained antimicrobial activity were identified, and the effective charge and hydrophobicity of the peptides were found to be inversely correlated.

New Insight into the Mechanism of Action of Wasp Mastoparan Peptides: Lytic Activity and Clustering Observed with Giant Vesicles

Antimicrobial peptides of the mastoparans family exert their bactericidal activity by binding to lipid membranes, inducing pores or defects and leaking the internal contents of vesicles and cells. However, this does not seem to be the only mechanism at play, and they might be important in the search for improved peptides with lower undesirable side effects. This work deals with three mastoparans peptides, Polybia-MP-1(MP-1), N2-Polybia-MP-1 (N-MP-1), and Mastoparan X (MPX), which exhibit high sequence homology. They all have three lysine residues and amidated C termini, but because of the presence of two, one, and no aspartic acid residues, respectively, they have +2, +3, and +4 net charges at physiological pH. Here we focus on the effects of these mastoparans peptides on anionic model membranes made of palmitoleyoilphosphatidylcholine (POPC) and palmitoleyoilphosphatidylglycerol (POPG) at 1:1 and 3:1 molar ratios in the presence and in the absence of saline buffer. Zeta potential experiments were carried out to measure the extent of the peptides' binding and accumulation at the vesicle surface, and CD spectra were acquired to quantify the helical structuring of the peptides upon binding. Giant unilamellar vesicles were observed under phase contrast and fluorescence microscopy. We found that the three peptides induced the leakage of GUVs at a gradual rate with many characteristics of the graded mode. This process was faster in the absence of saline buffer. Additionally, we observed that the peptides induced the formation of dense regions of phospholipids and peptides on the GUV surface. This phenomenon was easily observable for the more charged peptides (MPX > N-MP-1 > MP-1) and in the absence of added salt. Our data suggest that these mastoparans accumulate on the bilayer surface and induce a transient interruption to its barrier properties, leaking the vesicle contents. Next, the bilayer recovers its continuity, but this happens in an inhomogeneous way, forming a kind of ply with peptides sandwiched between two juxtaposed membranes. Eventually, a peptide-lipid aggregate forming a lump is formed at high peptide-to-lipid ratios.

Selective Acylation Enhances Membrane Charge Sensitivity of the Antimicrobial Peptide Mastoparan-X

The partitioning of the wasp venom peptide mastoparan-X (MPX) into neutral and negatively charged lipid membranes has been compared with two new synthetic analogs of MPX where the N α -terminal of MPX was acylated with propanoic acid (PA) and octanoic acid (OA). The acylation caused a considerable change in the membrane partitioning properties of MPX and it was found that the shorter acylation with PA gave improved affinity and selectivity toward negatively charged membranes, whereas OA decreased the selectivity. Based on these findings, we hypothesize that minor differences in the embedding and positioning of the peptide in the membrane caused by either PA or OA acylation play a critical role in the fine-tuning of the effective charge of the peptide and thereby the fine-tuning of the peptide's selectivity between neutral and negatively charged lipid membranes. This finding is unique compared to previous reports where peptide acylation enhanced membrane affinity but also resulted in impaired selectivity. Our result may provide a method of enhancing selectivity of antimicrobial peptides toward bacterial membranes due to their high negative charge—a finding that should be investigated for other, more potent antimicrobial peptides in future studies.

Site-Specific Hydration Status of an Amphipathic Peptide in AOT Reverse Micelles

Reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane (IO) and water have long been used as a means to provide a confined aqueous environment for various applications. In particular, AOT reverse micelles have often been used as a template to mimic membrane-water interfaces. While earlier studies have shown that membrane-binding peptides can indeed be incorporated into the polar cavity of AOT reverse micelles where they mostly fold into an alpha-helical structure, the underlying interactions leading to the ordered conformation are however not well understood. Herein, we have used circular dichroism (CD) and infrared (IR) spectroscopies in conjunction with a local IR marker (i.e., the CN group of a non-natural amino acid, p-cyano-phenylalanine) and a global IR reporter (i.e., the amide I' band of the peptide backbone) to probe the conformation as well as the hydration status of an antimicrobial peptide, mastoparan x (MPx), in AOT reverse micelles of different water contents. Our results show that at, w0=6, MPx adopts an alpha-helical conformation with both the backbone and hydrophobic side chains mostly dehydrated, whereas its backbone becomes partially hydrated at w0=20. In addition, our results suggest that the amphipathic alpha-helix so formed orients itself in such a manner that its positively charged, lysine-rich, hydrophilic face points toward the negatively charged AOT head groups, while its hydrophobic face is directed toward the polar interior of the water pool. This picture is in marked contrast to that observed for the binding of MPx to phospholipid bilayers wherein the hydrophobic surface of the bound alpha-helix is buried deeper into the membrane interior.

Structure of Tightly Membrane-Bound Mastoparan-X, a G-Protein-Activating Peptide, Determined by Solid-State NMR

The structure of mastoparan-X (MP-X), a G-protein activating peptide from wasp venom, in the state tightly bound to anionic phospholipid bilayers was determined by solid-state NMR spectroscopy. Carbon-13 and nitrogen-15 NMR signals of uniformly labeled MP-X were completely assigned by multidimensional intraresidue C–C, N–C α C β , and N–C α –C′, and interresidue C α –C α C β , N–C α C β , and N–C′–C α correlation experiments. The backbone torsion angles were predicted from the chemical shifts of 13C′, 13C α , 13C β , and 15N signals with the aid of protein NMR database programs. In addition, two 13C– 13C and three 13C– 15N distances between backbone nuclei were precisely measured by rotational resonance and REDOR experiments, respectively. The backbone structure of MP-X was determined from the 26 dihedral angle restraints and five distances with an average root-mean-square deviation of 0.6 Å. Peptide MP-X in the bilayer-bound state formed an amphiphilic α-helix for residues Trp 3−Leu 14 and adopted an extended conformation for Asn 2. This membrane-bound conformation is discussed in relation to the peptide’s activities to form pores in membranes and to activate G-proteins. This study demonstrates the power of multidimensional solid-state NMR of uniformly isotope-labeled molecules and distance measurements for determining the structures of peptides bound to lipid membranes.

Detection of Peptide−Phospholipid Interaction Sites in Bilayer Membranes by 13C NMR Spectroscopy: Observation of 2H/31P-Selective 1H-Depolarization under Magic-Angle Spinning

We have developed a solid-state NMR method for observing the signals due to 13C spins of a peptide in the close vicinity of 31P and 2H spins in deuterated phospholipid bilayers. The signal intensities in 13C high-resolution NMR spectra directly indicate the depolarization of 1H by 1H-31P and 1H-2H dipolar couplings under multiple-contact cross-polarization. This method was applied to a fully 13C-, 15N-labeled 14-residue peptide, mastoparan-X (MP-X), bound to phospholipid bilayers whose fatty acyl chains are deuterated. The 13C NMR spectra for the depolarization were simulated from the chemical shifts and structure of membrane-bound MP-X previously determined and the distribution of 2H and 31P spins in lipid bilayers. The minimization of RMSD between the simulated and the experimental spectra showed that the amphiphilic alpha-helix of MP-X was located in the interface between the water layer and the hydrophobic domain of the bilayer, with nonpolar residues facing the phosphorus atoms and alkyl chains of the lipids.

Crystallization and Preliminary X-Ray Diffraction Analysis of Three Mastoparans

Mastoparans are tetradecapeptides found to be the major component of wasp venoms. These peptides possess a variety of biological activities. Three related mastoparans, mastoparan from Polistes jadwagae (MP-PJ), mastoparanX (MP-X) and its carboxyl-free C-terminal form (MP-X-COO-), were crystallized. X-ray diffraction data for them were collected at resolutions of 1.2 A, 2.0 A and 3.3 A respectively.

G protein-bound conformation of mastoparan-X: heteronuclear multidimensional transferred nuclear overhauser effect analysis of peptide uniformly enriched with 13C and 15N.

Mastoparans, a family of tetradecapeptides from wasp venom, have been used as convenient low molecular weight models of receptors coupled to GTP-binding regulatory proteins (G proteins) for the understanding of the interaction between G proteins and receptors. Sukumar and Higashijima have analyzed the conformation of mastoparan-X (MP-X) bound to the G protein alpha-subunit using proton two-dimensional transferred nuclear Overhauser effect (TRNOE) spectroscopy [Sukumar, M., and Higashijima, T. (1992) J. Biol. Chem., 267, 21421-21424]. The resultant structure, however, was not well-defined due to severe overlap of peptide proton resonances. To determine the G protein-bound conformation of MP-X in detail, we have analyzed this interaction by heteronuclear multidimensional TRNOE experiments of MP-X uniformly enriched with 15N and/or 13C. By solving the overlap problem, we were able to determine the precise conformation of MP-X bound to Gi1alpha: the peptide adopts an amphiphilic alpha-helix from Trp3 to C-terminal Leu14, and the atomic root-mean-square deviation (rmsd) values in this portion about the averaged coordinates were 0.27 +/- 0.07 A for the backbone atoms (N, Calpha, C') and 0.84 +/- 0.16 A for all heavy atoms. These values are much smaller than the corresponding rmsd values of the structures obtained from the proton 2D TRNOE spectrum alone: 1.70 +/- 0.41 A for the backbone atoms (N, Calpha, C') and 2.84 +/- 0.51 A for all heavy atoms. Our results indicate that the heteronuclear multidimensional TRNOE experiments of peptides uniformly enriched with stable isotopes are a very powerful tool for analyzing the conformation of short peptides bound to large proteins. We will also discuss the structure-activity relationships of mastoparans in activating G proteins on the basis of the precise structure of MP-X bound to Gi1alpha.

Self-assembly of mastoparan X derivative having fluorescence probe in lipid bilayer membrane

A mastoparan X (MPX) derivative having an anthryl group at the C-terminal residue was synthesized (MPX-A), and its conformation, orientation and aggregation in phospholipid bilayer membrane were studied. The efficiency of intramolecular energy transfer from the Trp residue to the anthryl group at high peptide dilution suggested α-helical conformation in the lipid membrane, which is consistent with the previous report by NMR of MPX concentrated in the membrane. Either emission from the Trp residue or the anthryl group of MPX-A in the lipid membrane was quenched by 5-doxylstearic acid, suggesting that MPX-A is located at the membrane surface with the helix axis oriented parallel to the surface. The dependence of the excited energy transfer and the fluorescence depolarization of MPX-A on the peptide concentration revealed that MPX-A aggregated in the lipid membrane to form a defined structure.