Solution Synthesis,Conformational Analysis,and Antimicrobial Activity of Three Alamethicin F50/5 Analogs Bearing a Trifluoroacetyl Label

We prepared, by solution‐phase methods, and fully characterized three analogs of the membrane‐active peptaibiotic alamethicin F50/5, bearing a single trifluoroacetyl (Tfa) label at the N‐terminus, at position 9 (central region) or at position 19 (C‐terminus), and with the three Gln at positions 7, 18, and 19 replaced by Glu(OMe) residues. To add the Tfa label at position 9 or 19, a γ‐trifluoroacetylated α,γ‐diaminobutyric acid (Dab) residue was incorporated as a replacement for the original Val⁹ or Glu(OMe)¹⁹ amino acid. We performed a detailed conformational analysis of the three analogs (using FT‐IR absorption, CD, 2D‐NMR, and X‐ray diffraction), which clearly showed that Tfa labeling does not introduce any dramatic backbone modification in the predominantly α‐helical structure of the parent peptaibiotic. The results of an initial solid‐state ¹⁹F‐NMR study on one of the analogs favor the conclusion that the Tfa group is a very promising reporter for the analysis of peptaibiotic? membrane interactions. Finally, we found that the antimicrobial activities of the three newly synthesized analogs depend on the position of the Tfa label in the peptide sequence.     

Synthesis and preliminary conformational analysis of TOAC spin‐labeled analogues of the medium‐length peptaibiotic tylopeptin B

A set of analogues of the 14‐residue peptaibol tylopeptin B, containing the stable free‐radical 4‐amino‐1‐oxyl‐2,2,6,6,‐tetramethylpiperidine‐4‐carboxylic acid (TOAC) at one or two selected positions, was synthesized by the solid‐phase methodology. A solution conformational analysis performed by FTIR absorption and CD suggests that, in membrane‐mimicking solvents, the labeled tylopeptin B analogues preserve the helical propensity of the parent peptide, with a preference for the α‐helix or the 3₁₀‐helix type depending upon the nature of the solvent. In aqueous environment, the spin‐labeled analogues present a higher content of helical conformation as a consequence of the strong helix promoter effect of the conformationally constrained TOAC residue. We observed a progressive increase of the quenching effect of the nitroxyl radical on the fluorescence of the N‐terminal tryptophan as TOAC replaces the Aib residue at positions 13, 8, and 4, respectively. A membrane permeabilization assay performed on two selected analogues, TOAC⁸‐ and TOAC¹³‐tylopeptin B, showed that the labeled peptides exhibit membrane‐modifying properties comparable with those of the natural peptaibiotic. We conclude that our TOAC paramagnetic analogues of tylopeptin B are good models for a detailed ESR investigation of the mechanism of membrane permeabilization induced by medium‐length peptaibiotics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Self‐assembling organo‐peptide bolaphiles with KLK tripeptide head groups display selective antibacterial activity

In keeping with recent efforts to generate compounds for antibiotic and microbicide development, we focused on the creation of non‐natural organo‐peptide hybrids of antimicrobial peptide amides (KLK(L)_nKLK‐NH₂) derived from sapecin B and a self‐assembling oligoglycine organo‐peptide bolaphile containing an ω‐amino fatty acid residue. The hybrid organo‐peptide bolaphiles with two cationic KLK tripeptide motifs linked with an ω‐amino acid residue (penta‐, octa‐ or undecamethylene chain) maintained the self‐assembling properties of the root oligoglycine bolaphile. Electron microscopy clearly revealed complex supramolecular architectures for both sapecin B‐derived peptides and the hybrid analogues. FT‐IR spectroscopy indicated that the supramolecular structures were composed primarily of β‐sheets. CD revealed that the hybrid bolaphiles did not share the same secondary structures as the sapecin B peptides in solution. However, although secondary structures of antimicrobial peptides are central in the activity, the organo‐peptide bolaphiles also retained the potent antimicrobial activity of the leader sapecin B‐derived peptide against both Gram‐positive and Gram‐negative bacteria. In general, the hybrids were more selective than the sapecin B peptides, as they displayed little or no appreciable haemolytic activity. The results obtained herald a new approach for the design of purpose‐built hybrid organo‐peptide bolaphiles. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Structure‐activity relationship studies of shortened analogues of the antimicrobial peptide EeCentrocin 1 from the sea urchin Echinus esculentus

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide ( P6 ) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide ( P6 ) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide ( P6 ) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide ( P6 ) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.     

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G¹LLKR⁵IKT⁸LL‐NH₂, it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly¹, Arg⁵, and Thr⁸ and lipophilic groups with different lengths in the N‐terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF⁵IKK⁸LL‐NH₂ exhibited high activity against Gram‐negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N‐terminus increased the antimicrobial activity against Gram‐positive and Gram‐negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α‐helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Deletion of the carboxyl‐terminal residue disrupts the amino‐terminal folding,self‐association,and thermal stability of an amphipathic antimicrobial peptide

Understanding the complex relationship between amino acid sequence and protein behaviors, such as folding and self‐association, is a major goal of protein research. In the present work, we examined the effects of deleting a C‐terminal residue on the intrinsic properties of an amphapathic α‐helix of mastoparan‐B (MP‐B), an antimicrobial peptide with the sequence LKLKSIVSWAKKVL‐NH2. We used circular dichroism and nuclear magnetic resonance to demonstrate that the peptide MP‐B^[1‐13] displayed significant unwinding at the N‐terminal helix compared with the parent peptide of MP‐B, as the temperature increased when the residue at position 14 was deleted. Pulsed‐field gradient nuclear magnetic resonance data revealed that MP‐B forms a larger diffusion unit than MP‐B^[1‐13] at all experimental temperatures and continuously dissociates as the temperature increases. In contrast, the size of the diffusion unit of MP‐B^[1‐13] is almost independent of temperature. These findings suggest that deleting the flexible, hydrophobic amino acid from the C‐terminus of MP‐B is sufficient to change the intrinsic helical thermal stability and self‐association. This effect is most likely because of the modulation of enthalpic interactions and conformational freedom that are specified by this residue. Our results implicate terminal residues in the biological function of an antimicrobial peptide. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

IsCT‐based analogs intending better biological activity

IsCT1‐NH₂ is a cationic antimicrobial peptide isolated from the venom of the scorpion Opisthacanthus madagascariensis that has a tendency to form an α‐helical structure and shows potent antimicrobial activity and also inopportunely shows hemolytic effects. In this study, five IsCT1 (ILGKIWEGIKSLF)‐based analogs with amino acid modifications at positions 1, 3, 5, or 8 and one analog with three simultaneous substitutions at the 1, 5, and 8 positions were designed. The net charge of each analog was between +2 and +3. The peptides obtained were characterized by mass spectrometry and analyzed by circular dichroism for their structure in different media. Studies of antimicrobial activity, hemolytic activity, and stability against proteases were also carried out. Peptides with a substitution at position 3 or 5 ([L]³‐IsCT1‐NH₂, [K]³‐IsCT1‐NH₂, or [F]⁵‐IsCT1‐NH₂) showed no significant change in an activity relative to IsCT1‐NH₂. The addition of a proline residue at position 8 ([P]⁸‐IsCT1‐NH₂) reduced the hemolytic activity as well as the antimicrobial activity (MIC ranging 3.13‐50 μmol L^?1), and the addition of a tryptophan residue at position 1 ([W]¹‐IsCT1‐NH₂) increased the hemolytic activity (MHC = 1.56 μmol L^?1) without an improvement in antimicrobial activity. The analog [A]¹[F]⁵[K]⁸‐IsCT1‐NH₂, which carries three simultaneous modifications, presented increasing or equivalent values in antimicrobial activity (MIC approximately 0.38 and 12.5 μmol L^?1) with a reduction in hemolytic activity. In addition, this analog presented the best resistance against proteases. This kind of strategy can find functional hotspots in peptide molecules in an attempt to generate novel potent peptide antibiotics.     

Afp14 is involved in regulating the length of Anti‐feeding prophage (Afp)

The anti‐feeding prophage (Afp), a phage‐tail‐like particle that causes cessation of feeding in the New Zealand grass grub, Costelytra zealandica, is encoded by 18 open reading frames (afp1–18). C‐terminal truncations of afp14 resulted in shortened Afp particles, suggesting that Afp14 is involved in Afp length determination. We constructed an Afp assembly system (afp1–18), wherein Afp14 was truncated after the N‐terminal 88 residues. This construct, when expressed in trans in Escherichia coli expressing a N‐terminal 98‐amino acid Afp14 construct, yielded fully assembled Afp but no assembled Afp was detected in the case of a N‐terminal 96‐amino acid Afp14 construct. These results suggested that the 98 N‐terminal, amino acid residues of Afp14 is crucial for the initiation of Afp assembly via baseplate formation. Trans‐based expression of wild‐type afp14 resulted in Afp particles of varying lengths, all of which were shorter than the wild‐type Afp particle. On the other hand, similar expression of Afp14 harboring a C‐terminal extension (KLLEH₆) resulted in elongated Afp particles. This information, combined with bioinformatics data, allowed us to propose a model delineating the mechanism and role of Afp14 in the maturation of the Afp particle.     

Bactrocerin‐1: A novel inducible antimicrobial peptide from pupae of oriental fruit fly Bactrocera dorsalis Hendel

A novel antimicrobial peptide, Bactrocerin‐1, was purified and characterized from an immunized dipteran insect, Bactrocera dorsalis. Bactrocerin‐1 has 20 amino acid residues with a mass of 2,325.95 Da. The amino acid sequence of Bactrocerin‐1 showed very high similarity to the active fragment (46V‐65S‐NH₂) of Coleoptericin A. The composition of amino acid residues revealed that Bactrocerin‐1 is a hydrophobic, positively charged, and Lys/Ile/Gly‐rich peptide. Minimal growth inhibition concentration (MIC) measurements for synthesized Bactrocerin‐1 showed a very broad spectrum of anti‐microbial activity against Gram‐positive bacteria, Gram‐negative bacteria, and fungi. Bactrocerin‐1 did not show hemolytic activity toward mouse red blood cells even at a concentration of 50 µM. Analysis of the Helical‐wheel projection and the CD spectrum suggested that Bactrocerin‐1 contains the amphipathic α‐helix. © 2009 Wiley Periodicals, Inc.     

Antimicrobial activity and mechanism of action of a novel cationic α-helical dodecapeptide,a partial sequence of cyanate lyase from rice

CL(14-25), a dodecapeptide, that is a partial region near N-terminus of cyanate lyase (CL, EC 4.3.99.1) from rice (Oryza sativa L. japonica), contains three arginine and two lysine residues. It was a novel cationic α-helical antimicrobial peptide. The antimicrobial activity of CL(14-25) against Porphyromonas gingivalis, a periodontal pathogen, was quantitatively evaluated by a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentration of CL(14-25) against P. gingivalis cells was 145 μM. CL(14-25), even at a concentration of 800 μM, had no hemolytic activity. When giant unilamellar vesicles (GUVs) that mimic the membrane composition of Gram-negative bacteria were used, microscopy image analysis suggested that CL(14-25) disrupted GUVs in a detergent-like manner. Therefore, CL(14-25) appears to exhibit antimicrobial activity through membrane disruption. To investigate the contribution of cationic amino acid residues in CL(14-25) to its antimicrobial activity, we synthesized four truncated CL analogs, in which one or two cationic amino acid residues were deleted from the N- and C- termini of CL(14-25). The degrees of calcein leakage from large unilamellar vesicles (LUVs) and 3,3′-dipropylthiadicarbocyanine iodide (diSC₃-5) release from P. gingivalis cells induced by truncated CL analogs were closely related to their antimicrobial activities. CL analogs, which were truncated by removing an arginine residue from the N-terminus and a lysine residue from the C-terminus maintained their antimicrobial activity. However, CL analogs, which were further truncated by removing two arginine residues from the N-terminus, and an arginine and a lysine residue from the C-terminus, rarely exhibited antimicrobial activity.     

A novel defensin‐like peptide from salivary glands of the hard tick,Haemaphysalis longicornis

A novel defensin‐like antimicrobial peptide named longicornsin was isolated from the salivary glands of the hard tick, Haemaphysalis longicornis, using a 10‐kDa cut‐off Centriprep filter and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). Its amino acid sequence was determined as DFGCGQGMIFMCQRRCMRLYPGSTGFCRGFRCMCDTHIPLRPPFMVG by Edman degradation. The cDNA encoding longicornsin was cloned by cDNA library screening. The predicted protein from the cDNA sequence was composed of 78 amino acids including a mature longicornsin. It showed similarity with defensin‐like peptides from other ticks by BLAST search. Different from most other tick defensin‐like peptides, longicornsin had a C‐terminal extension. Purified longicornsin exerted potent antimicrobial activities against bacteria and fungi. Interestingly, it even showed strong antimicrobial ability against drug‐resistant microorganisms and Helicobacter pylori. The results of this study indicated that longicornsin is a potential candidate for novel antimicrobial drug design.     

The physiological target for LeuRS translational quality control is norvaline

The fidelity of protein synthesis depends on the capacity of aminoacyl‐tRNA synthetases (AARSs) to couple only cognate amino acid‐tRNA pairs. If amino acid selectivity is compromised, fidelity can be ensured by an inherent AARS editing activity that hydrolyses mischarged tRNAs. Here, we show that the editing activity of Escherichia coli leucyl‐tRNA synthetase (EcLeuRS) is not required to prevent incorrect isoleucine incorporation. Rather, as shown by kinetic, structural and in vivo approaches, the prime biological function of LeuRS editing is to prevent mis‐incorporation of the non‐standard amino acid norvaline. This conclusion follows from a reassessment of the discriminatory power of LeuRS against isoleucine and the demonstration that a LeuRS editing‐deficient E. coli strain grows normally in high concentrations of isoleucine but not under oxygen deprivation conditions when norvaline accumulates to substantial levels. Thus, AARS‐based translational quality control is a key feature for bacterial adaptive response to oxygen deprivation. The non‐essential role for editing under normal bacterial growth has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site.     

Synthesis,biological activity and solution structure of new analogues of the antimicrobial Gramicidin S

Gramicidin S (GS) is a cyclo‐decapeptide antibiotic isolated from Bacillus brevis. The structural studies have shown that GS forms a two‐stranded antiparallel β‐sheet imposed by two II′ β‐turns. Despite its wide Gram+ and Gram? antimicrobial spectrum, GS is useless in therapy because of its high hemotoxicity in humans. It was found, however, that the analogues of GS‐14 (GS with 14 amino acid residues) attained a better antimicrobial selectivity when their amphipatic moments were perturbed. In this study, we report effects of similar perturbations imposed on GS cyclo‐decapeptide analogues. Having solved their structures by NMR/molecular dynamics and having tested their activities/selectivities, we have concluded that the idea of perturbation of the amphipatic moment does not work for GS‐10_0 analogues. An innovative approach to the synthesis of head‐to‐tail cyclopeptides was used. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A novel lipid transfer protein from the dill Anethum graveolens L.: isolation,structure, heterologous expression,and functional characteristics

A novel lipid transfer protein, designated as Ag‐LTP, was isolated from aerial parts of the dill Anethum graveolens L. Structural, antimicrobial, and lipid binding properties of the protein were studied. Complete amino acid sequence of Ag‐LTP was determined. The protein has molecular mass of 9524.4 Da, consists of 93 amino acid residues including eight cysteines forming four disulfide bonds. The recombinant Ag‐LTP was overexpressed in Escherichia coli and purified. NMR investigation shows that the Ag‐LTP spatial structure contains four α ‐helices, forming the internal hydrophobic cavity, and a long C‐terminal tail. The measured volume of the Ag‐LTP hydrophobic cavity is equal to ~800 A³, which is much larger than those of other plant LTP1s. Ag‐LTP has weak antifungal activity and unpronounced lipid binding specificity but effectively binds plant hormone jasmonic acid. Our results afford further molecular insight into biological functions of LTP in plants. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Melectin MAPs: the influence of dendrimerization on antimicrobial and hemolytic activity

The recently described antimicrobial peptide melectin (MEP, GFLSILKKVLPKVMAHMK-NH₂) exhibits high antimicrobial activity against Gram-positive and Gram-negative bacteria. Here we describe the synthesis and biological activities of 23 new analogues of MEP. We studied the influence of dimerization and tetramerization (MAP-constructs of MEP) on the antimicrobial and hemolytic activities, as well as the role of Met in positions 14 and 17 of the peptide chain. Oxidation of the Met to Met(O) and Met(O₂) decreases antimicrobial activity of all tested bacteria if the peptide is in the monomeric form, however, only to Staphylococcus aureus if in the form of dimer or tetramer. Dimerization and tetramerization increase the undesirable hemolytic activity of the peptides. Interestingly, substitution of Leu for Val in position 6 leads to the decrease of hemolytic activity. Introduction of the isosteric amino acid Nle into positions 14 or 17 or both leads to slight increase of hemolytic activity under preservation of high antimicrobial activities. Unfortunately, dimerization again leads to an increase of hemolytic activity.     

Peptaibol,Secondary‐Metabolite,and Hydrophobin Pattern of Commercial Biocontrol Agents Formulated with Species of the Trichoderma harzianum Complex

The production of bioactive polypeptides (peptaibiotics) in vivo is a sophisticated adaptation strategy of both mycoparasitic and saprotrophic Trichoderma species for colonizing and defending their natural habitats. This feature is of major practical importance, as the detection of peptaibiotics in plant‐protective Trichoderma species, which are successfully used against economically relevant bacterial and fungal plant pathogens, certainly contributes to a better understanding of these complex antagonistic interactions. We analyzed five commercial biocontrol agents (BCAs), namely Canna^®, Trichosan^®, Vitalin^®, Promot^® WP, and TrichoMax^®, formulated with recently described species of the Trichoderma harzianum complex, viz. T. afroharzianum, T. simmonsii, and T. guizhouense. By using the well‐established, HPLC/MS‐based peptaibiomics approach, it could unequivocally be demonstrated that all of these formulations contained new and recurrent peptaibols, i.e., peptaibiotics carrying an acetylated N‐terminus, the C‐terminus of which is reduced to a 1,2‐amino alcohol. Their chain lengths, including the amino alcohol, were 11, 14, and 18 residues, respectively. Peptaibols were also to be the dominating secondary metabolites in plate cultures of the four strains obtained from four of the Trichoderma‐ based BCAs, contributing 95% of the UHPLC‐UV/VIS peak areas and 99% of the total ion count MS peak area from solid media. Furthermore, species‐specific hydrophobins, as well as non‐peptaibiotic secondary metabolites, were detected, the latter being known for their antifungal, siderophore, or plant‐growth‐promoting activities. Notably, none of the isolates produced low‐molecular weight mycotoxins.     

Regulatory mechanisms of SNAT2, an amino acid transporter,in L6 rat skeletal muscle cells by insulin,osmotic shock and amino acid deprivation

Several studies have demonstrated that the activity of system A is upregulated by insulin, osmotic shock and amino acid deprivation. However, the mechanisms are not clear. We carried out studies using L6 rat skeletal muscle cells to clarify the mechanisms of upregulation of system A activity by insulin, osmotic shock and amino acid deprivation. The upregulation was found to be due to an increase in V _max, not K _m. Chloroquine and wortmannin inhibited the upregulation induced by insulin stimulation and amino acid deprivation but not that induced by osmotic shock. On the other hand, cycloheximide and actinomycin D inhibited the upregulation by each stimulation. Moreover, PD98059 and SP600125 inhibited only amino acid deprivation-induced upregulation and SB202190 inhibited only insulin-induced upregulation. Our findings indicate that the mechanisms of upregulation of system A activity by insulin, osmotic shock and amino acid deprivation are different in L6 cells. Western blot and RT-PCR analysis showed an increase in system A at the protein and mRNA levels with each stimulation.