Antimicrobial Peptide Tachyplesin Research Articles

Rapid Formation of Intramolecular Disulfide Bridges using Light: An Efficient Method to Control the Conformation and Function of Bioactive Peptides.

Disulfide bonds are widely found in natural peptides and play a pivotal role in stabilizing their secondary structures, which are highly associated with their biological functions. Herein, we introduce a light-mediated strategy to effectively control the formation of disulfides. Our strategy is based on 2-nitroveratryl (oNv), a widely used photolabile motif, which serves both as a photocaging group and an oxidant (after photolysis). We demonstrated that irradiation of oNv-caged thiols with UV light could release free thiols that are rapidly oxidized by locally released byproduct nitrosoarene, leading to a "break-to-bond" fashion. This strategy is highlighted by the in situ restoration of the antimicrobial peptide tachyplesin I (TPI) from its external disulfide-caged analogue TPI-1. TPI-1 exhibits a distorted structure and a diminished function. However, upon irradiation, the β-hairpin structure and membrane activity of TPI were largely restored via rapid intramolecular disulfide formation. Our study proposes a powerful method to regulate the conformation and function of peptides in a spatiotemporal manner, which has significant potential for the design of disulfide-centered light-responsive systems.

Removal of Antimicrobial Peptides from Aqueous Solutions Using Carbon Nanotubes

The aim of this work is to study the possibility of removing antimicrobial peptides arenicin-1 and tachyplesin-1 from aqueous solutions using new highly hydrophobic nanomaterials—carbon nanotubes (CNTs) Taunit-M. Two types of CNTs, respectively, functionalized and nonfunctionalized with–COOH groups are studied. The RP HPLC method is used for the quantification of peptides in solutions before and after the sorption on the CNTs. It is revealed that tachyplesin-1 and arenicin-1 are actually not adsorbed on nonfunctionalized CNTs. A theoretical calculation of specific hydrophobicity and aliphatic index shows that both peptides have poorly expressed hydrophobic properties, and this can explain lack of their adsorption. On the contrary, using functionalized CNTs makes it possible to remove up to 89% of arenicin-1 and 92% of tachyplesin-1 from the solution. A comparative study of adsorption of the antimicrobial peptide tachyplesin- 1 on nonfunctionalized activated carbons and functionalized CNTs shows that the amount of peptide adsorbed on activated carbons is about three times less than that absorbed on CNTs. It is assumed that the high adsorption capacity of the functionalized CNTs toward the studied peptides results from the functionalization of the sorbent surface with the–COOH groups capable of forming ionic bonds with free–NH2 groups of peptides. This shows the prospects for using functionalized CNTs as sorbents for the removal of toxic preparations of peptide origin from aqueous solutions.

In Vitro Study of Antitumor Effect of Antimicrobial Peptide Tachyplesin I in Combination with Cisplatin.

We studied combined effect of β-hairpin antimicrobial peptide tachyplesin I and cytotoxic agent cisplatin on tumor and normal human cell lines. MTT assay and flow cytometry showed that tachyplesin I selectively sensitized cancer cells to cisplatin in specified concentration ratios. In vitro experiments demonstrated that combined use of tachyplesin I and cisplatin allows decreasing the effective dose of the cytostatic thus reducing nonspecific toxicity.

Effect of N- and C-Terminal Modifications on Cytotoxic Properties of Antimicrobial Peptide Tachyplesin I.

We analyze the effects of N-terminal acetylation and C-terminal amidation on the cytotoxic properties of β-hairpin antimicrobial peptide tachyplesin I. MTT-assay showed that modified tachyplesin I exhibited increased cytotoxicity toward both tumor and normal human cells. Hemolytic activity of modified tachyplesin I was also higher than that of the initial molecule. In contrast to non-modified tachyplesin I, the peptide with C- and N-terminal modifications is resistant to proteolytic degradation in fresh human serum. C- and N-terminal modifications make tachyplesin I more attractive prototype of anticancer drug due to its more potent cytotoxic effect and better pharmacokinetic properties.

Expression levels of antimicrobial peptide tachyplesin I in transgenic Ornithogalum lines affect the resistance to Pectobacterium infection

The genus Ornithogalum includes several ornamental species that suffer substantial losses from bacterial soft rot caused by Pectobacteria. The absence of effective control measures for use against soft rot bacteria led to the initiation of a project in which a small antimicrobial peptide from an Asian horseshoe crab, tachyplesin (tpnI), was introduced into two commercial cultivars: O. dubium and O. thyrsoides. Disease severity and bacterial colonization were examined in transgenic lines expressing this peptide. Disease resistance was evaluated in six lines of each species by measuring bacterial proliferation in the plant tissue. Three transgenic lines of each species were subjected to further analysis in which the expression level of the transgene was evaluated using RT-PCR and qRT-PCR. The development of disease symptoms and bacterial colonization of the plant tissue were also examined using GFP-expressing strain of P. carotovorum subsp. brasiliense Pcb3. Confocal-microscopy imaging revealed significantly reduced quantities of bacterial cells in the transgenic plant lines that had been challenged with the bacterium. The results clearly demonstrate that tpnI expression reduces bacterial proliferation, colonization and disease symptom (reduced by 95–100%) in the transgenic plant tissues. The quantity of tpnI transcripts, as measured by qRT-PCR, was negatively correlated with the protection afforded to the plants, as measured by the reduced severity of disease symptoms in the tissue.

Experimental Induction of Bacterial Resistance to the Antimicrobial Peptide Tachyplesin I and Investigation of the Resistance Mechanisms.

Tachyplesin I is a 17-amino-acid cationic antimicrobial peptide (AMP) with a typical cyclic antiparallel β-sheet structure that is a promising therapeutic for infections, tumors, and viruses. To date, no bacterial resistance to tachyplesin I has been reported. To explore the safety of tachyplesin I as an antibacterial drug for wide clinical application, we experimentally induced bacterial resistance to tachyplesin I by using two selection procedures and studied the preliminary resistance mechanisms. Aeromonas hydrophila XS91-4-1, Pseudomonas aeruginosa CGMCC1.2620, and Escherichia coli ATCC 25922 and F41 showed resistance to tachyplesin I under long-term selection pressure with continuously increasing concentrations of tachyplesin I. In addition, P. aeruginosa and E. coli exhibited resistance to tachyplesin I under UV mutagenesis selection conditions. Cell growth and colony morphology were slightly different between control strains and strains with induced resistance. Cross-resistance to tachyplesin I and antimicrobial agents (cefoperazone and amikacin) or other AMPs (pexiganan, tachyplesin III, and polyphemusin I) was observed in some resistant mutants. Previous studies showed that extracellular protease-mediated degradation of AMPs induced bacterial resistance to AMPs. Our results indicated that the resistance mechanism of P. aeruginosa was not entirely dependent on extracellular proteolytic degradation of tachyplesin I; however, tachyplesin I could induce increased proteolytic activity in P. aeruginosa Most importantly, our findings raise serious concerns about the long-term risks associated with the development and clinical use of tachyplesin I.

Improved strategy for recombinant production and purification of antimicrobial peptide tachyplesin I and its analogs with high cell selectivity.

Here, we report an efficient procedure for recombinant production and purification of tachyplesin I (THI) with a final yield of 17mg/L of the culture medium. The peptide was expressed in Escherichia coli as a part of the thioredoxin fusion protein. With the use of soluble expression followed by immobilized metal-ion affinity chromatography, the recombinant protein cleavage and reversed-phase high-performance liquid chromatography, a yield of THI did not exceed 6.5mg/L of the culture medium. Further optimization studies were carried out to improve the protein expression level and simplify purification procedure of the target peptide. To achieve better yield of the peptide, we used high-cell-density bacterial expression. The formed inclusion bodies were highly enriched with the fusion protein, which allowed us to perform direct chemical cleavage of the inclusion bodies solubilized in 6M guanidine-HCl with subsequent selective precipitation of proteins with trifluoroacetic acid. This enabled us to avoid an extra step of purification by immobilized metal-ion affinity chromatography. The developed procedure has made it possible to obtain biologically active THI and was used for screening a number of its mutant analogs. As a result, several selective and nonhemolytic analogs were developed. Significant reduction in hemolytic activity without losing antimicrobial activity was achieved by substitution of tyrosine or isoleucine residue in the β-turn region of the molecule with hydrophilic serine. The present study affords further insight into molecular mechanism of antimicrobial action of tachyplesin and gains a better understanding of structure-activity relationships in its analogs. This is aimed at searching for novel antibiotics on the basis of antimicrobial peptides with reduced cytotoxicity.

Corrigendum

The FEBS JournalVolume 282, Issue 24 p. 4822-4823 CorrigendumFree Access Corrigendum This article corrects the following: Marine antimicrobial peptide tachyplesin as an efficient nanocarrier for macromolecule delivery in plant and mammalian cells Aastha Jain, Bhoopesh K. Yadav, Archana Chugh, Volume 282Issue 4The FEBS Journal pages: 732-745 First Published online: January 8, 2015 First published: 20 October 2015 https://doi.org/10.1111/febs.13529AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat The authors of ‘Marine antimicrobial peptide tachyplesin as an efficient nanocarrier for macromolecule delivery in plant and mammalian cells’ in FEBS J 282 pages 732–745, wish to correct Fig. 2 and its legend, plus the legends for Figs 3, 5 and 8, as follows: Figure 2Open in figure viewerPowerPoint Internalization of Tpl in root tips of T. aestivum (A) control root tip (without addition of peptide), (B) 5 μm MTat, (C) 5 μm dextran sulfate, (D) 5 μm Tat, (E) 5 μm Tpl, (F) 10 μm Tpl and (G) 20 μm Tpl. The images were taken using epi-fluorescence microscope (Olympus, IX51). Figure legends with correction. Fig. 3. (A) Confocal microscopy images to determine the uptake of Tpl in HeLa cells (scale bar = 20 μm): (a) control (without any peptide treatment), (b) 5 μm MTat, (c) 5 μm dextran sulfate, (d) 5 μm Tat, (e) 30 μm Tpl. PMT voltage was adjusted for all samples to maximize image resolution and intensity (B) Quantitative fluorimetric estimation of uptake of Tpl in HeLa cells (RFU, relative fluorescence units) (C) The effect of Tpl on the viability of HeLa cells at varying concentrations is represented by MTT assay. Fig. 5. Increase in nuclear localization of Tpl as assessed by incubation with varying ratios of labelled: non-labelled Tpl in: (A) protoplasts where (a, a’) 0: 20 μm (labelled: non-labelled peptide), (a, b’) 5: 15, (a, c’) 10:10, (a, d’) 15:5, (a, e’) 20: 0 (B) HeLa cells where (a, a’) 0: 30 μm (labelled: non-labelled peptide), (a, b’) 10: 20, (a, c’) 20: 10, (a, d’) 30: 0. Scale bar = 20 μm. PMT voltage was adjusted for all samples to maximize image resolution and intensity. The images in Fig. 3A(e) and Fig. 5B(a, d’) overlap as they represent the same concentration and sample type. Fig. 8. Uptake of Tpl versus mutated Tpl in (A) protoplasts and (B) HeLa cells as determined by confocal microscopy and fluorimetric analysis. (a) Control (without peptide treatment), (b) MTpl-1, (c) MTpl-2, (d) MTpl-3 and (e) Tpl. Scale bar = 20 μm. PMT voltage was adjusted for all samples to maximize image resolution and intensity. Reference 1Jain A, Yadav BK & Chugh A (2015) Marine antimicrobial peptide tachyplesin as an efficient nanocarrier for macromolecule delivery in plant and mammalian cells. FEBS J 282, 732– 745. doi: 10.1111/febs.13178. Volume282, Issue24December 2015Pages 4822-4823 FiguresReferencesRelatedInformation

Marine antimicrobial peptide tachyplesin as an efficient nanocarrier for macromolecule delivery in plant and mammalian cells.

Membrane-active peptides can be classified as cell-penetrating peptides and antimicrobial peptides (AMPs) that are known to play interchangeable roles. In this study, this dual behaviour was studied for the marine AMP, tachyplesin. It is a well-established cyclic peptide known to possess antimicrobial properties and was investigated for its cell-penetrating property and cargo delivery ability. Because of its derivation from a marine organism as well as cyclic nature, it has been shown to possess higher stability in vitro. In this study, its internalization as a cell-penetrating peptide was established and characterized in both plant and mammalian systems. It was shown to deliver cargo molecules in both living systems, emerging as an efficient nonviral macromolecule nanocarrier.

Diaminodiacid‐Based Solid‐Phase Synthesis of Peptide Disulfide Bond Mimics

The antimicrobial peptide tachyplesin I was used as a model to apply the title strategy, which was developed for the preparation of peptidic macrocycles with double disulfide surrogates. The folding and activity of the tachyplesin I analogues were found to be sensitive to the structure of the disulfide surrogates, thus underlining the necessity of a flexible synthetic route for generating disulfide bond surrogates with high structural diversity.

3P024 カブトガニ由来抗菌ペプチドTachyplesin Iとキチン結合能に関する研究(01B.蛋白質:構造機能相関,ポスター,日本生物物理学会年会第51回(2013年度))

3P024 カブトガニ由来抗菌ペプチドTachyplesin Iとキチン結合能に関する研究(01B.蛋白質:構造機能相関,ポスター,日本生物物理学会年会第51回(2013年度))

Development and Characterization of a Mouse Monoclonal Antibody Against Antimicrobial Peptide Tachyplesin I

Monoclonal antibodies against tachyplesin I (TP I) were developed to study its mechanisms of activity, a kind of cationic antimicrobial peptides (AMPs), in vivo or in vitro, and to purify TP I from expression products. The synthesized TP I was chemically conjugated with the carrier protein BSA and then injected into BALB/c mice. Positive hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using TP I and subcloned three times with limiting dilution. Five MAbs effective in detecting the native TP I (named 2D8, 3B8, 5H2, 6B12, and 8F5) were obtained. Isotyping of all obtained MAbs indicated that MAbs 2D8, 3B8, 5H2, and 8F5 belong to IgG1, and MAb 6B12 belongs to IgG2a. Specificity assay showed that MAb 8F5 had almost the same level of specificity to natural TP I, recombinant TP I, and synthesized TP I and TP II, but did not cross-react with control peptides. These results suggest that the synthetic AMP conjugates can elicit antibodies against native AMPs and can be used to detect antimicrobial peptides.

Eukaryotic expression and antimicrobial spectrum determination of the peptide tachyplesin II

Ta0-a, the gene encoding the mature antimicrobial peptide tachyplesin II, was engineered to optimize the coding sequence according to codon usage bias in yeast. Ta0-a was efficiently expressed in the methylotrophic yeast Pichia pastoris strain SMD1168. The recombinant peptide Ta0 reached 150mg/L after methanol induction for 6 d. Ta0 was rapidly purified to homogeneity by a single step of size-exclusion chromatography. The minimal lethal concentrations of Ta0 to the Escherichia coli strain K12 was 30μg/mL. Ta0 exhibited a wide range of antimicrobial activity: the growth of 26 bacterial and fungal strains, including some typical food/feed spoilage microorganisms, was all substantially inhibited. This result indicates the potential practical application of the recombinant peptide in various industrial products.

Action mechanism of PEGylated magainin 2 analogue peptide

PEGylation is frequently used to improve the efficacy of protein and peptide drugs. Recently, we investigated its effects on the action mechanism of the cyclic β-sheet antimicrobial peptide tachyplesin I isolated from Tachypleus tridentatus [Y. Imura, M. Nishida, Y. Ogawa, Y. Takakura, K. Matsuzaki, Action Mechanism of Tachyplesin I and Effects of PEGylation, Biochim. Biophys. Acta 1768 (2007) 1160–1169]. PEGylation did not change the basic mechanism behind the membrane-permeabilizing effect of the peptide on liposomes, however, it decreased the antimicrobial activity and cytotoxicity. To obtain further information on the effects of PEGylation on the activities of antimicrobial peptides, we designed another structurally different PEGylated antimicrobial peptide (PEG-F5W, E19Q-magainin 2-amide) based on the α-helical peptide magainin 2 isolated from the African clawed frog Xenopus laevis. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles, however, the activity was weaker than that of the control peptides. The PEGylated peptide induced lipid flip-flop coupled to the leakage and was translocated into the inner leaflet of the bilayer, indicating that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. The cytotoxicity of the non-PEGylated peptides was nullified by PEGylation. At the same time, the antimicrobial activity was weakened only by 4 fold. The effects of PEGylation on the activity of magainin were compared with those for tachyplesin.

Membrane-Bound Conformation and Topology of the Antimicrobial Peptide Tachyplesin I by Solid-State NMR

The conformation and membrane topology of the disulfide-stabilized antimicrobial peptide tachyplesin I (TP) in lipid bilayers are determined by solid-state NMR spectroscopy. The backbone (phi and psi) torsion angles of Val(6) are found to be -133 degrees and 142 degrees , respectively, and the Val(6) CO-Phe(8) H(N) distance is 4.6 A. These constrain the middle of the N-terminal strand to a relatively ideal antiparallel beta-sheet conformation. In contrast, the phi angle of Gly(10) is +/-85 degrees , consistent with a beta-turn conformation. Thus, TP adopts a beta-hairpin conformation with straight strands, similar to its structure in aqueous solution but different from a recently reported structure in DPC micelles where bending of the two beta-strands was observed. The Val(6) and Gly(10) CO groups are both 6.8 A from the lipid (31)P, while the Val(6) side chain is in (1)H spin diffusion contact with the lipid acyl chains. These results suggest that TP is immersed in the glycerol backbone region of the membrane and is oriented roughly parallel to the plane of the membrane. This depth of insertion and orientation differs from those of the analogous beta-sheet antimicrobial peptide protegrin-1 and suggest the importance of structural amphiphilicity in determining the location and orientation of membrane peptides in lipid bilayers.

Peptide–lipid interactions of the β-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR

The peptide–lipid interaction of a β-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. 31P and 2H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid–peptide interactions between TP-1 and other β-hairpin antimicrobial peptides are discussed in terms of their molecular structure.

An antimicrobial peptide tachyplesin acts as a secondary secretagogue and amplifies lipopolysaccharide‐induced hemocyte exocytosis

In the horseshoe crab, bacterial lipopolysaccharide (LPS) induces exocytosis by granular hemocytes, resulting in the secretion of various defense molecules, such as lectins and antimicrobial peptides, via a G protein-mediating signaling pathway. This response is a key component of the horseshoe crab innate immune response against infectious microorganisms. Here, we report an endogenous amplification mechanism for LPS-induced hemocytes exocytosis. The concentration of LPS required for maximal secretion decreased in proportion to the density of hemocytes, suggesting the presence of a positive feedback mechanism for secretion via a mediator secreted from hemocytes. The exocytosed fluid of hemocytes was found able to induce hemocyte exocytosis in the absence of LPS. Furthermore, tachyplesin, a major antimicrobial peptide of hemocytes, was able to trigger exocytosis in an LPS-independent manner, which was inhibited by a phospholipase C inhibitor, U-73122, and a G protein inhibitor, pertussis toxin. Surface plasmon resonance analysis showed that tachyplesin directly interacts with bovine G protein. These findings suggest that the tachyplesin-induced hemocyte exocytosis also occurs via a G protein-mediating signaling pathway. We concluded that tachyplesin functions not only as an antimicrobial substance, but also as a secondary secretagogue of LPS-induced hemocyte exocytosis, leading to the amplification of the innate immune reaction at sites of injury.

Specific interactions of the antimicrobial peptide cyclic β-sheet tachyplesin I with lipopolysaccharides

The cyclic β-sheet antimicrobial peptide tachyplesin I (T-SS) was found to show 280-fold higher affinity for lipopolysaccharides (LPS) compared with acidic phospholipids, whereas the linear α-helical peptide F5W-magainin 2 (MG2) could not discriminate between LPS and acidic phospholipids. The recognition site was the lipid A moiety and the cyclic structure was crucial to this specific binding. The cyclic structure also endowed the peptide with very rapid outer membrane (OM) permeabilization.